Enzymatic Acrolein Production System and Its Impact on Human Cells.

Acrolein is an environmental toxicant and is also generated by microbial metabolism in the intestinal tract. Aqueous acrolein rapidly dissipates from standard human cell culture media with nondetectable levels after 8 h, hindering cell-based studies to understand its biological impacts. Thus, we developed an extracellular acrolein biosynthesis system to continuously produce acrolein compatible with human cell culture conditions. The approach uses spermine as a precursor, amine oxidase found in fetal calf serum, and catalase to remove the hydrogen peroxide byproduct. We confirmed amine oxidase activity of calf serum using a colorimetric assay and further tested the requirement for catalase in the system to mitigate hydrogen peroxide-induced cytotoxicity. We calibrated responses of human colon cells to this enzymatic acrolein production system by comparing transcriptional responses, DNA adduct formation and cytotoxicity responses to either this system or pure acrolein exposures in a human colon cell line. Several genes related to oxidative stress including HMOX1, and the colorectal cancer-related gene SEMA4A were upregulated similarly between the enzymatic acrolein production system or pure acrolein. The acrolein-DNA adduct γ-OH-Acr-dG increased in a dose-dependent manner with spermine in the enzymatic acrolein production system, producing a maximum of 1065 adducts per 108 nucleosides when 400 µM spermine was used. This biosynthetic production method provides a relevant model for controlled acrolein exposure in cultured human cells and overcomes current limitations due to its physical properties and limited availability.

Microrheological comparison of melanoma cells by atomic force microscopy.

Melanoma is one of the most severe cancers due to its great potential to form metastasis. Recent studies showed the importance of mechanical property assessment in metastasis formation which depends on the cytoskeleton dynamics and cell migration. Although cells are considered purely elastic, they are viscoelastic entities. Microrheology atomic force microscopy (AFM) enables the assessment of elasticity and viscous properties, which are relevant to cell behavior regulation. The current work compares the mechanical properties of human neonatal primary melanocytes (HNPMs) with two melanoma cell lines (WM793B and 1205LU cells), using microrheology AFM. Immunocytochemistry of F-actin filaments and phosphorylated focal adhesion kinase (p-FAK) and cell migration assays were performed to understand the differences found in microrheology AFM regarding the tumor cell lines tested. AFM revealed that HNPMs and tumor cell lines had distinct mechanical properties. HNPMs were softer, less viscous, presenting a higher power-law than melanoma cells. Immunostaining showed that metastatic 1205LU cells expressed more p-FAK than WM793B cells. Melanoma cell migration assays showed that WM73B did not close the gap, in contrast to 1205LU cells, which closed the gap at the end of 23 h. These data seem to corroborate the high migratory behavior of 1205LU cells. Microrheology AFM applied to HNPMs and melanoma cells allowed the quantification of elasticity, viscous properties, glassy phase, and power-law properties, which have an impact in cell migration and metastasis formation. AFM study is important since it can be used as a biomarker of the different stages of the disease in melanoma.

[Factors associated with caregiver burden in primary caregivers of older adults with type2 diabetes]. / Factores asociados a la sobrecarga del cuidador en cuidadores primarios de personas adultas mayores con diabetes tipo2.

OBJECTIVE: To characterize the profile of the informal primary caregiver (IPC) of adult patients with type2 diabetes (T2D) and the possible factors associated with caregiver collapse (CC). DESIGN: Observational, descriptive, cross-sectional and analytical study. SITE: Ambulatory Care Medical Unit. PARTICIPANTS: Mexican CPIs of adult patients with T2D. MAIN MEASUREMENTS: Data were collected through a prolective design using the Zarit scale and a structured survey on sociodemographic factors. A descriptive statistical analysis and univariate and multivariate logistic regression models were performed. RESULTS: The CPI profile is assumed by: women, people aged 36-58, daughters, people with a secondary and high school educational level, married, Catholic, with income <8,900 Mexican pesos, own home, inhabited by a maximum of 5 inhabitants, with support networks, who have dedicated >5years to the care of their patient, without training and with chronic diseases. The risk factors that increase the risk of CC are: being a woman (OR=11.03; 95%CI: 1.49-81.95), having a history of more than 5years of having assumed the role of caregiver (OR=2, 65; 95%CI: 1.07-6.55), living in one's own house (OR=3.03; 95%CI: 1.04-8.82), with 6 or more inhabitants (OR=2.41; 95%CI: 1.08-5.38). The support of other family members and/or friends was associated as a protective factor (OR=0.15; 95%CI: 0.07-0.33). CONCLUSIONS: Prevention programs are required to avoid CC and complications, as well as interventions to improve the quality of life of the CPI and patients in care, incorporating strategies to generate and/or increase their family and social support networks.

scFv biofunctionalized nanoparticles to effective and safe targeting of CEA-expressing colorectal cancer cells.

