In situ activation of therapeutics through bioorthogonal catalysis

Abstract

Bioorthogonal chemistry refers to any chemical reactions that can occur inside of living systems without interfering with native biochemical processes, which has become a promising strategy for modulating biological processes. The development of synthetic metal-based catalysts to perform bioorthogonal reactions has significantly expanded the toolkit of bioorthogonal chemistry for medicinal chemistry and synthetic biology. A wide range of homogeneous and heterogeneous transition metal catalysts (TMCs) have been reported, mediating different transformations such as cycloaddition reactions, as well as bond forming and cleaving reactions. However, the direct application of ‘naked’ TMCs in complex biological media poses numerous challenges, including poor water solubility, toxicity and catalyst deactivation. Incorporating TMCs into nanomaterials to create bioorthogonal nanocatalysts can solubilize and stabilize catalyst molecules, with the decoration of the nanocatalysts used to provide spatiotemporal control of catalysis. This review presents an overview of the advances in the creation of bioorthogonal nanocatalysts, highlighting different choice of nano-scaffolds, and the therapeutic and diagnostic applications.

Graphical abstract

1. Introduction

Bioorthogonal chemistry refers to reactions that can occur in living systems without interfering with native bioprocesses, which offers a versatile approach for modulating biological systems.^1,2,3 Bioorthogonal catalysis enables the generation of imaging and therapeutic agents dynamically from inactive components within the living cell.⁴ This activation of prodrugs and pro-imaging agents using bioorthogonal catalysis is particularly promising for biomedical applications, opening new pathways for treating diseases through reactions that native enzymes cannot catalyze.^4,5

Transition metal catalysts (TMCs) are excellent candidates for bioorthogonal catalysis due to their high catalytic efficiency, a key requirement for in situ generation of therapeutics.⁶ This in situ activation can provide the spatial and temporal control of therapeutic activation, minimizing off-target effects.⁷ However, the use of “naked” TMCs for activation of prodrugs faces several challenges, including low water solubility, low biocompatibility, and poor catalytic stability in the biological environment.⁸ Engineering of the metal complexes provides one strategy to address these issues, however, protection and controlled delivery of TMCs remain a significant challenge.

TMCs can be incorporated into nanomaterial scaffolds to generate bioorthogonal nanocatalysts that enhance catalyst stability and biocompatibility.⁸ Focusing on the integrated properties, these nanocatalysts have the potential to be functionalized as bioorthogonal nanozymes when recapitulating key enzymatic aspects, such as Michaelis-Menten kinetics or allosteric regulation.^4,9,10,11 Appropriate design of nanomaterial can be used to regulate cell penetration,¹² provide endogenous/exogenous stimuli responsiveness,^13,14,15,16 and localize catalysis at therapeutically important targets.^17,18,19 Clearly, these bioorthogonal nanocatalysts provide the capability to serve as in situ “drug factories” that continuously generate drugs at target sites within complex microenvironments.

In this review, we focus on the development of TMC-based nanocatalysts for biomedical applications. Since bioorthogonal catalysis allows for the highly controlled generation of therapeutics, it represents an alternative approach for dynamic drug delivery and expands the possibilities for applying different drug delivery strategies. We will discuss key progress in the in situ activation of therapeutics and imaging agents through metal-catalyzed bioorthogonal reactions, and explore the interplay between TMCs and nanomaterials in the development of nanocatalysts. We also emphasize in vitro and in vivo applications of nanocatalysts, and finally discuss both the promise and challenges of these bioorthogonal systems for biomedical applications.

2. Copper-catalyzed bioorthogonal reactions

The Cu (I)-catalyzed [3+2] azide-alkyne cycloaddition (CuAAC) ‘click’ reaction has had a profound impact on the development of organic synthesis,^20,21,22,23 material science,^24,25,26,27 as well as chemical biology^28,29 since being discovered by Sharpless and Meldal et al.^30,31 Currently, the CuAAC reaction is the most broadly employed reaction in click chemistry due to the short reaction times and high yields under mild (physiological) conditions.³² In biomedical applications, this reaction has great potential in target identification,^33,34,35 drug discovery,^36,37,38 and drug delivery.^{39,40,41,42,43} However, due to the inherent toxicity of Cu ions,^44,45 it is essential to develop safer Cu-based catalytic systems. Immobilization of Cu in/on to nanomaterials slows the nonspecific release of Cu ions reducing acute toxicity.⁴⁶

2.1. Cu-mediated cycloaddition reaction

In situ conversion of Cu (I) from Cu (0) biocompatible nanoparticles has been widely used in CuAAC. In a recent research, Qu et al. grew Cu (0) nanoparticles on mesoporous carbon nanospheres (MCNs) to provide CuAAC nanocatalysts (Figure 2A).⁴⁷ Under the near-infrared (NIR) irradiation, MCNs induced the generation of reactive oxygen species (ROS) that converted Cu (0) into Cu (I), thereby generating active catalyst (Figure 2, A and B). Meanwhile, the photothermal property of MCNs allowed a higher local temperature, further accelerating the catalytic process. They applied this nanocatalyst for in situ synthesis of resveratrol analogue (Figure 2C), obtaining promising antitumor efficacy with the assistance of NIR irradiation (Figure 2, D and E). Similarly, Zhang et al. doped Cu (0) nanoparticles into cross-linked lipoic acid nanoparticles to prepare morphology-changeable, biocompatible Cu nanocatalysts.⁴⁸ Metal-organic framework (MOF) systems provide an alternative way to stabilize and protect Cu catalysts.⁴⁹ Qu et al. anchored ultrasmall Cu (0) nanoparticles throughout a MOF scaffold and decorated the material with mitochondria-targeted triphenyl phosphonium (TPP) vector (Figure 3A). After endocytosis by breast cancer Michigan Cancer Foundation-7 (MCF-7) cells and relocation in mitochondria, this nanocatalyst assembled the two inactive precursors, and in situ synthesized the antitumor drug resveratrol, which induced apoptosis through mitochondrial damage (Figure 3B). In the Hepatoma 22 mice model, this heterogeneous MOF-Cu catalyst had good biocompatibility and efficient antitumor efficacy.

Figure 2.

(A) The construction of MCNs-Cu nanocatalyst. (B) Catalysis by MCNs-Cu with or without NIR irradiation. (C) In situ synthesis of the resveratrol analogue with NIR irradiation. (D) The change of tumor volumes and (E) photos of excised tumors after different treatment and control conditions. (Reprinted with permission from ref. 47. Copyright 2020, American Chemical Society.)

Figure 3.

