GMP-compliant extracellular vesicles derived from umbilical cord mesenchymal stromal cells: manufacturing and pre-clinical evaluation in ARDS treatment.

BACKGROUND AIMS: Extracellular vesicles (EVs) represent a new axis of intercellular communication that can be harnessed for therapeutic purposes, as cell-free therapies. The clinical application of mesenchymal stromal cell (MSC)-derived EVs, however, is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution, toxicity and potential efficacy of hUCMSC-EVs in pre-clinical studies using the LPS-induced acute lung injury model. METHODS: hUCMSCs were isolated from a cord tissue, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive mice after intravenous administration of the product. Efficacy was evaluated in an LPS-induced acute lung injury model. RESULTS: hUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Biodistribution studies demonstrated a major concentration in liver, spleen and lungs. HUCMSC-EVs reduced lung damage and inflammation in a model of LPS-induced acute lung injury. CONCLUSIONS: hUCMSC-EVs were successfully obtained following a cGMP-compliant protocol, with consistent characteristics and pre-clinical safety profile, supporting their future clinical development as cell-free therapies.

Automated macular segmentation can distinguish glaucomatous from compressive optic neuropathy.

PURPOSE: To compare macular damage in glaucomatous optic neuropathy (GON) and compressive optic neuropathy (CON) and assess its diagnostic accuracy in distinguishing between diseases. METHODS: Observational, cross-sectional, single-center study. Patients with GON, CON, and healthy controls were included according to the eligibility criteria. An automated spectral-domain optical coherence tomography (SD-OCT) algorithm was used to segment the circumpapilary retinal nerve fiber layer (cpRNFL) and macula. The layer thickness was measured in each sector according to the Early Treatment Diabetic Retinopathy Study and the 6-sector Garway-Heath-based grids. Data was compared across all study groups, and the significance level was set at 0.05. RESULTS: Seventy-five eyes of 75 participants, 25 with GON, 25 with CON, and 25 healthy controls (CG), were included. Macular thickness was diminished in the ganglion cell complex of GON and CON patients compared to CG (p<0.05). The best Garway-Heath-based grid parameters for distinguishing GON and CON were the nasal-inferior (NI) and nasal-superior sectors and the NI/temporal inferior (TI) damage ratios in the macular ganglion cell (mGCL) and inner plexiform (IPL) layers. Moreover, the combination of the NI sector and NI/TI damage ratios in both layers had higher discriminative power (AUC 0.909; 95% CI 0.830-0.988; p<0.001) than combining parameters in each layer separately. CONCLUSION: Our findings suggest that the evaluation of macular segmented layers damage by SD-OCT may be a helpful add-on tool in the differential diagnosis between GON and CON.

Intracranial thrombosis after ventriculoperitoneal shunting.

We report a case of intracranial thrombosis (IT) after ventriculoperitoneal shunting (VPS). We reviewed the literature to highlight the importance of considering the possibility of cerebral venous thrombosis following VPS, even though it is a rarely reported complication. A 14-year-old boy underwent distal catheter replacement due to its short size that was detected during a routine consultation. Five days postoperatively, he experienced nausea, vomiting, seizures, and headache. Although a diagnosis of meningitis was considered, diagnosis of IT was eventually confirmed through computed tomography venography and gadolinium magnetic angioresonance. The patient subsequently underwent anticoagulant therapy, which led to complete resolution of symptoms. In this report, we suggest a possible association between VPS and IT, which is a complication that is not commonly reported. The literature suggests that decreased blood flow due to over-drainage of CSF after VPS causes decreased head pressure, culminating in venous stasis and consequent thrombosis. In addition, the literature describes associations between IT and lumbar puncture (LP); therefore, it is important to consider the possibility of IT following these procedures.

Fetal epidural hematoma secondary to a mild blunt prenatal trauma: case report and review of the literature.

Fetal intracranial hemorrhage affects 1 in every 10,000 pregnancies. In most cases, the etiology of the bleeding is multifactorial, and they can be either related to the mother or the fetus. Blunt prenatal trauma was occasionally associated with these hemorrhages, nevertheless, reports of hematomas secondary to mild traumas are rare. Within the prenatal intracranial bleedings, the most frequent are the subarachnoid hematoma and intraparenchymal, scarcely ever the epidural hematoma. Treating these bleedings is challenging due to the ongoing pregnancy. Thus, the prognosis is often reserved, with a mortality rate of 43% and 25% of neurological sequelae. Here, we report a singular case of a fetal epidural hematoma secondary to a mild blunt trauma at the third trimester with a good outcome.

Docetaxel Loaded in Copaiba Oil-Nanostructured Lipid Carriers as a Promising DDS for Breast Cancer Treatment.

