Leveraging intracellular ALDH1A1 activity for selective cancer stem-like cell labeling and targeted treatment via in vivo click reaction.

Inhibition of overexpressed enzymes is among the most promising approaches for targeted cancer treatment. However, many cancer-expressed enzymes are "nonlethal," in that the inhibition of the enzymes' activity is insufficient to kill cancer cells. Conventional antibody-based therapeutics can mediate efficient treatment by targeting extracellular nonlethal targets but can hardly target intracellular enzymes. Herein, we report a cancer targeting and treatment strategy to utilize intracellular nonlethal enzymes through a combination of selective cancer stem-like cell (CSC) labeling and Click chemistry-mediated drug delivery. A de novo designed compound, AAMCHO [N-(3,4,6-triacetyl- N-azidoacetylmannosamine)-cis-2-ethyl-3-formylacrylamideglycoside], selectively labeled cancer CSCs in vitro and in vivo through enzymatic oxidation by intracellular aldehyde dehydrogenase 1A1. Notably, azide labeling is more efficient in identifying tumorigenic cell populations than endogenous markers such as CD44. A dibenzocyclooctyne (DBCO)-toxin conjugate, DBCO-MMAE (Monomethylauristatin E), could next target the labeled CSCs in vivo via bioorthogonal Click reaction to achieve excellent anticancer efficacy against a series of tumor models, including orthotopic xenograft, drug-resistant tumor, and lung metastasis with low toxicity. A 5/7 complete remission was observed after single-cycle treatment of an advanced triple-negative breast cancer xenograft (~500 mm3).

Novel Liposomal Azido Mannosamine Lipids on Metabolic Cell Labeling and Imaging via Cu-Free Click Chemistry.

In comparison with the popular Ac4ManNAz applied as cell labels via Cu-free click chemistry, two novel azido mannosamine lipids with C6 and C12 esters on anomeric hydroxyl groups were prepared and encapsulated in a liposome delivery system, which enhanced chemical stabilities and showed good cell-metabolizable labeling efficiency on MDA-MB-231 cells with strong fluorescence after the treatment of DBCO-Cy5 by triazole formation via click chemistry.

Selective in vivo metabolic cell-labeling-mediated cancer targeting.

Distinguishing cancer cells from normal cells through surface receptors is vital for cancer diagnosis and targeted therapy. Metabolic glycoengineering of unnatural sugars provides a powerful tool to manually introduce chemical receptors onto the cell surface; however, cancer-selective labeling still remains a great challenge. Herein we report the design of sugars that can selectively label cancer cells both in vitro and in vivo. Specifically, we inhibit the cell-labeling activity of tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) by converting its anomeric acetyl group to a caged ether bond that can be selectively cleaved by cancer-overexpressed enzymes and thus enables the overexpression of azido groups on the surface of cancer cells. Histone deacetylase and cathepsin L-responsive acetylated azidomannosamine, one such enzymatically activatable Ac4ManAz analog developed, mediated cancer-selective labeling in vivo, which enhanced tumor accumulation of a dibenzocyclooctyne-doxorubicin conjugate via click chemistry and enabled targeted therapy against LS174T colon cancer, MDA-MB-231 triple-negative breast cancer and 4T1 metastatic breast cancer in mice.

Pamidronate functionalized nanoconjugates for targeted therapy of focal skeletal malignant osteolysis.

