Bioorthogonal Click and Release Reaction of Iminosydnones with Cycloalkynes

We report the discovery of a new bioorthogonal click‐and‐release reaction involving iminosydnones and strained alkynes. This transformation leads to two products resulting from the ligation and fragmentation of iminosydnones under physiological conditions. Optimized iminosydnones were successfully used to design innovative cleavable linkers for protein modification, thus opening up new areas in the fields of drug release and target‐fishing applications. This click‐and‐release technology offers the possibility of exchanging tags on proteins for functionalized cyclooctynes under mild and bioorthogonal conditions.     

Tetrazine Carbon Nanotubes for Pretargeted In Vivo “Click‐to‐Release” Bioorthogonal Tumour Imaging

The bioorthogonal inverse‐electron‐demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans‐cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single‐walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO‐caged molecule was used to deliver active effector molecules. To optimize a turn‐on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near‐infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real‐time, non‐invasive tumour visualization with a high target‐to‐background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine‐functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off‐site activation of fluorophore/drug.     

Bioorthogonal Ligation and Cleavage by Reactions of Chloroquinoxalines with ortho‐Dithiophenols

A bioorthogonal ligation and cleavage method via reactions of chloroquinoxalines (CQ) and ortho‐dithiophenols (DT) is presented. Double nucleophilic substitutions of ortho‐dithiophenols to chloroquinoxalines provide conjugates containing tetracyclic benzo[5,6][1,4]dithiino[2,3‐b]quinoxaline with strong built‐in fluorescence together with release of the other functional molecules. Three cleavable linkers were designed and successfully used in release of the molecules containing biotin from the protein conjugates. The CQ‐DT bioorthogonal reactions can be applied for the bioorthogonal ligations, bioorthogonal cleavages, and trans‐tagging of proteins, and show advantages of readily accessible unnatural orthogonal groups, appealing reaction kinetics (k₂≈1.3 m ^?1 s^?1), excellent biocompatibility of orthogonal groups, and high stability of conjugates. This complements previous bioorthogonal reactions and is a new route for protein‐fishing applications and in‐gel fluorescence analysis.     

A General Strategy for Macrotheranostic Prodrug Activation: Synergy between the Acidic Tumor Microenvironment and Bioorthogonal Chemistry

Prodrugs activated by endogenous stimuli face the problem of tumor heterogeneity. Bioorthogonal prodrug activation that utilizes an exogenous click reaction has the potential to solve this problem, but most of the strategies currently used rely on the presence of endogenous receptors or overexpressed enzymes. We herein integrate the acidic, extracellular microenvironment of a tumor and a click reaction as a general strategy for prodrug activation. This was achieved by using a tumor pH‐responsive polymer containing tetrazine groups, which formed unreactive micelles in the blood but disassembled in response to tumor pH. The vinyl ether group on the macrotheranostic prodrug (CyPVE) is activated by the tetrazine groups, which was confirmed by tumor‐specific fluorescence activation and phototoxicity restoration. Therefore, the bioorthogonal reactions in the context of the ubiquitous acidic tumor microenvironment can provide a general strategy for bioorthogonal prodrug activation.     

Conditional Singlet Oxygen Generation through a Bioorthogonal DNA‐targeted Tetrazine Reaction

We report the use of bioorthogonal reactions as an original strategy in photodynamic therapy to achieve conditional phototoxicity and specific subcellular localization simultaneously. Our novel halogenated BODIPY‐tetrazine probes only become efficient photosensitizers (Φ_Δ≈0.50) through an intracellular inverse‐electron‐demand Diels–Alder reaction with a suitable dienophile. Ab initio computations reveal an activation‐dependent change in decay channels that controls ¹O₂ generation. Our bioorthogonal approach also enables spatial control. As a proof‐of‐concept, we demonstrate the feasibility of the selective activation of our dormant photosensitizer in cellular nuclei, causing cancer cell death upon irradiation. Thus, our dual biorthogonal, activatable photosensitizers open new venues to combat current limitations of photodynamic therapy.     

