Bioorthogonal reaction mediated size transformation clustered nanosystems for potentiating bioimaging and drug delivery

Abstract

Continuous advances have been witnessed in the booming of size variable nanosystems for molecular imaging and therapy. These nanosystems usually exhibit in situ size transformation, which promotes optimized biodistribution, retention and penetration in lesions. Bioorthogonal reactions have been introduced as a useful tool to develop size variable nanosystems. In general, researchers modify controllable block (including pH adjustment, disulfide reduction, and/or enzymatic hydrolysis) masked bioorthogonal handles on the nanoparticles or small molecules to develop biocompatible size variable nanosystems. These nanosystems undergo precise click cycloaddition and self-assemble into nanoaggregates in situ, showing enhanced tissue accumulation and retention. These advantages have demonstrated great promise in improving imaging quality and therapeutic outcomes with high effectiveness and controllability. To date, this strategy has been widely introduced to construct bioimaging probes or nanomedicines. To gain a comprehensive understanding of the strategy, in this review, we focus on bioorthogonal reaction mediated size variable nanosystems reported in the last five years, present their application in bioimaging and therapy, and elucidate the mechanism of bioorthogonal reactions. Based on these efforts, challenges and future research directions in this area are also discussed at the end.

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Juan Li

Juan Li is currently a postgraduate student at School of Life and Health Sciences, Hubei University of Technology. Her research focuses on the application of bioorthogonal reactions in nanodrug delivery.

Cao Li

Cao Li obtained his PhD degree under the supervision of Prof. Xian-Zheng Zhang from Wuhan University in 2012. He is now a full professor in Hubei University of Technology. His research interests focus on the development of polymeric nanodrugs for disease treatments.

Qi Yu

Qi Yu obtained her PhD degree from Nanjing University of Posts & Telecommunications in 2017 and then started as a postdoctoral fellow at Singapore University of Technology and Design. She is currently an associate professor in Hubei University of Technology. Her research interests focus on the development of functional luminescent nanoplatforms for bioimaging and cancer therapy.

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1. Introduction

To date, many functional inorganic and organic materials have been extensively developed as potential platforms for diagnosing and/or combating urgent health concerns including cancer, bacterial infection, and Alzheimer's disease as well as other life-threatening diseases.^1,2 In these platforms, organic materials suffer from low bioavailability and fast clearance,³ thus they are usually encapsulated in liposomes, micelles or polymeric nanoparticles for imaging and therapy.^4–6 Similar to inorganic materials (such as mesoporous silica nanoparticles and metal organic frameworks), most functional nanosystems rely on enhanced permeability and retention (EPR) effects to be transported to targeted tissues.^7–9 However, these diagnostic and therapeutic platforms mainly stay around blood vessels or non-targeted organs. For example, a limited injected dose (only 0.7%) of anticancer drugs has been found in targeted tumor sites.¹⁰ Although several studies have utilized specific peptide motifs and antibodies to improve targeting ability, the targeting efficacy is largely dependent on the levels of protein expression at the targeted sites resulting in heterogeneity among individual cells, tissues and bodies, leading to the off-targeting phenomenon.^11,12 Regarding this issue, efforts have been made in nanosystem design to overcome the dilemma of prolonged circulation and enhanced tissue accumulation.^13,14

In general, the size of nanostructures affects their accumulation and penetration in targeted lesions. Designing of size variable nanosystems that enable transforming their size in situ has become an apparent and steerable strategy.^15,16 As the renal filtration size is below 5.5 nm, on the one hand, nanosystems designed should be larger than 5.5 nm to allow time for diagnosis and treatment and avoid rapid renal clearance.^17–19 On the other hand, nanosystems are preferable to be degraded to ultrasmall nanoparticles below 5.5 nm, which guarantees the easy clearance from the body and avoids the long-term toxicity post-diagnosis or therapy.^20,21 The upper limit of parameters of nanosystems differ from hundreds of nanometers to several micrometers, which are largely affected by the vascular endothelial space in various types of tissues.²² Additionally, the size of the nanostructures also plays a crucial role in the phagocytosis from reticuloendothelial system (liver and spleen) and the exudation of blood vessels.²³ Apart from the tissue accumulation and circulation, the promotion of penetration should be taken into consideration because small-sized nanoparticles (<20 nm) are more conducive to penetrate the lesions.¹⁴ In view of this idea, the fabrication of size transformation clustered nanosystems, which are amenable to smart control of their size in situ, is an appropriate way to prolong drug circulation, promote tissue accumulation and penetration.

Bioorthogonal reactions that occur within biological organisms without the intervention of their normal biochemical processes refer to a highly selective and efficient chemical tool. Pioneered by Bertozzi et al. in 2000,²⁴ bioorthogonal reactions, involving the copper-catalyzed ones and copper free ones, have been well developed and widely used in specific protein labelling, molecular imaging and therapy due to the improved reaction rate and effectiveness.²⁵ Currently, bioorthogonal reactions have been employed to design size variable nanosystems through the modification of the corresponding bioorthogonal reactive motifs on the molecules or nanoparticles.^26,27 The intramolecular interaction facilitates the occurrence of in situ nano-to-cluster aggregation, which is beneficial to prolong the circulation time and retention time in targeted tissues. Moreover, these nanosystems can be further engineered using intelligent materials that are responsive in the presence of internal or external stimuli, such as overexpressed enzymes, light and slight acidity.²⁸ The functionalization not only efficiently improves high specificity and precise controllability to accumulate at targeted sites, but also has potential to show secondary cluster-to-nano size transformation in nanocarriers for deep penetration in tissues. Precisely speaking, using bioorthogonal reaction-mediated size variable nanosystems is considered as an apparent and steerable strategy for benefiting optimal site-specific accumulation during diagnosis and therapy.

In this review, we will focus on bioorthogonal reaction-mediated size variable nanosystems reported in the last five years, and present an overview of the versatile strategies to optimize the biodistribution and site-specific accumulation of these nanosystems for improved molecular imaging and drug delivery (Scheme 1). In contrast to the passive targeting strategy, the in situ size variable strategy offers precise controllability of enhanced retention and accumulation of nanosystems. In the molecular imaging section, the design strategies of these contrast agents, improved signal to noise ratios (SNR) and applications in various bioimaging modes will be summarized and discussed. Furthermore, the improved retention and accumulation in targeted sites also facilitate the effective drug release and improved therapeutic efficacy. Similarly, the design strategies of these intelligent nanodrugs and improving drug delivery efficiency for the treatment of cancer, bacterial infection and other life-threatening diseases will be presented and discussed. In the end, we discuss the trends of the employment of size variable nanosystems as diagnostic and/or therapeutic platforms, followed by giving our opinions on their future direction addressing the challenges. We expect that this review will be helpful in developing more strategies to achieve precise controllability of disease diagnosis and drug delivery.

Scheme 1 Schematic illustration of size transformation nanosystems for potentiating bioimaging and drug delivery.

2. Size impact on bioimaging and drug delivery

The size of nanosystems significantly affects drug delivery efficiency and bioimaging performance throughout their in vivo biological cycle, biodistribution, target tissue accumulation, and deep penetration. Upon entering the body, small-sized nanoparticles (<5.5 nm) evade capture from the mononuclear phagocyte system and are readily cleaned up through glomerular filtration.^17,29 Because of hepatic sinusoid retention with vascular fenestrations of 50–100 nm, particles below 50 nm are prone to penetrate vascular endothelium and be trapped in the liver.^30,31 On the other hand, following the principle of the EPR effect in accordance with tumor vascular pores of 200–1200 nm, large particles exhibit prolonged retention.^32,33 Large-sized nanoparticles are prohibited to enter the deep tissue matrix, and only stay within tumorous adjacent regions due to the poor penetration capability.^34,35 Nevertheless, essential small particles penetrate deeply and undergo interstitial fluid washout.^36,37

Apart from the entry into target tissues, particle size also has an important role in cellular uptake. Cells have their endocytosis and exocytosis processes, which usually have a size-dependent reverse mechanism to work on cellular uptake.³⁸ Large-sized nanoparticles (>500 nm) tend to enter cells via phagocytosis,³⁹ but are hard to be eliminated from the cells via exocytosis.⁴⁰ Small ones are preferrable to internalize cells via endocytosis. For instance, nanoparticles with a parameter of 20–100 nm and 120–150 nm enter cells via caveolae and clathrin-dependent endocytosis, respectively.^41,42 Based on these, a cellular uptake study should also take the size impact of nanoparticles into consideration.

3. Mechanism of bioorthogonal reactions

Bioorthogonal chemistry refers to chemical reactions that are efficiently and selectively carried out in biological systems without interference from innate biomolecules and offers relatively rapid kinetics under physiological conditions.^43–45 Because of their efficient, specific and rapid procedure, bioorthogonal reactions have been applied for in vivo synthesis. To date, the developed bioorthogonal reactions mainly include Staudinger ligation, copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC), strain-promoted azide–alkyne cycloaddition (SPAAC), inverse electron demand Diels–Alder reactions (iEDDA), 1,2-aminothiol-2-cyanobenzothiazole (CBT) click reactions, strain-promoted sydnone-alkyne cycloaddition (SPSAC) and nitrile imine-alkyne cycloaddition.^24,46–53 Through decorating the functional bioorthogonal pairs on molecules or nanoparticles, bioorthogonal reactions would occur and trigger intramolecular addition, in situ assembly or molecule-to-nano clustered transformation, which is advantageous for long-lasting imaging and therapy in vivo. Furthermore, these bioorthogonal functional groups can be protected by biomarker-responsive moieties to reinforce the specificity and sensitivity of bioorthogonal reactions, thus efficiently establishing relationship ties between biomarkers and theragnostic results. Currently reported strategies mainly used CuAAC, SPAAC, iEDDA, 1,2-aminothiol-CBT click reactions and SPSAC. In this section, we will briefly introduce these mechanisms to assist the understanding of the following specific strategies for imaging and therapy.

3.1. CuAAC

Discovered by Meldal and Sharpless in 2002, CuAAC involves Cu(I) catalyzed formation of a 1,2,3-disubstituted triazole ring between azide (–N₃) and terminal alkyne (–C≡CH) functional groups.^46,47 The possible accepted mechanism explains the construction of a terminal Cu(I) acetylide (RC≡C–Cu), followed by the formation of binuclear Cu(I) intermediates (Fig. 1A).⁵⁴ Then, the occurrence of the stepwise annulation events promoted the completion of the [3+2] cycloaddition to yield the 1,4-disubstituted 1,2,3-triazole product. The introduction of a Cu(I) catalyst efficiently increases the reaction rate up to 10⁸-fold (10–100 M⁻¹ s⁻¹).^46,47,55 Since azide and alkyne are inert in a biological environment, CuAAC is a versatile tool for in vivo synthesis. However, the potential copper cytotoxicity cannot be ignored in living organisms. Efforts have been made to develop metal free catalyzed bioorthogonal reactions.

Fig. 1 Mechanism of bioorthogonal reactions: (A) CuAAC, (B) SPAAC, (C) iEDDA, (D) 1,2-amino thiol-CBT click reaction and (E) SPSAC.

3.2. SPAAC

In order to overcome the cytotoxicity of catalyst Cu in the CuAAC reaction, researchers are committed to developing metal free catalyzed bioorthogonal reactions. Notably, copper-free SPAAC has emerged through the reaction between trans-cyclooctene (TCO) derivatives and azide (Fig. 1B).⁴⁸ In TCO derivatives, alkyne is strained in an eight membered ring, resulting in massive bond angle deformation to 163°. The ring strain of TCO facilitates the [3+2] cycloaddition between azide and alkynes and accelerates the reaction rate compared to unstrained alkynes.⁵⁶ Although SPAAC provides a relatively small reaction rate constant (k) (0.1–10 M⁻¹ s⁻¹),⁵⁷ researchers prefer to use more biocompatible SPAAC in contrast to CuAAC.

3.3. iEDDA

iEDDA involves the reaction between an electron-deficient diene, such as 1,2,4,5-tetrazine (Tz) and an electron-rich dienophile, such as TCO, to generate a six-membered ring.⁴⁹ Taking the reaction of Tz and TCO as an example, the reaction mechanism is as follows: [4+2] cycloaddition occurs between the –C=C– bond of TCO and the –C=N–N=C– diene system of Tz, forming a bicyclic intermediate (Fig. 1C). Then, retro [4+2] cycloaddition is conducted to yield the corresponding 4,5-dihydropyridazine and nitrogen (N₂), followed by 1,3-prototropic isomerization to obtain the stable 1,4-dihydropyridazine.⁵⁸ The reaction is metal-free and has a relatively high reaction rate constant (between 1–10⁶ M⁻¹ s⁻¹),⁵⁹ which is more cell-friendly than CuAAC.

3.4. 1,2-Aminothiol-CBT click reaction

1,2-Aminothiol-CBT click reaction originates from the biosynthesis of D-cysteine (D-Cys) and CBT in a firefly, and is a key step to afford luciferin.⁶⁰ Pioneered by Rao’ group, this reaction was applied for protein labelling, proving the non-metal nature, and the reaction requires no complicated purification steps.⁵⁰ Significantly, the 1,2-aminothiol-CBT click reaction possesses superfast kinetics with a second-order rate constant of 9.19 M⁻¹ s⁻¹,⁵⁰ opening a new direction of bioorthogonal chemistry. The specific reaction mechanism is: under physiological conditions, pH 7.4, the thiol group in Cys attacks the carbon atom of the cyano group (–CN) in CBT molecules, forming a thioether intermediate (Fig. 1D). The nitrogen atom in the cyano group further captures the proton of the sulfhydryl group to yield an enamine containing intermediate. The amino group-participated proton transfer and intramolecular reactions forms a thiazole ring structure releasing ammonia gas, resulting the final product, amino luciferin.⁶¹

3.5. SPSAC

Compared to azide, heterocyclic sydnones exhibit high stability in biological media and have been utilized to participate in an efficient and clean cycloaddition reaction with strained cycloalkynes (cyclooctyne BCN), denoted as the strain-promoted sydnone–alkyne cycloaddition (SPSAC).⁶² The SPSAC mechanism primarily involves a [3+2] cycloaddition followed by a retro-Diels–Alder reaction, accompanied by CO₂ release and the formation of a pyrazole cycloadduct via structural rearrangement (Fig. 1E). The [3+2] cycloaddition step has been demonstrated to be the rate-limiting process of the entire SPSAC reaction. For example, the initially reported reaction system between N-phenylsydnone and BCN was approximately 5-fold slower than analogous SPAAC reactions.⁶³ The introduction of electron-withdrawing substituents on the N-aryl group and halogen (particularly fluorine) at the C4 position facilitated optimization of the sydnone scaffold, which could significantly increase the reaction rate (exceeding 40 M⁻¹ s⁻¹).^51,63

4. Bioorthogonal reaction-mediated size variable nanosystems for bioimaging

Imaging modalities are popular methodologies in both research and clinical fields. The imaging probes are considered as the most pivotal elements to improve imaging quality, acquire sufficient disease-associated information and evaluate therapeutic outcomes. Accordingly, probes have been booming in recent years, but insufficient accumulation at the targeted sites and the lack of specific connection between probes and endogenous biomolecules are the major issues needed to be addressed. Toward this end, bioorthogonal reactive motifs have been recently introduced to functionalize contrast agents to obtain various probes with improved specificity and tissue retention. This section will introduce size transformation-clustered bioorthogonal active nanoprobes for targeted imaging (Table 1).