Malignant osteolysis associated with inoperable primary bone tumors and multifocal skeletal metastases remains a challenging clinical problem in cancer patients. Nanomedicine that is able to target and deliver therapeutic agents to diseased bone sites could potentially provide an effective treatment option for different types of skeletal cancers. Here, we report the development of polylactide nanoparticles (NPs) loaded with doxorubicin (Doxo) and coated with bone-seeking pamidronate (Pam) for the targeted treatment of malignant skeletal tumors. In vivo biodistribution of radiolabeled targeted Pam-NPs demonstrated enhanced bone tumor accumulation and prolonged retention compared with nontargeted NPs. In a murine model of focal malignant osteolysis, Pam-functionalized, Doxo-loaded NPs (Pam-Doxo-NPs) significantly attenuated localized osteosarcoma (OS) progression compared with nontargeted Doxo-NPs. Importantly, we report on the first evaluation to our knowlege of Pam-Doxo-NPs in dogs with OS, which possess tumors of anatomic size and physiology comparable to those in humans. The repeat dosing of Pam-Doxo-NPs in dogs with naturally occurring OS indicated the therapeutic was well tolerated without hematologic, nonhematologic, and cardiac toxicities. By nuclear scintigraphy, the biodistribution of Pam-Doxo-NPs demonstrated malignant bone-targeting capability and exerted measurable anticancer activities as confirmed with percent tumor necrosis histopathology assessment.

Macrophage-Membrane-Coated Nanoparticles for Tumor-Targeted Chemotherapy.

Various delivery vectors have been integrated within biologically derived membrane systems to extend their residential time and reduce their reticuloendothelial system (RES) clearance during systemic circulation. However, rational design is still needed to further improve the in situ penetration efficiency of chemo-drug-loaded membrane delivery-system formulations and their release profiles at the tumor site. Here, a macrophage-membrane-coated nanoparticle is developed for tumor-targeted chemotherapy delivery with a controlled release profile in response to tumor microenvironment stimuli. Upon fulfilling its mission of tumor homing and RES evasion, the macrophage-membrane coating can be shed via morphological changes driven by extracellular microenvironment stimuli. The nanoparticles discharged from the outer membrane coating show penetration efficiency enhanced by their size advantage and surface modifications. After internalization by the tumor cells, the loaded drug is quickly released from the nanoparticles in response to the endosome pH. The designed macrophage-membrane-coated nanoparticle (cskc-PPiP/PTX@Ma) exhibits an enhanced therapeutic effect inherited from both membrane-derived tumor homing and step-by-step controlled drug release. Thus, the combination of a biomimetic cell membrane and a cascade-responsive polymeric nanoparticle embodies an effective drug delivery system tailored to the tumor microenvironment.

High Drug Loading and Sub-Quantitative Loading Efficiency of Polymeric Micelles Driven by Donor-Receptor Coordination Interactions.

Polymeric micelles are extensively used for the delivery of hydrophobic drugs, which, however, suffer from unsatisfactory drug loading, colloidal uniformity, formulation stability, and drug release. Herein, we demonstrate a convenient strategy to prepare micelles with ultrahigh drug loading via the incorporation of polymer-drug coordination interactions. An amphiphilic copolymer containing pendant phenylboronic acid as electron acceptor unit was synthesized, which afforded donor-acceptor coordination with doxorubicin to obtain micelles with ultrahigh drug loading (∼50%), nearly quantitative loading efficiency (>95%), uniform size, and colloidal stability. Besides, the encapsulated drug can be effectively and selectively released in response to the high reactive oxygen species levels in cancer cells, which potentiated the anticancer efficacy and reduced systemic toxicity. Apart from doxorubicin, the current platform could be extended to other drugs with electron-donating groups (e.g., epirubicin and irinotecan), rendering a simple and robust strategy for enabling high drug loading in polymeric micelles and cancer-specific drug release.

Dynamic Ureas with Fast and pH-Independent Hydrolytic Kinetics.

Low cost, high performance hydrolysable polymers are of great importance in biomedical applications and materials industries. While many applications require materials to have a degradation profile insensitive to external pH to achieve consistent release profiles under varying conditions, hydrolysable chemistry techniques developed so far have pH-dependent hydrolytic kinetics. This work reports the design and synthesis of a new type of hydrolysable polymer that has identical hydrolysis kinetics from pH 3 to 11. The unprecedented pH independent hydrolytic kinetics of the aryl ureas were shown to be related to the dynamic bond dissociation controlled hydrolysis mechanism; the resulting hindered poly(aryl urea) can be degraded with a hydrolysis half-life of 10 min in solution. More importantly, these fast degradable hindered aromatic polyureas can be easily prepared by addition polymerization from commercially available monomers and are resistant to hydrolysis in solid form for months under ambient storage conditions. The combined features of good stability in solid state and fast hydrolysis at various pH values is unprecedented in polyurea material, and will have implications for materials design and applications, such as sacrificial coatings and biomaterials.

