An Activatable Nano-Prodrug for Treating Tyrosine-Kinase-Inhibitor-Resistant Non-Small Cell Lung Cancer and for Optoacoustic and Fluorescent Imaging.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the cause of high rate of mortality. The epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors are used to treat NSCLC, yet their curative effects are usually compromised by drug resistance. This study demonstrates a nanodrug for treating tyrosine-kinase-inhibitor-resistant NSCLC through inhibiting upstream and downstream EGFR signaling pathways. The main molecule of the nanodrug is synthesized by linking a tyrosine kinase inhibitor gefitinib and a near-infrared dye (NIR) on each side of a disulfide via carbonate bonds, and the nanodrug is then obtained through nanoparticle formation of the main molecule in aqueous medium and concomitant encapsulation of a serine threonine protein kinase (Akt) inhibitor celastrol. Upon administration, the nanodrug accumulates at the tumor region of NSCLC-bearing mice and releases the drugs for tumor inhibition, and the dye for fluorescence and optoacoustic imaging. Through suppressing the phosphorylation of upstream EGFR and downstream Akt in the EGFR pathway by gefitinib and celastrol, respectively, the nanodrug exhibits high inhibition efficacy against orthotopic NSCLC in mouse models.

Diagnosing Drug-Induced Liver Injury by Multispectral Optoacoustic Tomography and Fluorescence Imaging Using a Leucine-Aminopeptidase-Activated Probe.

Drug-induced liver injury (DILI) is a frequent cause of hepatic dysfunction as well as the single most frequent reason for removing approved medications from the market, and multispectral optoacoustic tomography (MSOT) is an emerging and noninvasive imaging modality for diagnosing and monitoring diseases. Herein, we report an activatable optoacoustic probe for imaging DILI through detecting the activity of leucine aminopeptidase (LAP). In this probe, an N-terminal leucyl moiety serving as the LAP recognition element is linked with a chromene-benzoindolium chromophore via 4-aminobenzylalcohol group. The elevated expression of hepatic LAP as a result of DILI cleaves the leucyl moiety and causes the red-shift of the probe's absorption band, thereby generating prominent optoacoustic signals for MSOT imaging. During this process, the probe also exhibits prominent NIR fluorescence, which can be utilized for fluorescent imaging. More importantly, by rendering stacks of cross-sectional images as maximal intensity projection (MIP) images, we could precisely locate the focus of drug-induced liver injury in mice. This probe is expected to serve a powerful tool for studying physiological and pathological processes related to LAP.

A Gold Nanocage/Cluster Hybrid Structure for Whole-Body Multispectral Optoacoustic Tomography Imaging, EGFR Inhibitor Delivery, and Photothermal Therapy.

Gold nanocages (AuNCs) and gold nanoclusters (AuClusters) are two classes of advantageous nanostructures with special optical properties, and many other attractive properties. Integrating them into one nanosystem may achieve greater and smarter performance. Herein, a hybrid gold nanostructure for fluorescent and optoacoustic tomography imaging, controlled release of drugs, and photothermal therapy (PTT) is demonstrated. For this nanodrug (EA-AB), an epidermal growth factor receptor (EGFR) inhibitor erlotinib (EB) is loaded into AuNCs, which are then capped and functionalized by biocompatible AuCluster@BSA (BSA = bovine serum albumin) conjugates via electrostatic interaction. Upon cell internalization, the lysosomal proteases and low pH cause the release of EB from EA-AB, and also induce fluorescence restoration of the AuCluster for imaging. Irradiation with near-infrared light further promotes the drug release and affords a PTT effect as well. The AuNC-based nanodrug is optoacoustically active, and its biodistribution and metabolic process have been successfully monitored by whole-body and 3D multispectral optoacoustic tomography imaging. Owing to the combined actions of PTT and EGFR pathway blockage, EA-AB exhibits marked tumor inhibition efficacy in vivo.

Oligo(ethylene glycol)-Functionalized Squaraine Fluorophore as a Near-Infrared-Fluorescent Probe for the In Vivo Detection of Diagnostic Enzymes.