Table 1 Bioorthogonal reaction-based nanosystems for molecular imaging

Imaging modalities	Probe	Responsive agent	The impact of size-variable probes on imaging	Reaction type	Size transformation, incubation time	Ref.
FL	Cbz-GPC(StBu)K(Cou)-CBT	FAP-α/GSH	“Turn-on” coumarin excimer emission, FL SNR↑	CBT-Cys	From molecules to 72.3 ± 9.5 nm nanoaggregates, 4 h	71
	Cou-D/L-CBT	GSH	Enabling long-time and high-contrast fluorescence imaging	CBT-Cys	From molecules to nanotubes with an outer diameter of 156 nm, 12 h	72
	Ala-Biotin-QMT	GSH/LAP	“Turn-on” the “dual AIE” fluorescence signal	CBT-Cys	From molecules to 140.9 ± 15.6 nm nanoaggregates, 6 h	73
	QMT-CBT	Caspase-1/GSH	“Dual-AIE” fluorescence signal enhanced imaging of AD in vivo	CBT-Cys	From molecules to 185.5 ± 18.8 nm nanoaggregates, 4 h	74
	AuNPs-Cy5.5-A&C	AEP	AEP triggered aggregation and emit strong fluorescence	CBT-Cys	From 50–60 nm to 422.2 ± 9.69 nm, 12 h	75
	CyNAP-SS-FK	GSH/Cat B	FL off–on, FL SNR↑	CBT-Cys	From molecules to ∼100 nm nanoaggregates, 12 h	76
PET	[^18F]1	GSH	Good imaging contrast and long retention time	CBT-Cys	From molecules to 138.2 ± 16.3 nm nanoaggregates	81
	[^18F]SF-DEVD	GSH/Caspase-3	Improve the in situ assembly efficiency and sensitive PET imaging of caspase-3	CBT-Cys	From molecules to ∼150 nm nanoaggregates	82
	[^18F]SF-Glu	GGT/GSH	Improve the in situ assembly efficiency and sensitive PET imaging of GGT activity	CBT-Cys	—	82
	[^18F]-C-SNAT4	GSH/Caspase-3	Enhanced retention to improve PET imaging contrasts	CBT-Cys	From molecules to 200 nm nanoaggregates, overnight	83
	[^68Ga]NOTA-SFCVM [^68Ga]NOTA-SFCVHEM	GSH/Cat B	Enhance tumor retention and PET signal intensity	CBT-Cys	—	85
	CBT-NODA/CBT-NODA-Ga/CBT-NODA-^68Ga	GSH/Furin	Prolonged tumor retention and amplified the microPET signal	CBT-Cys	From molecules to 356.8 ± 95.9 nm nanoaggregates, 7 h	86
PA	Cypate-CBT	GSH/CTSB	Enhanced retention to improve the PA signal	CBT-Cys	From molecules to 207.7 ± 15.9 nm nanoaggregates, 2 h	92
	NI-C-CBT	GSH/NTR	Triggered “on” and “enhanced” PA signals via intra-and intermolecular fluorescence quenching	CBT-Cys	From molecules to 117.9 ± 15.6 nm nanoaggregates, 5 h	93
MRI	DEVDCS-Gd-CBT	GSH/Caspase-3	Effectively improved MRI sensitivity under low magnetic fields	CBT-Cys	From molecules to 85.1 ± 15.1 nm nanoaggregates, 4.5 h	99
RI	nanoSABER	GSH/Legumain	Enhanced the intracellular accumulation, and improved and long-lasting Raman signals	CBT-Cys	From molecules to 147 ± 20 nm nanoaggregates, 3 h	105
	Yne-CBT	GSH/Cat B	Long retention in cell and relative Raman intensity↑	CBT-Cys	—	106
QPI	P-SiO2 NPs	GSH/Legumain	In situ high-contrast refractive index imaging of enzyme activity	CBT-Cys	—	110
NIR-II FL/PA	AuNNP@DEVD-IR1048	GSH/Caspase-3	Turn on both NIR-II FL and PA imaging signals	CBT-Cys	From 41.5 nm to 365.1 nm, 5 h	118
PET/PA	[^18F]-IR780-1	GSH/Caspase-3	Prolong probe retention and accumulation to enhance PA and PET signals	CBT-Cys	From molecules to 122 nmnanoaggregates, 1 h	121
PA/MRI	Gd-IR 780	GSH/Caspase-3	In situ self-assembly enhanced PAI and MRI signals	CBT-Cys	From molecules to 110 ± 23 nm nanoaggregates, 2 h	122

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4.1. Fluorescence imaging

Fluorescence imaging (FL) exhibits attractive advantages, including excellent sensitivity, non-invasiveness and no radiation damage, and has been applied from disease diagnosis, drug tracking to prognostic assessment.⁶⁴ For example, a fluorescent probe, Cytalux has been approved by FDA for imaging guided surgery.⁶⁵ Especially, fluorescence imaging in the NIR window (700–1700 nm) shows reduced autofluorescence, improved penetration and enhanced temporal resolution, pinpointing its powerful potential to be shifted from bench to bedside.⁶⁶ Non-specific labelling tissues of interest usually bring out “false-positive” signal interference. Instead, activatable signals that are sensitive to specific stimuli are preferrable to obtain disease-associated information. To date, activatable fluorescent probes have been rapidly explored to translate disease information to optical output,^67–70 but the small molecular nature of fluorescent probes renders them to be rapidly cleaned from lesions, which limits their application for real-time imaging. Hence, introduction of bioorthogonal reactions to promote the self-assembly or nanosized transformation in vivo is favorable to overcome the bottleneck of these activatable fluorescent probes.

Gao et al. combined CBT-Cys cycloaddition with excimer formation to prepare a fibroblast-activated protein-α (FAP-α)-responsive “turn-on” fluorescent probe (Cbz-GPC(StBu)K(Cou)-CBT) (Fig. 2A).⁷¹ Coumarin with a planar polycyclic aromatic structure was employed as the emissive excimer fluorophore. FAP-α cleavable Cbz-Gly-Pro peptide sequence, and StBu-protected Cys and CBT units were also included in the probe. In the tumor microenvironment, FAP-α cleavage of the peptide segment and glutathione (GSH) reduction generated a Cys intermediate, which subsequently triggered intermolecular CBT-Cys click reactions to form cyclic dimers and in situ self-assembly into nanoparticles (Cou-CBT-NPs). As seen in Fig. 2B, the probe exhibited a fluorescence peak located at 475 nm, corresponding to the emission of the coumarin monomer, while the presence of 50 U mL⁻¹ FAP-α red-shifted the emission to 550 nm, revealing the formation of the excimer. The excimer was ascribed to two coumarin molecules with a close proximity in the rigid cyclic dimer. The occurrence of in situ self-assembly led to the generation of nanoparticles with a diameter of 72.3 ± 9.5 nm via the H-aggregates (Fig. 2C). FAP-α-positive MIA PaCa-2 tumor cells and FAP-α-negative L929 normal cells were introduced to be incubated with the fluorescent probe for imaging. A time-dependent shift of the fluorescence signal from the blue to green channel was observed in MIA PaCa-2 cells due to the transition of monomer-to-excimer of the coumarin moiety (Fig. 2D). On the other hand, L929 cells only showed the enhanced fluorescence at the monomer channel. These results proved the probe sensitivity to FAP-α. Time-course fluorescence imaging of MIA PaCa-2 tumor-bearing BALB/c nude mice exhibited “turn-on” fluorescence and reached the maximum value at 6 h after the treatment with the probe, while no fluorescence was detected when the control probe removed the FAP-α sensitive peptide sequence. Additionally, Yang et al. reported a D, L alternated peptide containing probe Cys(StBu)-D-Glu-Lys-(coumarin)-D-Glu-CBT (Cou-D/L-CBT), which can react with GSH to trigger CBT-Cys cycloaddition to generate a fluorescent excimer of coumarin (Fig. 2E).⁷² Different from Cbz-GPC(StBu)K(Cou)-CBT, Cou-D/L-CBT included D-glutamic acid (D-Glu) that effectively underwent protonation in the lysosomal acidic environment (pH 4.5–5.5). The presence of alternated D,L-Glu in the probe facilitated the assembly into nanotubes, instead of nanoparticles. The performance prolonged the retention time of the probe in cells. The retention rates of Cou-D/L-CBT at 24 h and 36 h timepoint were 7.5 and 2.5 times higher than those of Cou-L-CBT, respectively, and the cell retention half-life was extended from 6 h (Cou-L-CBT) to 29 h (Cou-L-CBT). (Fig. 2F).

Fig. 2 (A) Schematic diagram of the mechanism of a FAP-α-responsive “turn-on” fluorescent probe (Cbz-GPC(StBu)K(Cou)-CBT). (B) Fluorescence spectra of Cbz-GPC(StBu)K(Cou)-CBT treated with or without FAP-α. Inset: Corresponding pictures illuminated by an UV lamp. (C) TEM image of Cou-CBT-NPs. (D) Confocal fluorescence images of FAP-α-overexpressing MIA PaCa-2 cells or FAP-α-deficient L929 cells after incubation with Cbz-GPC(StBu)K(Cou)-CBT for 0.5 h and 2 h, adapted with permission from ref. 71. Copyright 2022, American Chemical Society. (E) Mechanism of a D, L alternated peptide containing probe Cou-D/L-CBT. (F) Intracellular fluorescence intensity over different time periods after probe incubation, adapted with permission from ref. 72. Copyright 2025, American Chemical Society.

Commonly used fluorescent probes compromise the signal attenuation during in vivo imaging due to the aggregation-caused quenching effects at high concentrations. The emerging aggregation-induced emission luminogens (AIEgens) via limiting the molecular rotation resolve the issue. Hence, the development of an activatable fluorescent probe based on AIEgens has good potential for fluorescence imaging. Current AIEgen-based activatable probes usually form aggregates in the presence of specific targets, but the limited “turn-on” signal obtained results in compromised imaging sensitivity. Deng et al. developed an activatable AIEgen β-tBu-Ala-Cys(StBu)-Lys(Biotin)-Pra(QMT)-CBT (denoted as Ala-Biotin-QMT) to realize “two-step” aggregation in situ for enhanced fluorescence imaging (Fig. 3A).⁷³ This probe consisted of four functional modules: β-tBu-Ala as a leucine aminopeptidase (LAP)-specific recognition site, a StBu-protected CBT-Cys click reaction group, a biotin tumor-targeting ligand, and the NIR-emissive AIE luminogen QMT. The probe targeted cell membranes through biotin–receptor interactions, followed by GSH-mediated thiol release and LAP enzymatic hydrolysis within tumor cells to generate the active intermediate Biotin-QMT. Biotin-QMT underwent a CBT-Cys click reaction to form cyclic dimers and in situ self-assembly, which completed “two-step” aggregation in cells. Experimental data validated that Ala-Biotin-QMT enhanced 4.8-fold and 7.9-fold fluorescence in HepG2 cells and HepG2 tumor-bearing mouse models in contrast to “biotin + LAP inhibitor” pre-treated control groups.

Fig. 3 (A) Schematic diagram of activatable Ala-Biotin-QMT for in situ “two-step” aggregation, adapted with permission from ref. 73. Copyright 2024, American Chemical Society. (B) Schematic diagram of QMT-CBT for enhanced AD imaging. (C) Fluorescence spectra of QMT-CBT and QMT-CBT-Ctrl treated with or without caspase-1. (D) Schematic diagram of a QMT-dimer in an aqueous environment via molecular dynamics simulations. (E) Quantitative fluorescence signals of QMT-CBT and its control group in WT and AD mouse brain, adapted with permission from ref. 74. Copyright 2023, American Chemical Society.