Investigating the optimal size of anticancer nanomedicine.

Nanomedicines (NMs) offer new solutions for cancer diagnosis and therapy. However, extension of progression-free interval and overall survival time achieved by Food and Drug Administration-approved NMs remain modest. To develop next generation NMs to achieve superior anticancer activities, it is crucial to investigate and understand the correlation between the physicochemical properties of NMs (particle size in particular) and their interactions with biological systems to establish criteria for NM optimization. Here, we systematically evaluated the size-dependent biological profiles of three monodisperse drug-silica nanoconjugates (NCs; 20, 50, and 200 nm) through both experiments and mathematical modeling and aimed to identify the optimal size for the most effective anticancer drug delivery. Among the three NCs investigated, the 50-nm NC shows the highest tumor tissue retention integrated over time, which is the collective outcome of deep tumor tissue penetration and efficient cancer cell internalization as well as slow tumor clearance, and thus, the highest efficacy against both primary and metastatic tumors in vivo.

Integrating Display and Delivery Functionality with a Cell Penetrating Peptide Mimic as a Scaffold for Intracellular Multivalent Multitargeting.

The construction of a multivalent ligand is an effective way to increase affinity and selectivity toward biomolecular targets with multiple-ligand binding sites. Adopting this strategy, we used a known cell-penetrating peptide (CPP) mimic as a scaffold to develop a series of multivalent ligand constructs that bind to the expanded dCTG (CTG(exp)) and rCUG nucleotide repeats (CUG(exp)) known to cause myotonic dystrophy type I (DM1), an incurable neuromuscular disease. By assembling this polyvalent construct, the hydrophobic ligands are solubilized and delivered into cell nuclei, and their enhanced binding affinity leads to the inhibition of ribonuclear foci formation and a reversal of splicing defects, all at low concentrations. Some of the multivalent ligands are shown to inhibit selectively the in vitro transcription of (CTG·CAG)74, to reduce the concentration of the toxic CUG RNA in DM1 model cells, and to show phenotypic improvement in vivo in a Drosophila model of DM1. This strategy may be useful in drug design for other trinucleotide repeat disorders and more broadly for intracellular multivalent targeting.

Dimeric drug polymeric nanoparticles with exceptionally high drug loading and quantitative loading efficiency.

Encapsulation of small-molecule drugs in hydrophobic polymers or amphiphilic copolymers has been extensively used for preparing polymeric nanoparticles (NPs). The loadings and loading efficiencies of a wide range of drugs in polymeric NPs, however, tend to be very low. In this Communication, we report a strategy to prepare polymeric NPs with exceptionally high drug loading (>50%) and quantitative loading efficiency. Specifically, a dimeric drug conjugate bearing a trigger-responsive domain was designed and used as the core-constructing unit of the NPs. Upon co-precipitation of the dimeric drug and methoxypoly(ethylene glycol)-block-polylactide (mPEG-PLA), NPs with a dimeric drug core and a polymer shell were formed. The high-drug-loading NPs showed excellent stability in physiological conditions. No premature drug or prodrug release was observed in PBS solution without triggering, while external triggering led to controlled release of drug in its authentic form.