Gold‐Triggered Uncaging Chemistry in Living Systems

Recent advances in bioorthogonal catalysis are increasing the capacity of researchers to manipulate the fate of molecules in complex biological systems. A bioorthogonal uncaging strategy is presented, which is triggered by heterogeneous gold catalysis and facilitates the activation of a structurally diverse range of therapeutics in cancer cell culture. Furthermore, this solid‐supported catalytic system enabled locally controlled release of a fluorescent dye into the brain of a zebrafish for the first time, offering a novel way to modulate the activity of bioorthogonal reagents in the most fragile and complex organs.     

In Situ Probing Intracellular Drug Release from Redox‐Responsive Micelles by United FRET and AIE

Redox‐responsive micelles are versatile nanoplatforms for on‐demand drug delivery, but the in situ evaluation of drug release is challenging. Fluorescence resonance energy transfer (FRET) technique shows potential for addressing this, while the aggregation‐caused quenching effect limits the assay sensitivity. The aim of the current work is to combine aggregation‐induced emission (AIE) probe with FRET to realize drug release assessment from micelles. Tetraphenylethene (TPE) is selected as AIE dye and curcumin (Cur) is chosen as the model drug as well as FRET receptor. The drug is covalently linked to a block copolymer via the disulfide bond linker and TPE is also chemically linked to the polymer via an amide bond; the obtained amphiphilic polymer conjugate self‐assembles into micelles with a hydrodynamic size of ≈125 nm. Upon the supplement of glutathione or tris(2‐carboxyethyl)phosphine) trigger (10 × 10⁻³m ), the drug release induces the fluorescence increase of both TPE and Cur. Accompanied with the FRET decay, absorption enhancement and particle size increase are observed. The same phenomenon is observed in MCF‐7 cells. The FRET–AIE approach can be a useful addition to the spectrum of available methods for monitoring drug release from stimuli‐responsive nanomedicine.     

Polymer Micelles for Delayed Release of Therapeutics from Drug‐Releasing Surfaces with Nanotubular Structures

A new approach to engineer a local drug delivery system with delayed release using nanostructured surface with nanotube arrays is presented. TNT arrays electrochemically generated on a titanium surface are used as a model substrate. Polymer micelles as drug carriers encapsulated with drug are loaded at the bottom of the TNT structure and their delayed release is obtained by loading blank micelles (without drug) on the top. The delayed and time‐controlled drug release is successfully demonstrated by controlling the ratio of blank and drug loaded‐micelles. The concept is verified using four different polymer micelles (regular and inverted) loaded with water‐insoluble (indomethacin) and water‐soluble drugs (gentamicin).

     

Tetrazine Carbon Nanotubes for Pretargeted In Vivo “Click-to-Release” Bioorthogonal Tumour Imaging

The bioorthogonal inverse-electron-demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans-cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single-walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO-caged molecule was used to deliver active effector molecules. To optimize a turn-on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near-infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real-time, non-invasive tumour visualization with a high target-to-background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine-functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off-site activation of fluorophore/drug.     

Ferrocenyl amphiphilic Janus dendrimers as redox‐responsive micellar carriers

Amphiphilic Janus dendrimers have attracted increasing attention due to their asymmetric structures and various functional properties compared to the conventional symmetric macromolecules. Herein, a novel ferrocenyl‐terminated amphiphilic Janus dendrimer containing nine hydrophilic triethylene glycol branches was synthesized by two synthetic routes, namely the typical chemo selective coupling method and the mixed modular approach. Chemical redox triggers, namely Fe₂(SO₄)₃ as oxidant and ascorbic acid as reductant, could regulate the self‐assembly behavior of the Janus dendrimer in water through the redox‐switching between ferrocene and ferricinium cations, and the change of micelles formed were investigated and confirmed through scanning electron microscopy and dynamic light scattering. The cargo‐loading property of the micelles self‐assembled by the Janus dendrimer was further proved by the successful fabrication of Rhodamine B (RhB)‐loaded micelles, and the oxidation‐triggered release behavior of the encapsulated RhB could be mediated by changing the concentration of oxidants. This work provides an effective approach to prepare ferrocenyl‐terminated amphiphilic Janus dendrimers and the self‐assembled micelles might be used as a promising molecular carrier in areas such as drug delivery and catalysis.     