AIEgen-based fluorescence probes have been applied for Alzheimer's disease (AD) imaging. For instance, Ac-Trp-Glu-His-Asp-Cys(StBu)-Pra(QMT)-CBT (QMT-CBT) for enhanced AD imaging was prepared.⁷⁴ As illustrated in Fig. 3B, QMT-CBT underwent GSH-triggered reduction of the disulfide bond and caspase-1(a key biomarker of AD neuroinflammation)-mediated peptide hydrolysis so that the probe induced the formation of cyclic dimers (primary aggregation) via CBT-Cys cross-linking and self-assembled nanoparticles (secondary aggregation) via hydrophobic interactions. The “two-step” aggregation efficiently turned on the AIE fluorescence in the presence of caspase-1. Most importantly, the “two-step” aggregation-based strategy led to 15.7-fold enhancement of fluorescence, which was more sensitive than that of the control group (Ac-Trp-Glu-His-Asp-Cys(tBu)-Pra(QMT)-CBT, QMT-CBT-Ctrl) with signal aggregation (Fig. 3C). Theoretical calculations demonstrated that this phenomenon was ascribed to tighter stacked QMT in “two-step” aggregation forming nanoparticles compared to QMT-CBT-Ctrl, resulting in a stronger role of limited intramolecular motions (Fig. 3D). After pre-treatment of cyclosporine to overcome the blood–brain barrier, QMT-CBT was employed for in vivo imaging of caspase-1-associated neuroinflammation containing AD mouse brain. A NIR fluorescence signal was observed in QMT-CBT treated AD mice which was 1.4-fold superior to that in the QMT-CBT-Ctrl control group (Fig. 3E). Another example is an asparagine endopeptidase (AEP)-activatable two-component nanoprobe system (AuNPs-Cy5.5-A&C) comprising functionalized gold nanoparticles AuNPs-Cy5.5-AK (containing the enzyme-cleavable peptide Ala-Ala-Asn-Cys-Lys) and AuNPs-Cy5.5-CBT. AEP-dependent click cycloaddition facilitated the utilization of the nanoprobe for AD imaging in transgenic APPswe/PS1dE9 mice.⁷⁵

Apart from AIE and excimer formation, intramolecular charge transfer (ICT) is a strategy to be used for the activation of a fluorescent probe. Xu et al. constructed a GSH/cathepsin B (Cat B) dual-activatable nitrile-aminothiol (NAT) bioorthogonal probe, CyNA_P-SS-FK, for specific fluorescence diagnosis of hepatocellular carcinoma (HCC) (Fig. 4A).⁷⁶ Different from traditional bioorthogonal fluorogenic luminophores, this NAT one introduced a strong electron-withdrawing nitrile group in the asymmetric hemicyanine structure to obtain a donor–π–acceptor (D–π–A), thus resulting in the blocking of ICT and fluorescence quenching. The NAT luminophore was further modified with a GSH-responsive disulfide bond and a Cat B-reactive peptide moiety (Ac-FK) to obtain an activatable probe. To realize rational design of this NAT biorthogonal fluorogenic luminophore, a series of nitrile-substituted hemicyanines (CyN-X) were prepared and investigated the fluorescence. The C-6 position is preferrable to introduce electron-withdrawing groups to achieve optimized fluorescence. The spacer between reactive units and the indole ring was also investigated by either attaching an alkyl chain or a short PEG chain. The favorable electron transfer capability of the compound decorated with the flexible PEG chain (CyNA_P) was demonstrated to be preferable to facilitate intramolecular macrocyclization. Consequently, CyNA_P was identified for further investigation due to its superior intramolecular cyclization kinetics and highest fluorescence recovery efficiency. The first-order reaction rate constant for CyNA_P's intramolecular cyclization (7.97 × 10⁻⁵ s⁻¹) was significantly higher than its intermolecular reaction rate constant with free Cys. The rapid kinetics promoted the intramolecular cyclization pathway instead of intermolecular condensation, thereby enabling self-assembly into nanoaggregates with “turn-on” fluorescence. In vitro experiments demonstrated that CyNA_P-SS-FK generated 3.9-fold higher signal intensity in HCC-LM3 cells compared to GSH inhibitor-pretreated groups. CyNA_P-SS-FK was further used for real-time imaging orthotopic HCC in living mice. CyNA_P-SS-FK facilitated to detect cancerous lesions with a diameter of ∼2 mm, and exhibited an improved signal to background ratio (SBR), which is ∼5-fold enhancement relative to that of the “always-on” control probe CyNA_P-T (Fig. 4B). An extended detection window (∼36 h) was also detected (Fig. 4C). This CyNA_P-SS-FK is different from the previously reported bioorthogonal CBT-Cys reaction mediated probes, that are intrinsically fluorescent prior to in situ self-assembly. Instead, CyNA_P-SS-FK shows the “off-to-on” mode of signal response, which is beneficial to overcome the “false-positive” output and improve sensitivity and the SBR.

Fig. 4 (A) Schematic illustration of CyNA_P-SS-FK for specific fluorescence diagnosis of HCC. (B) SBRs of CyNA_P-SS-FK or CyNA_P-T. (C) Fluorescence intensity in livers of CyNA_P-SS-FK or CyNA_P-T injected mice at different timepoints, adapted with permission from ref. 76. Copyright 2025, Springer Nature.

4.2. Positron emission tomography imaging

Positron emission tomography (PET) imaging gains great interest due to ultra-high sensitivity (detecting picomolar concentration), limitless tissue penetration ability and quantification capability, and plays an irreplaceable role in the precise diagnosis and treatment of diseases, especially in the nuclear medicine field.⁷⁷ It can dynamically and noninvasively visualize biomolecular events via detecting the annihilation radiation γ photons released by radionuclide labelled probes.⁷⁸ Currently, the commonly used PET radioisotopes include ¹⁸F, ⁶⁸Ga, ⁸⁹Zr, etc.⁷⁹¹⁸F has become the most widely used one in clinic because of its long half-life (109.8 minutes) and the diversity of labelling chemistry.^78,80 Qiu et al. developed an intelligent ¹⁸F labelled PET probe based on bioorthogonal click chemistry [¹⁸F]1 (Fig. 5A).⁸¹ The probe is favorable to target the cancer overexpressing biotin receptor (BR) and undergo a bioorthogonal CBT-Cys click reaction in the presence of GSH. The preferable intramolecular cycloaddition, evidenced by negative changes of Gibbs free energy, led to the further self-assembly into nanoparticles with an average diameter of 138.2 ± 16.3 nm. This bioorthogonal self-assembly mechanism enabled the probe to have both the rapid penetration ability of small molecules and the long retention properties of nanoparticles. As a control, [¹⁸F]1a without a biotin targeting group and [¹⁸F]1b without a disulfide bond were prepared for the intracellular uptake experiments. The uptake rates of [¹⁸F]1 in BR positive tumor cells A549 and HeLa cells at 4 h were 5.19 ± 0.25% and 4.12 ± 0.23%, which were significantly higher than those of [¹⁸F]1a (0.81 ± 0.15% in HeLa cells) and [¹⁸F]1b (1.34 ± 0.31% in HeLa cells). PET imaging demonstrated higher uptake of [¹⁸F]1 in biotin-overexpressing HeLa tumors in vivo in contrast to the biotin-pretreated blocking groups (Fig. 5B). The tumor-to-muscle uptake ratio was about 3 at 10 min and reached 4 at 1 h (Fig. 5C), proving good imaging contrast and a long retention time.

Fig. 5 (A) Chemical structures of 1, 1a, and 1b. (B) MicroPET imaging of [¹⁸F]1 and [¹⁸F]1 + Block (pretreated with biotin) in HeLa tumor-bearing mice. (C) Tumor and muscle uptake of [¹⁸F]1 in HeLa tumor bearing mice at different times, adapted with permission from ref. 81. Copyright 2021, American Chemical Society. (D) Chemical structures of SF, adapted with permission from ref. 82. Copyright 2022, American Chemical Society. (E) Chemical structures of PET tracers [¹⁸F]SF-DEVD and [¹⁸F]SF-Glu.

Another example is a novel molecule SF that was introduced with two benzyl groups attached on the abovementioned intramolecular CBT-Cys macrocylization scaffold.⁸² For SF, 4-(aminomethyl) benzoic acid was selected as a rigid linker and a flexible glycine residue was employed to adjust the macrocycle size (Fig. 5D). The synergetic role of both moieties optimized the intramolecular CBT-Cys cycloaddition kinetics, and improved the reaction rate, resulting in the reaction completion within 1 min and forming a stable macrocyclic structure (SF-C) under physiological conditions. The intramolecular reactivity not only eliminates the high-concentration dependency of traditional probes but also exhibits robust resistance to free Cys interference, consequently improving both in situ assembly efficacy and imaging specificity. Furthermore, SF was further modified with the radiolabelled precursor [¹⁸F]AmBF₃ (¹⁸F labelled aminomethyl trifluoroborate) and an enzyme specific substrate, including a caspase-3 or γ-glutamyltranspeptidase (GGT) responsive DEVD peptide and a Glu motif to obtain two PET tracers [¹⁸F]SF-DEVD and [¹⁸F]SF-Glu (Fig. 5E). Both PET tracers have been applied for imaging of enzymatic activity in vivo. Taking caspase-3 detection as an example, HeLa tumor bearing mice were intratumorally injected with DOX to induce apoptosis for the detection of caspase-3 activity (Fig. 6A). Compared with the saline treated control group, DOX treated tumors showed significantly higher radioactivity (7.74 ± 1.56% ID per mL, 15 min post-injection). The further treatment of non-radioactive probe, [¹⁸F]SF-DEVD, further enhanced tumor uptake to 10.29 ± 1.11% ID per mL and prolonged signal retention at 30 min. The tumor-to-muscle ratio was 2.42 ± 0.54 and 6.91 ± 0.66 in [¹⁸F]SF-DEVD and the co-injection group. Compared to the previously reported [¹⁸F] tracer without a benzyl linker, [¹⁸F]SF-DEVD achieved excellent tumor uptake (7.74 ± 1.56% vs. 4.25 ± 0.97% ID per mL), and the acquiring dose of nonradioactive compounds was reduced 16 times (25 nmol vs. 400 nmol), which was attributed to the rapid intramolecular reaction mechanism of SF, enabling concentration independent self-assembly and enhancing the retention of nanoparticles in tumors. Chen et al. developed a caspase-3-sensitive PET tracer ([¹⁸F]-C-SNAT4) (Fig. 6B), in which the luciferin motif was replaced with 2-pyrimidinecarbonitrile and a benzyl linker to increase serum stability.⁸³ In the presence of caspase-3, the occurrence of the thiol-nitrile intramolecular reaction promoted the in situ self-assembly into nanoparticles with an average size of around 200 nm. [¹⁸F]-C-SNAT4 was used to detect cell death in cisplatin-treated drug-sensitive NCI-H460 non-small cell lung cancer cells. In NCI-H460 (drug-sensitive) and NCI-H1299 (drug-resistant) tumor-bearing mouse models, PET signal intensity in NCI-H460 tumors exhibited a positive correlation with cisplatin dosage. Significantly, using PET apoptotic signals the chemotherapeutic outcomes can be predicted, obtaining consistent results with tumor volume changes. Again, the tracer successfully differentiated checkpoint inhibitor responders from non-responders in a colorectal cancer immunotherapy model, confirming its capability to predict therapeutic efficacy.

Fig. 6 (A) MicroPET images of HeLa-tumor-bearing mice with or without DOX treatment, adapted with permission from ref. 82. Copyright 2022, American Chemical Society. (B) Schematic illustration of the mechanism of caspase-3-sensitive PET tracer [¹⁸F]-C-SNAT4, adapted with permission from ref. 83. Copyright 2021, Springer Nature. (C) Schematic diagram of GSH and Cat B-responsive PET probes [⁶⁸Ga]NOTA-SFCVM and [⁶⁸Ga]NOTA-SFCVHEM, adapted with permission from ref. 85. Copyright 2024, American Chemical Society. (D) Chemical structures of the unlabelled precursor CBT-NODA, the non-radioactive gallium complex CBT-NODA-Ga, and the ⁶⁸Ga-labeled radioactive tracer CBT-NODA-⁶⁸Ga, adapted with permission from ref. 86. Copyright 2021, American Chemical Society.

Apart from ¹⁸F, ⁶⁸Ga labelling PET probes have been used due to its matchable radioactive half-life (67.7 min) with the pharmacokinetics of biological molecules, such as peptides and oligonucleotides.⁸⁴ Li et al. developed two Cat B-responsive PET probes, [⁶⁸Ga]NOTA-SFCVM and [⁶⁸Ga]NOTA-SFCVHEM (Fig. 6C).⁸⁵ Both probes were integrated with lysosome-targeting morpholine and Cat B/GSH dual activatable modules for the CBT-Cys click condensation reaction, but only [⁶⁸Ga]NOTA-SFCVHEM included a histidine–glutamate–histidine–glutamate–histidine–glutamate sequence (HEHEHE) to promote tumor uptake and hepatic metabolism. In vitro experiments confirmed both probes specifically recognized tumor cell lines (U87 and A549) with differential Cat B expression levels, and co-localization studies demonstrated selective localization in lysosomes. In vivo PET imaging revealed that [⁶⁸Ga]NOTA-SFCVHEM and [⁶⁸Ga]NOTA-SFCVM exhibited superior tumor uptake in Cat B-positive ones. HEHEHE containing [⁶⁸Ga]NOTA-SFCVHEM exhibited remarkable lower liver uptake compared to [⁶⁸Ga]NOTA-SFCVM. Chen et al. developed three compounds, containing a furin sensitive RVRR substrate, and StBu and CBT motifs, denoted as the unlabelled precursor AcRVRRC(StBu)K(NODAGA)-CBT (CBT-NODA), the non- radioactive gallium complex AcRVRRC(StBu)K(NODAGA-Ga)-CBT (CBT-NODA-Ga), and the ⁶⁸Ga-labelled radioactive tracer AcRVRRC(StBu)K(NODAGA-⁶⁸Ga)-CBT (CBT-NODA-⁶⁸Ga) (Fig. 6D).⁸⁶ These compounds have been demonstrated to activate the CBT-Cys click reaction in a furin-rich tumor microenvironment. Experiments demonstrated that sole injection of the radioactive tracer CBT-NODA-⁶⁸Ga resulted in nonspecific condensation between unpurified residual precursors and endogenous Cys in tumor cells, thereby limiting nanoparticle generation efficiency. Instead, co-injection of the ⁶⁸Ga-labelled PET tracer and its nonradioactive analogue blocked nonspecific interactions, ensuring efficient synthesis of hybrid ⁶⁸Ga nanoparticles within tumor cells, which prolonged tumor retention and amplified the microPET signal.