Colorectal cancer (CRC) is one of the deadliest cancers worldwide, with the 5 year survival rate in metastatic cases limited to 12%. The design of targeted and effective therapeutics remains a major unmet clinical need in CRC treatment. Carcinoembryonic antigen (CEA), a glycoprotein overexpressed in most colorectal tumors, may constitute a promising molecule for generating novel CEA-targeted therapeutic strategies for CRC treatment. Here, we developed a smart nanoplatform based on chemical conjugation of an anti-CEA single-chain variable fragment (scFv), MFE-23, with PLGA-PEG polymers to deliver the standard 5-Fluorouracil (5-FU) chemotherapy to CRC cells. We confirmed the specificity of the developed CEA-targeted NPs on the internalization by CEA-expressing CRC cells, with an enhance of threefold in the cell uptake. Additionally, CEA-targeted NPs loaded with 5-FU induced higher cytotoxicity in CEA-expressing cells, after 24 h and 48 h of treatment, reinforcing the specificity of the targeted NPs. Lastly, the safety of CEA-targeted NPs loaded with 5-FU was evaluated in donor-isolated macrophages, with no relevant impact on their metabolic activity nor polarization. Altogether, this proof of concept supports the CEA-mediated internalization of targeted NPs as a promising chemotherapeutic strategy for further investigation in different CEA-associated cancers and respective metastatic sites.Authors: Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 1 Given name: [Maria José] Last name [Silveira]. Author 7 Given name: [Maria José] Last name [Oliveira]. Also, kindly confirm the details in the metadata are correctokAffiliations: Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary.ok.

Biological Evaluation and Mechanistic Studies of Quinolin-(1H)-Imines as a New Chemotype against Leishmaniasis.

Leishmaniasis is one of the most challenging neglected tropical diseases and remains a global threat to public health. Currently available therapies for leishmaniases present significant drawbacks and are rendered increasingly inefficient due to parasite resistance, making the need for more effective, safer, and less expensive drugs an urgent one. In our efforts to identify novel chemical scaffolds for the development of antileishmanial agents, we have screened in-house antiplasmodial libraries against axenic and intracellular forms of Leishmania infantum, Leishmania amazonensis, and Leishmania major. Several of the screened compounds showed half-maximal inhibitory concentrations (IC50s) against intracellular L. infantum parasites in the submicromolar range (compounds 1h, IC50 = 0.9 µM, and 1n, IC50 = 0.7 µM) and selectivity indexes of 11 and 9.7, respectively. Compounds also displayed activity against L. amazonensis and L. major parasites, albeit in the low micromolar range. Mechanistic studies revealed that compound 1n efficiently inhibits oxygen consumption and significantly decreases the mitochondrial membrane potential in L. infantum axenic amastigotes, suggesting that this chemotype acts, at least in part, by interfering with mitochondrial function. Structure-activity analysis suggests that compound 1n is a promising antileishmanial lead and emphasizes the potential of the quinoline-(1H)-imine chemotype for the future development of new antileishmanial agents.

Cationic Carbosilane Dendrimers Prevent Abnormal α-Synuclein Accumulation in Parkinson's Disease Patient-Specific Dopamine Neurons.

Accumulation of misfolded α-synuclein (α-syn) is a hallmark of Parkinson's disease (PD) thought to play important roles in the pathophysiology of the disease. Dendritic systems, able to modulate the folding of proteins, have emerged as promising new therapeutic strategies for PD treatment. Dendrimers have been shown to be effective at inhibiting α-syn aggregation in cell-free systems and in cell lines. Here, we set out to investigate the effects of dendrimers on endogenous α-syn accumulation in disease-relevant cell types from PD patients. For this purpose, we chose cationic carbosilane dendrimers of bow-tie topology based on their performance at inhibiting α-syn aggregation in vitro. Dopamine neurons were differentiated from induced pluripotent stem cell (iPSC) lines generated from PD patients carrying the LRRK2G2019S mutation, which reportedly display abnormal accumulation of α-syn, and from healthy individuals as controls. Treatment of PD dopamine neurons with non-cytotoxic concentrations of dendrimers was effective at preventing abnormal accumulation and aggregation of α-syn. Our results in a genuinely human experimental model of PD highlight the therapeutic potential of dendritic systems and open the way to developing safe and efficient therapies for delaying or even halting PD progression.

