Chemical Strategies for the Detection and Elimination of Senescent Cells.

Cellular senescence can be defined as an irreversible stopping of cell proliferation that arises in response to various stress signals. Cellular senescence is involved in diverse physiological and pathological processes in different tissues, exerting effects on processes as differentiated as embryogenesis, tissue repair and remodeling, cancer, aging, and tissue fibrosis. In addition, the development of some pathologies, aging, cancer, and other age-related diseases has been related to senescent cell accumulation. Due to the complexity of the senescence phenotype, targeting senescent cells is not trivial, is challenging, and is especially relevant for in vivo detection in age-related diseases and tissue samples. Despite the elimination of senescent cells (senolysis) using specific drugs (senolytics) that have been shown to be effective in numerous preclinical disease models, the clinical translation is still limited due to the off-target effects of current senolytics and associated toxicities. Therefore, the development of new chemical strategies aimed at detecting and eliminating senescent cells for the prevention and selective treatment of senescence-associated diseases is of great interest. Such strategies not only will contribute to a deeper understanding of this rapidly evolving field but also will delineate and inspire new possibilities for future research.In this Account, we report our recent research in the development of new chemical approaches for the detection and elimination of senescent cells based on new probes, nanoparticles, and prodrugs. The designed systems take advantage of the over-representation in senescent cells of certain biomarkers such as ß-galactosidase and lipofuscin. One- and two-photon probes, for higher tissue penetration, have been developed. Moreover, we also present a renal clearable fluorogenic probe for the in vivo detection of the ß-galactosidase activity, allowing for correlation with the senescent burden in living animals. Moreover, as an alternative to molecular-based probes, we also developed nanoparticles for senescence detection. Besides, we describe advances in new therapeutic agents to selectively eradicate senescent cells using ß-galactosidase activity-sensitive gated nanoparticles loaded with cytotoxic or senolytic agents or new prodrugs aiming to increase the selectivity and reduction of off-target toxicities of current drugs. Moreover, new advances therapies have been applied in vitro and in vivo. Studies with the probes, nanoparticles, and prodrugs have been applied in several in vitro and in vivo models of cancer, fibrosis, aging, and drug-induced cardiotoxicity in which senescence plays an important role. We discuss the benefits of these chemical strategies toward the development of more specific and sophisticated probes, nanoparticles, and prodrugs targeting senescent cells.

A navitoclax-loaded nanodevice targeting matrix metalloproteinase-3 for the selective elimination of senescent cells.

Cellular senescence is implicated in the occurrence and progression of multiple age-related disorders. In this context, the selective elimination of senescent cells, senolysis, has emerged as an effective therapeutic strategy. However, the heterogeneous senescent phenotype hinders the discovery of a universal and robust senescence biomarker that limits the effective of senolytic with off-target toxic effects. Therefore, the development of more selective strategies represents a promising approach to increase the specificity of senolytic therapy. In this study, we have developed an innovative nanodevice for the selective elimination of senescent cells (SCs) based on the specific enzymatic activity of the senescent secretome. The results revealed that when senescence is induced in proliferating WI-38 by ionizing radiation (IR), the cells secrete high levels of matrix metalloproteinase-3 (MMP-3). Based on this result, mesoporous silica nanoparticles (MSNs) were loaded with the senolytic navitoclax (Nav) and coated with a specific peptide which is substrate of MMP-3 (NPs(Nav)@MMP-3). Studies in cells confirmed the preferential release of cargo in IR-induced senescent cells compared to proliferating cells, depending on MMP-3 levels. Moreover, treatment with NPs(Nav)@MMP-3 induced a selective decrease in the viability of SCs as well as a protective effect on non-proliferating cells. These results demonstrate the potential use of NPs to develop enhanced senolytic therapies based on specific enzymatic activity in the senescent microenvironment, with potential clinical relevance. STATEMENT OF SIGNIFICANCE: The common ß-galactosidase activity has been exploited to develop nanoparticles for the selective elimination of senescent cells. However, the identification of new senescent biomarkers is a key factor for the development of improved strategies. In this scenario, we report for the first time the development of NPs targeting senescent cells based on specific enzymatic activity of the senescent secretome. We report a navitoclax-loaded nanodevice responsive to the matrix metalloproteinase-3 (MMP-3) associated with the senescent phenotype. Our nanosystem achieves the selective release of navitoclax in an MMP-3-dependent manner while limiting off-target effects on non-senescent cells. This opens the possibility of using nanoparticles able to detect an altered senescent environment and selectively release its content, thus enhancing the efficacy of senolytic therapies.