Reversal of Chemoresistance via Staged Liberation of Chemodrug and siRNA in Hierarchical Response to ROS Gradient.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) often leads to the failure of antitumor chemotherapy, and codelivery of chemodrug with P-gp siRNA (siP-gp) represents a promising approach for treating chemoresistant tumors. To maximize the antitumor efficacy, it is desired that the chemodrug be latently released upon completion of siP-gp-mediated gene silencing, which however, largely remains an unmet demand. Herein, core-shell nanocomplexes (NCs) are developed to overcome MDR via staged liberation of siP-gp and chemodrug (doxorubicin, Dox) in hierarchical response to reactive oxygen species (ROS) concentration gradients. The NCs are constructed from mesoporous silica nanoparticles (MSNs) surface-decorated with cRGD-modified, PEGylated, ditellurium-crosslinked polyethylenimine (RPPT), wherein thioketal-linked dimeric doxorubicin (TK-Dox2) and photosensitizer are coencapsulated inside MSNs while siP-gp is embedded in the RPPT polymeric layer. RPPT with ultrahigh ROS-sensitivity can be efficiently degraded by the low-concentration ROS inside cancer cells to trigger siP-gp release. Upon siP-gp-mediated gene silencing and MDR reversal, light irradiation is performed to generate high-concentration, lethal amount of ROS, which cleaves thioketal with low ROS-sensitivity to liberate the monomeric Dox. Such a latent release profile greatly enhances Dox accumulation in Dox-resistant cancer cells (MCF-7/ADR) in vitro and in vivo, which cooperates with the generated ROS to efficiently eradicate MCF-7/ADR xenograft tumors.

Digital ray: enhancing cataractous fundus images using style transfer generative adversarial networks to improve retinopathy detection.

BACKGROUND/AIMS: The aim of this study was to develop and evaluate digital ray, based on preoperative and postoperative image pairs using style transfer generative adversarial networks (GANs), to enhance cataractous fundus images for improved retinopathy detection. METHODS: For eligible cataract patients, preoperative and postoperative colour fundus photographs (CFP) and ultra-wide field (UWF) images were captured. Then, both the original CycleGAN and a modified CycleGAN (C2ycleGAN) framework were adopted for image generation and quantitatively compared using Frechet Inception Distance (FID) and Kernel Inception Distance (KID). Additionally, CFP and UWF images from another cataract cohort were used to test model performances. Different panels of ophthalmologists evaluated the quality, authenticity and diagnostic efficacy of the generated images. RESULTS: A total of 959 CFP and 1009 UWF image pairs were included in model development. FID and KID indicated that images generated by C2ycleGAN presented significantly improved quality. Based on ophthalmologists' average ratings, the percentages of inadequate-quality images decreased from 32% to 18.8% for CFP, and from 18.7% to 14.7% for UWF. Only 24.8% and 13.8% of generated CFP and UWF images could be recognised as synthetic. The accuracy of retinopathy detection significantly increased from 78% to 91% for CFP and from 91% to 93% for UWF. For retinopathy subtype diagnosis, the accuracies also increased from 87%-94% to 91%-100% for CFP and from 87%-95% to 93%-97% for UWF. CONCLUSION: Digital ray could generate realistic postoperative CFP and UWF images with enhanced quality and accuracy for overall detection and subtype diagnosis of retinopathies, especially for CFP.\ TRIAL REGISTRATION NUMBER: This study was registered with ClinicalTrials.gov (NCT05491798).

Cancer cell-targeted cisplatin prodrug delivery in vivo via metabolic labeling and bioorthogonal click reaction.

The discrepancy of surface receptors on cancerous and non-cancerous cells has been regarded as the mainstay of cancer-targeted therapy. However, due to the heterogeneity of tumor cells and the insufficient levels of receptors on the tumor cell surface, the success of cancer cell-targeted therapies is largely limited. Histone deacetylase/cathepsin l-responsive acetylated azidomannose (DCL-AAM) was previously developed to effectively and selectively label cancer cell surfaces with reactive azido groups via sugar metabolism. Herein, the labeling kinetics and generality of DCL-AAM were systematically investigated in varieties of tumor cells in vitro and in SKOV3 xenograft tumors in vivo. Based on this, dibenzocyclooctyne-cisplatin (DBCO-Pt) prodrug was developed, and DCL-AAM-mediated metabolic labeling of SKOV3 cells enhanced the tumor accumulation of DBCO-Pt ∼2 fold via bioorthogonal click chemistry, potentiating the anti-tumor efficacy of cisplatin yet alleviating the systemic toxicity. This work, therefore, provides the experimental and theoretical support for the future design of sugar metabolism-based targeted delivery systems and may provide a promising candidate for the treatment of cancers lacking appropriate biomarkers.