Drug release and biocompatibility of self‐assembled micelles prepared from poly (ɛ‐caprolactone/glycolide)‐poly (ethylene glycol) block copolymers

A series of poly(?‐caprolactone/glycolide)‐poly(ethylene glycol) (P(CL/GA)‐PEG) diblock copolymers were prepared by ring opening polymerization of a mixture of ?‐caprolactone and glycolide using mPEG as macro‐initiator and stannous octoate as catalyst. Self‐assembled micelles were prepared from the copolymers using nanoprecipitation method. The micelles were spherical in shape. The micelle size was larger for copolymers with longer PEG blocks. In contrast, the critical micelle concentration of copolymers increased with decreasing the overall hydrophobic block length. Drug loading and drug release studies were performed under in vitro conditions, using paclitaxel as a hydrophobic model drug. Higher drug loading was obtained for micelles with longer poly(ε‐caprolactone) blocks. Faster drug release was obtained for micelles of mPEG2000 initiated copolymers than those of mPEG5000 initiated ones. Higher GA content in the copolymers led to faster drug release. Moreover, drug release rate was enhanced in the presence of lipase from Pseudomonas sp., indicating that drug release is facilitated by copolymer degradation. The biocompatibility of copolymers was evaluated from hemolysis, dynamic clotting time, and plasma recalcification time tests, as well as MTT assay and agar diffusion test. Data showed that copolymer micelles present outstanding hemocompatibility and cytocompatibility, thus suggesting that P(CL/GA)‐PEG micelles are promising for prolonged release of hydrophobic drugs.     

K+‐Responsive Block Copolymer Micelles for Targeted Intracellular Drug Delivery

In this work, a novel type of block copolymer micelles with K⁺‐responsive characteristics for targeted intracellular drug delivery is developed. The proposed smart micelles are prepared by self‐assembly of poly(ethylene glycol)‐b‐poly(N‐isopropylacry‐lamide‐co‐benzo‐18‐crown‐6‐acrylamide) (PEG‐b‐P(NIPAM‐co‐B18C6Am)) block copolymers. Prednisolone acetate (PA) is successfully loaded into the micelles as the model drug, with loading content of 4.7 wt%. The PA‐loaded micelles display a significantly boosted drug release in simulated intracellular fluid with a high K⁺ concentration of 150 × 10⁻³m , as compared with that in simulated extracellular fluid. Moreover, the in vitro cell experiments indicate that the fluorescent molecules encapsulated in the micelles can be delivered and specifically released inside the HSC‐T6 and HepG2 cells responding to the increase of K⁺ concentration in intracellular compartments, which confirms the successful endocytosis and efficient K⁺‐induced intracellular release. Such K⁺‐responsive block copolymer micelles are highly potential as new‐generation of smart nanocarriers for targeted intracellular delivery of drugs.     

Terminal Alkenes as Versatile Chemical Reporter Groups for Metabolic Oligosaccharide Engineering

The Diels–Alder reaction with inverse electron demand (DAinv reaction) of 1,2,4,5‐tetrazines with electron rich or strained alkenes was proven to be a bioorthogonal ligation reaction that proceeds fast and with high yields. An important application of the DAinv reaction is metabolic oligosaccharide engineering (MOE) which allows the visualization of glycoconjugates in living cells. In this approach, a sugar derivative bearing a chemical reporter group is metabolically incorporated into cellular glycoconjugates and subsequently derivatized with a probe by means of a bioorthogonal ligation reaction. Here, we investigated a series of new mannosamine and glucosamine derivatives with carbamate‐linked side chains of varying length terminated by alkene groups and their suitability for labeling cell‐surface glycans. Kinetic investigations showed that the reactivity of the alkenes in DAinv reactions increases with growing chain length. When applied to MOE, one of the compounds, peracetylated N‐butenyloxycarbonylmannosamine, was especially well suited for labeling cell‐surface glycans. Obviously, the length of its side chain represents the optimal balance between incorporation efficiency and speed of the labeling reaction. Sialidase treatment of the cells before the bioorthogonal labeling reaction showed that this sugar derivative is attached to the glycans in form of the corresponding sialic acid derivative and not epimerized to another hexosamine derivative to a considerable extent.     

