Systemically Injectable Enzyme‐Loaded Polyion Complex Vesicles as In Vivo Nanoreactors Functioning in Tumors

The design and construction of nanoreactors are important for biomedical applications of enzymes, but lipid‐ and polymeric‐vesicle‐based nanoreactors have some practical limitations. We have succeeded in preparing enzyme‐loaded polyion complex vesicles (PICsomes) through a facile protein‐loading method. The preservation of enzyme activity was confirmed even after cross‐linking of the PICsomes. The cross‐linked β‐galactosidase‐loaded PICsomes (β‐gal@PICsomes) selectively accumulated in the tumor tissue of mice. Moreover, a model prodrug, HMDER‐βGal, was successfully converted into a highly fluorescent product, HMDER, at the tumor site, even 4 days after administration of the β‐gal@PICsomes. Intravital confocal microscopy showed continuous production of HMDER and its distribution throughout the tumor tissues. Thus, enzyme‐loaded PICsomes are useful for prodrug activation at the tumor site and could be a versatile platform for enzyme delivery in enzyme prodrug therapy.     

A Novel Ratiometric Fluorescent Probe for Highly Sensitive and Selective Detection of β‐Galactosidase in Living Cells

β‐Galactosidase, a glycoside hydrolase enzyme, has been proved to be an important biomarker of cell senescence and primary ovarian cancer. Effective detection of β‐galactosidase has attracted wide attention. Herein, one ratiometric fluorescent probe has been successfully synthesized for detecting the β‐galactosidase in living cells. The as‐prepared probe exhibits two emission peaks at 490 nm and 530 nm, respectively, and the ratio of fluorescence intensities from the two emission peaks could be utilized to monitor the β‐galactosidase. This present ratiometric fluorescent probe is, therefore, very promising for effective, sensitive, and selective detection of the β‐galactosidase in living cells.     

Lysosome‐Targeting Amplifiers of Reactive Oxygen Species as Anticancer Prodrugs

Cancer cells produce elevated levels of reactive oxygen species, which has been used to design cancer specific prodrugs. Their activation relies on at least a bimolecular process, in which a prodrug reacts with ROS. However, at low micromolar concentrations of the prodrugs and ROS, the activation is usually inefficient. Herein, we propose and validate a potentially general approach for solving this intrinsic problem of ROS‐dependent prodrugs. In particular, known prodrug 4‐(N ‐ferrocenyl‐N ‐benzylaminocarbonyloxymethyl)phenylboronic acid pinacol ester was converted into its lysosome‐specific analogue. Since lysosomes contain a higher concentration of active ROS than the cytoplasm, activation of the prodrug was facilitated with respect to the parent compound. Moreover, it was found to exhibit high anticancer activity in a variety of cancer cell lines (IC₅₀=3.5–7.2 μm ) and in vivo (40 mg kg⁻¹, NK/Ly murine model) but remained weakly toxic towards non‐malignant cells (IC₅₀=15–30 μm ).     

Selective Ablation of β‐Galactosidase‐Expressing Cells with a Rationally Designed Activatable Photosensitizer

We have developed an activatable photosensitizer capable of specifically inducing the death of β‐galactosidase‐expressing cells in response to photoirradiation. By using a selenium‐substituted rhodol scaffold bearing β‐galactoside as a targeting substituent, we designed and synthesized HMDESeR‐βGal, which has a non‐phototoxic spirocyclic structure owing to the presence of the galactoside moiety. However, β‐galactosidase efficiently converted HMDESeR‐βGal into phototoxic HMDESeR, which exists predominantly in the open xanthene form. This structural change resulted in drastic recovery of visible‐wavelength absorption and the ability to generate singlet oxygen (¹O₂). When HMDESeR‐βGal was applied to larval Drosophila melanogaster wing disks, which express β‐galactosidase only in the posterior region, photoirradiation induced cell death in the β‐galactosidase‐expressing region with high specificity.     