Time-Tested Strategies in Modern Context: A Bibliometric Study of Chemotherapy's Continued Importance in Colorectal Cancer Treatment.

In the landscape of colorectal cancer treatment, classical chemotherapeutic agents such as 5-fluorouracil, capecitabine, irinotecan, oxaliplatin, trifluridine, and tipiracil have historically played a pivotal role. This study presents a comprehensive bibliometric analysis of the top 100 most influential articles focusing on these classic chemotherapy drugs in the management of colorectal cancer. With this, we shed light on their current importance, despite the emergence of new therapeutic targets and treatments in the field of oncology. Systematically evaluating research outputs, this analysis reveals a prevalence of co-authorship among institutions, countries (led by the United States, China, and Europe), and researchers highlighting the global and collaborative nature of efforts in research, utilization, and development of these drugs. Three thematic axes lead the research: pharmacogenetics, the development of new pharmaceutical forms, and the use of adjuvants. This research serves as a foundation for future endeavors, aiding researchers, clinicians, and policymakers in making informed decisions about the direction of research and development in the dynamic field of colorectal cancer therapy.

Targeted Delivery of the Pan-Inflammasome Inhibitor MM01 as an Alternative Approach to Acute Lung Injury Therapy.

Acute lung injury (ALI) is a severe pulmonary disorder responsible for high percentage of mortality and morbidity in intensive care unit patients. Current treatments are ineffective, so the development of efficient and specific therapies is an unmet medical need. The activation of NLPR3 inflammasome during ALI produces the release of proinflammatory factors and pyroptosis, a proinflammatory form of cell death that contributes to lung damage spreading. Herein, it is demonstrated that modulating inflammasome activation through inhibition of ASC oligomerization by the recently described MM01 compound can be an alternative pharmacotherapy against ALI. Besides, the added efficacy of using a drug delivery nanosystem designed to target the inflamed lungs is determined. The MM01 drug is incorporated into mesoporous silica nanoparticles capped with a peptide (TNFR-MM01-MSNs) to target tumor necrosis factor receptor-1 (TNFR-1) to proinflammatory macrophages. The prepared nanoparticles can deliver the cargo in a controlled manner after the preferential uptake by proinflammatory macrophages and exhibit anti-inflammatory activity. Finally, the therapeutic effect of MM01 free or nanoparticulated to inhibit inflammatory response and lung injury is successfully demonstrated in lipopolysaccharide-mouse model of ALI. The results suggest the potential of pan-inflammasome inhibitors as candidates for ALI therapy and the use of nanoparticles for targeted lung delivery.

Lipofuscin labeling through biorthogonal strain-promoted azide-alkyne cycloaddition for the detection of senescent cells.

A new method for senescent cell detection is described, which is based on lipofuscin labeling with a fluorescent reporter through a biorthogonal strain-promoted azide-alkyne cycloaddition. The sensing protocol involves a first step where the interaction of lipofuscin with a Sudan Black B derivative containing an azide moiety (SBB-N3 ) is carried out. In the final step, the azide moiety reacts with a fluorophore containing a cyclooctene ring (BODIPY). The efficacy of this two-step protocol is assessed in senescent melanoma SK-MEL-103 cells, senescent triple-negative breast cancer MDA-MB-231 cells and senescent WI-38 fibroblasts. In all cases, a clear fluorescence pattern was observed in senescent cells, compared to proliferative cells, only when the SBB-N3 -BODIPY probe was formed. Our results provide an alternative tool for the detection of senescent cells, based on an in situ bio-orthogonal reaction for lipofuscin labeling.

