Chemical Strategies for the Detection and Elimination of Senescent Cells.

ConspectusCellular 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.

Remarkable enhancement of cinnamaldehyde antimicrobial activity encapsulated in capped mesoporous nanoparticles: A new "nanokiller" approach in the era of antimicrobial resistance.

Combating antimicrobial resistance is one of the biggest health challenges because of the ineffectiveness of standard biocide treatments. This challenge could be approached using natural products, which have demonstrated powerful therapeutics against multidrug-resistant microbes. In the present work, a nanodevice consisting of mesoporous silica nanoparticles loaded with an essential oil component (cinnamaldehyde) and functionalized with the polypeptide ε-poly-l-lysine is developed and used as an antimicrobial agent. In the presence of the corresponding stimuli (i.e., exogenous proteolytic enzymes from bacteria or fungi), the polypeptide is hydrolyzed, and the cinnamaldehyde delivery is enhanced. The nanodevice's release mechanism and efficacy are evaluated in vitro against the pathogenic microorganisms Escherichia coli, Staphylococcus aureus, and Candida albicans. The results demonstrate that the new device increases the delivery of the cinnamaldehyde via a biocontrolled uncapping mechanism triggered by proteolytic enzymes. Moreover, the nanodevice notably improves the antimicrobial efficacy of cinnamaldehyde when compared to the free compound, ca. 52-fold for E. coli, ca. 60-fold for S. aureus, and ca. 7-fold for C. albicans. The enhancement of the antimicrobial activity of the essential oil component is attributed to the decrease of its volatility due to its encapsulation in the porous silica matrix and the increase of its local concentration when released due to the presence of microorganisms.

Synergistic antimicrobial photodynamic therapy using gated mesoporous silica nanoparticles containing curcumin and polymyxin B.

Photodynamic Therapy is a therapy based on combining a non-toxic compound, known as photosensitizer (PS), and irradiation with light of the appropriate wavelength to excite the PS molecule. The photon absorption by the PS leads to reactive oxygen species generation and a subsequent oxidative burst that causes cell damage and death. In this work, we report an antimicrobial nanodevice that uses the activity of curcumin (Cur) as a PS for antimicrobial Photodynamic Therapy (aPDT), based on mesoporous silica nanoparticles in which the action of the classical antibiotic PMB is synergistically combined with the aPDT properties of curcumin to combat bacteria. The synergistic effect of the designed gated device in combination with irradiation with blue LED light (470 nm) is evaluated against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus epidermidis. The results show that the nanodevice exhibits a noteworthy antibacterial activity against these microorganisms, a much more significant effect than free Cur and PMB at equivalent concentrations. Thus, 0.1 µg/mL of MSNs-Cur-PMB eliminates a bacterial concentration of about 105 CFU/mL of E. coli, while 1 µg/mL of MSNs-Cur-PMB is required for P. aeruginosa and S. epidermidis. In addition, antibiofilm activity against the selected bacteria was also tested. We found that 0.1 mg/mL of MSNs-Cur-PMB inhibited 99 % biofilm formation for E. coli, and 1 mg/mL of MSNs-Cur-PMB achieved 90 % and 100 % inhibition of biofilm formation for S. epidermidis and P. aeruginosa, respectively.

Cellular senescence promotes progenitor cell expansion during axolotl limb regeneration.

Axolotl limb regeneration is accompanied by the transient induction of cellular senescence within the blastema, the structure that nucleates regeneration. The precise role of this blastemal senescent cell (bSC) population, however, remains unknown. Here, through a combination of gain- and loss-of-function assays, we elucidate the functions and molecular features of cellular senescence in vivo. We demonstrate that cellular senescence plays a positive role during axolotl regeneration by creating a pro-proliferative niche that supports progenitor cell expansion and blastema outgrowth. Senescent cells impact their microenvironment via Wnt pathway modulation. Further, we identify a link between Wnt signaling and senescence induction and propose that bSC-derived Wnt signals facilitate the proliferation of neighboring cells in part by preventing their induction into senescence. This work defines the roles of cellular senescence in the regeneration of complex structures.