4.3. Photoacoustic imaging

Photoacoustic (PA) imaging has received widespread attention due to its high spatial-temporal resolution and deep penetration.⁸⁷ Its mechanism involves PA-activatable reagents (i.e. endogenous biomolecules or exogenous PA chromophore-containing probes) artificially shifting absorbed NIR light to heat, thus further elevating the local temperature to produce ultrasonic waves.^88,89 Most fluorescent probes suffer from aggregation-caused quenching effects during in vivo imaging, but their probability of non-irradiation relaxation is favorable to enhance PA signals.^90,91 This motivates the design of probes as PA imaging contrasts via in situ biomarker-induced aggregation. Wang et al. developed a PA probe Val-CitCys(Set)-Lys(Cypate)-CBT(Cypate-CBT) (Fig. 7A).⁹² In the presence of Cat B, Cypate-CBT underwent the intramolecular CBT-Cys crosslinking to yield its dimer and self-assembled into Cypate-CBT-NPs with a diameter of 207.7 ± 15.9 nm. PA phantom images of Cypate-CBT were observed to be increased after 2 h-incubation with Cat B, with the corresponding fluorescence quenching. Good sensitivity and selectivity of Cypate-CBT towards Cat B were also demonstrated. Cypate-CBT was used in Cat B-overexpressing MDA-MB-231 cells and MDA-MB-231 cells bearing tumors, showing 4.9-fold or 4.7-fold PA signal enhancement, respectively (Fig. 7B and C).

Fig. 7 (A) The mechanism of PA probe Cypate-CBT. (B) Time-dependent PA intensity of probes in each group after incubation with MDA-MB-231 cells. (C) Time-dependent PA intensity after injection of probes in MDA-MB-231 tumors, adapted with permission from ref. 92. Copyright 2021, Wiley-VCH GmbH. (D) Schematic diagram of the mechanism of NI-C-CBT, adapted with permission from ref. 93. Copyright 2023, Wiley-VCH GmbH.

Another example is a nitroreductase (NTR)-responsive smart photoacoustic probe, NI-C-CBT, comprising 2-nitroimidazole (NI), StBu-protected cysteine-diaminopimelic acid (Cys(StBu)-Dap), near-infrared (NIR) chromophore IR780 and CBT (Fig. 7D).⁹³ In the hypoxic tumor microenvironment, elevated GSH and NTR synergistically triggered the reductive cleavage of NI-C-CBT to release C-CBT, and underwent the CBT-Cys bioorthogonal click reaction. Dimerization and self-assembly of the probe subsequently induced intramolecular and intermolecular fluorescence quenching of IR780, switching on and amplifying the PA signal. In vitro experiments demonstrated a 1.9-fold overall PA signal enhancement in hypoxic HeLa cells compared to normoxic cells. Further investigation demonstrated highly sensitive and specific photoacoustic imaging of tumor hypoxia in a HeLa tumor-bearing mouse model.

4.4. Magnetic resonance imaging

Magnetic resonance imaging (MRI) is a noninvasive imaging technique and has been widely clinically used.⁹⁴ It obtains 3D anatomical images with high-resolution, which can provide rich morphological and functional information from soft tissues.⁹⁵ Clinical MRI imaging usually relies on the transverse (T₂) relaxation time of protons (¹H) in human tissues to collect signals. Nevertheless, the limited relaxation time of ¹H imposed the injection of contrast agents to enhance ¹H MRI signals.⁹⁶ According to the ratio of transverse to longitudinal relaxation rates (r₂/r₁), contrast agents can be classified to T₁- or T₂-type ones.⁹⁷ Clinical T₁-type ones, such as Gd-DOTA and Gd-DTPA, are preferrable because they light up the targeted area.⁹⁸ Xu et al. introduced Gd-DOTA as the contrast agent conjugated with caspase-3-responsive CBT-Cys cycloaddition moieties (DEVD peptide, CBT and StBu-protected Cys) as a self-assembling probe (DEVDC_S-Gd-CBT) for T₁-weighed MRI imaging of apoptosis (Fig. 8A).⁹⁹ Caspase-3 treatment of DEVDC_S-Gd-CBT led to 1.14-fold enhancement of the r₁ value compared to nontreated one, while the r₂ value showed a small change from 4.13 to 4.52 mM⁻¹ s⁻¹. The r₂/r₁ value was calculated to be 0.82, which is suitable for its use as a T₁ contrast agent at 0.5 T. The probe was further applied for T₁-weighed of MRI imaging of cis-dichlorodiamine platinum-induced apoptosis in cells and in tail-amputation-simulated apoptotic zebrafish. The apoptotic zebrafish model showed a bright signal in the lesion near the amputation and the highest lesion to body ratio was 1.33, which was 1.84-fold of the PBS treated control group (Fig. 8B).

Fig. 8 (A) Schematic illustration of caspase-3-responsive probe DEVDC_S-Gd-CBT for T₁-weighed MRI imaging of apoptosis. (B) T₁-weighted MR images and the lesion/body grayscale value ratios of MR images of zebrafish in different groups, adapted with permission from ref. 99. Copyright 2023, American Chemical Society.

4.5. Raman imaging

As an attractive optical imaging modality, Raman imaging (RI) has gained researchers’ interest because it offers high spectroscopic sensitivity, exquisite molecular specificity and resistance to photobleaching.^100,101 More importantly, the introduction of plasmonic nanomaterials to amplify Raman signals by 10⁵–10¹⁵ orders of magnitude, which is called surface enhancement of Raman scattering (SERS), has boosted the development of RI.¹⁰² Currently, RI has been applied for quantitative mapping of molecules, even single molecules, in complex physiological activities. Bioorthogonal CBT-Cys cycloaddition provides the reactive nitrile group (C≡N) on the CBT motif, and shows a representative Raman peak at 2227 cm⁻¹, which is located in the cell silent region (1800–2800 cm⁻¹).¹⁰³ This means CBT-Cys cycloaddition-based probes can be regarded as natural candidates for RI. Moreover, cycloaddition mediated self-assembly facilitates the specific accumulation in the targeted site, which is advantageous to promote the clinical translation of RI.¹⁰⁴ For example, Barman et al. proposed a novel self-assembling bioorthogonal enzyme recognition probe (nanoSABER) for targeted tumor RI imaging (Fig. 9A and B).¹⁰⁵ NanoSABER consisted of a polyarginine oligomer (R₆) with six repeating units to enhance cell membrane penetration, a legumain-responsive substrate (alanine–alanine–asparagine (AAN)), a Raman-detectable vibrational tag alkyne group (2120 cm⁻¹) from propargylglycine (Pra) and a nitrile group (2230 cm⁻¹) from CBT-modified lysine. The tumor specific CBT-Cys reaction resulted in the gradual reduction of the Raman signal of the nitrile peak (2230 cm⁻¹), but the alkyne peak (2120 cm⁻¹) remained constant. Through the tumor-overexpressing legumain triggered reaction, nanoSABER achieved distinct increase in the ratio of the alkyne to nitrile Raman signals ranging from 1.5 to 4.5 in DU145 cells (Fig. 9C). In contrast, LNCaP with a low expression level of legumain did not show significant changes in the Raman ratio. The enzyme-mediated self-assembly strategy was demonstrated to generate a Raman signal with long retention. The imaging enzyme activity was further exceeded in vivo and ex vivo. In combination with machine learning models, nanoSABER demonstrated a 100% specificity within DU145 tumor bearing mice (Fig. 9D).

Fig. 9 (A and B) Schematic diagram of nanoSABER for targeted tumor RI imaging. (C) Ratio of the alkyne to nitrile Raman peak intensities of DU145, LNCaP and RWPE1 cells treated with nanoSABER. (D) Score plot of MCR2 versus MCR3 for the DU145 and LNCaP cells, adapted from ref. 105, under the license CC-BY, published by Wiley-VCH GmbH. (E) Schematic illustration of Yne-CBT for the detection of intracellular enzyme activity. (F) Thermogram of CTSB enzyme activity in a single MDA-MB-231 cell, adapted with permission from ref. 106. Copyright 2024, American Chemical Society.

Wang et al. designed a self-referencing Raman probe, Val-Cit-Cys(StBu)-Pra-Gly-CBT (Yne-CBT), for the detection of intracellular enzyme activity (Fig. 9E).¹⁰⁶ The integration of Yne-CBT with intracellular overexpressing Cat B formed long-retained cyclic dimers, which displayed a distinct increase in the ratio of the alkyne to nitrile Raman signals. Additionally, uniform Au@SiO₂ NPs were used to enhance the Raman signal with a detection limit of 61.4 U L⁻¹. Using a custom microfluidic channel coupled with confocal Raman microscopy, real-time monitoring of MDA-MB-231 cells (high Cat B expression) co-incubated with Yne-CBT and Au@SiO₂ NPs demonstrated dynamic shifts in relative signal intensities at 2120 and 2227 cm⁻¹, confirming Cat B-activated CBT-Cys click reactions. Enzymatic activity heatmaps revealed significant Raman signal variability due to cellular heterogeneity (Fig. 9F).

4.6. Quantitative phase imaging

Quantitative phase imaging (QPI) detects the refractive index instead of exogenous contrast agents to monitor cellular behavior, which has been demonstrated as a label-free three-dimensional optical imaging technique.^107,108 However, its molecular imaging capability remained constrained by the bottleneck that rarely are direct refractive index variations associated with intracellular molecular dynamics, such as enzymatic catalytic processes.¹⁰⁹ Tanwar et al. developed bioorthogonal click chemistry-based QPI nanoprobes (P-SiO₂ NPs) for label-free, high-contrast visualization of intracellular enzyme activity.¹¹⁰ Silica nanoparticles (SiO₂ NPs) with a relative higher refractive index of 1.435 ± 0.006 compared to intracellular background (1.350 ± 0.002), were introduced as scaffolds. SiO₂ NPs were functionalized with peptide sequences Ac-Arg-Arg-Arg-Arg-Arg-Arg-Ala-Ala-Asn-Cys(StBu)-Pra-Lys-CBT (P), containing polyarginine cell-penetrating motifs, legumain cleavage sites (AAN), and CBT groups (Fig. 10A). GSH/legumain dual responses promoted CBT-Cys intermolecular condensation and SiO₂ aggregation. This phenomenon was revealed by optical diffraction tomography showing that the refractive index range spanned from 1.40 to 1.48. P-SiO₂ NPs selectively aggregated in legumain-overexpressing DU145 prostate cancer cells (Fig. 10B and C), while SiO₂ NPs maintained mono-dispersion in low-expressing LNCaP cells. Aggregation significantly increased local nanoparticle density, forming high-refractive index regions (1.40–1.50) against the cellular background (refractive index = 1.350 ± 0.002), thereby leading to high-contrast refractive index images. Confocal fluorescence imaging of FITC-labelled probes further validated aggregation specificity. Compared to conventional fluorescence techniques, QPI eliminated phototoxicity and photobleaching. The developed biorthogonal QPI probes further enhanced signal specificity through enzyme-triggered refractive index amplification, enabling three-dimensional spatiotemporal visualization of enzyme activity at single-cell resolution.

Fig. 10 (A) Schematic diagram of P-SiO₂ NPs for QPI. Volume percentage of the refractive index between 1.4 and 1.5 in DU145 (B) and LNCaP (C) cells treated with different groups, adapted with permission from ref. 110. Copyright 2023, American Chemical Society.

4.7. Multimodal imaging

Molecular imaging usually employs molecular probes, especially biomarker-activatable probes as imaging contrasts and achieves high-resolution images about specific biological targets, enzyme activities and real-time quantitative monitoring of pathological processes.^111,112 In the past few decades, biomarker-activatable molecular probes have been widely explored and applied for FL, PET, MRI or PA imaging.^113,114 However, these probes have their own merits and demerits, such as high sensitivity but low penetration for fluorescent ones, deep penetration but poor spatial resolution and high cost for PET ones, and high spatial resolution but low sensitivity for MRI ones. Instead of molecular probes for single modal imaging, the development of multimodal probes that integrate two or more complementary imaging techniques is advantageous to address inherent deficiencies in single-modality systems and achieve comprehensive diagnostic information.^115–117 Fu et al. developed a caspase-3 activated second near-infrared (NIR-II) fluorescent/PA bimodal nanoprobe IR1048-Asp-Glu-Val-Asp-Cys(StBu)-(AuNNP)-cyanobenzothiazole(CBT) (AuNNP@DEVD-IR1048) for imaging of apoptosis (Fig. 11A).¹¹⁸ In this nanoprobe, core–shell structured nanogapped Au NPs were introduced as a PA imaging contrast agent because of broad absorption in the NIR-II window. IR1048 was used as NIR-II fluorescence imaging agent. Au NPs were modified with azido and thiolated PEG, and then covalently linked with IR1048-DEVD-alkyne to obtain the bimodal probe. Caspase-3 and GSH addition promoted the hydrolysis of DEVD, released IR1048 and formed Au NP nanoclusters through CBT-Cys cycloaddition, which switched on NIR-II and PA signals simultaneously. In vitro experiments showed that the NIR-II fluorescence intensity of the probe gradually enhanced with the incubation with increased caspase-3. The red-shift absorption in the NIR-II window triggered the enhancement of the PA₁₂₅₀ to PA₆₈₀ ratio in the presence of caspase-3. Moreover, AuNNP@DEVD-IR1048 was used as a radiosensitizer for radiotherapy and could realize self-monitoring of radiotherapy-triggered apoptotic signals via PA and NIR-II fluorescence imaging. With the increase of the X-ray dose (2–8 Gy), AuNNP@DEVD-IR1048 treated HepG2 cells showed AuNNP aggregates in biological transmission electron microscopy (bio TEM) (Fig. 11B). “Turn-on” PA and NIR-II fluorescence signals were detected (Fig. 11C and D), which were in accordance with apoptosis results from cleaved caspase-3 expression in western blotting (Fig. 11E). The self-evaluation capability of AuNNP@DEVD-IR1048 was used in a HepG2 tumor bearing mouse model and a luciferase-transfected MC38 orthotopic liver cancer model. Significantly, in an orthotopic liver cancer model, bioluminescence intensity associated tumor volume was strongly correlated with caspase-3 expression levels in mice under radiotherapy treatment, showing Pearson's correlation coefficient of −0.9637 (Fig. 11F). ΔPA and ΔFL intensity also correlated well with tumor volume changes, showing Pearson's correlation coefficient of −0.9433 and −0.9577, respectively. These results demonstrated that this “treatment feedback” mechanism has successfully established a quantitative correlation model between the radiotherapy effect and the real-time imaging parameters, providing a visualization evaluation tool for early prediction of the clinical treatment effect.