ß-Galactosidase-Activatable Nile Blue-Based NIR Senoprobe for the Real-Time Detection of Cellular Senescence.

Cellular senescence is a stable cell cycle arrest in response to stress or other damage stimuli to maintain tissue homeostasis. However, the accumulation of senescent cells can lead to the progression of various senescence-related disorders. In this paper, we describe the development of a ß-galactosidase-activatable near-infrared (NIR) senoprobe, NBGal, for the detection of senescent cells based on the use of the FDA-approved Nile blue (NB) fluorophore. NBGal was validated in chemotherapeutic-induced senescence cancer models in vitro using SK-Mel 103 and 4T1 cell lines. In vivo monitoring of cellular senescence was evaluated in orthotopic triple-negative breast cancer-bearing mice treated with palbociclib to induce senescence. In all cases, NBGal exhibited a selective tracking of senescent cells mainly ascribed to the overexpressed ß-galactosidase enzyme responsible for hydrolyzing the NBGal probe generating the highly emissive NB fluorophore. In this way, NBGal has proven to be a qualitative, rapid, and minimally invasive probe that allows the direct detection of senescent cells in vivo.

Combination of palbociclib with navitoclax based-therapies enhances in vivo antitumoral activity in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer with a poor prognosis and limited effective therapeutic options. Induction of senescence, arrest of cell proliferation, has been explored as an effective method to limit tumor progression in metastatic breast cancer. However, relapses occur in some patients, possibly as a result of the accumulation of senescent tumor cells in the body after treatment, which promote metastasis. In this study, we explored the combination of senescence induction and the subsequent removal of senescent cells (senolysis) as an alternative approach to improve outcomes in TNBC patients. We demonstrate that a combination treatment, using the senescence-inducer palbociclib and the senolytic agent navitoclax, delays tumor growth and reduces metastases in a mouse xenograft model of aggressive human TNBC (hTNBC). Furthermore, considering the off-target effects and toxicity derived from the use of navitoclax, we propose a strategy aimed at minimizing the associated side effects. We use a galacto-conjugated navitoclax (nav-Gal) as a senolytic prodrug that can preferentially be activated by ß-galactosidase overexpressed in senescent cells. Concomitant treatment with palbociclib and nav-Gal in vivo results in the eradication of senescent hTNBC cells with consequent reduction of tumor growth, while reducing the cytotoxicity of navitoclax. Taken together, our results support the efficacy of combination therapy of senescence-induction with senolysis for hTNBC, as well as the development of a targeted approach as an effective and safer therapeutic opportunity.

Enhancement of photoactivity and cellular uptake of (Bu4N)2[Mo6I8(CH3COO)6] complex by loading on porous MCM-41 support. Photodynamic studies as an anticancer agent.

The incorporation by ionic assembly of the hexanuclear molybdenum cluster (Bu4N)2[Mo6I8(CH3CO2)6] (1) in amino-decorated mesoporous silica nanoparticles MCM-41, has yielded the new molybdenum-based hybrid photosensitizer 1@MCM-41. The new photoactive material presents a high porosity, due to the intrinsic high specific surface area of MCM-41 nanoparticles (989 m2 g-1) which is responsible for the good dispersion of the hexamolybdenum clusters on the nanoparticles surface, as observed by STEM analysis. The hybrid photosensitizer can generate efficiently singlet oxygen, which was demonstrated by using the benchmark photooxygenation reaction of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) in water. The photodynamic therapy activity has been tested using LED light as an irradiation source (λirr ~ 400-700 nm; 15.6 mW/cm2). The results show a good activity of the hybrid photosensitizer against human cervical cancer (HeLa) cells, reducing up to 70 % their viability after 20 min of irradiation, whereas low cytotoxicity is detected in the darkness. The main finding of this research is that the incorporation of molybdenum complexes at porous MCM-41 supports enhances their photoactivity and improves cellular uptake, compared to free clusters.