Non-invasive biomarkers for mild cognitive impairment and Alzheimer's disease.

Alzheimer's disease is the most common type of dementia in the elderly. It is a progressive degenerative disorder that may begin to develop up to 15 years before clinical symptoms appear. The identification of early biomarkers is crucial to enable a prompt diagnosis and to start effective interventions. In this work, we conducted a metabolomic study using proton Nuclear Magnetic Resonance (1H NMR) spectroscopy in serum samples from patients with neuropathologically confirmed Alzheimer's disease (AD, n = 51), mild cognitive impairment (MCI, n = 27), and cognitively healthy controls (HC, n = 50) to search for metabolites that could be used as biomarkers. Patients and controls underwent yearly clinical follow-ups for up to six years. MCI group included samples from three subgroups of subjects with different disease progression rates. The first subgroup included subjects that remained clinically stable at the MCI stage during the period of study (stable MCI, S-MCI, n = 9). The second subgroup accounted for subjects which were diagnosed with MCI at the moment of blood extraction, but progressed to clinical dementia in subsequent years (MCI-to-dementia, MCI-D, n = 14). The last subgroup was composed of subjects that had been diagnosed as dementia for the first time at the moment of sample collection (incipient dementia, Incp-D, n = 4). Partial Least Square Discriminant Analysis (PLS-DA) models were developed. Three models were obtained, one to discriminate between AD and HC samples with high sensitivity (93.75%) and specificity (94.75%), another model to discriminate between AD and MCI samples (100% sensitivity and 82.35% specificity), and a last model to discriminate HC and MCI with lower sensitivity and specificity (67% and 50%). Differences within the MCI group were further studied in an attempt to determine those MCI subjects that could develop AD-type dementia in the future. The relative concentration of metabolites, and metabolic pathways were studied. Alterations in the pathways of alanine, aspartate and glutamate metabolism, pantothenate and CoA biosynthesis, and beta-alanine metabolism, were found when HC and MCI- D patients were compared. In contrast, no pathway was found disturbed in the comparison of S-MCI with HC groups. These results highlight the potential of 1H NMR metabolomics to support the diagnosis of dementia in a less invasive way, and set a starting point for the study of potential biomarkers to identify MCI or HC subjects at risk of developing AD in the future.

Gated Organonanoclays for Large Biomolecules: Controlled Release Triggered by Surfactant Stimulus.

The low toxicity and high adsorption capacities of clay minerals make them attractive for controlled delivery applications. However, the number of controlled-release studies in the literature using clay minerals is still scarce. In this work, three different clays from the smectite group (Kunipia F, montmorillonite; Sumecton SA, saponite; and Sumecton SWN, hectorite) were successfully loaded with rhodamine B dye and functionalized with oleic acid as a gatekeeper to produce organonanoclays for active and controlled payload-release. Moreover, hematin and cyanocobalamin have also been encapsulated in hectorite gated clay. These organonanoclays were able to confine the entrapped cargos in an aqueous environment, and effectively release them in the presence of surfactants (as bile salts). A controlled delivery of 49 ± 6 µg hematin/mg solid and 32.7 ± 1.5 µg cyanocobalamin/mg solid was reached. The cargo release profiles of all of the organonanoclays were adjusted to three different release-kinetic models, demonstrating the Korsmeyer-Peppas model with release dependence on (i) the organic-inorganic hybrid system, and (ii) the nature of loaded molecules and their interaction with the support. Furthermore, in vitro cell viability assays were carried out with Caco-2 cells, demonstrating that the organonanoclays are well tolerated by cells at particle concentrations of ca. 50 µg/mL.

Lactose-Gated Mesoporous Silica Particles for Intestinal Controlled Delivery of Essential Oil Components: An In Vitro and In Vivo Study.