A H_2S-triggered two-photon ratiometric fluorescent theranostic prodrug for bio-imaging

Hydrogen sulfide(H_2 S) is a signaling molecule that plays important roles in biological systems.The exploration of H_2 S as a new drug release trigger and its related fluorescent theranostic system is crucial for cancer bio-imaging and therapy.Herein,we designed a new two-photon ratiometric fluorescent theranostic prodrug(compound 1) and studied its spectroscopic properties and application in in vivo imaging.Compound 1 specifically reacted with H_2 S and released the free active therapeutic component of 7-ethyl-10-hydroxycamptothecin,which was accompanied with a red-shift fluorescence emission signal from 460 nm to 545 nm.The exogenous and endogenous H_2 S in living cells were imaged by compound 1 under one-photon and two-photon excitation.Furthermore,compound 1 monitored the H_2 S concentration changes in Caenorhabditis elegans by fluorescence imaging.Additionally,it showed effective drug release activation in situ tumor with exogenous and endogenous H_2 S as the trigger.The H_2 S-sensitive activation and drug-release properties highlight the potential of theranostic compound 1 in future cancer treatment and therapy.     

Detection of LacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

The LacZ gene, which encodes Escherichia coli β‐galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ‐positive cells in living organisms or tissues at single‐cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β‐galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single‐cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.     

Development of Luminescent Coelenterazine Derivatives Activatable by β‐Galactosidase for Monitoring Dual Gene Expression

Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect β‐galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing β‐galactose caging groups to block the auto‐oxidation of coelenterazine. Both probes contain β‐galactosidase cleavable caging groups at the carbonyl group of the imidazo–pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for β‐galactosidase over other glycoside hydrolases. bGalN‐oCoel could detect β‐galactosidase activity in living HEK‐293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by β‐galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual‐enzyme substrate and detect enzyme activity across two separate cell populations.     

Theranostic hyaluronic acid prodrug micelles with aggregation-induced emission characteristics for targeted drug delivery

Theranostic hyaluronic acid (HA) prodrug micelles with pH-responsive drug release and aggregation-induced emission (AIE) properties were prepared by chemical graft of biomimetic phosphorylcholine (PC), anticancer drug doxorubicin (DOX) and AIE fluorogen tetraphenylene (TPE) to the HA backbone. DOX was conjugated to the HA backbone by a hydrazone bond which can be hydrolyzed under acidic environment and result in pH-triggered smart release of DOX. The TPE units with typical AIE characteristics were applied for real time drug tracking in cancer cells. The HA-based prodrugs could self-assemble into micelles in aqueous solution as confirmed by the dynamic light scattering (DLS) and transmission electron microscopy (TEM). The intracellular distribution of HA prodrug micelles could be clearly observed by fluorescence microscopy based on the strong fluorescence of TPE. Moreover, after treated with the micelles, stronger fluorescence of TPE in CD44 overexpressed MDA-MB-231 cancer cells was observed, compared to the CD44 negative cell line, NIH3T3 cells, suggesting efficient cell uptake of HA prodrug micelles by receptor-mediated endocytosis. The cell viability results indicated that the prodrug micelles could inhibit the proliferation of the cancer cells effectively. Such pH-triggered theranostic drug delivery system with AIE features can provide a new platform for targeted and image-guided cancer therapy.     

The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme

The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.     

Monochalcoplatin: An Actively Transported,Quickly Reducible,and Highly Potent PtIV Anticancer Prodrug

Recently, Pt^IV prodrugs have attracted much attention as the next generation of platinum‐based antineoplastic drug candidates. Here we report the discovery and evaluation of monochalcoplatin, a monocarboxylated Pt^IV prodrug that is among the most cytotoxic Pt^IV prodrugs to date. Compared with its dicarboxylated counterpart chalcoplatin, monochalcoplatin accumulates astonishingly effectively and rapidly in cancer cells, which is not ascribed to its lipophilicity. The prodrug is quickly reduced, causes DNA damage, and induces apoptosis, resulting in superior cytotoxicity with IC₅₀ values in the nanomolar range in both cisplatin‐sensitive and ‐resistant cells; these IC₅₀ values are up to 422‐fold higher than that of cisplatin. A detailed mechanistic study reveals that monochalcoplatin actively enters cells through a transporter‐mediated process. Moreover, monochalcoplatin shows significant antitumor activity in an in vivo colorectal tumor model. Our study implies a practical strategy for the design of more effective Pt^IV prodrugs to conquer drug resistance by tuning both cellular uptake pathways and activation processes.     