Facile Click-Mediated Cell Imaging Strategy of Liposomal Azido Mannosamine Lipids via Metabolic or Nonmetabolic Glycoengineering.

Two Ac4ManNAz (AAM) derivatives with octadecanoic ester (C18 ester) and octadecyl ether (C18 ether) attached to the anomeric hydroxyl groups were synthesized and used in preparation of liposomes. Both liposomes show strong cell-labeling efficiencies on MDA-MB-231 cancer cells. The cell surface-anchored azide group can react with DBCO-Cy5 via Cu-free click chemistry. The two liposomes exhibit different azide placement mechanisms; C18-ether-AAM-treated cells have azido placement through direct insertion, while C18-ester-AAM-treated cells express azido more through metabolic glycoengineering.

Induction of a higher-ordered architecture in glatiramer acetate improves its biological efficiency in an animal model of multiple sclerosis.

Glatiramer acetate (GA), a linear random copolypeptide, is a first-line treatment for multiple sclerosis (MS). A major concern, however, is that GA treatment is associated with adverse effects and poor patient adherence due to the need for frequent injections. Here we describe improved performance of the polymeric drug, even at low doses with less-frequent injections, through the modification of its architecture into a star-shaped GA (sGA). In a sGA, multiple GAs are covalently linked onto a core, which greatly changes their properties such as molecular weight, size, and shape. The spherical sGA is retained longer in the body after intraperitoneal injection, and is more readily internalized by RAW 264.7 macrophage cells and bone marrow-derived dendritic cells than GA. In C57BL/6 mice induced with experimental autoimmune encephalitis, a mouse model for MS, sGA treatment exerts disease amelioration effect that is significantly better than that of GA despite a lower dose and less frequent injection. Moreover, spinal cord pathologies of demyelination and leukocyte infiltration are dramatically less pronounced in the sGA treatment condition compared to the GA treatment condition. Thus, we propose that sGA with a higher-ordered architecture offers an attractive and potentially viable treatment option for MS patients.

Targeting infected host cells in vivo via responsive azido-sugar mediated metabolic cell labeling followed by click reaction.

The early in vivo diagnosis of infectious disease foci is largely hindered by invasion and concealment of pathogens in host cells, making it difficult for conventional probes to detect and analyze intracellular pathogens. Taking advantage of the excessively produced reactive oxygen species (ROS) within host cells, herein we report the design of thiol-hemiketal blocked N-azidoacetyl galactosamine (Ac3GalNAzSP), an azido unnatural sugar bearing an unprecedent designed ROS-responsive moiety for targeted labelling of infected host cells. Ac3GalNAzSP showed great stability under physiological conditions, specifically released active unnatural sugar in host cells overproducing ROS, metabolically labeled infected host cells with azido groups, and enabled targeting in vivo infection sites by subsequent Click Chemistry reactions, substantiating an unprecedented approach for targeting infected host cells. This technique could be a powerful tool for early in vivo diagnosis and targeted treatment of infectious disease.

In vivo cancer targeting via glycopolyester nanoparticle mediated metabolic cell labeling followed by click reaction.

We developed glycopolyesters (GPs) via azido-sugar initiated ring-opening polymerization of O-carboxyanhydrides (OCAs) and achieved efficient in vivo cancer targeting via GP-nanoparticle (GP-NP) mediated metabolic cell labeling followed by Click reaction. GP-NP shows controlled release of azido-sugars and can efficiently label LS174T colon cancer cells with azido groups in tumor-bearing mice. The exogenously introduced azido groups render excellent in vivo cancer targeting and retention of dibenzocyclooctyne-Cy5 (DBCO-Cy5) with an increasing tumor retention enhancement over time (68% at 6 h, 105% at 24 h, and 191% at 48 h) compared to control mice without azido labeling. The tumor accumulation of DBCO-doxorubicin is also significantly enhanced in GP-NP pretreated mice, resulting in improved in vivo anticancer efficacy. This study, for the first time, proposes the use of azido-sugar initiated polymerization of OCAs to form sugar delivery vehicles with high stability and controlled release, and demonstrates the increasing tumor targeting effect of DBCO-cargo over time by azido-modified tumor cells.