Mesoporous silica microparticles functionalized with lactose for the specific release of essential oil components (EOCs) in the small intestine are presented. In vitro and in vivo intestinal models were applied to validate the microparticles (M41-EOC-L), in which the presence of lactase acts as the triggering stimulus for the controlled release of EOCs. Among the different microdevices prepared (containing thymol, eugenol and cinnamaldehyde), the one loaded with cinnamaldehyde showed the most significant Caco-2 cell viability reduction. On the other hand, interaction of the particles with enterocyte-like monolayers showed a reduction of EOCs permeability when protected into the designed microdevices. Then, a microdevice loaded with cinnamaldehyde was applied in the in vivo model of Wistar rat. The results showed a reduction in cinnamaldehyde plasma levels and an increase in its concentration in the lumen of the gastrointestinal tract (GIT). The absence of payload release in the stomach, the progressive release throughout the intestine and the prolonged stay of the payload in the GIT-lumen increased the bioavailability of the encapsulated compound at the site of the desired action. These innovative results, based on the specific intestinal controlled delivery, suggest that the M41-payload-L could be a potential hybrid microdevice for the protection and administration of bioactive molecules in the small intestine and colon.

Secreted Enzyme-Responsive System for Controlled Antifungal Agent Release.

Essential oil components (EOCs) such as eugenol play a significant role in plant antimicrobial defense. Due to the volatility and general reactivity of these molecules, plants have evolved smart systems for their storage and release, which are key prerequisites for their efficient use. In this study, biomimetic systems for the controlled release of eugenol, inspired by natural plant defense mechanisms, were prepared and their antifungal activity is described. Delivery and antifungal studies of mesoporous silica nanoparticles (MSN) loaded with eugenol and capped with different saccharide gates-starch, maltodextrin, maltose and glucose-against fungus Aspergillus niger-were performed. The maltodextrin- and maltose-capped systems show very low eugenol release in the absence of the fungus Aspergillus niger but high cargo delivery in its presence. The anchored saccharides are degraded by exogenous enzymes, resulting in eugenol release and efficient inhibition of fungal growth.

Towards the Enhancement of Essential Oil Components' Antimicrobial Activity Using New Zein Protein-Gated Mesoporous Silica Microdevices.

The development of new food preservatives is essential to prevent foodborne outbreaks or food spoilage due to microbial growth, enzymatic activity or oxidation. Furthermore, new compounds that substitute the commonly used synthetic food preservatives are needed to stifle the rising problem of microbial resistance. In this scenario, we report herein, as far as we know, for the first time the use of the zein protein as a gating moiety and its application for the controlled release of essential oil components (EOCs). The design of microdevices consist of mesoporous silica particles loaded with essential oils components (thymol, carvacrol and cinnamaldehyde) and functionalized with the zein (prolamin) protein found in corn as a molecular gate. The zein protein grafted on the synthesized microdevices is degraded by the proteolytic action of bacterial enzymatic secretions with the consequent release of the loaded essential oil components efficiently inhibiting bacterial growth. The results allow us to conclude that the new microdevice presented here loaded with the essential oil component cinnamaldehyde improved the antimicrobial properties of the free compound by decreasing volatility and increasing local concentration.

Surfactant-Triggered Molecular Gate Tested on Different Mesoporous Silica Supports for Gastrointestinal Controlled Delivery.

In recent decades, the versatility of mesoporous silica particles and their relevance to develop controlled release systems have been demonstrated. Within them, gated materials able to modulate payload delivery represent great advantages. However, the role played by the porous matrix in this kind of systems is scarce. In this work, different mesoporous silica materials (MCM-41, MCM-48, SBA-15 and UVM-7) are functionalized with oleic acid as a molecular gate. All systems are fully characterized and their ability to confine the entrapped cargo and release it in the presence of bile salts is validated with release assays and in vitro digestion experiments. The cargo release profile of each synthesized support is studied, paying attention to the inorganic scaffold. Obtained release profiles fit to Korsmeyer-Peppas model, which explains the differences among the studied supports. Based on the results, UVM-7 material was the most appropriate system for duodenal delivery and was tested in an in vivo model of the Wistar rat. Payload confinement and its complete release after gastric emptying is achieved, establishing the possible use of mesoporous silica particles as protection and direct release agents into the duodenum and, hence, demonstrating that these systems could serve as an alternative to the administration methods employed until now.