Squaraine dyes have excellent photostability with intensive absorption and strong fluorescence in the near-infrared (NIR) region. However, they display a strong tendency to aggregate in aqueous media because of their poor water solubility, often causing fluorescence quenching that severely limits their in vivo applications, especially for detecting or imaging diagnostic enzymes. In this work, an oligo(ethylene glycol)-functionalized squaraine fluorophore has been developed as an NIR-fluorescent probe that can detect and image the activities of a diagnostic enzyme (leucine aminopeptidase) both in vitro and in vivo. The probe shows near-infrared absorption and emission, a low detection limit (0.61 ng/mL), relatively good aqueous solubility, high selectivity, and little toxicity. In addition, the probe herein was successfully used to track endogenous leucine aminopeptidase both in vitro and in vivo with a nude-mouse model.

A Fluorescent Probe for Early Detection of Melanoma and Its Metastasis by Specifically Imaging Tyrosinase Activity in a Mouse Model.

Melanoma is a type of highly malignant and metastatic skin cancer, and early detection of melanoma by analyzing the level of its biomarker may decrease the likelihood of mortality. In this study, a fluorescent probe called NBR-AP for detecting tyrosinase (a biomarker of melanoma) has been created by incorporating a hydroxyphenylurea group (as a substrate for the enzyme) onto a fluorescent dye phenoxazine derivative (as an activatable signal reporter). This probe can be activated to generate fluorescence through a tyrosinase-mediated oxidation followed by hydrolysis of the urea linkage. The probe is able to detect the endogenous tyrosinase level in live cells and in zebrafish sensitively and selectively. Moreover, by imaging the tyrosinase activity, NBR-AP has been successfully applied to diagnose the melanoma and its metastasis in xenogeneic mouse models established via subcutaneous injection of B16F10 cells.

Macrophage membrane-biomimetic adhesive polycaprolactone nanocamptothecin for improving cancer-targeting efficiency and impairing metastasis.

The recent remarkable success and safety of mRNA lipid nanoparticle technology for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has stimulated intensive efforts to expand nanoparticle strategies to treat various diseases. Numerous synthetic nanoparticles have been developed for pharmaceutical delivery and cancer treatment. However, only a limited number of nanotherapies have enter clinical trials or are clinically approved. Systemically administered nanotherapies are likely to be sequestered by host mononuclear phagocyte system (MPS), resulting in suboptimal pharmacokinetics and insufficient drug concentrations in tumors. Bioinspired drug-delivery formulations have emerged as an alternative approach to evade the MPS and show potential to improve drug therapeutic efficacy. Here we developed a biodegradable polymer-conjugated camptothecin prodrug encapsulated in the plasma membrane of lipopolysaccharide-stimulated macrophages. Polymer conjugation revived the parent camptothecin agent (e.g., 7-ethyl-10-hydroxy-camptothecin), enabling lipid nanoparticle encapsulation. Furthermore, macrophage membrane cloaking transformed the nonadhesive lipid nanoparticles into bioadhesive nanocamptothecin, increasing the cellular uptake and tumor-tropic effects of this biomimetic therapy. When tested in a preclinical murine model of breast cancer, macrophage-camouflaged nanocamptothecin exhibited a higher level of tumor accumulation than uncoated nanoparticles. Furthermore, intravenous administration of the therapy effectively suppressed tumor growth and the metastatic burden without causing systematic toxicity. Our study describes a combinatorial strategy that uses polymeric prodrug design and cell membrane cloaking to achieve therapeutics with high efficacy and low toxicity. This approach might also be generally applicable to formulate other therapeutic candidates that are not compatible or miscible with biomimetic delivery carriers.

Tumor-overexpressed enzyme responsive amphiphiles small molecular self-assembly nano-prodrug for the chemo-phototherapy against non-small-cell lung cancer.

Rational design of self-assembly drug amphiphiles can provide a promising strategy for constructing nano-prodrug with high drug loading, smart stimuli-responsive drug release and high tumor selectivity. Herein, we report a small molecular amphiphile prodrug that can self-assemble into multifunctional nano-prodrug for enhanced anticancer effect by the combination of chemotherapy and phototherapy (PDT/PTT). In this prodrug, the simple insertion of quinone propionate into hydrophilic drug Irinotecan (Ir) generates suitable amphiphiles that endow a good self-assembly behavior of the prodrug and transform it into a stable and uniform nanoparticle. Interestingly, this excellent self-assembly behavior can load phototherapy agent ICG to form a multifunctional nano-prodrug, thereby enhancing the chemotherapeutic effect with PDT/PTT. Importantly, the quinone propionic acid moiety in the prodrug showed a high sensitivity to the overexpressed NAD(P)H:quinone oxidoreductase-1 (NQO1) in non-small cell lung cancer (NSCLC) cells, and this sensitivity enables the disassembly of nano-prodrug and efficient NQO1-responsive drug release. To further enhance the drug accumulation on tumor tissue and migrate the blood clearance, a biomimetic nano-prodrug has been successfully explored by coating hybrid membrane on the above nano-prodrug, which displays high selective inhibition of tumor growth and metastasis on NSCLC mice model. Our findings provide new insights into the rational design of tumor-overexpressed enzyme responsive nano-prodrug for cancer combinational therapy.