(A) Construction of a biocompatible heterogeneous MOF-Cu catalyst. (B) In situ drug synthesis in mitochondria via the CuAAC reaction. (Reprinted with permission from ref. 49. Copyright 2019, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.) (C) Synthesis of a bifunctional molecule for disintegration of Aβ aggregates and chelation of Cu ions. (D) In situ synthesis of the bifunctional molecule for disassembly of Aβ-Cu aggregates. (Reprinted with permission from ref.57. Copyright 2019, The Royal Society of Chemistry.)

Instead of oxidation, reduction from Cu (II) to Cu (I) provides an alternative strategy. Hydrazine has been applied to reduce Cu (II) due to the high reduction activity and convenience. Bradley et al. developed heterogeneous and highly biocompatible microcatalysts (E-Cu-NPs) by entrapping hydrazine-reduced Cu nanoparticles in TentaGel resin.⁵⁰ Using this microcatalyst, they first reported in situ extracellular synthesis of the triazole-containing combretastatin A4 analogue via CuAAC click reaction. The E-Cu-NPs drove the conjugation of two inactive precursors to produce the active substance that induced the apoptosis⁵¹ of ovarian adenocarcinoma SKOV-3 cells. These E-Cu-NPs were tested in vivo by implanting them into zebrafish, maintaining efficient kinetics and reduced toxicity.⁵⁰

Apart from hydrazine, ascorbate is another popular reducing agent. Zimmerman and coworkers developed series of Cu-containing single-chain polymer nanoparticles for CuAAC. Cu was covalently attached on the polymer backbone and reduced by ascorbate for catalysis. Polymeric scaffolds provide biocompatibility to Cu and mimic the structure of natural enzymes. Using these platforms, they established enzyme-like kinetics,⁵² substrate selectivity,⁵³ live cell imaging,⁵⁴ antimicrobial activation,⁵⁵ and anticancer drug screening.⁵³ Copper source for CuAAC can be exogenous or endogenous. Qu et al. reported a DNAzyme-encapsulated exogenous Cu/Zn bimetallic MOF for synergistic chemo-gene therapy.⁵⁶ Cu(II)-doped zeolitic imidazolate framework-8 (Cu/ZIF-8) not only protected the DNAzyme from premature degradation (DNAzyme@Cu/ZIF-8), but also had an acid-responsive disassembly behavior for intracellular release of these three components. Cu(II) was reduced by sodium ascorbate to catalyze synthesis of the resveratrol analogue for chemotherapy, while Zn(II) functioned as the cofactor of DNAzyme to trigger the gene therapy. Distinct inhibition of tumor growth and metastasis was observed in vivo, confirming the therapeutic potential of this combinational therapy. Besides exogenous Cu source, they also utilized endogenous Cu(II) ions to assemble drug molecules for the Alzheimer’s disease (AD) treatment.^57,58 Based on amyloid-β (Aβ) aggregation and Cu dysregulation in AD,^59,60 they designed a bifunctional molecule consisting of azide-thioflavin T (yellow quarter circle) and propargylglycine (red quarter circle) (Figure 3C). In this design, the thioflavin T unit selectively photo-oxygenated Aβ aggregates for disintegration, while the triazole-glycine structure served as the Cu chelator to prevent Aβ-Cu coordination. These two segments and reductive ascorbic acid were co-loaded into H₂O₂-responsive mesoporous silica nanoparticles (MSN-IgG). Intracellular Aβ-Cu aggregate-induced H₂O₂ promoted release of cargoes, initiating the assembly of drug molecules catalyzed by Cu accumulated in Aβ plaques. Therefore, by Cu chelation and UV light-triggered oxygenation, the synthesized drug effectively disintegrated Aβ-Cu aggregates, and neurotoxicity was mitigated (Figure 3D). Through the above synergetic mechanism, the bifunctional molecule exerted its protective effect in pheochromocytoma PC12 cells, a transgenic AD model of Caenorhabditis elegans CL2006, and ex vivo brain slices of triple transgenic AD mice model.

In conclusion, the classic CuAAC has been developed from test tubes to biological systems owing to the protection and enhanced biocompatibility of nano-scaffolds. Currently, the application of CuAAC has been expended to bioimaging and therapeutic activation for Alzheimer’s disease and cancer treatments. Considering high efficiency and relatively low toxicity of copper, the CuAAC has the potential for other biomedical applications, such as wounding healing and autoimmune diseases.

2.2. Cu-mediated bond cleavage

Copper is capable of catalyzing both cycloaddition and cleavage reactions in bioorthogonal catalysis. Recently, Chen and coworkers⁶¹ reported a free catalyst Cu(I)-triggered bioorthogonal reaction for extracellular linker cleavage. The linker was used on antibody-drug conjugates (ADCs) and genetically encoded protein, thus the cleavage enabled the release therapeutics right from targeting antibodies and reversible cell modification. They established a high-throughput screening platform to systematically study the reactivity between different metal catalysts and disubstituted propargyl coumarin derivatives. Based on this platform, two new bioorthogonal cleavage pairs (catalysis/cleavage functional group) were identified, i.e. Cu(I)-BTTAA/dual-substituted propargyloxycarbonyl (dspoc) for amines and Cu(I)-BTTAA/dual-substituted propargyl (dspra) for phenols, respectively. The bioorthogonal feasibility of these two pairs was further confirmed in prodrug activation, reversible cell surface modification, and modulation of receptor-ligand interactions on cell membranes.

3. Ruthenium-catalyzed bioorthogonal reactions

Ruthenium catalysts are versatile due to their wide scope of stable oxidation states (−II to +VIII) and control of catalytic processes through ligand geometry.^62,63 Additionally, some ruthenium catalysts maintain high catalytic activity in aqueous media^64,65 and resist deactivation by thiols.^66,67,68

3.1. Ru-mediated bond cleavage

In 2006, the Meggers group reported the use of a free ruthenium catalyst in bioorthogonal catalysis.⁶⁹ The commercially available catalyst Cp*Ru(COD)CI (Cp* = pentamethylcyclopentadienyl, COD = 1,5-cyclooctadiene) efficiently cleaved allylcarbamate protected rhodamine to release fluorophore in cell lysate and living cells. In further studies, Meggers and coworkers designed 2-quinolinecarboxylate and 8-hydroxyquinoline derivatives as ligands to ruthenium catalysts.^70,71 These catalysts exhibited high catalytic activity under biological conditions, and free catalysts were used to uncage allylcarbamate-protected doxorubicin (Dox) for chemotherapy in HeLa cells. Hsu et al. used free 2-quinolinecarboxylate-Ru catalysts to activate luciferin in luciferase-expressed 4T1 breast cancer cells for visualization of bioluminescence.⁷² Based on these prior designs and applications, Mascareñas and coworkers attached mitochondria targeting element on 2-quinolinecarboxylate-Ru catalysts to accomplish efficient bioorthogonal catalysis in mitochondria.⁷³

Okamoto et al. incorporated Ru catalyst into the streptavidin scaffold to protect catalyst against detrimental cellular components.⁷⁴ Biotin has a strong affinity to streptavidin, thus, researchers linked biotin to 2-quinolinecarboxylate-Ru catalysts for the successful incorporation. They also attached a biotinylated cell penetrating peptide to enhance cellular uptake. As a result, the artificial metalloenzymes (ArM, protein-catalyst complex) performed intracellular activation of a thyroid hormone, leading to the expression of secreted nanoluc, a potent bioluminescence reporter. This research showed the potential of bioorthogonal catalysis in modulating cellular behavior.