Mesoporous Silica Materials as an Emerging Tool for Cancer Immunotherapy.

Cancer immunotherapy has emerged in the past decade as a promising strategy for treating many forms of cancer by stimulating the patient's immune system. Although immunotherapy has achieved some promising results in clinics, more efforts are required to improve the limitations of current treatments related to lack of effective and targeted cancer antigens delivery to immune cells, dose-limiting toxicity, and immune-mediated adverse effects, among others. In recent years, the use of nanomaterials has proven promising to enhance cancer immunotherapy efficacy and reduce side effects. Among nanomaterials, attention has been recently paid to mesoporous silica nanoparticles (MSNs) as a potential multiplatform for enhancing cancer immunotherapy by considering their unique properties, such as high porosity, and good biocompatibility, facile surface modification, and self-adjuvanticity. This review explores the role of MSN and other nano/micro-materials as an emerging tool to enhance cancer immunotherapy, and it comprehensively summarizes the different immunotherapeutic strategies addressed to date by using MSN.

Pharmacological senolysis reduces doxorubicin-induced cardiotoxicity and improves cardiac function in mice.

Many anticancer agents used in clinics induce premature senescence in healthy tissues generating accelerated aging processes and adverse side-effects in patients. Cardiotoxicity is a well-known limiting factor of anticancer treatment with doxorubicin (DOX), a very effective anthracycline widely used as antitumoral therapy in clinical practice, that leads to long-term morbidity and mortality. DOX exposure severely affects the population of cardiac cells in both mice and human hearts by inducing premature senescence, which may represent the molecular basis of DOX-induced cardiomyopathy. Here, we demonstrate that senescence induction in the heart contributes to impaired cardiac function in mice upon DOX treatment. Concomitant elimination of senescent cells with the senolytic Navitoclax in different formulations produces a significant decrease in senescence and cardiotoxicity markers together with the restoration of the cardiac function in mice followed by echocardiography. These results evidence the potential clinical use of senolytic therapies to alleviate cardiotoxicities induced in chemotherapy-treated patients.

Horseradish Peroxidase-Functionalized Gold Nanoconjugates for Breast Cancer Treatment Based on Enzyme Prodrug Therapy.

INTRODUCTION: Breast cancer has the highest mortality rate among cancers in women. Patients suffering from certain breast cancers, such as triple-negative breast cancer (TNBC), lack effective treatments. This represents a clinical concern due to the associated poor prognosis and high mortality. As an approach to succeed over conventional therapy limitations, we present herein the design and evaluation of a novel nanodevice based on enzyme-functionalized gold nanoparticles to efficiently perform enzyme prodrug therapy (EPT) in breast cancer cells. RESULTS: In particular, the enzyme horseradish peroxidase (HRP) - which oxidizes the prodrug indole-3-acetic acid (IAA) to release toxic oxidative species - is incorporated on gold nanoconjugates (HRP-AuNCs), obtaining an efficient nanoplatform for EPT. The nanodevice is biocompatible and effectively internalized by breast cancer cell lines. Remarkably, co-treatment with HRP-AuNCs and IAA (HRP-AuNCs/IAA) reduces the viability of breast cancer cells below 5%. Interestingly, 3D tumor models (multicellular tumor spheroid-like cultures) co-treated with HRP-AuNCs/IAA exhibit a 74% reduction of cell viability, whereas the free formulated components (HRP, IAA) have no effect. CONCLUSION: Altogether, our results demonstrate that the designed HRP-AuNCs nanoformulation shows a remarkable therapeutic performance. These findings might help to bypass the clinical limitations of current tumor enzyme therapies and advance towards the use of nanoformulations for EPT in breast cancer.