Preclinical antitumor efficacy of senescence-inducing chemotherapy combined with a nanoSenolytic.

The induction of senescence produces a stable cell cycle arrest in cancer cells, thereby inhibiting tumor growth; however, the incomplete immune cell-mediated clearance of senescent cells may favor tumor relapse, limiting the long-term anti-tumorigenic effect of such drugs. A combination of senescence induction and the elimination of senescent cells may, therefore, represent an efficient means to inhibit tumor relapse. In this study, we explored the antitumor efficacy of a combinatory senogenic and targeted senolytic therapy in an immunocompetent orthotopic mouse model of the aggressive triple negative breast cancer subtype. Following palbociclib-induced senogenesis and senolysis by treatment with nano-encapsulated senolytic agent navitoclax, we observed inhibited tumor growth, reduced metastases, and a reduction in the systemic toxicity of navitoclax. We believe that this combination treatment approach may have relevance to other senescence-inducing chemotherapeutic drugs and additional tumor types. SIGNIFICANCE: While the application of senescence inducers represents a successful treatment strategy in breast cancer patients, some patients still relapse, perhaps due to the subsequent accumulation of senescent cells in the body that can promote tumor recurrence. We now demonstrate that a combination treatment of a senescence inducer and a senolytic nanoparticle selectively eliminates senescent cells, delays tumor growth, and reduces metastases in a mouse model of aggressive breast cancer. Collectively, our results support targeted senolysis as a new therapeutic opportunity to improve outcomes in breast cancer patients.

Galacto-conjugation of Navitoclax as an efficient strategy to increase senolytic specificity and reduce platelet toxicity.

Pharmacologically active compounds with preferential cytotoxic activity for senescent cells, known as senolytics, can ameliorate or even revert pathological manifestations of senescence in numerous preclinical mouse disease models, including cancer models. However, translation of senolytic therapies to human disease is hampered by their suboptimal specificity for senescent cells and important toxicities that narrow their therapeutic windows. We have previously shown that the high levels of senescence-associated lysosomal ß-galactosidase (SA-ß-gal) found within senescent cells can be exploited to specifically release tracers and cytotoxic cargoes from galactose-encapsulated nanoparticles within these cells. Here, we show that galacto-conjugation of the BCL-2 family inhibitor Navitoclax results in a potent senolytic prodrug (Nav-Gal), that can be preferentially activated by SA-ß-gal activity in a wide range of cell types. Nav-Gal selectively induces senescent cell apoptosis and has a higher senolytic index than Navitoclax (through reduced activation in nonsenescent cells). Nav-Gal enhances the cytotoxicity of standard senescence-inducing chemotherapy (cisplatin) in human A549 lung cancer cells. Concomitant treatment with cisplatin and Nav-Gal in vivo results in the eradication of senescent lung cancer cells and significantly reduces tumour growth. Importantly, galacto-conjugation reduces Navitoclax-induced platelet apoptosis in human and murine blood samples treated ex vivo, and thrombocytopenia at therapeutically effective concentrations in murine lung cancer models. Taken together, we provide a potentially versatile strategy for generating effective senolytic prodrugs with reduced toxicities.

New Oleic Acid-Capped Mesoporous Silica Particles as Surfactant-Responsive Delivery Systems.

A new delivery microdevice, based on hydrophobic oleic acid-capped mesoporous silica particles and able to payload release in the presence of surfactants, has been developed. The oleic acid functionalization confers to the system a high hydrophobic character, which avoids cargo release unless surfactant molecules are present. The performance of this oleic-acid capped microdevice in the presence of different surfactants is presented and its zero-release operation in the absence of surfactants is demonstrated.