The versatility and utility of Ru in bioorthogonal catalysis was further extended by the incorporation of catalysts into nanoparticle scaffolds. Rotello et al. encapsulated Ru catalysts inside the monolayer of small (2nm core) gold nanoparticles (AuNPs) to enhance their stability in biological environments. The resulting nanoreactors could catalyze bioorthogonal reactions and followed Michaelis-Menten enzymatic kinetics, thus named as bioorthogonal nanozymes. Supramolecular decoration of these nanozymes was used to impart stimuli-responsive behavior.¹⁶ AuNPs were decorated with functional monolayers, which included three structural units: a hydrophobic alkane chain on the inside for encapsulation of Ru catalysts, a hydrophilic tetra(ethylene glycol) spacer to improve biocompatibility, and a dimethylbenzylammonium group for binding with curcubit[7]uril (CB[7]) through host-guest interactions (Figure 4 A–C). The structure of nanozyme completely blocked the catalytic process between substrate and catalyst by steric hindrance of CB[7] complexed to the AuNP ligand (Figure 4B). However, after the addition of 1-adamantylamine (ADA) as a competitive guest, CB[7] was dissociated from the particle surface, and substrates could access the catalyst and undergo catalytic reaction (Figure 4 C). By encapsulating Ru or Pd catalyst into this nanozyme, they achieved controllable bioorthogonal activation of bis-allyloxycarbonyl-protected rhodamine 110 (Ru, Figure 4 A to C) and propargyl-caged 5FU (Pd, Figure 4 E and F) in living cells for potential applications in imaging and therapeutics.

Figure 4.

(A) Intracellular catalysis by bioorthogonal nanozymes. (B) Inhibition of catalytic activity by CB[7]-induced steric hindrance. (C) Recovery of nanozymed activity after addition of competitive ADA. (D) Cell viability of 5- fluorouracil (5FU) and the prodrug. (E) Intracellular activation of 5FU using stimuli-responsive bioorthogonal nanozymes.

In further research, Rotello decorated AuNP nanozymes with pH-sensitive groups to generate switchable nanozymes for imaging of bacterial biofilms (Figure 5A).¹⁸ The alkoxyphenylacyl sulfonamide-functionalized nanozyme (pKa ~ 6.5, Figure 5B) was constructed by encapsulating Ru catalyst into the hydrophobic ligand layer on the AuNP surface. In acidic microenvironments such as those found in bacterial biofilms these nanozymes were protonated, with a concomitant change in surface charge from neutral (zwitterionic) to cationic. In this protonated cationic form, these particles interacted strongly with negatively-charged bacterial biofilms. Selectivity of the nanozymes towards the biofilms was established using a bacterial biofilm-mammalian cell co-culture model, where high selectivity was observed for activation of pro-fluorophore for the biofilm relative to fibroblasts. However, the catalytic activity of Ru catalysts was not high enough to generate antibiotic for bacteria killing. The ability to control nanozyme localization in cellular systems by surface charge was further demonstrated through programmed intra- or extra-cellular bioorthogonal catalysis (Figure 5C).¹⁷ Positively charged nanozymes were uptaken into cells while zwitterionic sulfobetaine moieties remained in extracellular space. These two Ru catalyst nanozymes provided activation of pro-fluorophores and allylcarbamate-caged Dox inside and outside cells, respectively (Figure 5D) The result emphasized the importance of intracellular localization of nanozymes due to enhanced therapeutic efficacy by bioorthogonal catalysis.

Figure 5.

(A) and (B) pH-switchable nanozymes for biofilm imaging. (Reprinted with permission from ref. 18. Copyright 2018, American Chemical Society.) (C) Schematic presentation of intracellular catalysis and extracellular catalysis through engineering the surface functionalization of bioorthogonal nanozymes. (D) Pro-Dox activation by surface engineered bioorthogonal nanozymes. (Reprinted with permission from ref. 17. Copyright 2019, American Chemical Society.) (E) and (F) Intracellular activation of bioorthogonal nanozymes through the endosomal proteolysis process. (Reprinted with permission from ref. 15. Copyright 2020, American Chemical Society.)

The selective activation of therapeutics within cells provides a potential strategy for minimizing off target effects.¹⁷ In a recent work, Rotello made use of endosomal proteolysis to realize reactivation of intracellular nanozymes (Figure 5E).¹⁵ Specifically, hard corona formation induced nanozyme aggregation and caused complete loss of activity, while soft corona did not lead to aggregation and only partially inhibited the nanozyme activity. After internalization by cells, activity of both hard and soft corona-coated nanozymes was restored through endogenous protease-mediated protein corona degradation in endosomes/lysosomes(Figure 5F). More recently, polymer-based ‘polyzymes’ that used polymeric self-assemblies as scaffolds to protect Ru catalysts were shown to resist deactivation from serum and activate anticancer drug mitoxantrone in cells.¹²

3.2. Olefin metathesis

Grubbs catalysts, popular in organic chemistry, have been attractive in chemical biology for protein modification.⁷⁵ However, the biological thiols poison catalysts and hamper their direct use in bioorthogonal catalysis. Ward and Panke reported the protection of biotinylated Hoveyda–Grubbs second-generation catalyst by streptavidin. The resulting ArM performed ring-closing metathesis leading to the generation of fluorophore both in tube and in living bacteria. They also engineered the protein scaffold to establish the substrate preference.^76,77

Instead of using streptavidin, Tanaka et al. utilized engineered albumin to protect Grubbs catalysts.⁷⁸ The catalyst was anchored by the interaction of coumarin with albumin, and the resulting ArM resisted to glutathione poisoning. The scientists then attached complex N-glycans on ArM (to obtain GArM) for efficient cellular uptake. Thus, GArM entered cells and in situ activated fluorophore through ring-closing metathesis. For therapeutic applications, both ArM and GArM activated an umbelliprenin prodrug, leading to the death of cancer cells. Interestingly, GArM showed much more effective cell killing, which indicated intracellular prodrug activation led to a higher therapeutic effect. Recently, the bioorthogonality of free Ru catalysts has been expanded to include cycloaddition^79,80,81 and isomerization.⁸². These systems have good catalytic efficiency, and are awaiting demonstration of therapeutic potential.