Monochalcoplatin: An Actively Transported,Quickly Reducible,and Highly Potent PtIV Anticancer Prodrug

Recently, Pt^IV prodrugs have attracted much attention as the next generation of platinum‐based antineoplastic drug candidates. Here we report the discovery and evaluation of monochalcoplatin, a monocarboxylated Pt^IV prodrug that is among the most cytotoxic Pt^IV prodrugs to date. Compared with its dicarboxylated counterpart chalcoplatin, monochalcoplatin accumulates astonishingly effectively and rapidly in cancer cells, which is not ascribed to its lipophilicity. The prodrug is quickly reduced, causes DNA damage, and induces apoptosis, resulting in superior cytotoxicity with IC₅₀ values in the nanomolar range in both cisplatin‐sensitive and ‐resistant cells; these IC₅₀ values are up to 422‐fold higher than that of cisplatin. A detailed mechanistic study reveals that monochalcoplatin actively enters cells through a transporter‐mediated process. Moreover, monochalcoplatin shows significant antitumor activity in an in vivo colorectal tumor model. Our study implies a practical strategy for the design of more effective Pt^IV prodrugs to conquer drug resistance by tuning both cellular uptake pathways and activation processes.     

Cyclization-activated prodrugs

Many drugs suffer from an extensive first-pass metabolism leading to drug inactivation and/or production of toxic metabolites, which makes them attractive targets for prodrug design. The classical prodrug approach, which involves enzyme-sensitive covalent linkage between the parent drug and a carrier moiety, is a well established strategy to overcome bioavailability/toxicity issues. However, the development of prodrugs that can regenerate the parent drug through non-enzymatic pathways has emerged as an alternative approach in which prodrug activation is not influenced by inter- and intraindividual variability that affects enzymatic activity. Cyclization-activated prodrugs have been capturing the attention of medicinal chemists since the middle-1980s, and reached maturity in prodrug design in the late 1990 s. Many different strategies have been exploited in recent years concerning the development of intramoleculary-activated prodrugs spanning from analgesics to anti-HIV therapeutic agents. Intramolecular pathways have also a key role in two-step prodrug activation, where an initial enzymatic cleavage step is followed by a cyclization-elimination reaction that releases the active drug. This work is a brief overview of research on cyclization-activated prodrugs from the last two decades.     

NIR‐Light‐Activated Combination Therapy with a Precise Ratio of Photosensitizer and Prodrug Using a Host–Guest Strategy

The co‐delivery of photosensitizers with prodrugs sensitive to reactive oxygen species (ROS) for light‐triggered ROS generation and cascaded prodrug activation has drawn tremendous attention. However, the absence of a feasible method to deliver the two components at a precise ratio has impaired the application potential. Herein, we report an efficient method to produce a nanosized platform for the delivery of an optimized ratio of the two components by the means of host–guest strategy for maximizing the combination therapy efficacy of cancer treatment. The key features of this host–guest strategy for the combination therapy are that the ratio between photosensitizer and ROS‐sensitive prodrug can be easily tuned, near‐infrared (NIR) irradiation can sensitize the photosensitizer and activate the paclitaxel prodrug for its release, and the accumulation process can be tracked by NIR imaging to maximize the efficacy of photodynamic and chemotherapy.     

Real‐Time In Vivo Detection of Cellular Senescence through the Controlled Release of the NIR Fluorescent Dye Nile Blue

In vivo detection of cellular senescence is accomplished by using mesoporous silica nanoparticles loaded with the NIR‐FDA approved Nile blue (NB) dye and capped with a galactohexasaccharide ( S3 ). NB emission at 672 nm is highly quenched inside S3 , yet a remarkable emission enhancement is observed upon cap hydrolysis in the presence of β‐galactosidase and dye release. The efficacy of the probe to detect cellular senescence is tested in vitro in melanoma SK‐Mel‐103 and breast cancer 4T1 cells and in vivo in palbociclib‐treated BALB/cByJ mice bearing breast cancer tumor.     