Determination of organic pollutants in meconium and its relationship with fetal growth. Case control study in Northwestern Spain.

OBJECTIVES: Antenatal exposure to organic pollutants is a leading public health problem. Meconium is a unique matrix to perform prenatal studies because it enables us to retrospectively evaluate fetal exposure accumulated during the second and third trimester. The aim of the present study was to evaluate associations between organic pollutant levels in meconium and birth weight in NW Spain. METHODS: In this study, we quantify the concentrations of 50 organic pollutants together with the total values of the most important chemical groups in meconium using gas chromatography coupled to tandem mass spectrometry. RESULTS: Organochlorine pesticides, polychlorinated biphenyls and polybrominated diphenyl ethers were detected with the highest levels in meconium from small for gestational age newborns. It was estimated that several congeners were statistically significant (p<0.05). However, organophosphorus pesticides attained higher concentrations in newborns with an appropriate weight. CONCLUSIONS: The occurrence of transplacental transfer can be confirmed. Prenatal exposure to organic pollutants was associated with a decrease in birth weight and, therefore, organic pollutants could have an impact on fetal growth. Nevertheless, these results need validation in larger sample sized studies.

Interaction of Cationic Carbosilane Dendrimers and Their siRNA Complexes with MCF-7 Cells.

The application of siRNA in gene therapy is mainly limited because of the problems with its transport into cells. Utilization of cationic dendrimers as siRNA carriers seems to be a promising solution in overcoming these issues, due to their positive charge and ability to penetrate cell membranes. The following two types of carbosilane dendrimers were examined: CBD-1 and CBD-2. Dendrimers were complexed with pro-apoptotic siRNA (Mcl-1 and Bcl-2) and the complexes were characterized by measuring their zeta potential, circular dichroism and fluorescence of ethidium bromide associated with dendrimers. CBD-2/siRNA complexes were also examined by agarose gel electrophoresis. Both dendrimers form complexes with siRNA. Moreover, the cellular uptake and influence on the cell viability of the dendrimers and dendriplexes were evaluated using microscopic methods and XTT assay on MCF-7 cells. Microscopy showed that both dendrimers can transport siRNA into cells; however, a cytotoxicity assay showed differences in the toxicity of these dendrimers.

Cationic Carbosilane Dendritic Systems as Promising Anti-Amyloid Agents in Type†2 Diabetes.

The most common denominator of many of the neurodegenerative diseases is badly folded protein accumulation, which results in the formation of insoluble protein deposits located in different parts of the organism, causing cell death and tissue degeneration. Dendritic systems have turned out to be a promising new therapeutic approach for the treatment of these diseases due to their ability to modulate the folding of these proteins. With this perspective, and focused on type 2 diabetes (T2D), characterized by the presence of deposits containing the amyloidogenic islet amyloid polypeptide (IAPP), we demonstrate how different topologies of cationic carbosilane dendrimers inhibit the formation of insoluble protein deposits in pancreatic islets isolated from transgenic Tg-hIAPP mice. Also, the results obtained by the modification of dendritic carbosilane wedges with the chemical chaperone 4-phenylbutyric acid (4-PBA) at the focal point confirmed their potential as anti-amyloid agents with a concentration efficiency in their therapeutic action five orders of magnitude lower than that observed for free 4-PBA. Computational studies, which determined the main interaction between IAPP and dendrimers at the atomic level, support the experimental work.

Evaluation of pH-dependent amphiphilic carbosilane dendrons in micelle formation, drug loading and HIV-1 infection.