4. Palladium-catalyzed bioorthogonal reactions

Palladium is one of the most versatile metals for TMC-driven chemistry,⁸³ effectively mediating both bond forming and bond cleavage reactions. ⁸⁴ Examples of the former include the use of Pd-catalyzed Suzuki-Miyaura reaction for protein modification in 2009 by Davis and coworkers,.⁸⁵ Later, Lin et al. used Pd-mediated Sonogashira reaction to functionalize alkyne-encoded proteins in both aqueous solution and bacteria.⁸⁶

4.1. Pd-mediated bond cleavage

Depropargylation is perhaps the most widely used Pd-mediated decaging reaction. Unciti-Broceta et al. have reported a series of Pd-catalyzed reactions that lead to drug release based on depropargylation intracellularly or extracellularly. The model drugs (gemcitabine,⁸⁷ 5-fluorouracil,⁸⁸ floxuridine,⁸⁹ and vorinostat⁹⁰) were caged with different propargyl-bearing moieties on the pharmacophore^91,92 to form inactive precursors (Figure 6, A). Biocompatible Pd(0)-functionalized resins were prepared to trigger cleavage reactions, thus effectively releasing the active compounds for antitumor effects. Similarly, in 2018, they reported the activation of Dox prodrug by Pd microcatalysts with tunable size and activity.⁹³ Optimized Pd catalysts were precisely guided to the tumor site by ultrasound (Figure 6B) in the prostatic cancer mice model, providing localized cancer therapy to minimize off-target effects of chemotherapy. They also adapted this strategy to bioorthogonally activate camptothecin derivatives (Figure 6C).⁹⁴ The active metabolite of irinotecan (SN-38) was caged at C₁₀ hydroxyl to mask its cytotoxicity, which restored its antitumor activity through heterogeneous Pd catalysts against colorectal carcinoma HCT116 and glioma U87/U251 cells (Figure 6C). Combined with their previous work on 5FU prodrug,⁸⁸88 dual prodrug activation with the same bioorthogonal microcatalyst resulted in synergistic anticancer activity in vitro.

Figure 6.

(A) Pd-catalyzed decaging reactions for antitumor prodrug activation, including 5-fluorouracil, floxuridine, gemcitabine, and vorinostat. Reprinted with the permission from Ref. 87. Copyright 2014, Springer Nature. (B) In situ activation of Dox through ultrasound-guided Pd devices. Reprinted with permission from Ref. 93. Copyright 2018, The Royal Society of Chemistry (C) Activation of pro-SN-38 by Pd resins for antitumor therapy. Reprinted with the permission from Ref. 94. Copyright 2018, Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim.

In a recent report, Unciti-Broceta’s group prepared Pd-loaded exosomes (Exo) for intracellular prodrug activation.¹⁹ Cancer-derived exosomes Exo^A549 were obtained from non-small cell lung carcinoma A549 cell culture that showed selective uptake by cancer cells. After purification, Pd²⁺ catalysts were loaded into these exosomes to form Pd²⁺-Exo^A549. Subsequently, CO reduced Pd²⁺ to Pd⁰ to obtain Pd-Exo^A549. Panobinostat, a histone deacetylase (HDAC) inhibitor, was selected as a model drug to validate the catalytic properties of Pd-Exo^A549. Cell experiments revealed that these modified exosomes exhibited preferential tropism for their progenitor cells and activated panobinostat, thereby realizing selectivity for tumor cells and in situ drug production.

Mascareñas stabled Pd catalyst by histidine residue on the peptide. The peptide-Pd complex activated pro-fluorophore in living cells. They further engineered integrin-targeting motif RGD (Arg-Gly-Asp) peptide with di-histidine on each side for Pd chelation. The modifted-RGD-Pd maintained cell type selectivity while performing bioorthogonal catalysis.⁹⁵ This research broadens the scaffold design with inherent imidazole functional group for Pd stabilization. Very recently, Mascareñas in collaboration of del Pino reported the application of ZIF-8 to protect Pd nanocubes for bioorthogonal catalysis. They successfully activated imaging agents in living cells and in 3D tumor spheroids.⁹⁶

In 2017, Weissleder et al. designed highly efficient palladium catalysts by screening complexes formed between Pd and electron-poor phosphine ligands. They created a biocompatible Pd nanocatalyst (Pd-NP) by encapsulating the identified Pd(II)-containing (bis[tri(2-furyl)phosphine] palladium (II) dichloride, PdCl₂(TFP)₂) into poly(lactic-co-glycolic acid)-polyethyleneglycol (PLGA-PEG) nanoparticles.⁹⁷ In vivo studies proved these Pd-NPs could effectively accumulate at the tumor sites via the EPR effect, and locally activated a doxorubicin prodrug to suppress tumor growth. However, the antitumor efficacy was limited because of insufficient activition of the prodrug that required higher dosing, and the relatively poor encapsulation efficiency of prodrug in PLGA-PEG NPs. To overcome these limitations, they next designed a modular nanoplatform for local prodrug activation inside the tumor.⁹⁸ An analogue of 4-aminomandelic acid was functionalized as a three-branched self-immolative linker (SIL) that combined a Pd-cleavable allyloxycarbonyl (Alloc) group, a nanoencapsulation anchor (C₁₆), and a drug molecule (Monomethyl auristatin E, MMAE, or Dox) together (Figure 7B). Introduction of the aliphatic anchor greatly facilitated the encapsulation efficiency of the prodrug into the PLGA-PEG polymeric micelle to form prodrug-NPs. In the presence of Pd-NP, the Alloc caging group on the trigger was cleaved, leading to the drug release from self-immolative linker (Figure 7A). Significantly, the Pd-activated MMAE showed comparable efficacy to free drug MMAE (Figure 7C). The combination of prodrug-NP and Pd-NP was confirmed to safely and effectively inhibit tumor growth (Figure 7, D and E), and this antitumor efficacy was further enhanced by local radiation therapy (Figure 7F). The biodegradable platform (PLGA-PEG) has potential to reduce the toxicity from concomitant accumulation of Pd, but this hypothesis is yet to be proven.

Figure 7.

(A) Modular nanoplatform for Pd-catalyzed prodrug activation. (B) Chemical structures of MMAE and Dox prodrug. (C) In vitro cytotoxicity of MMAE prodrug with different concentrations of Pd-NP. The antitumor efficacy of MMAE prodrug and Pd-NP in (D) HT1080 and (E) MC38 tumor models. (F) The enhanced antitumor efficacy with assistance of local tumor irradiation in HT1080 tumor model. (Reprinted with permission from ref. 98. Copyright 2018, American Chemical Society.)