Fig. 11 (A) Schematic diagram of AuNNP@DEVD-IR1048 for imaging of apoptosis. (B) Bio-TEM images of AuNNP@DEVD-IR1048-treated HepG2 cells under varying X-ray doses (2–8 Gy). Photoacoustic (C) and NIR-II fluorescence (D) intensity of AuNNP@DEVD-IR1048-treated HepG2 cells under varying X-ray doses. (E) Western blot analysis of cleaved caspase-3 expression as a marker of apoptotic activation. (F) Pearson's correlation coefficient of the relationship between the relative tumor volume changes and the caspase-3 expression, the ΔPA changes or the ΔFL changes after RT, adapted with permission from ref. 118. Copyright 2021, Wiley-VCH GmbH.

The 2-cyano-6-hydroxyquinoline (CHQ)-Cys macrocyclization reaction has been commonly used to design activatable probes based on Cys-Luc-CHQ or Cys-Ben-PMN scaffolds for signal mode molecular imaging, such as PET, FL, or PA imaging.^119,120 However, the introduction of these scaffolds to design probes for multimodal imaging is challenging due to the difficulty in installing two imaging tags together in one molecule. Also, the two imaging tags introduced would amplify the molecular size and induce steric hindrance that slows down the macrocyclization kinetics. To address this issue, Wang et al. designed a caspase-3 activatable probe ([¹⁸F]-IR780-1) based on the triazole IR780 scaffold for PA/PET bimodal imaging of apoptosis (Fig. 12A).¹²¹ [¹⁸F]-IR780-1 modified CBT-Cys reaction pairs and ¹⁸F labelled zwitterionic trifluoromethyl borate ([¹⁸F]-AMBF₃) onto the IR780 scaffold to enable reliable PA/PET dual-modal imaging. To optimize macrocyclization kinetics, different acyclic precursors that were differed in linkers containing no glycine residue (8a) or one glycine residue (8b) on the D-Cys sites were screened and designed. Experimental results revealed that the macrocyclization kinetics of 8a (first-order rate constant k = (3.9 ± 0.1) × 10⁻³ s⁻¹) were significantly faster than that of 8b (k = (2.6 ± 0.2) × 10⁻³ s⁻¹) (Fig. 12B). The half-life of 8a was comparable to those of previously reported scaffolds like Cys-Luc-CHQ or Cys-Ben-PMN. In [¹⁸F]-IR780-1, the caspase-3 cleavage collaborated with GSH reduction facilitated in situ self-assembly into nanoparticles, which quenched the fluorescence of IR780 and enhanced the PA signal. The cold compound of [¹⁸F]-IR780-1, IR780-1 has demonstrated 4-fold enhancement of the PA signal at 855 nm (Fig. 12C). In U87MG tumor-bearing mice, pretreatment with DOX and [¹⁸F]-IR780-1 significantly enhanced the PET signal and PA intensity in tumors, compared to the saline group, and the group preinjected with a caspase-3 inhibitor, Z-VAD-fmk (Fig. 12D). Moreover, the PET tracer in the IR780 fluorophore based bimodal probe can be replaced by Gd-DOTA for PA and MRI imaging of tumor apoptosis (Fig. 13A).¹²² The caspase-3 triggered self-assembly concurrently turned on the PA signal (∼4.3-fold at 855 nm) (Fig. 13B) and increased r₁ from 7.98 to 19.66 mM⁻¹ s⁻¹ in MRI at 0.5 T (Fig. 13C).

Fig. 12 (A) Schematic diagram of [¹⁸F]-IR780-1 based on the triazole IR780 fluorophore for PA/PET bimodal imaging of apoptosis. (B) Calculation of first-order constants of intramolecular macrocyclization of 8a and 8b. (C) PA images (insets) and normalized PA intensities acquired at 790 and 855 nm before and after incubation of IR780-1 with caspase-3. (D) Representative axial and coronal PET images and transverse PA images of U87MG tumors in living mice after different treatments, adapted with permission from ref. 121. Copyright 2022, Wiley-VCH GmbH.

Fig. 13 (A) Schematic diagram of Gd-IR780 for PA and MRI imaging of tumor apoptosis. (B) PA images (insets) and normalized PA intensities acquired at 790 and 855 nm before and after incubation of Gd-IR780 with caspase-3. (C) Comparative r₁ analysis of Gd-IR780 based on 1/T₁versus Gd concentration plots before and after caspase-3 incubation, adapted with permission from ref. 122. Copyright 2022, Elsevier.

5. Bioorthogonal reaction-mediated size variable nanosystems for therapy

On the basis of the well-known bioorthogonal reaction mechanism, many drug delivery systems have been widely developed to prolong the retention of drugs in targeted sites with improved selectivity and enhanced therapeutic performance.^123,124 Generally, these drug delivery systems are classified into organic molecule-to-nano in situ self-assembled systems and nano-to-cluster aggregate systems. These systems possess great application potential in various treatment modes, verifying the strong practicality of bioorthogonal reactions. In this section, we present and review both design principles and discussion of the latest research progress with an emphasis on the bioorthogonal reaction-mediated drug delivery systems to enhance therapeutic effects (Table 2), which are expected to assist the understanding of this research field.

Table 2 Bioorthogonal reaction based nanosystems for therapy

Treatment mode	Material	Responsive agent	Reaction type	Disease	Size transformation, incubation time	Ref.
Chemotherapy	SPr-CPT@PEG	GSH	CBT-Cys	4T1 tumor	From molecules to fusiform in shape with a length of 400 nm and a width of 20 nm	131
	iCPDNDBCO/iCPDNN3	pH 6.5	SPAAC	4T1 tumor	From 107 nm to 1216 nm, 15 min; from 1216 nm to 10 nm, 24 h	134
	NP@DOXDBCO + iCPPAN3	pH 6.5	SPAAC	4T1 tumor	From 112.0 nm to 848.1 nm, 15 min; from 848.1 nm to 10 nm, 12 h	135
	AuNPs-D&H-R&C	Furin	CBT-Cys	MCF-7/ADR tumor	From 38.2 ± 1.2 nm to 263.2 ± 10.6 nm, 48 h	136
	Cip-CBT-Ada/CD-M	GSH/Caspase-1	CBT-Cys	S. aureus infection	From molecules to 20.0 ± 2.5 nm nanoaggregates, 18 h	137
	Ag-P&C NPs	pH 6.5	CBT-Cys	MRSA-nfected wounds, anti-biofilm models, periodontitis	From ∼50 nm to ∼400 nm, 24 h	140
	MEM/DON/INS ICV	Legumain	CBT-Cys	SAMP8 mice	From 198–203 nm to 800 nm–4.5 μm, 6 h	141
Photothermal therapy	AuNP@1	GSH/Furin	CBT-Cys	MDA-MB-468 tumor	From 25.7 ± 10.2 nm to 103.5 ± 12.3 nm, 10 h	147
	SPIO@1NPs	GSH/Furin	CBT-Cys	MDA-MB-468 tumor	From ∼50 nm to 139.72 ± 24.69 nm, 6 h	148
	AuNPs-ImLND and AuNPs-DBCO-RGD	DBCO	BCR	4T1 tumor	From 16–24 nm to over 1000 nm, 12 h	149
	Cu-POM NCs, antibacterial molecule 6	Cu-based catalysts	CuAAC	S. aureus and E. coli infected biofilm	From 7–10 nm to 500 nm (pH 4.5), 22.5 h	150
	CNDs(CNDs@N3 and CNDs@C≡C)	Cu-based catalysts	CuAAC	VRE bacterial infection	From 6 ± 4 nm to 40 ± 20 nm	151
Radiotherapy	MnAuNP-C&B	GSH	CBT-Cys	CT26 tumor	From 29 nm to over 200 nm, 24 h	156
	[^131I]IM(HE)3 AAN	Legumain/GSH	CBT-Cys	HCT116 tumor	From molecules to 86 ± 9 nm	157
Photodynamic therapy	P-FFGd-TCO + 775NP-Tz + SA-Tz	ALP	iEDDA	HeLa tumor	From 40–240 nm to ∼1 μm, 15 min	162
Chemodynamic therapy	cPFCDBCO and cPFCN3	pH 6.5	SPAAC	4T1 tumor	From ∼100 nm to 995.07 nm, 15 min; from 995.07 nm to 10 nm, 12 h	167
Synergistic therapy	D-NP and C-NP	pH 6.5	CBT-Cys	4T1 tumor	From ∼65 nm to 2611 ± 115 nm, 24 h	169
	SIA-αTSLs	CTSB	CBT-Cys	CT26 tumor	From 148.13 ± 4.24 nm to 498.70 ± 23.89 nm, 2 h; from ∼600 nm to small sized nanoparticles, 10 min	172
	NGOPC@PTX	Legumain	CBT-Cys	4T1 tumor	From ∼60 nm to ∼900 nm, 24 h	173
	Ce6-Leu@Mn^2+	LAP/GSH	CBT-Cys	HepG2 tumor	From molecules to ∼80 nm aggregates, from ∼80 nm to ∼23 nm	174
	NPCe6-DBCO and TK-PAMAMPR104A-N3	pH 6.5	SPAAC	4T1 tumor	From ∼100 nm to ∼740 nm; from 740 nm to 10 nm, 0.5 h	175

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5.1. Chemotherapy

Chemotherapeutics, which deliver drugs to targeted sites via blood circulation and combat disease, are the most commonly used strategy in disease treatment.^125,126 Currently available drugs encounter a few challenges such as drug hydrophobicity and insufficient accumulation, causing poor therapeutic effects and inducing systemic toxicity.¹²⁷ Nanocarriers utilize nanotechnology to encapsulate hydrophobic drugs and modify active targeting units to resolve the abovementioned problem.^128,129 Nevertheless, varied receptor expression degrees, and complicated and dynamic intracellular environments limit the recognition capability and compromise therapeutic efficacy.¹³⁰ Utilization of bioorthogonal reactions to label drugs allows selective self-assembly of them in targeted sites, which has exhibited increasing potential to realize precise delivery and controlled drug release with improved efficiency. For example, Zhong's group developed a bioorthogonal reaction-participated lysine dendrimer, SPr-G2, as a peptide dendritic agent for cancer therapy.¹³¹ SPr-G2 included a CBT motif and a disulfide-protected cysteine residue for the GSH-responsive bioorthogonal CBT-Cys click reaction (Fig. 14A). SPr-G2 underwent in situ polymerization to generate “hand-in-hand” dendrimers and can be self-assembled into nanoparticles via π–π stacking interactions. The self-assembled SPr-G2 selectively accumulated in lysosomes through a strong binding affinity between the sufficient positively charged periphery NH₂ group and the negative lysosomal membrane. SPr-G2 disrupted the lysosomal integrity and induced cathepsin release through investigation of the lysosomal size and co-staining assay with acridine orange and Magic Red MR-(RR)2. The GSH-activated in situ polymerization triggered cell death in different types of cancer cells, including MDA-MB-231, MCF-7/ADR and A549/T, while exhibited low cytotoxicity in HUVECs and 3T3 cells, which was ascribed to the high-level GSH in cancer cells facilitating the specific self-assembly in cancer cells. The SPr-G2 triggered lysosome dysfunction prevented its degradation of histocompatibility complex class I (MHC-I), which exhibited 4.2-fold enhancement of the MHC-1 expression level on 4T1 cell compared to that in control cells (Fig. 14B). Additionally, SPr-G2 was conjugated with chemotherapeutic drug camptothecin (CPT) and coated with methoxy polyethylene glycol-aldehyde (mPEG-CHO) via acid-responsive benzoic imine bonds to obtain fusiform-like SPr-CPT@PEG (Fig. 14C). In the acidic tumor microenvironment, the shedding PEG shell of SPr-CPT@PEG will be removed. The further entry of GSH-rich cancer cells induced self-assembly in lysosomes and enabled CPT escaping from lysosomes to synergistically amplify antitumor immunity. To investigate the anti-tumor immune effects induced by SPr-CPT@PEG during in vivo therapy, bilateral tumor models were established. Compared to control groups, SPr-CPT@PEG suppressed both primary and distant tumor growth, corresponding to the observation of a decreased tumor weight (Fig. 14D).

Fig. 14 (A) Schematic diagram of the SPr-G2 dendrimer and the mechanism of self-assembly. (B) MHC-I expression on 4T1 cells. (C) Schematic illustration of the synthesis of SPr-CPT@PEG. (D) Primary and distant tumor weights after treatment with different groups, adapted with permission from ref. 131. Copyright 2024, Wiley-VCH GmbH. (E) Schematic diagram of tumor acidity and a bioorthogonal reaction dual-responsive nanosystem to co-deliver NO donor and chemotherapeutic drug DOX for enhanced chemotherapy. (F) FL images of 4T1 murine models after i.v. administration of: (I) iCPDN_Cy5.5 + iCPDN_DBCO/Cy5.5 and (II) iCPDN_N3/Cy5.5 + iCPDN_DBCO/Cy5.5 at different time points. (G) Quantification of the DOX concentration in major organs and tumor tissues at 24 h post-injection with DOX, iCPDN+ iCPDN_DBCO, and iCPDN_N3 + iCPDN_DBCO, adapted with permission from ref. 134. Copyright 2022, American Chemical Society.