New amphiphilic carbosilane dendrons with pH-dependent behaviour based on the presence of carboxylate (propionate or succinate) groups at their peripheries and a fatty acid at the focal point were developed. In the presence of salts, they were able to form micelles with critical aggregation concentrations increasing with increasing dendron generation. Their thermodynamic parameters were calculated from surface tension measurements and their diameters at different pHs were measured by dynamic light scattering. These micelles were stable at basic pH but degraded under acidic conditions. No significant differences were found for the propionate and succinate based dendron micelles at basic or acidic pH, but the succinate dendron assemblies were more stable at neutral pH. The properties of these systems as drug nano-carriers were studied using both hydrophilic and hydrophobic molecules, and the drug loading varied with the structure and charge of the drug. In addition, due to the presence of multiple negative charges, the dendrons exhibited anti-HIV activity. Higher generation dendrons with more peripheral carboxylates that were not assembled into micelles were more active than micelles composed of lower generation dendrons having fewer peripheral carboxylates.

New Ionic Carbosilane Dendrons Possessing Fluorinated Tails at Different Locations on the Skeleton.

The fluorination of dendritic structures has attracted special attention in terms of self-assembly processes and biological applications. The presence of fluorine increases the hydrophobicity of the molecule, resulting in a better interaction with biological membranes and viability. In addition, the development of 19F magnetic resonance imaging (19F-MRI) has greatly increased interest in the design of new fluorinated structures with specific properties. Here, we present the synthesis of new water-soluble fluorinated carbosilane dendrons containing fluorinated chains in different positions on the skeleton, focal point or surface, and their preliminary supramolecular aggregation studies. These new dendritic systems could be considered as potential systems to be employed in drug delivery or gene therapy and monitored by 19F-MRI.

Adoptive Transfer of IL13Rα2-Specific Chimeric Antigen Receptor T Cells Creates a Pro-inflammatory Environment in Glioblastoma.

In order to fully harness the potential of immunotherapy with chimeric antigen receptor (CAR)-modified T cells, pre-clinical studies must be conducted in immunocompetent animal models that closely mimic the immunosuppressive malignant glioma (MG) microenvironment. Thus, the goal of this project was to study the in vivo fate of T cells expressing CARs specific for the MG antigen IL13Rα2 (IL13Rα2-CARs) in immunocompetent MG models. Murine T cells expressing IL13Rα2-CARs with a CD28.ζ (IL13Rα2-CAR.CD28.ζ) or truncated signaling domain (IL13Rα2-CAR.Δ) were generated by retroviral transduction, and their effector function was evaluated both in vitro and in vivo. IL13Rα2-CAR.CD28.ζ T cells' specificity toward IL13Rα2 was confirmed through cytokine production and cytolytic activity. In vivo, a single intratumoral injection of IL13Rα2-CAR.CD28.ζ T cells significantly extended the survival of IL13Rα2-expressing GL261 and SMA560 glioma-bearing mice; long-term survivors were resistant to re-challenge with IL13Rα2-negative and IL13Rα2-positive tumors. IL13Rα2-CAR.CD28.ζ T cells proliferated, produced cytokines (IFNγ, TNF-α), and promoted a phenotypically pro-inflammatory glioma microenvironment by inducing a significant increase in the number of CD4+ and CD8+ T cells and CD8α+ dendritic cells and a decrease in Ly6G+ myeloid-derived suppressor cells (MDSCs). Our data underline the significance of CAR T cell studies in immunocompetent hosts and further validate IL13Rα2-CAR T cells as an efficacious therapeutic strategy for MG.

Mitochondrial peroxiredoxin functions as crucial chaperone reservoir in Leishmania infantum.

Cytosolic eukaryotic 2-Cys-peroxiredoxins have been widely reported to act as dual-function proteins, either detoxifying reactive oxygen species or acting as chaperones to prevent protein aggregation. Several stimuli, including peroxide-mediated sulfinic acid formation at the active site cysteine, have been proposed to trigger the chaperone activity. However, the mechanism underlying this activation and the extent to which the chaperone function is crucial under physiological conditions in vivo remained unknown. Here we demonstrate that in the vector-borne protozoan parasite Leishmania infantum, mitochondrial peroxiredoxin (Prx) exerts intrinsic ATP-independent chaperone activity, protecting a wide variety of different proteins against heat stress-mediated unfolding in vitro and in vivo. Activation of the chaperone function appears to be induced by temperature-mediated restructuring of the reduced decamers, promoting binding of unfolding client proteins in the center of Prx's ringlike structure. Client proteins are maintained in a folding-competent conformation until restoration of nonstress conditions, upon which they are released and transferred to ATP-dependent chaperones for refolding. Interference with client binding impairs parasite infectivity, providing compelling evidence for the in vivo importance of Prx's chaperone function. Our results suggest that reduced Prx provides a mitochondrial chaperone reservoir, which allows L. infantum to deal successfully with protein unfolding conditions during the transition from insect to the mammalian hosts and to generate viable parasites capable of perpetuating infection.