Focusing on molecular targeting of bioorthogonal catalysis, Bernardes and coworkers designed a Pd-cleavable bifunctional linker containing thioether for protein modification and propargyl carbamate for small-molecule conjugation.⁹⁹ Using this strategy, the authors were able to improve the water solubility of prodrugs by functionalizing PEG on the thioether terminus of the linker (Figure 8A). With the addition of Pd complex (Pd(COD)Cl₂), this prodrug was decaged efficiently and released free Dox to kill human embryonic kidney (HEK) 293 cells. Significantly, the thioether moiety could also be used to conjugate prodrugs to antibodies for generating ADCs. (Figure 8B).¹⁰⁰ In the presence of the extracellular Pd catalyst, this ADC displayed comparable cytotoxicity to free Dox in HER2-positive human breast adenocarcinoma MCF-7 cells, indicating the successful decaging for Dox activation.

Figure 8.

(A) Pd-catalyzed Dox release from Dox-PEG conjugate. (B) The design of Pd-cleavable ADC. (Reprinted with permission from ref. 99. Copyright 2018, The Royal Society of Chemistry.) (C) The reaction between Pd ligand and bifunctional molecule for imaging and tumor therapy.

The ability of bioorthogonal catalysis to generate both therapeutics and imaging agents provides a versatile platform for theranostic applications.¹⁰¹ Wu et al. generated a prodrug (Pro-Cou-NCl) consisting of a propargyl-modified coumarin derivative for in situ imaging and nitrogen mustard (NCl) for chemotherapy. In the presence of Pd catalysts (Pd-DPPF), the prodrug released coumarin and NCl simultaneously (Figure 8C). Both Pd catalysts and substrates were loaded into liposomes separately and injected at tumor site intratumorally. Once inside tumor cells, the prodrug and Pd catalyst were released, thus triggering the decaging process. The Pd-DPPF catalyzed the cleavage of propargyl to trigger subsequent reaction. This strategy efficiently inhibited tumor growth and provided in situ tumor imaging.

Embedding Pd into mesoporous silica nanoparticles (MSNs) not only enhanced biocompatibility but also broadens up the design space for stimuli responsive applications. Qu and coworkers reported a reversible light-controlled bioorthogonal nanocatalyst system through the interaction of cyclodextrin (CD) and azobenzene.¹³ After reducing Pd in situ inside MSNs, the researchers fabricated trans-azobenzene on the surface of MSNs, which further non-covalently bonded with CD. The bulky CD served as a “gate keeper” blocking the access to catalytic center. However, after shining UV light, trans-azobenzene converted to cis-azobenzene and lost the strong affinity to CD. The releasing of CD triggered the substrates to interact with inner Pd catalysts for the conversion. Visible light, on the other hand, reversed the process and inhibited the catalysis. They demonstrated the reversibility both in test tubes and in cells through activation of a pro-fluorophore. The therapeutic application was through activation of pro-5FU leading to the death of cancer cells.

Doping Pd with magnetic nanoparticles compliment bioorthogonal catalysis with mobility under magnetic field. Hoop et al. prepared a hybrid nanowire containing Pd and Fe.¹⁰² Nanowires were able to localize around the magnet and activated pro-5FU leading to the death of cancer cells. They further performed in vivo study to confirm that the activation of therapeutics by Pd-Fe nanowire reduced the tumor growth rate.

4.2. Pd-catalyzed Suzuki-Miyaura reaction

Going beyond bond cleavage-based decaging reactions, heterogeneous Pd nanocatalysts can also be applied to generate therapeutics through bond forming reactions such as the Suzuki-Miyaura cross-coupling reaction. In a recent study, Bradley et al. successfully developed a multifunctional Pd catalyst for tumor-targeting and intracellular prodrug activation.¹⁰³ Pd nanoparticle-functionalized fluorescent microspheres were conjugated with cyclic-RGD peptides onto their surfaces for targeting purposes. These fluorescent microspheres exhibited high uptake in α_vβ₃-positive human glioblastoma cells and maintained their catalytic activity. Crucially, two antitumor agents, 5FU and PP-121 (a multi-targeted kinase inhibitor),^104,105,106 were activated by these catalysts simultaneously through two different mechanisms, namely decaging and cross-coupling (Figure 9A), resulting in a significant reduction in cell viability (Figure 9B). Mascareñas and coworkers also fabricated efficient Pd nanoreactors for depropargylation and cross-coupling.¹⁰⁷ These nanocatalysts were hollow microspheres composed of mesoporous silica nanoshells and Pd nanoparticles at their inner layers (Figure 9, C and D). In vitro experiments validated their dual catalytic activities of O-depropargylation of phenols and intermolecular cross-coupling. Significantly, the bioorthogonality of depropargylation was practicable within Verda Reno (Vero) cells as shown by Pd-triggered fluorescence recovery.

Figure 9.

(A) The process of simultaneous activation of two prodrugs. (B) Cell viability after various treatments. (Reprinted with permission from ref. 103. Copyright 2017, Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim.) (C) The fabrication of hollow Pd nanoreactors and (D) their reactive tunability. (Reprinted with permission from ref. 107. Copyright 2019, The Royal Society of Chemistry.)

4.3. Pd-induced asymmetric hydrogenation

TMCs have been used to effect intracellular hydrogenation of unsaturated prodrugs.^108,109,110 Recently, Qu et al. constructed a chiral Pd catalytic system to activate prodrug in situ through asymmetric transfer hydrogenation (ATH) reaction.¹¹¹ Mesoporous silica nanoparticles (MSNs) were used to immobilize Pd nanoparticles (MSN-Pd), with enantioselectivity provided using cinchonidine (CD) as a chiral ligand (MSN-Pd/CD). These assemblies were then coated with neutrophil membrane (MSN-Pd/CD@Neu), providing targeting of asymmetric catalysis to sites of inflammation. (Figure 10A). Using formate as the hydrogen donor, MSN-Pd/CD converted achiral atropic acid (AA) to chiral hydratropic acid (HAA) (Figure 10 B and 10C) with a conversion of 99 % and an enantiomeric excess of 78 % (the biologically-active S-enantiomer). Qu et al. chose ibuprofen (IBU) as the model drug to demonstrate the utility of chiral bioorthogonal hydrogenation. In RAW264.7 macrophage cells, the prodrug (preIBU) was activated to S-IBU by MSN-Pd/CD-mediated ATH, and effectively relieved lipopolysaccharide (LPS)-induced inflammation (Figure 10D). Furthermore, the targeting capability of MSN-Pd/CD@Neu was also validated by its preferential accumulation in inflamed cells. In vivo studies demonstrated a decrease in the mouse paw inflammation over time (Figure 10 B and 10 E).

Figure 10.

(A) Preparation of chiral MSN-Pd/CD@Neu catalyst. (B) Targeted activation of chiral IBU to relief inflammation in vivo. (C) Conversion of AA to HAA mediated by chiral MSN-Pd catalysts. (D) Intracellular synthesis of IBU through chiral MSN-Pd-catalyzed ATH. (E) ROS imaging of inflamed paws. (Reprinted with permission from ref. 111. Copyright 2020, Elsevier Inc.)