MUC1 Aptamer-Capped Mesoporous Silica Nanoparticles for Navitoclax Resistance Overcoming in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. In the last years, navitoclax has emerged as a possible treatment for TNBC. Nevertheless, rapid navitoclax resistance onset has been observed thorough Mcl-1 overexpression. As a strategy to overcome Mcl-1-mediated resistance, herein we present a controlled drug co-delivery system based on mesoporous silica nanoparticles (MSNs) targeted to TNBC cells. The nanocarrier is loaded with navitoclax and the Mcl-1 inhibitor S63845 and capped with a MUC1-targeting aptamer (apMUC1-MSNs(Nav/S63845)). The apMUC1-capped nanoparticles effectively target TNBC cell lines and successfully induce apoptosis, overcoming navitoclax resistance. Moreover, navitoclax encapsulation protects platelets against apoptosis. These results point apMUC1-gated MSNs as suitable BH3 mimetics nanocarriers in the targeted treatment of MUC1-expressing TNBC.

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 ß-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.

Stimulus-responsive nanomotors based on gated enzyme-powered Janus Au-mesoporous silica nanoparticles for enhanced cargo delivery.

Here we report functional stimulus-responsive nanomotors based on Janus Au-mesoporous silica nanoparticles capable of self-propelling via the biocatalytic conversion of chemical fuel, that read information from the environment (the presence of glutathione) and accordingly deliver a cargo.

Janus nanocarrier powered by bi-enzymatic cascade system for smart delivery.

We report herein the assembly of an integrated nanodevice with bi-enzymatic cascade control for on-command cargo release. This nanocarrier is based on Au-mesoporous silica Janus nanoparticles capped at the mesoporous face with benzimidazole/ß-cyclodextrin-glucose oxidase pH-sensitive gate-like ensembles and functionalized with invertase on the gold face. The rationale for this delivery mechanism is based on the invertase-mediated hydrolysis of sucrose yielding glucose, which is further transformed into gluconic acid by glucose oxidase causing the disruption of the pH-sensitive supramolecular gates at the Janus nanoparticles. This enzyme-powered device was successfully employed in the autonomous and on-demand delivery of doxorubicin in HeLa cancer cells.

Glucose-Responsive Enzyme-Controlled Mesoporous Nanomachine with a Layer-by-Layer Supramolecular Architecture.

Here we describe the construction of an integrated and pH-sensitive nanomachine with layer-by-layer supramolecular design and enzymatic control for on-command delivery. The nanodevice comprises a first layer of ß-cyclodextrin-coated gold nanoparticles as capping element of benzimidazole functionalized mesoporous silica nanoparticles, and a second control layer based on an adatamantane-modified glucose oxidase derivative. The nanomachine was selectively fuelled by glucose and successfully employed for the autonomous release of doxorubicin in HeLa cancer cells.

Biocompatible Phenylboronic-Acid-Capped ZnS Nanocrystals Designed As Caps in Mesoporous Silica Hybrid Materials for on-Demand pH-Triggered Release In Cancer Cells.

Biocompatible ZnS-based nanocrystals capped with 4-mercaptophenylboronic acid (ZnS@B) have been size-designed as excellent pH-responsive gatekeepers on mesoporous silica nanoparticles (MSNs), which encapsulate fluorophore safranin O (S2-Saf) or anticancer drug epirubicin hydrochloride (S2-Epi) for delivery applications in cancer cells. In this novel hybrid system, the gate mechanism consists of reversible pH-sensitive boronate ester moieties linking the nanocrystals directly to the alcohol groups from silica surface scaffold, avoiding tedious intermediate functionalization steps. The ∼3 nm size of the ZnS@B nanocrystals was tailored to allow efficient sealing of the pore voids and achieve a "zero premature cargo release" at neutral pH (7.4). The system selectively released the cargo in acidic conditions (pH 5.4 and 3.0) because of the hydrolysis of the boronate esters, which unblocked the pore voids. Delivery of the cargo by off-on cycles was demonstrated by changes in pH from 7.4 to 3.0, showing its potential pH-switching behavior. Cellular uptake of these nanocarriers within human cervix adenocarcinoma (HeLa) cells was achieved and the controlled release of the chemotherapeutic drug epirubicin was shown to occur within the endogenous endosomal/lysosomal acidified cancer cell microenvironment and further diffused into the cytosol. Cytotoxicity tests done on the mesoporous support without cargo and covalently linked with ZnS@B nanocrystals as caps were negative, suggesting that the proposed system is biocompatible and can be considered as a very promising drug nanocarrier.