B-Raf regulation of integrin α4ß1-mediated resistance to shear stress through changes in cell spreading and cytoskeletal association in T cells.

The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4ß1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4ß1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4ß1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4ß1 integrin and not other integrins, such as α5ß1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4ß1 integrin-mediated adhesion.

T cells redirected to interleukin-13Rα2 with interleukin-13 mutein--chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Rα1.

BACKGROUND AIMS: Outcomes for patients with glioblastoma remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL) 13Rα2, human epidermal growth factor receptor 2, epidermal growth factor variant III or erythropoietin-producing hepatocellular carcinoma A2 has shown promise for the treatment of glioma in preclinical models. On the basis of IL13Rα2 immunotoxins that contain IL13 molecules with one or two amino acid substitutions (IL13 muteins) to confer specificity to IL13Rα2, investigators have constructed CARS with IL13 muteins as antigen-binding domains. Whereas the specificity of IL13 muteins in the context of immunotoxins is well characterized, limited information is available for CAR T cells. METHODS: We constructed four second-generation CARs with IL13 muteins with one or two amino acid substitutions, and evaluated the effector function of IL13-mutein CAR T cells in vitro and in vivo. RESULTS: T cells expressing all four CARs recognized IL13Rα1 or IL13Rα2 recombinant protein in contrast to control protein (IL4R) as judged by interferon-γ production. IL13 protein produced significantly more IL2, indicating that IL13 mutein-CAR T cells have a higher affinity to IL13Rα2 than to IL13Rα1. In cytotoxicity assays, CAR T cells killed IL13Rα1- and/or IL13Rα2-positive cells in contrast to IL13Rα1- and IL13Rα2-negative controls. Although we observed no significant differences between IL13 mutein-CAR T cells in vitro, only T cells expressing IL13 mutein-CARs with an E13K amino acid substitution had anti-tumor activity in vivo that resulted in a survival advantage of treated animals. CONCLUSIONS: Our study highlights that the specificity/avidity of ligands is context-dependent and that evaluating CAR T cells in preclinical animal model is critical to assess their potential benefit.

Anterior Hyperfunction Syndrome: Literature Review and Conceptual Model.

Combined Kelly syndrome, also known as anterior hyperfunction syndrome, is a complex pathological condition of the stomatognathic system, originally established by five characteristics but primarily triggered by edentulism, specifically, the absence of the upper and posterior mandibular teeth. This condition is characterized by a series of clinical features, such as bone loss, tuberosity growth, enamel wear, periodontal damage, muscle fatigue, pain, and temporomandibular joint issues. However, these features are not unique and rather reflect an oral hyperfunction state. There is a lack of consensus on the best way to assess and diagnose this condition, which is proposed to be understood as an "oral hyperfunction state" rather than a syndrome. This study aims to conduct a literature review to analyze the available information on anterior hyperfunction syndrome in dentistry, with the goal of proposing a conceptual model of the etiological risk factors that contribute to early diagnosis and the prevention of complications. This approach has important clinical implications, as it would allow for the early identification and management of risk factors, thus improving the quality of life of patients and preventing malpractice that could compromise their oral health.

Gut commensals require Peyer's patches to induce protective systemic IgA responses.

Gut educated IgA secreting plasma cells that disseminate beyond the mucosa and into systemic tissues have been described as providing beneficial effects from disease in several contexts. Several bacteria have been implicated in the induction of systemic IgA, however the mechanisms that result in differential levels of induction by each bacterial species are still unknown. Here we show, the commensal bacteria, Bacteroides fragilis (Bf), is an efficient inducer of systemic IgA responses. The ability of Bf to induce the production of bone marrow IgA plasma cells and high levels of serum IgA relied on high levels of gut colonization in a dose-dependent manner. Colonization induced Bf-specific IgA responses were severely diminished in the absence of Peyer's patches, but not the murine cecal patch. Colonization of mice with Bf, a natural human commensal, resulted in few changes within the microbiome and the host transcriptional profile in the gut, suggesting a commensal relationship with the host. Bf colonization did benefit the mice by inducing systemic IgA that led to increased protection in a bowel perforation model resulting in lower peritoneal abscess formation. These findings demonstrate a critical role for bacterial colonization and Peyer's patches in the induction of robust systemic IgA responses that confer protection from bacterial dissemination outside of the gut.