4.4. Pd-catalyzed cyclization

Electrophilic Pd(II) can activate aromatic C-H bond to give σ-aryl-Pd complex leading to the coupling of arenes with carbon-carbon unsaturated bonds.¹¹² Fujiwara et al. reported synthesis of coumarin derivative via Pd-catalyzed intramolecular cyclization in mild condition.¹¹³ Inspired by this, Lee and coworkers reported a magnetothermia-induced nanoreactor through growing Pd nanocrystals on iron oxide nanoparticles.¹¹⁴ In the presence of an adjusted magnetic field (AMF), nanoreactor generated heat and accelerated the synthesis of coumarin derivative through cyclization by Pd. In vitro imaging showed the potential of this remote operable nanoreactor in biomedical applications.

5. Gold-catalyzed bioorthogonal reactions

The noble metal gold is a strong Lewis acid in its oxidized form, with relatively stable oxidation states +I and +III that can coordinate with alkyne groups. In 2010, Kim et al. reported the use of free gold(III) ion in living cells to generate fluorophores through hydroarylation reaction.¹¹⁵ Mascareñas et al. reported that free catalyst 1,3,5-triaza-7-phosphaadamantante (PTA)-Au(III)Cl catalyzed carbocyclization reaction leading to intracellular generation of fluorophore.¹¹⁶ Tanaka et al. used glycoalbumin-bound Au(III) complexes for organ-targeted, localized amidation of propargyl ester-based probes with nearby proteins in living mice.¹¹⁷ However, the strong interaction between gold and biological thiols hampers the potential for the activation of therapeutics. Tanaka and coworkers engineered albumin to protect Au catalyst from thiols and developed gold triggered-cyclization for therapeutic activation.^118,119 Unciti-Broceta et al. embedded Au clusters in a solid support for Au-catalyzed decaging (Figure 11 A).¹²⁰ The solid support protected Au clusters from thiol-rich biomacromolecules, while allowing alkyne-functionalized pro-fluorophore and prodrugs to enter and undergo the decaging process. Au clusters were grown within a PEG-grafted low-cross-linked polystyrene matrix to prepare the solid-supported gold catalysts ([Au]-resins). These catalysts showcased bioorthogonal catalytic activity in physiological conditions, with strong fluorescence release of Rhodamine 110 upon O-depropargylation (Figure 11 B). For medical application, three different prodrugs were used to study this gold-triggered drug release in A549 (lung) cells. The study confirmed that these active drugs were released in situ and gained in the presence of heterogeneous gold catalysts (Figure 11 C). In this work, a single [Au]-resin was successfully transplanted into the brain of a zebrafish, which realized locally controlled release of fluorescence via the bioorthogonal catalysis by the [Au]-resin. (Figure 11 D).

Figure 11.

(A) Au-catalyzed uncaging reactions for molecular release. (B) The green fluorescence release of rhodamine 110 from its precursor catalyzed by [Au]-resins. (C) Au-catalyzed prodrug activation of three antitumor agents. (D) Au-catalyzed bioorthogonal fluorescence recovery in the brain of a zebrafish. (Reprinted with permission from ref. 120. Copyright 2017, Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim.)

6. Platinum-catalyzed bioorthogonal reaction

Platinum is used widely as a catalyst in organic chemistry, however applications of Pt in bioorthogonal chemistry remain underexplored. Recently, Bernardes and coworkers used small-molecule Pt-catalyzed decaging through an intramolecular cyclization mechanism. Free Pt complexes (dipotassium tetrachloroplatinate or cisplatin) were employed to mediate the decaging of pentynoyl tertiary amides and N-propargyls. ¹²¹ Pentynoyl amide derivative of MMAE and the N-propargyl substituted 5FU were synthesized as prodrugs. These prodrugs were partially activated in HeLa cells using non-toxic concentrations of Pt salts. For in vivo studies, they chose cisplatin to depropargylate the 5FU prodrug in HCT116 zebrafish xenografts. The prodrug-cisplatin treatment significantly induced cell apoptosis and reduced tumor size compared to single treatments at 3-day post treatment. As the duration of treatment increased, they observed a synergistic effect from the activation of 5FU and the inherent toxicity from cisplatin. Inspired by a similar concept, Huang et al. used the synergy of free cisplatin and in situ generation of nitric oxide (NO) to induce apoptosis in cancer cells cancer cells.¹²² The Pt catalyst was generated as Pt (IV), which was inactive to prodrugs. However, after internalization by cancer cells, Pt (IV) was reduced to Pt (II) cisplatin in the cytoplasm. This cisplatin further cleaved propargyl bonds on the prodrug, leading to the release of NO. Their system exhibited high toxicity to cancer cells and low toxicity to normal cells, with activity also observed in the A2780 zebrafish xenograft model.

7. Iron-catalyzed bioorthogonal reactions

Iron is present in high abundance in humans and hence biocompatible, making it an attractive alternative to heavy metal catalysts. In 2012, Meggers et al. reported the use of free catalyst Fe(III)-containing 5,10,15,20-tetraphenyl-21H,23H-porphine (TPP) complex, [Fe(TPP)Cl] to efficiently catalyze the reduction of aromatic azides to their amines under biologically relevant in vitro conditions.¹²³ During this reaction, thiols were required converting the Fe (IV)-nitrene intermediate to the reduced amine. Green fluorescence of rhodamine 110 recovered quickly from the nonfluorescent bisazide pro-fluorophore after the addition of [Fe(TPP)Cl] in HeLa cells, demonstrating the successful application of this catalyst.

Rotello et al. reported the encapsulation of Fe(TPP)Cl into the monolayer of AuNPs to obtain thermally responsive bioorthogonal nanozymes (Figure 12 A).¹⁴ Due to the confined environment in the monolayer, Fe(TPP)Cl catalysts stacked at room temperature, blocking access to the catalytic center. When heated up to 37°C, Fe(TPP)Cl catalysts de-aggregated and redistributed in the monolayer, allowing the substrate access to the catalytic center. The fully reversible aggregation-redistribution process was monitored by the characteristic absorption band of aggregated porphyrin.. The therapeutic potential of these thermally gated nanozymes was demonstrated by the activation of aryl azide protected moxifloxacin against the E. coli bacteria biofilm. As shown in Figure 12 B, the addition of nanozymes with pro-Mox had no effect on biofilms at 25 °C, and killed bacteria in the biofilms at 37 °C.

Figure 12.