Click and Release: A Chemical Strategy toward Developing Gasotransmitter Prodrugs by Using an Intramolecular Diels–Alder Reaction

Prodrug strategies have been proven to be a very effective way of addressing delivery problems. Much of the chemistry in prodrug development relies on the ability to mask an appropriate functional group, which can be removed under appropriate conditions. However, developing organic prodrugs of gasotransmitters represent unique challenges. This is especially true with carbon monoxide, which does not have an easy “handle” for bioreversible derivatization. By taking advantage of an intramolecular Diels–Alder reaction, we have developed a prodrug strategy for preparations of organic CO prodrugs that are stable during synthesis and storage, and yet readily release CO with tunable release rates under near physiological conditions. The effectiveness of the CO prodrug system in delivering a sufficient quantity of CO for possible therapeutic applications has been studied using a cell culture anti‐inflammatory assay and a colitis animal model. These studies fully demonstrate the proof of concept, and lay a strong foundation for further medicinal chemistry work in developing organic CO prodrugs.     

Magnetically Guided Protein Transduction by Hybrid Nanogel Chaperones with Iron Oxide Nanoparticles

Protein pharmaceuticals show great therapeutic promise, but effective intracellular delivery remains challenging. To address the need for efficient protein transduction systems, we used a magnetic nanogel chaperone (MC): a hybrid of a polysaccharide nanogel, a protein carrier with molecular chaperone‐like properties, and iron oxide nanoparticles, enabling magnetically guided delivery. The MC complexed with model proteins, such as BSA and insulin, and was not cytotoxic. Cargo proteins were delivered to the target HeLa cell cytosol using a magnetic field to promote movement of the protein complex toward the cells. Delivery was confirmed by fluorescence microscopy and flow cytometry. Delivered β‐galactosidase, inactive within the MC complex, became enzymatically active within cells to convert a prodrug. Thus, cargo proteins were released from MC complexes through exchange interactions with cytosolic proteins. The MC is a promising tool for realizing the therapeutic potential of proteins.     

Liver‐Targeted Small‐Molecule Inhibitors of Proprotein Convertase Subtilisin/Kexin Type 9 Synthesis

Targeting of the human ribosome is an unprecedented therapeutic modality with a genome‐wide selectivity challenge. A liver‐targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesterase 1. The synthesis of select tetrazole prodrugs was crucial. A cell‐free in vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. In vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography ¹⁸F‐isotopologue validated our liver‐targeting approach.     

An Esterase‐Sensitive Prodrug Approach for Controllable Delivery of Persulfide Species

A strategy to deliver a well‐defined persulfide species in a biological medium is described. Under near physiological conditions, the persulfide prodrug can be activated by an esterase to generate a “hydroxymethyl persulfide” intermediate, which rapidly collapses to form a defined persulfide. Such persulfide prodrugs can be used either as chemical tools to study persulfide chemistry and biology or for future development as H₂S‐based therapeutic reagents. Using the persulfide prodrugs developed in this study, the reactivity between S ‐methyl methanethiosulfonate (MMTS) with persulfide was unambiguously demonstrated. Furthermore, a representative prodrug exhibited potent cardioprotective effects in a murine model of myocardial ischemia‐reperfusion (MI/R) injury with a bell shape therapeutic profile.     

Activation of Platinum(IV) Prodrugs By Motexafin Gadolinium as a Redox Mediator

Water‐soluble platinum(IV) prodrugs, which proved kinetically stable to reduction in the presence of physiological concentration of ascorbate, were quickly reduced to their active form, oxaliplatin, when co‐incubated with a macrocycle metallotexaphyrin (i.e., Motexafin Gadolinium (MGd)). The reduction of Pt^IV to Pt^II promoted by MGd occurs in cell culture as well, leading to an increase in the antiproliferative activity of the Pt^IV species in question. The mediated effect is proportional to the concentration of MGd and gives rise to an enhancement when the prodrug is relatively hydrophilic. MGd is known to localize/accumulate preferentially in tumor tissues. Thus, the present “activation by reduction” approach may allow for the cancer‐selective enhancement in the cytotoxicity of Pt^IV prodrugs.