Bioorthogonal Ligation and Cleavage by Reactions of Chloroquinoxalines with ortho-Dithiophenols

A bioorthogonal ligation and cleavage method via reactions of chloroquinoxalines (CQ) and ortho-dithiophenols (DT) is presented. Double nucleophilic substitutions of ortho-dithiophenols to chloroquinoxalines provide conjugates containing tetracyclic benzo[5,6][1,4]dithiino[2,3-b]quinoxaline with strong built-in fluorescence together with release of the other functional molecules. Three cleavable linkers were designed and successfully used in release of the molecules containing biotin from the protein conjugates. The CQ-DT bioorthogonal reactions can be applied for the bioorthogonal ligations, bioorthogonal cleavages, and trans-tagging of proteins, and show advantages of readily accessible unnatural orthogonal groups, appealing reaction kinetics (k₂≈1.3 m ⁻¹ s⁻¹), excellent biocompatibility of orthogonal groups, and high stability of conjugates. This complements previous bioorthogonal reactions and is a new route for protein-fishing applications and in-gel fluorescence analysis.     

Self‐assembly of thermo‐ and pH‐responsive poly(acrylic acid)‐b‐poly(N‐isopropylacrylamide) micelles for drug delivery

Self‐assembled thermo‐ and pH‐responsive poly(acrylic acid)‐b‐poly(N‐isopropylacrylamide) (PAA‐b‐PNIPAM) micelles for entrapment and release of doxorubicin (DOX) was described. Block copolymer PAA‐b‐PNIPAM associated into core‐shell micelles in aqueous solution with collapsed PNIPAM block or protonated PAA block as the core on changing temperature or pH. Complexation of DOX with PAA‐b‐PNIPAM triggered by the electrostatic interaction and release of DOX from the complexes due to the changing of pH or temperature were studied. Complex micelles incorporated with DOX exhibited pH‐responsive and thermoresponsive drug release profile. The release of DOX from micelles was suppressed at pH 7.2 and accelerated at pH 4.0 due to the protonation of carboxyl groups. Furthermore, the cumulative release of DOX from complex micelles was enhanced around LCST ascribed to the structure deformation of the micelles. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 5028–5035, 2008     

A Double‐Clicking Bis‐Azide Fluorogenic Dye for Bioorthogonal Self‐Labeling Peptide Tags

Herein, we give the very first example for the development of a fluorogenic molecular probe that combines the two‐point binding specificity of biarsenical‐based dyes with the robustness of bioorthogonal click‐chemistry. This proof‐of‐principle study reports on the synthesis and fluorogenic characterization of a new, double‐quenched, bis‐azide fluorogenic probe suitable for bioorthogonal two‐point tagging of small peptide tags by double strain‐promoted azide–alkyne cycloaddition. The presented probe exhibits remarkable increase in fluorescence intensity when reacted with bis‐cyclooctynylated peptide sequences, which could also serve as possible self‐labeling small peptide tag motifs.     

Boronic Acids as Bioorthogonal Probes for Site‐Selective Labeling of Proteins

Over the past two decades, bioorthogonal chemistry has become a preferred tool to achieve site‐selective modifications of proteins. However, there are only a handful of commonly applied bioorthogonal reactions and they display some limitations, such as slow rates, use of unstable or cytotoxic reagents, and side reactions. Hence, there is significant interest in expanding the bioorthogonal chemistry toolbox. In this regard, boronic acids have recently been introduced in bioorthogonal chemistry and are exploited in three different strategies: 1) boronic ester formation between a boronic acid and a 1,2‐cis diol; 2) iminoboronate formation between 2‐acetyl/formyl‐arylboronic acids and hydrazine/hydroxylamine/semicarbazide derivatives; 3) use of boronic acids as transient groups in a Suzuki–Miyaura cross‐coupling or other reactions that leave the boronyl group off the conjugation product. In this Review, we summarize progress made in the use of boronic acids in bioorthogonal chemistry to enable site‐selective labeling of proteins and compare these methods with the most commonly utilized bioorthogonal reactions.     