(A) Schematic presentation of thermally gated nanozymes. (B) E. coli biofilm treated with pro-Mox and thermally gated nanozymes at 37°C and 25°C. (Reprinted with permission from ref. 14. Copyright 2020, Elsevier Inc.) (C) The scheme of in situ activation of pro-fluorophore and prodrugs by polyzymes. (D) Viability of E. coli biofilm and P. aeruginosa biofilm treated with polyzymes and pro-Mox and pro-Cip, respectively. (Reprinted with permission from ref. 124. Copyright 2020, American Chemical Society.)

In further studies, Rotello and coworkers developed polymer-protected “polyzymes” based on the use of Fe(TPP)Cl catalysts (Figure 12 C).¹²⁴ These polyzymes used a self-assembled polymer with cationic quaternary ammonium terminal groups to facilitate penetration into the biofilm. Catalytic activity was confirmed through activation of aryl azide-protected resorufin in solution and in biofilm. Two antimicrobial fluoroquinolones, moxifloxacin (Mox) and ciprofloxacin (Cip), were masked with aryl azide carbamate linkers on their secondary amine groups. In the presence of polyzymes, these two prodrugs were effectively unmasked to regain their antibacterial activity, and a significantly decreased viability was observed in E. coli (CD-2) and P. aeruginosa (ATCC-27853) biofilms, roughly equivalent to that of free drugs (Figure 12 D). Meanwhile, this nanocatalyst showed no acute cytotoxicity in 3T3 fibroblast cells, indicating its biosafety to mammalian cells in eliminating bacterial biofilms.

8. Emerging metals

The previously mentioned transition metals exhibit excellent interactions with unsaturated caging moieties via π-π interactions via π-back-bonding either from the metal to the ligand or the ligand to the metal center, depending on their electronic structure.¹²⁵ Due to these interactions, metal catalysts containing an iron-, ruthenium-, or palladium-center show a great affinity towards unsaturated double- or triple-bonds with a high π -electron density. However, transition metals with a higher occupation of d-orbitals such as silver can engage in metal-ligand interactions as π donors and have the potential to activate molecules caged with π-acceptor ligands. Depending on the mode of action, these features can be utilized to design novel catalysts with enhanced catalytic activity. For instance, rhodium-, silver-, and iridium-catalysts have shown the potential to rapidly catalyze azide-alkyne or azide-ynamide cycloaddition under mild conditions.^126–133 Therefore, these transition metals present new potential tools for bioorthogonal applications.

9. Conclusions and perspective

Bioorthogonal chemistry provides in situ generation of imaging and therapeutic agents in biological environments using reactions unattainable to nature. TMC-mediated bioorthogonal catalysis is a versatile strategy; loading TMCs in/on nanomaterials enhances the water solubility, biocompatibility and biological stability of these catalysts. These nanocatalysts can provide in situ generation of therapeutics through bond cleavage and formation reactions. These chemical capabilities have been used for anticancer and antimicrobial imaging and therapeutic applications with both in vitro and in vivo models. The unique capabilities of these catalysts can be complemented by targeting and stimuli-responsive nanocatalysts to provide enhanced spatiotemporal control of drug activation, rendering localized therapy to reduce the off-target effects (Table 1).¹³⁴

Table 1.

Summary table of TMCs and their applications.