Breast cancer is the neoplasia of highest incidence in women worldwide. Docetaxel (DTX), a taxoid used to treat breast cancer, is a BCS-class-IV compound (low oral bioavailability, solubility and intestinal permeability). Nanotechnological strategies can improve chemotherapy effectiveness by promoting sustained release and reducing systemic toxicity. Nanostructured lipid carriers (NLC) encapsulate hydrophobic drugs in their blend-of-lipids matrix, and imperfections prevent drug expulsion during storage. This work describes the preparation, by design of experiments (23 factorial design) of a novel NLC formulation containing copaiba oil (CO) as a functional excipient. The optimized formulation (NLCDTX) showed approximately 100% DTX encapsulation efficiency and was characterized by different techniques (DLS, NTA, TEM/FE-SEM, DSC and XRD) and was stable for 12 months of storage, at 25 °C. Incorporation into the NLC prolonged drug release for 54 h, compared to commercial DTX (10 h). In vitro cytotoxicity tests revealed the antiproliferative effect of CO and NLCDTX, by reducing the cell viability of breast cancer (4T1/MCF-7) and healthy (NIH-3T3) cells more than commercial DTX. NLCDTX thus emerges as a promising drug delivery system of remarkable anticancer effect, (strengthened by CO) and sustained release that, in clinics, may decrease systemic toxicity at lower DTX doses.

Exploring mechanisms underlying learning from deliberate reflection: An experimental study.

INTRODUCTION: Previous research suggests that, relative to generating a differential diagnosis, deliberate reflection during practice with clinical cases fosters learning from a subsequently studied scientific text and promotes interest in the subject matter. The present experiment aimed to replicate these findings and to examine whether motivational or cognitive mechanisms, or both, underlie the positive effects of reflection. METHODS: A total of 101 5th-year medical students participated in an experiment containing four phases: Students (a) diagnosed two clinical cases of jaundice-related diseases either through deliberate reflection or differential diagnosis; (b) reported their situational interest and awareness of knowledge gaps; (c) studied a text about jaundice, either under free or restricted time; and (d) recalled the text. Outcome measures were text-recall, situational interest and awareness of knowledge gaps. RESULTS: A main effect of diagnostic approach on recall of the text was found, with the reflection group recalling more studied material than the differential diagnosis group (means: 72.56 vs 58.80; P = .01). No interaction between diagnostic approach and study time (free or restricted) emerged, nor was there a main effect of the latter. Relative to the differential diagnosis group, students who reflected upon the cases scored significantly higher on both situational interest (means: 4.45 vs 3.99, P < .001) and awareness of knowledge gaps (means: 4.13 vs 3.85, P < .01). DISCUSSION: Relative to generating differential diagnoses, reflection upon clinical cases increased learning outcomes on a subsequent study task, an effect that was independent of study time, suggesting that cognitive mechanisms underlie this effect, rather than increases in motivation to study. However, higher scores on situational interest and awareness of knowledge gaps and a tendency towards larger gains when time was free suggest that higher motivation may also contribute to learning from reflection.

Inhibition of αvß3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. METHODS: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvß3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. RESULTS: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvß3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvß3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. CONCLUSION: In summary, our findings demonstrate for the first time a key role of αvß3 integrin in cell-cell communication through SEVs. Video Abstract.

Bruch's Membrane Opening Minimum Rim Width in the Differential Diagnosis of Optic Neuropathies.

Observing optic disc pallor during a patient's first visit frequently raises a diagnostic challenge, particularly in regards to whether the cause is due to glaucoma or another form of optic neuropathy. Bruch's membrane opening (BMO) was recently discovered as the anatomical border of the optic disc. BMO minimum rim width (BMO-MRW) seems to be a reliable representation of the neuroretinal rim. In our study, we demonstrate the ability of BMO-MRWs to differentiate between glaucomatous and non-glaucomatous. Additionally, we propose an MRW ratio which may allow discrimination of open angle glaucoma from either non-arteritic anterior ischaemic optic neuropathy or compressive optic neuropathy.

Effects of deliberate reflection on students' engagement in learning and learning outcomes.