Mesoporous Silica-Based Materials with Bactericidal Properties.

Bacterial infections are the main cause of chronic infections and even mortality. In fact, due to extensive use of antibiotics and, then, emergence of antibiotic resistance, treatment of such infections by conventional antibiotics has become a major concern worldwide. One of the promising strategies to treat infection diseases is the use of nanomaterials. Among them, mesoporous silica materials (MSMs) have attracted burgeoning attention due to high surface area, tunable pore/particle size, and easy surface functionalization. This review discusses how one can exploit capacities of MSMs to design and fabricate multifunctional/controllable drug delivery systems (DDSs) to combat bacterial infections. At first, the emergency of bacterial and biofilm resistance toward conventional antimicrobials is described and then how nanoparticles exert their toxic effects upon pathogenic cells is discussed. Next, the main aspects of MSMs (e.g., physicochemical properties, multifunctionality, and biosafety) which one should consider in the design of MSM-based DDSs against bacterial infections are introduced. Finally, a comprehensive analysis of all the papers published dealing with the use of MSMs for delivery of antibacterial chemicals (antimicrobial agents functionalized/adsorbed on mesoporous silica (MS), MS-loaded with antimicrobial agents, gated MS-loaded with antimicrobial agents, MS with metal-based nanoparticles, and MS-loaded with metal ions) is provided.

Electrospun Antimicrobial Films of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Containing Eugenol Essential Oil Encapsulated in Mesoporous Silica Nanoparticles.

The main goal of this study was to develop poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films with long-term antimicrobial capacity of interest in food packaging applications. To this end, eugenol was first highly efficiently encapsulated at 50 wt.-% in the pores of mesoporous silica nanoparticles by vapor adsorption. The eugenol-containing nanoparticles were then loaded in the 2.5⁻20 wt.-% range into PHBV by electrospinning and the resultant electrospun composite fibers were annealed at 155 °C to produce continuous films. The characterization showed that the PHBV films filled with mesoporous silica nanoparticles containing eugenol present sufficient thermal resistance and enhanced mechanical strength and barrier performance to water vapor and limonene. The antimicrobial activity of the films was also evaluated against foodborne bacteria for 15 days in open vs. closed conditions in order to simulate real packaging conditions. The electrospun PHBV films with loadings above 10 wt.-% of mesoporous silica nanoparticles containing eugenol successfully inhibited the bacterial growth, whereas the active films stored in hermetically closed systems increased their antimicrobial activity after 15 days due to the volatile portion accumulated in the system's headspace and the sustained release capacity of the films. The resultant biopolymer films are, therefore, potential candidates to be applied in active food packaging applications to provide shelf life extension and food safety.

The relationship between structure and in vitro antistaphylococcal effect of plant-derived stilbenes.

Staphylococcus aureus is a major human pathogen that is responsible for both hospital- and community-acquired infections. Stilbenes are polyphenol compounds of plant origin known to possess a variety of pharmacological properties, such as antibacterial, antiviral, and antifungal effects. This study reports the in vitro growth-inhibitory potential of eight naturally occurring stilbenes against six standard strains and two clinical isolates of S. aureus, using a broth microdilution method, and expressing the results as minimum inhibitory concentrations (MICs). Pterostilbene (MICs = 32-128 µg/ml), piceatannol (MICs = 64-256 µg/ml), and pinostilbene (MICs = 128 µg/ml) are among the active compounds that possess the strongest activity against all microorganisms tested, followed by 3'-hydroxypterostilbene, isorhapontigenin, oxyresveratrol, and rhapontigenin with MICs 128-256 µg/ml. Resveratrol (MIC = 256 µg/ml) exhibited only weak inhibitory effect. Furthermore, structure-activity relationships were studied. Hydroxyl groups at ortho-position (B-3' and -4') played crucial roles for the inhibitory effect of hydroxystilbene piceatannol. Compounds with methoxy groups at ring A (3'-hydroxypterostilbene, pinostilbene, and pterostilbene) produced stronger effect against S. aureus than their analogues (isorhapontigenin and rhapontigenin) with methoxy groups at ring B. These findings provide arguments for further investigation of stilbenes as prospective leading structures for development of novel antistaphylococcal agents for topical treatment of skin infections.