Apart from insufficient retention, another main challenge in cancer chemotherapy is chemo-resistance.¹³² A tumor hypoxic environment upregulates hypoxia-inducible factor-1α (HIF-1α), which has been reported to enhance the expression level of P-glycoprotein, cause drug efflux and contribute to chemo-resistance.¹³³ Hence, researchers developed various strategies to overcome hypoxia and improve the treatment outcome of chemotherapy. For instance, Wang et al. employed a nitric oxide (NO) donor to increase oxygen (O₂) perfusion within a tumor to overcomer hypoxia and introduced a tumor acidity and bioorthogonal reaction dual-responsive nanosystem to co-deliver the NO donor and a chemotherapeutic drug, doxorubicin (DOX), for enhanced chemotherapy (Fig. 14E).¹³⁴ In this nanosystem, DOX was conjugated to polyamidoamine (PAMAM) via an acid-sensitive hydrazone bond to obtain PAMAM-DOX. PAMAM-DOX was further introduced with thiol groups and linked with tert-butyl nitrite to generate ultrasmall PAMAM-DOX/NO (PND) containing a pH sensitive DOX prodrug and a NO donor. PDN was cross-linked with pH-cleavable maleic acid amide to form clustered nanoparticles (iCPDN) (107 nm). iCPDN were modified by an azide (N₃) motif containing poly(ethylene glycol) (PEG) or a dibenzocyclooctyne (DBCO) motif containing poly(2-azepane ethyl methacrylate) (PAEMA). In a normal physiological pH environment, DBCO was shed in the nanoparticles, while in a tumorous environment, mild acidity slowly deprotonated PAEMA, then exposed the DBCO moiety, and bioorthogonally reacted with N₃ containing PEGylated iCPDN, thus allowing rapid formation of nanoaggregates (∼1216 nm) within 15 min. Subsequently, as the maleic acid amide moieties were cleaved, the size of nanoaggregates was shifted to 10 nm with the incubation time prolonged to 24 h. Cy5.5 labelled iCPDN_N3 and iCPDN_DBCO were introduced to track the biodistribution in the tumor bearing mice model (Fig. 14F). Markedly enhanced accumulation and a prolonged retention time within tumor tissue were observed in the iCPDN_N3/Cy5.5 + iCPDN_DBCO/Cy5.5 group. This demonstrated that the “two-step” size transformation facilitated superior tumor retention. The delivery of DOX to hypoxic tumor tissues is also revealed in Fig. 14G. The released NO could reverse hypoxia-induced chemo-resistance by downregulating HIF-1α levels, enhance the chemotherapeutic efficacy of DOX, and reprogram the tumor immune microenvironment to boost antitumor immune responses. Additionally, authors also utilized the same strategy to prepare DBCO masked DOX prodrug nanoparticles and N₃ PEGylated hypoxia-activated prodrugs PR104A to construct a “two-step” size-transformation nanosystem (Fig. 15A), which efficiently overcame hypoxia and enhanced chemotherapy efficacy.¹³⁵

Fig. 15 (A) Schematic diagram of a dual-responsive nanosystem designed to overcome hypoxia-induced intratumoral heterogeneity and improve chemotherapy efficacy, adapted with permission from ref. 135. Copyright 2022, Elsevier. (B) The mechanism of furin protease sensitive AuNPs-D&H-R&C, adapted with permission from ref. 136. Copyright 2021, Elsevier. (C) Schematic illustration of Cip-CBT-Ada/CD-M for combating S. aureus infections in macrophages. (D) Fluorescence microscopy imaging of normal RAW 264.7 cells incubated with NBD-CBT-Ada/CD-M, S. aureus-infected RAW 264.7 cells treated with different groups, adapted with permission from ref. 137. Copyright 2023, Wiley-VCH GmbH.

Different from the modulation of tumor microenvironments, the inhibition of cancerous pro-survival pathways is also a desirable strategy to decrease chemo-resistance. Xie et al. designed a tumorous overexpressed furin protease sensitive Au NP-based nanoplatform to co-deliver DOX and hydroxychloroquine (HCQ), which inhibited one of the pro-survival pathways, cell autophagy (Fig. 15B).¹³⁶ In the nanoplatform, Au NPs were co-loaded with DOX and HCQ, and were modified with the RK peptide (RVRRCK) and 2-cyanobenzothiazole-polyethylene (CBT-PE), respectively. Furin cleaved the RK peptide sequence and subsequently promoted the CBT-Cys reaction, thus leading to the exposure of 1,2-aminothiol groups on Cys to form AuNP aggregates. The efficient uptake of AuNP aggregates delivered sufficient HCQ and DOX to MCF-7/ADR cells. HCQ interfered cell autophagy and promoted autophagic degradation, indicated by the blocking of more LC3-II recycling to LC3-I and high expression of p62 protein. Furthermore, HCQ plays a synergistic role with DOX in intensifying DNA damage and apoptosis pressure by activating the p53 signalling pathway. The nanoplatform was further used in chemo-resistant MCF-7/ADR breast tumors in vivo, eliciting superiority over free drug delivery systems in decreasing systemic toxicity.

Bioorthogonal chemistry-mediated chemotherapy can be also applied for different kinds of disease treatments. For instance, Zhan et al. designed a supramolecular antibiotic delivery system, Cip-CBT-Ada/CD-M, for combating S. aureus infections in macrophages.¹³⁷ The antibiotic-peptide conjugate, Cip-CBT-Ada, consisted of ciprofloxacin (Cip), a caspase-1/GSH reactive bioorthogonal CBT-Cys module, and an adamantane (Ada) moiety (Fig. 15C). The antibiotic-peptide conjugate was linked with β-cyclodextrin-heptameric mannoside (CD-M) through guest–host recognition, and showed a strong affinity to macrophages due to abundant mannose receptors on the surface. Within infected macrophages, elevated GSH and caspase-1 exposed reactive Cys groups triggered CBT-Cys click reactions. Cyclic Cip-dimers were further formed and subsequently self-assembled into Cip nanoparticles (Nano-Cip, ∼20 nm) via hydrophobic interactions. The following esterase-mediated hydrolysis promoted the Cip release to eradicate intracellular S. aureus. 7-Nitro-1,2,3-benzoxadiazole (NBD)-labelled NBD-CBT-Ada was prepared to investigate the uptake of S. aureus-infected macrophages. Only infected cells exhibited NBD fluorescence at 2 h incubation (Fig. 15D). Mannose pretreatment (receptor blockade) or the CD-M motif-absent group decreased fluorescence by 75.8% and 65.8%, respectively, verifying mannose receptor-mediated endocytosis. The control group treated GSH inhibitors or caspase-1 inhibitors led to 62.1% or 85.8% decrease of fluorescence, confirming GSH/caspase-1-dependent activation of self-assembly. Cip-CBT-Ada/CD-M treated S. aureus-infected RAW264.7 cells and mouse infection models demonstrated superior bactericidal efficacy and inflammation alleviation capability. Silver nanoparticles (Ag NPs) are commonly used as antibiotics because they can release Ag⁺ to inhibit ATP generation and affect the membrane structure.^138,139 Delivering sufficient Ag NPs to the biofilm is beneficial to antibacterial efficacy. Cheng et al. developed pH-responsive Ag NPs (Ag-P&C NPs) based on bioorthogonal chemistry, and employed an intelligent size-regulation strategy for drug-resistant bacterial infections (Fig. 16A).¹⁴⁰ PEGylated Ag NPs were modified with a peptide sequence (NH₂-Lys-Arg₄-Gly-His₄-Cys-CM) and a CBT moiety, respectively. In the neutral environment of healthy tissues, the peptide chain folded into a U-shaped conformation via hydrogen bonding, shedding the active thiol in the Cys group. Upon entering the acidic microenvironment of bacterial infection sites, the protonation of histidine imidazole groups induced surface charge reversal and triggered the following CBT-Cys cycloaddition. The system has been proved to acquire efficient bactericidal effects through multiple mechanisms, including disrupting membrane integrity, inhibiting ATP synthesis, and generating ROS. The system was successfully applied in three animal infection models, including MRSA-infected wounds, and for eradication of biofilms established on the surface of bone implants, ameliorating osseointegration, and treating periodontitis.

Fig. 16 (A) Schematic illustration of pH-responsive Ag-P&C NPs, adapted with permission from ref. 140. Copyright 2022, Wiley-VCH GmbH. (B) Schematic diagram of bioorthogonal reaction-mediated crosslinked vesicles to intracerebrally co-deliver insulin, donepezil hydrochloride and memantine hydrochloride for AD treatment. (C) Distribution of fluorescent nanovesicles in biological tissues treated with either ^RhBVesicle_AK or ^RhBICV after 12 h, adapted from ref. 141, under the license CC-BY-NC-ND 4.0, published by Elsevier.

Another example is bioorthogonal reaction-mediated crosslinked vesicles to intracerebrally co-deliver insulin, donepezil hydrochloride (DON HCl) and memantine hydrochloride (MEM HCl) for Alzheimer's disease treatment.¹⁴¹ These vesicles are respectively coated with AD brain parenchyma specific expressed legumain cleavable AK peptide sequences (Ac-Ala-Ala-Asn-Cys-Asp) and the CBT unit, thus triggering the CBT-Cys cross-linking reaction and forming in situ aggregation, which effectively prevented the drug efflux pumps of the brain (Fig. 16B). Multi-drugs were validated to exhibit a neuroprotective effect and improve memory ability of SAMP8 mice. Most importantly, the cross-linking strategies improved the intracerebral retention in vivo and overcame the brain barrier efflux. The distribution of RhB labelling cross-linked vesicles revealed that the brain and liver total retention was more than 91% (Fig. 16C), while other major organ retention was less than 9% at 12 h, providing good potential for chemotherapy of the AD brain.

5.2. Photothermal therapy

Photothermal therapy (PTT) employs photothermal reagents to elevate the temperature of local tumor lesions under light-irradiation to ablate cancer cells.¹⁴² Owing to the spatiotemporal controllability and noninvasiveness, PTT effectively decreased hazardous damage to health tissues, which has been regarded as an alternative and complemented therapeutic modality for the clinic cancer treatment.¹⁴³ Metal nanoparticles show a unique localized surface plasmon resonance (LSPR) effect so that they can transfer the absorbed photon energy to heat, which makes them excellent candidates as photothermal reagents.^144,145 Bioorthogonal reaction is an effective tool to achieve the metal nano-to-cluster conversion, which induces a red shift of absorption spectra and enhance photothermal conversion efficiency of metal nanoparticles. For example, spherical homogeneous AuNPs decorated with organic molecules can undergo bioorthogonal reactions and lead to the morphology shift from small sized ones to aggregates (above 50 nm), resulting in the red shift of absorption in the NIR region and the enhancement of photothermal conversion efficiency.¹⁴⁶ Chen et al. proposed a novel gold nanoparticle platform (AuNP@1) that is capable of undergoing the tumorous overexpressed furin protease sensitive reaction for enhanced PTT in tumors.¹⁴⁷ In AuNP@1, peptide sequence 1 (Ac-Arg-Val-Arg-Arg-Cys(StBu)-Lys-CBT) was grafted on ultrasmall AuNPs (5 nm) through the conjugation between the side chain of the lysine motif and AuNPs (Fig. 17A). When AuNP@1 entered cancer cells, high concentrated GSH reduced the disulfide bonds and furin protease cleaved peptide sequence (Arg-Val-Arg-Arg), thus leading to the exposure of 1,2-aminothiol groups on Cys and subsequently triggering the CBT-Cys condensation reaction to form AuNP aggregates. As seen in Fig. 17B, these furin and GSH triggered CBT-Cys condensation reactions promoted the formation of AuNP aggregates with a hydrodynamic size of 103.5 ± 12.3 nm and increased the absorption in the NIR region (705–900 nm) (Fig. 17C). Upon laser irradiation at 808 nm for 5 min, the temperature of GSH + furin treated AuNP@1 was elevated faster and higher (ΔT_max = 29.4 °C) than that of AuNPs (ΔT_max = 12.5 °C) (Fig. 17D). The calculated photothermal conversion efficiency of AuNPs was also observed to be superior (3.02%) in the presence of GSH and furin. Furthermore, high furin-expressing MDA-MB-468 cells and low furin expressing A549 cells were, respectively, used to investigate the aggregation of AuNPs in cancer cells. TEM images of the AuNP@1-treated MDAMB-468 cells exhibited a large amount of aggregates, while AuNP@1-Scr (containing the scrambled peptide Ac-Arg-Lys-Arg-Cys(StBu)-Arg-Val-CBT) or furin inhibitor II (H-(D)Arg-ArgArg-Arg-Arg-Arg-NH₂) pre-treated cells did not show AuNP aggregates. Confocal fluorescence imaging demonstrated that AuNP@1 entered lysosomes via endocytosis and then accumulated at Golgi bodies in MDA-MB-468 cells due to the trans-Golgi protein convertase, furin, instructed aggregation. AuNP@1 were further used for PTT treatment in vivo, and exhibited best photothermal effects in MDAMB-468 tumors than other control groups. Moreover, the tumor-specific furin-induced self-aggregating nanoparticle system was not limited to AuNPs. Wang et al. prepared peptide 1 functionalized superparamagnetic iron oxide nanoparticles (SPIO@1 NPs), which allowed in situ aggregation to significantly enhance transverse molar relaxivity (r₂) and NIR absorption for enhanced MRI imaging and PTT in murine tumor tissues.¹⁴⁸ T₂ weighted imaging of mice showed that SPIO@1NP reached the tumor region at about 3 h post-injection, and the SNR ratio was elevated to be 39.73%, which was superior to that of the SPIO NP group (14.78%). The exposure of light irradiation elevated the local tumor temperature to 55.8 °C in the SPIO@1NP treated group, whereas the temperature of the naked SPIO NP group was 44.6 °C. The good photothermal conversion efficiency further facilitated the best therapeutic effects.

Fig. 17 (A) Schematic of the mechanism of AuNP@1 for enhanced PTT in tumors. (B) TEM images of AuNP@1 treated with GSH and furin. (C) Visible–NIR absorption spectra of AuNPs, AuNP@1 or AuNP@1-Scr treated with GSH and furin. (D) The temperature curves of different groups irradiated by 808 nm NIR laser, adapted with permission from ref. 147. Copyright 2020, Wiley-VCH GmbH. (E) Schematic illustration of a BCR reaction based two-component nanoplatform for synergistic PTT and chemotherapy. (F) The photothermal conversion efficiency of different AuNP aggregation. (G) PA imaging of different groups. (H) HPLC results of the LND released from aggregated AuNPs, adapted with permission from ref. 149. Copyright 2024, Wiley-VCH GmbH.