Combination therapy to overcome ferroptosis resistance by biomimetic self-assembly nano-prodrug.

Ferroptosis has emerged as a potent form of no-apoptotic cell death that offers a promising alternative to avoid the chemoresistance of apoptotic pathways and serves as a vulnerability of cancer. Herein, we have constructed a biomimetic self-assembly nano-prodrug system that enables the co-delivery of gefitinib (Gefi), ferrocene (Fc) and dihydroartemisinin (DHA) for the combined therapy of both ferroptosis and apoptosis. In the tumor microenvironment, this nano-prodrug is able to disassemble and trigger drug release under high levels of GSH. Interestingly, the released DHA can downregulate GPX4 level for the enhancement of intracellular ferroptosis from Fc, further executing tumor cell death with concomitant chemotherapy by Gefi. More importantly, this nano-prodrug provides highly homologous targeting ability by coating related cell membranes and exhibits outstanding inhibition of tumor growth and metastasis, as well as no noticeable side-effects during treatments. This simple small molecular self-assembled nano-prodrug provides a new reasonably designed modality for ferroptosis-combined chemotherapy.

De novo engineering of both an omega-3 fatty acid-derived nanocarrier host and a prodrug guest to potentiate drug efficacy against colorectal malignancies.

Drug-carrier compatibility impacts drug delivery efficiency and resulting therapeutic efficacy and tolerability. Although numerous biodegradable carrier materials have been pursued over the past decades, chemical strategies that are sought to tailor therapeutic structures and their carriers together in a concerted effort remain rare yet may be powerful. Based on the principle of improving the structural similarity between these central components, we developed an omega-3 fatty acid-conjugated poly(ethylene glycol) (PEG) nanocarrier host that is capable of supramolecular assembly of a cytotoxic prodrug guest. To demonstrate the proof of concept, we ligated two docosahexaenoic acid (DHA) molecules and one PEG chain via a d-lysine linkage to produce an amphiphilic matrix DHA2-PEG, which is suited for the encapsulation of active compounds, including a DHA monoconjugated camptothecin prodrug. The resulting DHA2-PEG-cloaked nanoassemblies show superior stability and rapid cellular uptake compared with those formulated in clinically approved materials. In a chemically induced mouse model of colitis-associated colorectal cancer, administration of the camptothecin nanoassemblies demonstrated notable inhibition of colon tumor growth. Furthermore, this new delivery platform has low systemic toxicity and immunotoxicity in animals and is appealing for further investigation and clinical translation. Thus, through rational engineering of the carrier biomaterials and drug derivatization, the in vivo performance of drug delivery systems can be improved. This approach also establishes a methodology for leveraging synthetic chemistry tools to optimize delivery systems for a broad range of drug classes.

A dopamine-precursor-based nanoprodrug for in-situ drug release and treatment of acute liver failure by inhibiting NLRP3 inflammasome and facilitating liver regeneration.

Acute liver failure (ALF) is a severe liver disease with high mortality rate. Inflammasome is a newly-found and promising target for effective treatment of immunity-associated diseases including liver disease, and dopamine has recently been proved as an inhibitor for NLRP3 inflammasome. This work demonstrates a diselenide-based nanodrug for ALF treatment through inhibiting NLRP3 inflammasome activation and enhancing liver regeneration. A diselenide-containing molecule (DSeSeD) has been synthesized via covalently linking two l-Dopa molecules to a diselenide linker, and the resultant molecules form stable nanoparticles in aqueous media and encapsulate SW033291 (an inhibitor of prostaglandin-degrading enzyme that hampers liver regeneration) to produce the nanodrug (SW@DSeSeD). As a nanoscale prodrug, SW@DSeSeD protects its payloads from decomposition in bloodstream upon administration, accumulates in liver of ALF mice, then responds to the overexpressed ROS and thereby releases SW033291 as well as a stable dopamine precursor that can transform into dopamine in hepatic cells, thus achieving significant therapeutic efficacy against ALF through inhibiting NLRP3 inflammasome activation and enhancing hepatic regeneration. Moreover, multiple contrast agents have been loaded onto the nanodrug to achieve fluorescence, optoacoustic and magnetic resonance imaging for nanodrug location and disease evaluation.