TMC-based bioorthogonal catalysis	Dimension	Formulation	Main advantages & applications	Stage	Ref.
Cu-based catalytic systems	Free small molecule	Cu(I)-BTTAA	Extracellular prodrug activation; reversible cell surface modifications; site-specific modulation of protein.	In vitro	[61]
	Nanoscale	CuNP-loaded MSNs	Accelerating CuAAC by dual synergistic mechanisms; intracellular drug synthesis.	In vivo	[47]
		CuNP-doped LANPs	Endogenous lipoic acid as supports; rugby-like morphology with better catalytic efficiency; intracellular drug synthesis.	In vitro	[48]
		CuNP-loaded MOFs	TPP functionalization for mitochondria-targeting; localized drug synthesis.	In vivo	[49]
		DNAzyme-encapsulated Cu/Zn MOFs	pH-responsive degradation; intracellular drug synthesis; synergistic therapeutic effects.	In vivo	[56]
		Drug fragment-loaded MSNs	Using endogenous neurotoxic Cu ions as TMCs; H2O2-responsive cargo release; intracellular drug synthesis.	In vivo	[57]
		Cu-loaded MSNs	biocompatible; implantable; acting as molecular-sieve-typed nanoreactors; intracellular and extracellular drug synthesis.	In vivo	[141]
		Cu-SCNPs	Metalloenzyme-mimicking activity; dual catalytic modes; intracellular or extracellular drug synthesis.	In vitro	[53–55]
			Selective localization by different delivery approaches	In vitro	[142]
			Cell membrane localization; Scaffold or substrate-guided product localization	In vitro	[143]
	Microscale	CuNP-entrapped resins	Biocompatible; implantable; extracellular drug synthesis.	In vivo	[50]
Ru-based catalytic systems	Free small molecule	Cp*Ru(COD) Cl and its derivatives	Well-tolerated in living cells; intracellular prodrug activation.	In vitro	[69–73]
	Nanoscale	Biotinylated Ru complex-incorporated streptavidin	As a cell-penetrating ArM; intracellular hormone activation.	In vitro	[74]
		Ru-SCNPs	Codelivery of Ru catalysts and exogenous enzymes; activating precursors in both concurrent and tandem modes; intracellular dual prodrug activation.	In vitro	[144]
		Cp*Ru(COD) Cl-encapsulated AuNPs	Reversible supramolecular control of catalytic activity.	In vitro	[16]
			pH-switchable nanozymes; selective targeting of acidic microenvironments.	In vitro	[18]
			Controllable nanozyme localization for intra- or extracellular bioorthogonal catalysis.	In vitro	[17]
		CpRu(8HQ)(allyl)PF6-encapsulated AuNPs	Controllable intracellular reactivation of nanozymes through endosomal proteolysis.	In vitro	[15]
		Cp*Ru(COD) Cl-encapsulated polymeric NPs	Protecting Ru catalysts from deactivation; intracellular prodrug activation.	In vitro	[12]
		Biotinylated Ru complex-incorporated streptavidin	Directed evolution into the ArM; periplasmic pro-fluorophore activation.	In vitro	[76]
		Ru-glycoalbumin complexes	Glycan functionalization for cell targeting; as a biocompatible ArM; protection from glutathione; intracellular prodrug activation.	In vitro	[78]
Pd-based catalytic systems	Free small molecule	Pd(COD)Cl2	Biocompatible; intracellular and extracellular prodrug activation.	In vitro	[99]
		Pd(dba)2	Intracellular release of NO.	In vitro	[145]
	Nanoscale	Pd(dppf)Cl2-encapsulated AuNPs	Reversible supramolecular control of catalytic activity; intracellular prodrug activation.	In vitro	[16]
		Pd nanosheet-loaded exosomes	Selective targeting to specific tumor cell types; intracellular prodrug activation.	In vitro	[19]
		Pd-chelated peptides	Enhanced cellular internalization; protecting Pd catalysts from deactivation; intracellular pro-fluorophore activation.	In vitro	[95,146]
		Pd nanocube-embedded MOFs	Protecting Pd nanocubes from passivation and deactivation; Intracellular controlled pro-fluorophore activation.	In vitro	[96]
		PdCl2(TFP)2-encapsulated PLGA-PEG NPs	Local efficient accumulation; intracellular prodrug activation.	In vivo	[97, 98]
		Pd(dppf)Cl2-encapsulated liposomes	Protecting Pd catalysts from premature reaction; intracellular prodrug activation; monitoring drug release by fluorescence.	In vivo	[101]
		Pd-Fe hybrid nanowires	Magnetic field-guided local prodrug activation.	In vivo	[102]
		PdNP-loaded fluorescent microspheres	cRGD functionalization for glioblastoma-targeting; simultaneous activation of two drugs intracellularly; synergistic therapeutic effects.	In vitro	[103]
		PdNP-equipped mesoporous SiO2 nanoshells	Biocompatible; tunable shell thickness and porosity; protecting PdNPs from leakage and passivation.	In vitro	[107]
		PdNP-embedded MSNs with azo-CD modification	Reversible light-controlled catalytic activity; intracellular prodrug activation.	In vitro	[13]
		PdNP-immobilized MSNs with chiral modification	Neutrophil membrane coating for inflammation-targeting; intracellular chiral drug synthesis.	In vivo	[111]
		Pd-SCNPs	Highly efficient; controlled intracellular localization	In vitro	[142,143]
		Pd nanocrystal-loaded Fe3O4 core with a hollow SiO2 nanoshell	Spatioselective Pd growth at the Fe3O4 surface; magnetothermia-induced catalysis; intracellular alkynyl-carbocyclization.	In vitro	[114]
	Microscale	PdNP-entrapped resins	Biocompatible; implantable; extracellular prodrug activation.	In vitro or in vivo	[87–90,93,94]
		Pd nanosheet-entrapped hydrogels	Biodegradable; extracellular prodrug activation.	In vitro	[147]
		PdNP-TiO2 nanosheet-incorporated microneedle array patches	Robust and removable; skin penetration; preventing PdNP leakage; extracelluar prodrug activation.	In vivo	[140]
Au-based catalytic systems	Free small molecule	HAuCl4	Au(III) ion mediated-hydroarylation; intracellular pro-fluorophore activation.	In vitro	[115]
		Au(I) and Au(III) complexes	Water-mediated activation of Au catalysts;intracellular pro-fluorophore activation; in parallel with the Ru-catalyzed decaging.	In vitro	[116]
		Au(I) and Au(III) complexes	Amenable Ayba groups for manipulating properties of prodrugs; orthogonal with other TMCs.	In vitro	[118]
		Au(I) complexes	Activated by transmetallation; catalytically active; cytotoxicity.	In vivo	[146]
	Nanoscale	Au(III)-glycoalbumin complexes	Glycan functionalization for organ targeting; liver and intestine-selective fluorescence labeling.	In vivo	[117]
		Au(I)-albumin complexes	As a biocompatible ArM; protection from glutathione; hydroamination for phenanthridinium-based drug synthesis.	In vitro	[119]
	Microscale	AuNP-embedded resins	Protecting AuNPs from thiol-rich biomolecules; extracellular prodrug activation.	In vivo	[120]
Pt-based catalytic systems	Free small molecule	K2PtCl4 or Cisplatin	Water-mediated activation of Pt catalysts; extracellular and intracellular prodrug activation; synergistic therapeutic effects.	In vivo	[121]
		Cisplatin	An integrated molecule with dual-prodrug strategy; intracellular activation of two prodrugs; synergistic therapeutic effects.	In vivo	[122]
Fe-based catalytic systems	Free small molecule	Fe(TPP)Cl	Using thiols as reductants; well-tolerated in biologically relevant conditions.	In vivo	[123]
	Nanoscale	Fe(TPP)-encapsulated AuNPs	Reversible thermos-responsive nanozymes; controlled activation of antimicrobial prodrugs.	In vitro	[14]
		Fe(TPP)Cl-encapsulated polymeric NPs	Protective hydrophobic environment for Fe catalysts; enhanced penetration ability; controlled activation of antimicrobial prodrugs.	In vitro	[124]

The future success of bioorthogonal catalysis in biomedicine relies on effective choice of catalysts and the prodrug design. The versatility of TMC nanocatalysts provides considerable design space, and there are multiple opportunities for developing and enhancing nanocatalysts as they move towards the clinic. Perhaps both the greatest opportunity and challenge in implementing these systems is the choice of metal for the TMC. Transition and noble metals (e.g. Ru, Pt, Pd and Au) have the potential for both acute toxicity and genotoxicity.¹³⁵ Taking advantage of the toxicity, TMCs can be designed to achieve direct biological effects as metallodrugs while maintaining promising catalytic activity for therapeutic activation, such as cisplatin.^{121, 122} On the other hand, concerns of inherent toxicity can be addressed by either improving catalyst activity for reduced dosing, or contemplating the application of other low-toxicity metal catalysts such as iron, nickel, cobalt, and titanium. Moreover, the metabolism, excretion, and long-term effects of TMCs are additional hurdles to be faced before moving to clinic.

Prodrug design can also highly impact the performance of bioorthogonal catalysts. Compared to parent drugs, prodrugs alter key physicochemical and pharmacokinetics (PK), such as solubility, membrane permeability, biodistribution and excretion, which can expand the application of drugs that are not suitable for the clinical use.⁹⁸ Activation of prodrugs can even overcome drug resistance by escaping from enzymatic degradation processes and reducing the interaction of efflux transporters.¹³⁶ Building on the success of endogenously-activated prodrugs,^137,138 bioorthogonal prodrugs broaden the design space with biologically inert groups. Bioorthogonal activation of prodrugs controls the generation of therapeutics in locally constrained manner and avoids traditional adverse effect including myelotoxicity and heart attack. At present, bioorthogonal prodrugs are majorly decorated with hydrophobic protecting groups on amino or hydroxyl groups with compromised solubility. Developing modifiable protecting groups is crucial for future development, where the tunable pharmacokinetics of prodrugs is key to the ultimate success of therapeutic strategies. Recently, bioorthogonal mediated-prodrug activation has entered into clinical trials((NCT04106492),¹³⁹ revealing the encouraging translational potential. Moreover, implants or patches can be other conceivable applications for localized therapy.¹⁴⁰ While still in the early stages of implementation, bioorthogonal catalysis present great promise for therapeutic applications.

Figure 1.

Schematic presentation of in situ activation of prodrugs or pro-imaging agents through TMC-mediated bioorthogonal catalysis.