Bioorthogonal “Click and Release” (BCR) reactions, in which an unstable intermediate is formed via cycloaddition and then rapidly decomposes to release a molecular motif, offer the opportunities to involve nano-to-cluster conversion and drug release simultaneously. Yan et al. developed a BCR reaction based two-component nanoplatform for synergistic PTT and chemotherapy.¹⁴⁹ In the nanoplatform, a specific BCR reaction was introduced based on [3+2] cycloaddition between a mesoionic prodrug iminosydnone-lonidamine (ImLND) and DBCO to trigger in situ nanoassembly of AuNPs and release chemotherapeutic drug lonidamine (LND) that can downregulate heat shock protein expression and avoid the cellular protection from hyperthermia damage (Fig. 17E). The two-component nanoplatform using HS-PEG₅₀₀₀-NH₂ as a linker is composed of ImLND grafted AuNPs and DBCO-RGD grafted AuNPs. In the therapeutic regimen, DBCO-RGD grafted AuNPs specifically located at tumor sites through the specific affinity between RGD and integrin αvβ3. Subsequently, ImLND grafted AuNPs were administered, so that the BCR reaction triggered [3+2] cycloaddition to promote the AuNP aggregation and simultaneous release of LND. The generation of nanoaggregates through the BCR reaction facilitated excellent photothermal effects with a photothermal conversion rate of 57.3% and activated a strong photoacoustic signal (Fig. 17F and G). On the other hand, the release profile of LND was revealed by HPLC and LC-MS with the observation of an absorption peak at an elution time of 7.903 min (Fig. 17H) and the corresponding peak m/z = 406.0819, respectively. The release of LND in cells downregulated the HSP expression, which was confirmed via western-blotting assay. The synergistic roles of released LND and aggregation enhanced PTT facilitated a robust tumor suppression effect in vitro and in vivo.

Cu(I) catalyzed bioorthogonal CuAAC reaction has been employed to design prodrugs, but the introduction of a Cu(I) catalyst is harmful to living cells or organisms and the instability of Cu(I) compromised its catalytic activity. Qu et al. developed copper-doped molybdenum-based polyoxometalate nanoclusters (Cu-POM NCs) (Fig. 18A), which selectively self-assembled into large aggregates in an acidic bacterial biofilm through the hydrogen bonding and efficiently catalyzed the CuAAC reaction to synthesize antibacterial molecule 6 for antibiofilm therapy.¹⁵⁰ Additionally, Cu-POM NCs consumed bacterial H₂S through the Mo^VI-to-Mo^V conversion and enhanced the NIR-II photothermal effect for PTT (Fig. 18B). TEM images exhibited monodispersed spherical Cu-POM NCs with a diameter of 7–10 nm at pH 7.2, whereas formed nanoaggregates with a diameter of ∼220 nm at pH 5.5. The aggregates did not affect the catalytic activity, which was demonstrated by the consistent results in the model CuAAC reaction between 3-azido-7-hydroxycoumarin and phenylacetylene at different pH buffers. The time-dependent absorption spectra of Cu-POM treated with H₂S showed gradually increased absorption ability in the NIR-II region. Acid/H₂S dual responsiveness promoted the excellent photothermal effects with a photothermal conversion efficiency of 49.2%. This platform integrates bioorthogonal catalysis with NIR-II photothermal effects, offering a synergistic strategy to combat biofilm infections and reduce bacterial tolerance.

Fig. 18 (A) Schematic illustration of Cu-POM NCs selectively self-assembled into large aggregates and efficiently catalyzed the CuAAC reaction to synthesize antibacterial molecule 6 for antibiofilm therapy. (B) NaHS-dependent temperature increases irradiation by 1064 nm NIR laser and the mechanism, adapted with permission from ref. 150. Copyright 2023, Wiley-VCH GmbH. (C) Schematic illustration of covalent assembly of CNDs for improved PTT. (D) The calculated HOMO and LUMO levels of CNDs@N₃, CNDs@C≡C and the W/O assembly, adapted with permission from ref. 151. Copyright 2022, American Chemical Society.

Another example is covalent assembly of carbon nanodots (CNDs) via a CuAAC reaction in a water-in-oil emulsion system for improved PTT.¹⁵¹ The system comprised azide-modified CNDs@N₃ and alkynylated CNDs@C≡C (Fig. 18C), which formed triazole covalent crosslinks through CuAAC to yield nanocomposites with a particle size of 40 ± 20 nm. Compared to free CNDs with a diameter of (6 ± 4 nm), covalent assembled CNDs reduced the electronic transition bandgap from 3.45 eV to 3.05 eV (Fig. 18D) and shifted the energy dissipation pathway from radiative decay to non-radiative decay, which resulted in fluorescence quenching and the enhancement of NIR absorption from 600 to 1100 nm. Upon laser irradiation at 808 nm, covalent assembled CNDs possessed a photothermal conversion efficiency of 32.34%. The assembled CNDs were further applied for in vitro and in vivo photothermal antibacterial efficacy. According to standard plate counting assays, the assembled CNDs showed 99% bactericidal efficiency against vancomycin-resistant enterococcus (VRE). In a subcutaneous VRE-infected mouse model, NIR irradiation plus assembled CND treatment led to almost eradication of the infected abscessed area on the third day, giving strong evidence of excellent photothermal antibacterial efficacy of the assembled CNDs in vivo.

5.3. Radiotherapy

Radiotherapy utilizes high-energy ionizing radiation to eliminate the primary or metastatic lesions, and is an important cancer modality for clinical cancer.^152,153 Radiotherapy is favorable to destroy tumor tissues via directly breaking DNA strands or indirectly undergoing radiolysis of water to generate reactive oxygen species (ROS).¹⁵⁴ Since the tissue absorption of X-ray photon energy is limited, high dose radiation is usually needed to obtain desirable therapy performance, but brings out unignorable adjacent damage to normal tissues.¹⁵⁵ Therefore, it is indispensable to develop strategies to achieve precision radiotherapy for efficient eradication of local tumors. The combination of bioorthogonal chemistry and tumor targeted delivery is an effective strategy. Wu et al. employed high atomic number Au NPs, which possess a high photoelectric absorption cross-section, as radiosensitizers, and developed an intelligent nanoplatform, MnAuNP-C&B (Fig. 19A).¹⁵⁶ MnAuNP-C&B was composed of MnAuNP-C and MnAuNP-B in a 1 : 1 ratio. MnAuNP-C and MnAuNP-B contained PEGylated CBT or GSH-protective Cys motifs, and Mn²⁺ anchored metal-phenolic networks. The nanoplatform induced in situ aggregation in the presence of GSH, which was highly advantageous to prolong the retention in tumor tissues. Furthermore, metal-phenolic networks contained pH-sensitive C=N bonds, allowing Mn²⁺ release in a tumor acidic environment. The released Mn²⁺ activated the cGAS-STING pathway, as evidenced by elevated expression levels of p-IRF3, p-STING, and p-TBK1 in western blotting assay. The combination of radiosensitization and STING pathway activation significantly inhibited tumor growth in vivo. Another example is peptide-based radiopharmaceutical, denoted as [¹³¹I]IM(HE)₃AAN (Fig. 19B), for targeted radiotherapy.¹⁵⁷ [¹³¹I]IM(HE)₃AAN was introduced with a hydrophilic peptide chain His-Glu-His-Glu-His-Glu ((HE)₃), which was sensitive to tumorous overexpressed legumain. In legumain-positive tumors, [¹³¹I]IM(HE)₃AAN is specifically cleaved by legumain, followed by GSH-mediated exposure of the 1,2-aminothiol group and the promotion of CBT-Cys cycloaddition. Since the hydrophilic HE₃ was removed, a hydrophobic heterodimer [¹³¹I]H-Dimer was formed in tumor sites. Ultimately, [¹³¹I]IM(HE)₃AAN demonstrated excellent tumor-inhibiting capabilities in legumain-positive HCT116 tumor models in vivo.

Fig. 19 (A) Schematic illustration of intelligent nanoplatform MnAuNP-C&B, adapted with permission from ref. 156. Copyright 2024, American Chemical Society. (B) Chemical structure and the mechanism of [¹³¹I]IM(HE)₃AAN.

5.4. Photodynamic therapy

Photodynamic therapy (PDT) that involves photoirradiation, photosensitizers and O₂ to generate cytotoxic ROS and kill cancer cells has been introduced clinically as a supplementary modality for malignant tumor treatment.^158,159 Although PDT is considered to be a promising regime due to the non-invasiveness and spatiotemporal controllability, it still encounters the challenges such as tumor hypoxia and insufficient enrichment of photosensitizers at tumor sites.^160,161 Wen et al. integrated alkaline phosphatase (ALP)-mediated in situ self-assembly with the bioorthogonal IEDDA reaction, and reported a tumor pre-targeting theranostic strategy to alleviate tumor hypoxia, achieving multimodal imaging-guided PDT (Fig. 20A).¹⁶² A phosphorylated probe, P-FFGd-TCO, was prepared by the conjugation of an ALP-responsive moiety, a MRI imaging reagent – the Gd complex and a TCO group. P-FFGd-TCO underwent ALP-catalyzed dephosphorylation and was self-assembled into nanoparticles (FMNPs-TCO) and anchored on tumor cell membranes. The dense TCO groups on FMNPs-TCO served as “artificial antigens” to efficiently capture subsequently injected tetrazine (Tz)-modified NIR photosensitizer nanoparticles (775 NP-Tz) and carbonic anhydrase (CA) inhibitor (SA-Tz) through bioorthogonal IEDDA reactions, regenerating micron-scale complexes (FMNP-775/SA). FMNP-775/SA was efficiently anchored on HeLa cell membranes, where the dual NIR fluorescence signals from mCy (710 nm) and NIR775 were observed in the P-FFGd-TCO + 775NP-Tz + SA-Tz treated groups (Fig. 20B). SEM imaging of HeLa cells further intuitively revealed the existence of many nanoparticles (∼200–300 nm) on the membranes after the treatment of P-FFGd-TCO (Fig. 20C). The further incubation of HeLa cells with 775NP-Tz + SA-Tz led to the observation of micro-size large particles on the membranes, evidencing the enrichment of FMNP-775/SA on HeLa cells. Western blotting and immunofluorescence staining proved the inhibition of CA IX and HIF-1α expression, confirming hypoxia alleviation by SA-Tz. The two-step pre-targeting process efficiently accumulated photosensitizers in tumors and overcame hypoxia, achieving complete HeLa tumor ablation without recurrence after treatment.

Fig. 20 (A) Schematic diagram of a tumor pre-targeting theranostic strategy to alleviate tumor hypoxia and achieve multimodal imaging-guided PDT. (B) Epifluorescence imaging of HeLa tumor cells in different treatment groups. I: P-FFGd-TCO, II: P-FFGd-TCO and 775NP-Tz (containing R6G) + SA-Tz; III: P-FFGd-TCO and 775NPs-OMe (containing R6G) + SA-Tz; IV: 775NP-Tz (containing R6G) + SA-Tz. (C) Bio-SEM images of blank HeLa cells, and HeLa cells incubated with P-FFGd-TCO or P-FFGd-TCO + 775NP-Tz + SA-Tz, adapted with permission from ref. 162. Copyright 2023, Wiley-VCH GmbH.

5.5. Chemodynamic therapy

Chemodynamic therapy (CDT) based on Fenton and Fenton-like reactions is a novel therapeutic regimen and has gained widespread attention.^163,164 They usually occur in a tumor acidic environment and involve a nanocatalyst and hydrogen and oxygen (H₂O₂) to generate one of the highly toxic ROS, hydroxyl radicals (˙OH).¹⁶⁵ In contrast to PDT, CDT is advantageous in avoiding the exposure of light irradiation so that tissue penetration is not the bottleneck of CDT. Instead, the performance of CDT is largely dependent on the acidic environment, endogenous H₂O₂ level and nanocatalytic ability.¹⁶⁶ Zhou et al. prepared PAMAM modified with cinnamaldehyde (CA) for H₂O₂ elevation and GSH depletion, and ferrocene (Ferr) as a Fenton reaction catalyst to obtain hybrid nanoparticles (PFC) (Fig. 21A).¹⁶⁷ PFC were further cross-linked with pH-cleavable maleic acid amide to form cPFC. To improve the enrichment of tumor accumulation, cPFC were anchored with PAEMA shedding DBCO and PEGylated N₃ motifs, respectively, to obtain functional polymeric nanoparticles, cPFC_DBCO and cPFC_N3. Through the rapid exposure of DBCO and a bioorthogonal reaction, cPFC_DBCO and cPFC_N3 formed stable drug depots within a 15-min incubation under tumoral acidity conditions (pH 6.5). The small size of PAMAM was monitored with a prolonged incubation time to 12 h due to the slow cleavage of the maleic acid amide linker. The depots elevated ˙OH levels under both normoxia (21% O₂) and hypoxia (1% O₂) (Fig. 21B). Western blotting and BODIPY^665/676 based fluorescence staining demonstrated that the depots triggered O₂-independent ferroptosis, revealing GSH consumption, inhibition of GPX4 activity, and decrease of lipid peroxide (LPO).

Fig. 21 (A) Schematic diagram of the mechanism of cPFC_DBCO and cPFC_N3 nanoparticles for enhanced tumor therapy. (B) Confocal fluorescence imaging of ˙OH generation in 4T1 cells treated with different groups under normal and hypoxic conditions, adapted with permission from ref. 167. Copyright 2022, Elsevier.

5.6. Synergistic therapy

Despite the advanced development of various noninvasive therapeutic modalities, multimodal synergistic therapy strategies have been gradually utilized to achieve a synergistic effect and improve therapeutic efficacy.¹⁶⁸ Recent progress has introduced bioorthogonal chemistry in synergistic therapy. Cao et al. reported bioorthogonal in situ assembled nanodepots for enhanced tumor accumulation and cocktail therapy (Fig. 22A).¹⁶⁹ The drug depots were encapsulated by the diblock polylactic acid (PLA) and (PEG) polymer (PLA-b-PEG). One depot@PLA-b-PEG was terminated with the Cys residue and pH-sensitive 2,3-dimethylmaleic anhydride (DA) (denoted as D-NP), another one was terminated with CBT (denoted as C-NP). The tumor acidic environment promoted the cleavage of the amide bond between DA and the Cys residue, and exposed a free 1,2-aminothiol group for CBT-Cys cross-linking. Under tumor acidic conditions (pH 6.5), self-assembled micrometer-sized particles (2611 ± 115 nm) were observed when C-NP and D-NP were mixed for 24 h. Real-time intravital confocal laser scanning imaging was further employed for the investigation of the self-assembly at tumor sites. Mice bearing an orthotopic GFP-4T1 tumor with dorsal skinfold window chambers were i.v. injected with Cy5 labelled C-NP and D-NP (Fig. 22B). The red fluorescence signals were visualized to move from the blood vessels at 10 min post-injection to the tumor site at 6 h post-injection, and the signals remained in the tumor site for 48 h, which was longer than that of the control group (containing acid-insensitive polymer). The results demonstrated the efficient accumulation of nanoparticles at tumor sites based on bioorthogonal chemistry-based strategy. The model drugs, including chemotherapeutic DOX, immunotherapy reagents NLG919, BLZ945 or extracellular matrix metalloproteinase inhibitor BB94 have been loaded in the depots, showing the advantages of cocktail therapy for remodelling of the tumor immune microenvironment and activation of anti-tumor immune responses.