Amphiphilic block poly(propylene carbonate)‐block‐allyloxypolyethyleneglycol copolymer based shell cross‐linked micelles for controlled release of drug

Amphiphilic block poly(propylene carbonate)‐block‐allyloxypolyethyleneglycol (PPC‐b‐APEG) copolymer was synthesized by the click chemistry, and its structure were characterized. PPC‐b‐APEG can self‐assemble into micelles without emulsifier in water. Shell cross‐linked micelles were obtained by the reaction of the allyloxy groups, which were exposed on the outer of the PPC‐b‐APEG micelles, and N‐vinylpyrrolidone (NVP). The morphology and size of the micelles before and after cross‐link reactions were characterized. The research result shows that the shell cross‐linking could improve the stability of micelles. The particle size of uncross‐linked micelle was about 800 nm. The size of cross‐linked micelles increased with increasing amount of cross‐linking degree. To better evaluate the release behavior of PPC‐b‐PEG copolymer, doxorubicin (DOX)‐loaded micelles were synthesized using DOX as the model drug. Results showed that the DOX releasing rate decreased with increasing of NVP. The shell cross‐linking do decrease the burst release behaviours of DOX and reduce the DOX release rate.     

Folate‐Conjugated and Dual Stimuli‐Responsive Mixed Micelles Loading Indocyanine Green for Photothermal and Photodynamic Therapy

A folic acid targeted mixed micelle system based on co‐assembly of poly(ε‐caprolactone)‐b‐poly(methoxytri(ethylene glycol) methacrylate‐co‐N‐(2‐methacrylamido)ethyl folatic amide) and poly(ε‐caprolactone)‐b‐poly(diethylene glycol monomethyl ether methacrylate) is developed to encapsulate indocyanine green (ICG) for photothermal therapy and photodynamic therapy. In this study, the use of folic acid is not only for specific cancer cell recognition, but also in virtue of the carboxylic acid on folic acid to regulate the pH‐dependent thermal phase transition of polymeric micelles for controlled drug release. The prepared ICG‐loaded mixed micelles possess several superior properties such as a preferable thermoresponsive behavior, excellent storage stability, and good local hyperthermia and reactive oxygen species generation under near‐infrared (NIR) irradiation. The photototoxicity induced by the ICG‐loaded micelles has efficiently suppressed the growth of HeLa cells (folate receptor positive cells) under NIR irradiation compared to that of HT‐29, which has low folate receptor expression. Hence, this new type of mixed micelles with excellent features could be a promising delivery system for controlled drug release, effective cancer cell targeting, and photoactivated therapy.     

Complex micelles formed from two diblock copolymers for applications in controlled drug release

Double‐responsive core‐shell‐corona complex micelles for applications in drug release were formed from self‐assembly of two diblock copolymers PtBA‐b‐ PNIPAM and PtBA‐b‐P4VP. The two diblock copolymers coaggregated into core‐shell complex micelles in acidic water with the hydrophobic PtBA blocks as the common core and soluble PNIPAM/P4VP blocks as the mixed shell. Increasing temperature or pH value, the micelles converted into core‐shell‐corona micelles because of the collapse of PNIPAM or P4VP blocks as the inner shell and soluble P4VP or PNIPAM chains stretching outside as the outer corona. The anti‐inflammation drug naproxen (NAP) was loaded as the model drug in micelles in acidic water and released because of the ionization of NAP in alkaline solutions. Compared with pure core‐shell micelles, release of NAP from core‐shell‐corona complex micelles avoided the burst diffusion and the release rate is more easily controlled by tuning the composition of the mixtures or by adjusting the pH of the medium. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1804–1810, 2009