A biopolymer-based and inflammation-responsive nanodrug for rheumatoid arthritis treatment via inhibiting JAK-STAT and JNK signalling pathways.

Rheumatoid arthritis (RA) is a common chronic autoimmune disease associated with progressive disability, systemic complications, and poor prognosis. The improved understanding of the roles of immune signaling pathway inhibitors has shed light on designing new and more effective approaches for RA treatment. In this work, an inflammation-responsive and molecularly targeted drug system has been developed for RA therapy. The drug carrier was synthesized by covalently grafting hydrophobic cholesterol (Chol) molecules onto a hydrophilic chondroitin sulfate (CS) chain via the inflammation-responsive diselenide bonds (SeSe). The resultant amphiphilic polymer CSSeSeChol readily forms nanoparticles (NPs) and encapsulates two kinase inhibitors tofacitinib and SP600125 in aqueous media. Upon administration into the RA mouse model, the nanodrug accumulates in RA lesions and releases the inhibitors for regulating the JAK-STAT and JNK pathways. As a result, the nanodrug exhibits satisfactory efficacy in RA treatment by suppressing the expression of relevant pro-inflammatory cytokines, blocking the activation of osteoclasts and providing protection for cartilage and joints.

Tetrazine-Mediated Bioorthogonal System for Prodrug Activation, Photothermal Therapy, and Optoacoustic Imaging.

Bioorthogonal "bond cleavage" reactions hold great promise in a variety of biological applications such as controlled activation of the drug and probe, while the application of these biocompatible reactions in living animals is still in its infancy and has yet to be further explored. Herein we demonstrate a nanosized and two-component bioorthogonal system for tumor inhibition through the combined action of chemo- and photothermal therapy. The trigger of the system was fabricated by immobilizing PEGylated tetrazine on the gold nanorods, and the bioorthogonal prodrug was synthesized by caging the drug camptothecin with vinyl ether, followed by encapsulating it with phospholipid liposomes. The tetrazine-based trigger effectively mediates the bioorthogonal reaction and triggers the release of camptothecin for chemotherapy, and the gold nanorods exhibit high photothermal capability for photothermal therapy and for three-dimensional optoacoustic imaging. Upon injection into tumor-bearing mice, the two components accumulate in the tumor region and carry out a bioorthogonal reaction therein, hence releasing the parent drug. The combined actions of chemo- and photothermal therapy greatly inhibited tumor growth in mice. This strategy may afford a promising approach for achieving controlled release of an active drug in vivo through an alternative external stimulus-a bioorthogonal reaction.

A two-photon-activated prodrug for therapy and drug release monitoring.

A light-activated cleavage strategy for the concomitant release of active drugs and generation of fluorescence changes is highly desirable. Herein a molecular prodrug featuring real-time monitoring of drug localization and release by manipulating fluorophores has been created by constructing a cleavable structure which comprises a photoremovable coumarinyl, an anticancer drug camptothecin, a cleavable linker and a near infrared fluorescent dye dicyanomethylene-4H-pyran (DCM). The fluorescence of coumarinyl and CPT is completely quenched by the DCM moiety via fluorescence resonance energy transfer (FRET). The internalization of the prodrug by cells and its subsequent intracellular location can be tracked by collecting the red fluorescence of DCM; while the release of active CPT as a result of one- or two-photon irradiation can be monitored by observing the newly emerged fluorescence of CPT under one- or two-photon excitation. The prodrug also shows highly controllable cytotoxicity toward HeLa cells and A549 cells, with low IC50 values of 4.01 and 2.53 µM, respectively, upon light irradiation and with much higher IC50 values (>40 µM) without light irradiation. This strategy may provide an approach for the development of light-activatable theranostic anticancer therapeutics.