Fig. 22 (A) Schematic diagram of bioorthogonal in situ assembled nanodepots for enhanced tumor accumulation and cocktail therapy. (B) Intravital CLSM real-time image of Cy5 labeled D-NP/C-NP and S-NP/C-NP in GFP-4T1 tumor-bearing mice, adapted from ref. 169, under the license CC-BY, published by Springer Nature. (C) Schematic diagram of a dual mode imaging guided nanotheranostic agent based on bioorthogonal click chemistry for precise cancer treatment. (D) Abemaciclib release curves for different groups. (E) Cell cycle arrest of CT26 cells treated with different groups, adapted from ref. 172, under the license CC-BY, published by Wiley-VCH GmbH.

PTT has been testified to trigger cells to release damage-associated molecular patterns (DAMPs), such as heat shock proteins (HSP), high mobility group protein B1 (HMGB1), and calreticulin (CRT), which can promote maturation of dendritic cells (DC) and motivate host's immune system to combat tumors.^170,171 Therefore, PTT can be combined with immunotherapy for cancer treatment. Sang et al. designed themoresponsive liposomes to codeliver magnetic Fe₃O₄ nanoparticles, chemotherapeutic drug Abemaciclib and photothermal reagents IR780 (Fig. 22C).¹⁷² The liposomes were modified with the ACKFRGD peptide sequence or CBT motif so that these liposomes could reach tumor sites and form aggregates in the presence of highly expressed Cat B via CBT-Cys cycloaddition. Upon laser irradiation, SPIONs/IR780 generated localized hyperthermia to trigger liposomal phase transition. Abemaciclib and IR780 were released in deep tumor tissues resulting in a synergistic photothermal/chemo/immuno therapeutic cascade. Experimental validation demonstrated that laser irradiation of in situ aggregated liposomes significantly enhanced Abemaciclib release and induced the highest rate of G1 phase arrest (49.42 ± 2.65%) (Fig. 22D and E), promoted release of CRT, HMGB1, ATP and increased T-cell infiltration, leading to potent tumor suppression and long-term immune memory. Moreover, magnetic resonance/NIR fluorescence imaging is beneficial to precisely localize the liposomes in vivo and guide a multimodal therapy. Another example is a legumain-responsive in situ self-assembled nanosystem containing a photothermal reagent, nano-graphene oxide (NGO) and paclitaxel (PTX) (Fig. 23A).¹⁷³ Legumain triggered the specific CBT-Cys cross-linking to generate large-size aggregates, which ensured prolonged retention and sustained PTX release. As illustrated in Fig. 23B, NGOPC@PTX exhibits a sustained drug release profile in the presence of 4T1 lysates. The 808 nm laser irradiation slightly enhanced PTX liberation by 9.5%, indicating a photothermal release behavior. Ex vivo fluorescence imaging of tumors and major organs of mice bearing IR783 labeled NGOPC revealed maximal fluorescence intensity localized within tumor regions, whereas a negligible signal was detected in liver, indicating that the formed aggregates augmented the tumor accumulation and benefited for drug retention (Fig. 23C). Accompanied by the blocking immune escaping role of PTX, the nanosystem promoted DC maturation via binding to Toll-like receptors (TLRs) on DC surfaces and selectively killed regulatory T cells (Tregs) through Bcl-2/Bax-mediated apoptosis, thereby promoting a shift from an immunosuppressive to immunostimulatory tumor microenvironment.

Fig. 23 (A) Schematic illustration of a legumain-responsive in situ self-assembled nanosystem. (B) In vitro PTX release curves of different groups. (C) Ex vivo fluorescence imaging of major organs and tumors in different groups, adapted with permission from ref. 173. Copyright 2020, Elsevier. (D) Schematic illustration of GSH and LAP-responsive size-transformable smart nanoprobe Ce6-Leu, adapted with permission from ref. 174. Copyright 2021, American Chemical Society.

PDT has also been commonly used to work with other modalities to enhance therapeutic efficacy. Wang et al. designed a size-transformable smart nanoprobe, Ce6-Leu, capable of specifically responding to leucine aminopeptidase (LAP) and GSH (Fig. 23D).¹⁷⁴ By chelating with Mn²⁺ to obtain Ce6-Leu@Mn²⁺, the probe realized efficient multimodal tumor imaging guided photodynamic/radiotherapy. Ce6-Leu@Mn²⁺ consists of three components: the photosensitizer Ce6, a leucine and disulfide bond unit, and a CBT group. The amphiphilic characteristics induced the probe to self-assemble as nanoparticles (∼80 nm) via the hydrophobic interactions and π–π stacking of Ce6, while the presence of LAP and GSH spontaneously cleaved the probe to yield its cyclic dimer via CBT-Cys cycloaddition with a diameter of ∼23 nm. More importantly, the Mn²⁺-chelation in the probe exhibited peroxidase-like catalytic ability to transfer endogenous H₂O₂ to O₂, which was beneficial to PDT and radiotherapy in a hypoxic environment. Jiang et al. reported DBCO masked photosensitizer Ce6 nanoparticles and N₃ functionalized hypoxia-activated prodrugs PR104A to construct “two-step” size-transformation nanodepots (Fig. 24A).¹⁷⁵ Bioorthogonal click reaction-induced macro-aggregates were formed within tumor adjacent regions, enabling effective co-delivery of prodrugs and photosensitizers. Upon laser irradiation, Ce6-generated ROS facilitated the transformation of N₃-functionalized PR104A into ultrasmall nanoparticles with enhanced permeability. Fluorometric analysis confirmed 60% release of RhB-labeled PAMAM from crosslinked depots upon 15 min post-irradiation (Fig. 24B), demonstrating efficient photo-triggered decrosslinking. Negligible release in non-irradiated control groups validated the design feasibility. Furthermore, in vitro experiments showed hypoxia-activated PR104A release in 4T1 cells by HPLC quantification (Fig. 24C). Using 4T1 multicellular spheroids (MCSs) as an in vitro model, photoirradiation/ROS-mediated permeation of PAMAM-PR104A was verified by the observation of red fluorescence at 50 μm depth in the nanodepot incubated group treated in an acidic environment and with light irradiation (Fig. 24D), ultimately achieving synergistic PDT and chemotherapy within hypoxic tumors.

Fig. 24 (A) Schematic illustration of the ultra-fast pH-responsive “two-step” size-transformation nanodepots achieving synergistic PDT and chemotherapy in hypoxic tumors. (B) Release of PAMAM_RhB upon 660 nm laser exposure of crosslinked depots. (C) HPLC analysis of hypoxia-triggered PR104A prodrug release from 4T1 cells at varying incubation times. (D) Confocal fluorescence image of the MCSs incubated with TK-PAMAM_RhB-N₃ and N_ce6-DBCO after different treatments at different depths, adapted with permission from ref. 175. Copyright 2022, Elsevier.

6. Conclusions

In summary, we discussed bioorthogonal reaction-based strategies to design size variable nanosystems for molecular imaging and therapy (Table 3). These nanosystems are usually decorated with bioorthogonal reactive pairs and undergo in situ size transformation upon the response of external or internal stimuli. Benefitting from bioorthogonal reactions, size variable nanosystems have been booming to avoid off-target biodistribution, prolong probe retention and ensure precise drug delivery, which hold immense potential for achieving optimized molecular imaging quality and therapeutic efficiency. Despite significant advances in improved imaging and therapeutic outcomes, several important and challenging issues remain to be solved.

Table 3 Summary list of size-variable nanosystems for bioimaging and drug delivery

	The type of size transformation	Applications
Small to large	From molecules to the dimer, the dimer self-assembles into nanoaggregates	FL, PET, PA, MRI, RI, QPI, multi modality, chemotherapy, and PDT
	From molecules to the dimer, the dimer self-assembles into nanotubes	FL
	From molecules to the dimer	PET, RI, and RT
	From nanoparticles to nanoaggregates	FL, chemotherapy, PTT, RT, and synergistic therapy
	From nanoparticles to nanoaggregates and release drugs	PTT
Multi-size transformation	From nanoparticles to nanoaggregates, and then release smaller particles	Chemotherapy, CDT, and synergistic therapy
	From molecules into nanoaggregates and then reassemble into dimers	Synergistic therapy

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Firstly, it is necessary to modulate bioorthogonal responsive units that allows elevating tissue selectivity. Most reported bioorthogonal responsive nanosystems rely on the responsiveness between probes and overexpressed biomarkers, such as caspase-1, Cat B and GSH, to be transferred from the inert to activated state. However, these biomarkers do not have very closely relationship with specific tissues. For example, overexpressed caspase-1 can be found in S. aureus infected RAW264.7 cells and is associated with AD neuroinflammation. GSH exists in both normal and tumorous tissues. Hence, it is advisable to build multiple biomarker responsive platforms to amplify the selectivity of bioorthogonal reactions. Additionally, “photo-triggered” bioorthogonal reactions are another good candidate. For example, the light driven reaction between tetrazine and norbornene provides a powerful tool owing to temporospatial controllability. The unique “photo-triggered” reactive units can combine with other bioorthogonal reactions and allow building an “AND” logic gate that can simultaneously or sequentially response to multiple stimuli for precise targeted imaging and drug release. Despite the above-mentioned strategies being helpful to improve the bioorthogonal reactions’ selectivity, the off-target effects persist in the execution of bioorthogonal reactions in complex in vivo physiological environments. First, the bioorthogonal functional groups may undergo unintended size reactions with endogenous substances within biological systems, such as Cys, thereby some bioorthogonal macrocyclization reactions with relatively slow kinetics decrease the targeting capability. Another unresolved issue is that the complex in vivo physiological environments induce off-target effects in bioorthogonal reactions. The in vivo environments with a high dilution factor exhibit decreased target concentrations and affect the reaction kinetics.

Secondly, enriching bioorthogonal reactive pairs is essential. Some bioorthogonal reaction rates are relatively low (bioorthogonal-induced nanoparticle aggregation finishes in 24 or 48 h) leading to the clearance of original nanocarriers before tumoral accumulation happens. Therefore, more stable, biocompatible and rapid bioorthogonal reactions needed to be explored. In general, reactions with rapid kinetics (iEDDA > CuAAC > 1,2-aminothiol-CBT click reactions ≥ SPAAC > SPSAC) are preferrable for biological applications,¹⁷⁶ but optimizing molecules with specific moieties facilitates to dramatically decrease the activation free energy and allows the reaction kinetics to be changed. For example, structural optimization of molecules significantly enhanced macrocyclization kinetics, achieving first-order reaction rates of 7.97 × 10⁻⁵ s⁻¹ for CyNA_P and (3.9 ± 0.1) × 10⁻³ s⁻¹ for [¹⁸F-IR780-1], respectively.^76,121 This accelerated reaction kinetics effectively prevents premature clearance of the original nanocarrier prior to targeted site accumulation. On the other hand, this process may be simplified in the future via the collaboration between artificial intelligence and organic chemistry. Then the optimization of the scheduling gap between the bioorthogonal reaction pairs, the administered dose, the site accumulation and in vivo clearance rates is still the main challenge needed to be resolved. Additionally, some derivatives of bioorthogonal reactive pairs can be introduced in size variable nanosystems to improve functionality. For example, dissociative bioorthogonal reactions should be considered to be integrated into nanocarriers. These dissociative bioorthogonal reactive motifs can be linked with prodrugs or protein activators for targeting theranostics. The green byproduct from bioorthogonal reactions can also participate in synergistic therapy. For example, the IEDDA click reaction between a thiocarbamate-functionalized TCO and a Tz can generate H₂S in cells, which opens up a new direction for the combination of H₂S gas therapy with other modalities.¹⁷⁷

Finally, the bioorthogonal chemistry mediated size variable nanosystems commonly induce the nano-to-cluster morphology transformation. The cascaded utilization of “small-to-large” and “large-to-small” size transformation may be advantageous due to the good distribution, penetration and metabolism. The long-term biosafety of these developed nanoplatforms should also be taken into consideration to achieve clinical translation. Instead of the well-developed CuAAC, copper-free alternatives, such as IEDDA, with faster kinetics and lower toxicity are preferrable to design size transformation systems for in vivo applications. Notably, a bioorthogonal reaction-mediated prodrug activation strategy, SQ3370, has entered Phase II clinical trials for tumor therapy.¹⁷⁸ This milestone has significantly heightened researcher interest in bioorthogonal reaction-mediated drug delivery systems.

All in all, this area is still in its infancy. We hope efficient and low-toxic bioorthogonal tools will aid in developing intelligent nanocarriers and speeding up the pace of nanocarrier innovations to clinical trials.

Author contributions

Juan Li: conceptualization and writing – original draft preparation. Yan Shan: validation and visualization. Jie Xu: validation and visualization. Cao Li: supervision, project administration, and funding acquisition. Qi Yu: conceptualization, writing – review and editing, supervision, project administration, and funding acquisition.

Conflicts of interest

There are no conflicts to declare.

Data availability

This narrative review does not present any new data. All the data discussed were derived from previously published studies, all of which are fully cited within the text. No new data were analyzed or produced for the purpose of this review.

Acknowledgements

This work is financially supported by the National Natural Science Foundation of China (51973053), the open research fund of State Key Laboratory of Organic Electronics and Information Displays, the Scientific Research Project of Department of Education, Hubei Province (Q20231403) and Research Foundation of Hubei University of Technology (GCCCT20220010).

Notes and references

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