Escherichia coli Strains Isolated from American Bison (Bison bison) Showed Uncommon Virulent Gene Patterns and Antimicrobial Multi-Resistance.

E. coli is considered one of the most important zoonotic pathogens worldwide. Highly virulent and antimicrobial-resistant strains of E. coli have been reported in recent years, making it essential to understand their ecological origins. In this study, we analyzed the characteristics of E. coli strains present in the natural population of American bison (Bison bison) in Mexico. We sampled 123 individuals and determined the presence of E. coli using standard bacteriological methods. The isolated strains were characterized using molecular techniques based on PCR. To evaluate the diversity of E. coli strains in this population, we analyzed 108 suggestive colonies from each fecal sample. From a total of 13,284 suggestive colonies, we isolated 33 E. coli strains that contained at least one virulence gene. The virotypes of these strains were highly varied, including strains with atypical patterns or combinations compared to classical pathotypes, such as the presence of escV, eae, bfpB, and ial genes in E. coli strain LMA-26-6-6, or stx2, eae, and ial genes in E. coli strain LMA-16-1-32. Genotype analysis of these strains revealed a previously undescribed phylogenetic group. Serotyping of all strains showed that serogroups O26 and O22 were the most abundant. Interestingly, strains belonging to these groups exhibited different patterns of virulence genes. Finally, the isolated E. coli strains demonstrated broad resistance to antimicrobials, including various beta-lactam antibiotics.

Carbosilane Dendritic Amphiphiles from Cholesterol or Vitamin E for Micelle Formation.

Cationic dendritic amphiphiles were prepared through the linkage of interesting hydrophobic molecules such as cholesterol or vitamin E to the focal point of carbosilane dendrons. These new dendritic systems self-assembled in saline, producing micellar aggregates with hydrodynamic diameters ranging from 6.5 to 9.2 nm, and critical micelle concentrations of approximately 5 and 10 µM for second- and third-generation systems, respectively. The assemblies were able to encapsulate drugs of different charges (anionic, neutral, and cationic). Surprisingly, a 92% encapsulation efficiency for diclofenac was achieved in micelles prepared from second-generation dendrons. Toxicity measurements on peripheral blood mononuclear cells indicated different behavior depending on the generation, corresponding to the micellar regime. In contrast to the third-generation system, the second-generation system was non-toxic up to 20 µM, opening a window for its use in a micellar regimen, thereby operating as a drug delivery system for different biomedical applications.

Multimodal evaluation of drug antibacterial activity reveals cinnamaldehyde analog anti-biofilm effects against Haemophilus influenzae.

Biofilm formation by the pathobiont Haemophilus influenzae is associated with human nasopharynx colonization, otitis media in children, and chronic respiratory infections in adults suffering from chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD). ß-lactam and quinolone antibiotics are commonly used to treat these infections. However, considering the resistance of biofilm-resident bacteria to antibiotic-mediated killing, the use of antibiotics may be insufficient and require being replaced or complemented with novel strategies. Moreover, unlike the standard minimal inhibitory concentration assay used to assess antibacterial activity against planktonic cells, standardization of methods to evaluate anti-biofilm drug activity is limited. In this work, we detail a panel of protocols for systematic analysis of drug antimicrobial effect on bacterial biofilms, customized to evaluate drug effects against H. influenzae biofilms. Testing of two cinnamaldehyde analogs, (E)-trans-2-nonenal and (E)-3-decen-2-one, demonstrated their effectiveness in both H. influenzae inhibition of biofilm formation and eradication or preformed biofilms. Assay complementarity allowed quantifying the dynamics and extent of the inhibitory effects, also observed for ampicillin resistant clinical strains forming biofilms refractory to this antibiotic. Moreover, cinnamaldehyde analog encapsulation into poly(lactic-co-glycolic acid) (PLGA) polymeric nanoparticles allowed drug vehiculization while maintaining efficacy. Overall, we demonstrate the usefulness of cinnamaldehyde analogs against H. influenzae biofilms, present a test panel that can be easily adapted to a wide range of pathogens and drugs, and highlight the benefits of drug nanoencapsulation towards safe controlled release.