Mesoporous Bioactive Glasses Equipped with Stimuli-Responsive Molecular Gates for Controlled Delivery of Levofloxacin against Bacteria.

An increase of bone diseases incidence has boosted the study of ceramic biomaterials as potential osteo-inductive scaffolds. In particular, mesoporous bioactive glasses have demonstrated to possess a broad application in the bone regeneration field, due their osteo-regenerative capability and their ability to release drugs from the mesoporous structure. These special features have been studied as an option to fight against bone infection, which is one of the most common problems regarding bone regeneration therapies. In this work, a mesoporous bioglass functionalized with polyamines and capped with adenosine triphosphate (ATP) as the molecular gate was developed for the controlled release of the antibiotic levofloxacin. Phosphate bonds of ATP were hydrolyzed in the presence of acid phosphatase (APase), the concentration of which is significantly increased in bone infection due to the activation of bone resorption processes. The solid was characterized and tested successfully against bacteria. The final gated solid induced bacterial death only in the presence of acid phosphatase. Additionally, it was demonstrated that the solid is not toxic against human cells. The double function of the prepared material as a drug delivery system and bone regeneration enhancer confirms the possible development of a new approach in the tissue engineering field, in which controlled release of therapeutic agents can be finely tuned and, at the same time, osteoinduction is favored.

Hybrid Mesoporous Nanocarriers Act by Processing Logic Tasks: Toward the Design of Nanobots Capable of Reading Information from the Environment.

Here, we present the design of smart nanodevices capable of reading molecular information from the environment and acting accordingly by processing Boolean logic tasks. As proof of concept, we prepared Au-mesoporous silica (MS) nanoparticles functionalized with the enzyme glucose dehydrogenase (GDH) on the Au surface and with supramolecular nanovalves as caps on the MS surface, which is loaded with a cargo (dye or drug). The nanodevice acts as an AND logic gate and reads information from the solution (presence of glucose and nicotinamide adenine dinucleotide (NAD+)), which results in cargo release. We show the possibility of coimmobilizing GDH and the enzyme urease on nanoparticles to mimic an INHIBIT logic gate, in which the AND gate is switched off by the presence of urea. We also show that such nanodevices can deliver cytotoxic drugs in cancer cells by recognizing intracellular NAD+ and the presence of glucose.

MUC1 aptamer-capped mesoporous silica nanoparticles for controlled drug delivery and radio-imaging applications.

Mucin 1 (MUC1) is a cell surface protein overexpressed in breast cancer. Mesoporous silica nanoparticles (MSNs) loaded with safranin O, functionalized with aminopropyl groups and gated with the negatively charged MUC1 aptamer have been prepared (S1-apMUC1) for specific targeting and cargo release in tumoral versus non-tumoral cells. Confocal microscopy studies showed that the S1-apMUC1 nanoparticles were internalized in MDA-MB-231 breast cancer cells that overexpress MUC1 receptor with subsequent pore opening and cargo release. Interestingly, the MCF-10-A non-tumorigenic breast epithelial cell line that do not overexpress MUC1, showed reduced (S1-apMUC1) internalization. Negligible internalization was also found for S1-ap nanoparticles that contained a scrambled DNA sequence as gatekeeper. S2-apMUC1 nanoparticles (similar to S1-apMUC1 but loaded with doxorubicin) internalized in MDA-MB-231 cells and induced a remarkable reduction in cell viability. Moreover, S1-apMUC1 nanoparticles radio-labeled with 99mTc (S1-apMUC1-Tc) showed a remarkable tumor targeting in in vivo studies with MDA-MB-231 tumor-bearing Balb/c mice.