Selective cancer treatment via photodynamic sensitization of hypoxia-responsive drug delivery.

The precise and selective delivery of chemodrugs into tumors represents a critical requirement for anti-cancer therapy. Intelligent delivery systems that are responsive to a single internal or external stimulus often lack sufficient cancer selectivity, which compromises the drug efficacy and induces undesired side effects. To overcome this dilemma, we herein report a cancer-targeting vehicle which allows highly cancer-selective drug release in response to cascaded external (light) and internal (hypoxia) dual triggers. In particular, doxorubicin (DOX)-loaded, hypoxia-dissociable nanoparticles (NPs) were prepared from self-assembled polyethylenimine-nitroimidazole (PEI-NI) micelles that were further co-assembled with hyaluronic acid-Ce6 (HC). Upon accumulation in tumor cells, tumor site-specific light irradiation (660 nm, 10 mW cm-2) generated high levels of reactive oxygen species (ROS) and greatly enhanced the hypoxic levels to induce NP dissociation and accordingly DOX release. A synergistic anti-cancer efficacy between DOX-mediated chemotherapy and Ce6-mediated photodynamic therapy (PDT) was thus achieved, resulting in reduced side effects to normal tissues/cells. This study therefore provides an effective method to control the cancer-specific drug delivery by responding to cascaded multiple triggers, and it renders promising applications for the programmed combination of chemotherapy and PDT toward cancer treatment.

A caged metabolic precursor for DT-diaphorase-responsive cell labeling.

In this study, we report incorporation of a covalent linker at the anomeric position of N-azidoacetylmannosamine (ManNAz) for caging its metabolic process. We synthesized a DT-diaphorase-responsive metabolic precursor, HQ-NN-AAM, using an optimized linker. The caged metabolite showed responsiveness to DT-diaphorase in vitro, resulting in metabolic incorporation of an azido sugar into the cell surface in multiple cell lines.

Dimeric Prodrug Self-Delivery Nanoparticles with Enhanced Drug Loading and Bioreduction Responsiveness for Targeted Cancer Therapy.

Efficient drug accumulation in tumor cells is essential for cancer therapy. Herein, we developed dimeric prodrug self-delivery nanoparticles (NPs) with enhanced drug loading and bioreduction responsiveness for triple negative breast cancer (TNBC) therapy. Specially designed camptothecin dimeric prodrug (CPTD) containing a disulfide bond was constructed to realize intracellular redox potential controlled drug release. Direct conjugation of hydrophobic CPTD to poly(ethylene glycol) PEG5000, a prodrug-based amphiphilic CPTD-PEG5000 co-polymer was synthesized, which could encapsulate parental CPTD prodrug spontaneously and form ultrastable NPs due to the highly analogous structure. Such dimeric prodrug self-delivery nanoparticles showed ultrahigh stability with critical micelle concentration as low as 0.75 µg/mL and remained intact during endocytosis. In addition, neurotensin (NT), a 13 amino acid ligand, was further modified on the nanoparticles for triple negative breast cancer (TNBC) targeting. Optimized NT-CPTD NPs showed improved pharmacokinetics profile and increased drug accumulation in TNBC lesions than free CPT, which largely reduced the systemic toxicity and presented an improved anticancer efficacy in vivo. In summary, with advantages of extremely high drug loading capacity, tumor microenvironmental redox responsiveness, and targeted TNBC accumulation, NT-CPTD NPs showed their potential for effective triple negative breast cancer therapy.