CONTEXT: Reflection in practice is assumed to enhance interest in knowing more about a topic, increasing engagement in learning and learning outcomes. However, this claim lacks empirical evidence, particularly in medical education. The authors investigated the effects of deliberate reflection upon clinical cases on medical students' engagement in a learning activity and learning outcomes. METHODS: A three-task (diagnostic task; learning activity; test) experiment was conducted in August 2017. Seventy-two fourth-year students from UNIFENAS-BH Medical School, Brazil, diagnosed two clinical cases with jaundice as the chief complaint, either by following a deliberate reflection procedure or making differential diagnosis. Subsequently, all participants received the same study material on the diagnosis of jaundice. Finally, they took a recall test on the study material. Outcome measurements were study time and test scores. RESULTS: There was a significant effect of experimental condition on students' engagement in the learning activity and on learning outcomes. Students who deliberately reflected upon the cases invested more time in studying the material than those who made a differential diagnosis (respectively, mean = 254.97, standard deviation = 115.45 versus mean = 194.96, standard deviation = 111.68; p = 0.02; d = 0.53). Deliberate reflection was also related to higher scores in the test relative to differential diagnosis (respectively, mean = 22.08, standard deviation = 14.94 versus mean = 15.75, standard deviation = 9.24; p = 0.03; d = 0.51). Medium effect sizes (Cohen's d) were observed in both measurements. CONCLUSIONS: Relative to making differential diagnosis, deliberate reflection while diagnosing cases fostered medical students' engagement in learning and increased learning outcomes. Teachers can employ this relatively easy procedure, possibly both with simulated and real scenarios, to motivate their students and help them expand their knowledge, an important requirement for their professional development.

Liposomal-based lidocaine formulation for the improvement of infiltrative buccal anaesthesia.

This study describes the encapsulation of the local anaesthetic lidocaine (LDC) in large unilamellar liposomes (LUV) prepared in a scalable procedure, with hydrogenated soybean phosphatidylcholine, cholesterol and mannitol. Structural properties of the liposomes were assessed by dynamic light scattering, nanoparticle tracking analysis and transmission electron microscopy. A modified, two-compartment Franz-cell system was used to evaluate the release kinetics of LDC from the liposomes. The in vivo anaesthetic effect of liposomal LDC 2% (LUVLDC) was compared to LDC 2% solution without (LDCPLAIN) or with the vasoconstrictor epinephrine (1:100 000) (LDCVASO), in rat infraorbital nerve blockade model. The structural characterization revealed liposomes with spherical shape, average size distribution of 250 nm and low polydispersity even after LDC incorporation. Zeta potential laid around -30 mV and the number of suspended liposomal particles was in the range of 1012 vesicles/mL. Also the addition of cryoprotectant (mannitol) did not provoke structural changes in liposomes properties. In vitro release profile of LDC from LUV fits well with a biexponential model, in which the LDC encapsulated (EE% = 24%) was responsible for an increase of 67% in the release time in relation to LDCPLAIN (p < 0.05). Also, the liposomal formulation prolonged the sensorial nervous blockade duration (∼70 min), in comparison with LDCPLAIN (45 min), but less than LDCVASO (130 min). In this context, this study showed that the liposomal formulations prepared by scalable procedure were suitable to promote longer and safer buccal anaesthesia, avoiding side effects of the use of vasoconstrictors.

Sustained Release from Ionic-Gradient Liposomes Significantly Decreases ETIDOCAINE Cytotoxicity.

PURPOSE: Etidocaine (EDC) is a long lasting local anesthetic, which alleged toxicity has restricted its clinical use. Liposomes can prolong the analgesia time and reduce the toxicity of local anesthetics. Ionic gradient liposomes (IGL) have been proposed to increase the upload and prolong the drug release, from liposomes. METHODS: First, a HPLC method for EDC quantification was validated. Then, large unilamellar vesicles composed of hydrogenated soy phosphatidylcholine:cholesterol with 250 mM (NH4)2SO4 - inside gradient - were prepared for the encapsulation of 0.5% EDC. Dynamic light scattering, nanotracking analysis, transmission electron microscopy and electron paramagnetic resonance were used to characterize: nanoparticles size, polydispersity, zeta potential, concentration, morphology and membrane fluidity. Release kinetics and in vitro cytotoxicity tests were also performed. RESULTS: IGLEDC showed average diameters of 172.3 ± 2.6 nm, low PDI (0.12 ± 0.01), mean particle concentration of 6.3 ± 0.5 × 1012/mL and negative zeta values (-10.2 ± 0.4 mV); parameters that remain stable during storage at 4°C. The formulation, with 40% encapsulation efficiency, induced the sustained release of EDC (ca. 24 h), while reducing its toxicity to human fibroblasts. CONCLUSION: A novel formulation is proposed for etidocaine that promotes sustained release and reduces its cytotoxicity. IGLEDC can come to be a tool to reintroduce etidocaine in clinical use.

Effect of reflection on medical students' situational interest: an experimental study.