A versatile drug delivery system targeting senescent cells.

Senescent cells accumulate in multiple aging-associated diseases, and eliminating these cells has recently emerged as a promising therapeutic approach. Here, we take advantage of the high lysosomal ß-galactosidase activity of senescent cells to design a drug delivery system based on the encapsulation of drugs with galacto-oligosaccharides. We show that gal-encapsulated fluorophores are preferentially released within senescent cells in mice. In a model of chemotherapy-induced senescence, gal-encapsulated cytotoxic drugs target senescent tumor cells and improve tumor xenograft regression in combination with palbociclib. Moreover, in a model of pulmonary fibrosis in mice, gal-encapsulated cytotoxics target senescent cells, reducing collagen deposition and restoring pulmonary function. Finally, gal-encapsulation reduces the toxic side effects of the cytotoxic drugs. Drug delivery into senescent cells opens new diagnostic and therapeutic applications for senescence-associated disorders.

Two New Fluorogenic Aptasensors Based on Capped Mesoporous Silica Nanoparticles to Detect Ochratoxin†A.

Aptamers have been used as recognition elements for several molecules due to their great affinity and selectivity. Additionally, mesoporous nanomaterials have demonstrated great potential in sensing applications. Based on these concepts, we report herein the use of two aptamer-capped mesoporous silica materials for the selective detection of ochratoxin A (OTA). A specific aptamer for OTA was used to block the pores of rhodamine B-loaded mesoporous silica nanoparticles. Two solids were prepared in which the aptamer capped the porous scaffolds by using a covalent or electrostatic approach. Whereas the prepared materials remained capped in water, dye delivery was selectively observed in the presence of OTA. The protocol showed excellent analytical performance in terms of sensitivity (limit of detection: 0.5-0.05 nm), reproducibility, and selectivity. Moreover, the aptasensors were tested for OTA detection in commercial foodstuff matrices, which demonstrated their potential applicability in real samples.

An OFF-ON Two-Photon Fluorescent Probe for Tracking Cell Senescence in Vivo.

A naphthalimide-based two-photon probe (AHGa) for the detection of cell senescence is designed. The probe contains a naphthalimide core, an l-histidine methyl ester linker, and an acetylated galactose bonded to one of the aromatic nitrogen atoms of the l-histidine through a hydrolyzable N-glycosidic bond. Probe AHGa is transformed into AH in senescent cells resulting in an enhanced fluorescent emission intensity. In vivo detection of senescence is validated in mice bearing tumor xenografts treated with senescence-inducing chemotherapy.

Enhanced antimicrobial activity of essential oil components immobilized on silica particles.

The antimicrobial activity of essential oils components (EOCs) is well-known. However, their high volatility and powerful aroma limit their application in the formulation of a wide range of food products. In this context, the antimicrobial activity of carvacrol, eugenol, thymol and vanillin grafted onto the surface of three silica supports with different morphologies, textural properties and chemical reactivities (fumed silica, amorphous silica and MCM-41) was evaluated herein. Materials characterization revealed a good immobilization yield and all the devices showed a micro-scale particle size. Sensory evaluation revealed that sensory perception of EOCs decreases after covalent immobilization. Moreover, immobilization greatly enhanced the antimicrobial activity of the essential oil components against Listeria innocua and Escherichia coli compared to free components. The incorporation of EOCs immobilized on silica particles into pasteurized milk inoculated with L. innocua demonstrated their effectiveness not only for in vitro conditions, but also in a real food system.