CONTEXT: Reflection has been considered crucial to learning. Engaging in reflection while solving problems is expected to foster identification of knowledge gaps and interest in learning more about them, the latter being a major motivational force in learning. Although theoretically sound, this assumption still lacks empirical evidence. This experiment investigated whether reflection while diagnosing clinical cases of different levels of difficulty influences medical students' awareness of knowledge gaps and situational interest. METHODS: Forty-two fourth-year students from a Brazilian medical school were randomly allocated to diagnose six clinical cases (three difficult; three easy), either by following a structured reflection procedure (reflection group) or by giving alternative diagnoses (control group). Subsequently, for each case, all students rated their situational interest and awareness of knowledge gaps. RESULTS: Situational interest was significantly higher in the reflection group than in the control group (mean = 4.10, standard deviation = 0.50 versus mean = 3.65, standard deviation = 0.48, respectively; p = 0.003; range, 1-5). The effect size was large (d = 0.92). Awareness of knowledge gaps was higher in the reflection group than in the control group, but the difference was not significant. Case difficulty influenced both situational interest, which was significantly higher on easy than on difficult cases (mean = 3.96, standard deviation = 0.56 versus mean = 3.80, standard deviation = 0.55, respectively; p = 0.004), and awareness of knowledge gaps, with higher scores observed on difficult compared with easy cases (mean = 3.99, standard deviation = 0.46 versus mean = 3.66, standard deviation = 0.53, respectively; p < .001). No interaction between experimental condition and case difficulty emerged. CONCLUSION: Relative to providing alternative diagnoses while solving cases, structured reflection increased medical students' interest and may therefore be a useful tool for teachers concerned with enhancing students' motivation for learning. Surprisingly, easy cases promoted higher situational interest despite the higher awareness of knowledge gaps on difficult cases. This suggests the potential for case difficulty to inhibit students' interest in learning, a possibility that demands further investigation.

Therapeutic Effects of Essential Oils and Their Bioactive Compounds on Prostate Cancer Treatment.

Since prostate cancer (PCa) relies on limited therapies, more effective alternatives are required. Essential oils (EOs) and their bioactive compounds are natural products that have many properties including anticancer activity. This review covers studies published between 2000 and 2023 and discusses the anti-prostate cancer mechanisms of the EOs from several plant species and their main bioactive compounds. It also provides a critical perspective regarding the challenges to be overcome until they reach the market. EOs from chamomile, cinnamon, Citrus species, turmeric, Cymbopogon species, ginger, lavender, Mentha species, rosemary, Salvia species, thyme and other species have been tested in different PCa cell lines and have shown excellent results, including the inhibition of cell growth and migration, the induction of apoptosis, modulation in the expression of apoptotic and anti-apoptotic genes and the suppression of angiogenesis. The most challenging aspects of EOs, which limit their clinical uses, are their highly lipophilic nature, physicochemical instability, photosensitivity, high volatility and composition variability. The processing of EO-based products in the pharmaceutical field may be an interesting alternative to circumvent EOs' limitations, resulting in several benefits in their further clinical use. Identifying their bioactive compounds, therapeutic effects and chemical structures could open new perspectives for innovative developments in the field. Moreover, this could be helpful in obtaining versatile chemical synthesis routes and/or biotechnological drug production strategies, providing an accurate, safe and sustainable source of these bioactive compounds, while looking at their use as gold-standard therapy in the close future.

Antimicrobial Activity of Essential-Oil-Based Nanostructured Lipid Carriers against Campylobacter Spp. Isolated from Chicken Carcasses.

Campylobacter is a virulent Gram-negative bacterial genus mainly found in the intestines of poultry. The indiscriminate use of traditional antibiotics has led to drug resistance in these pathogens, necessitating the development of more efficient and less toxic therapies. Despite their complex biologically active structures, the clinical applications of essential oils (EOs) remain limited. Therefore, this study aimed to increase the bioavailability, stability, and biocompatibility and decrease the photodegradation and toxicity of EO using nanotechnology. The diffusion disk test revealed the potent anti-Campylobacter activity of cinnamon, lemongrass, clove, geranium, and oregano EOs (>50 mm). These were subsequently used to prepare nanostructured lipid carriers (NLCs). Formulations containing these EOs inhibited Campylobacter spp. growth at low concentrations (0.2 mg/mL). The particle size, polydispersity index, and zeta potential of these systems were monitored, confirming its physicochemical stability for 210 days at 25 °C. FTIR-ATR and DSC analyses confirmed excellent miscibility among the excipients, and FE-SEM elucidated a spherical shape with well-delimited contours of nanoparticles. The best NLCs were tested regarding nanotoxicity in a chicken embryo model. These results indicate that the NLC-based geranium EO is the most promising and safe system for the control and treatment of multidrug-resistant strains of Campylobacter spp.

Real-World Evidence of the Long-Term Effectiveness of 0.2 µg/Day Fluocinolone Acetonide Implant in Persistent and Recurrent Diabetic Macular Edema - A Single Center Study.

Purpose: To report the long-term functional, anatomical and safety outcomes of 0.2 µg/day fluocinolone acetonide 0.19mg in patients with persistent or recurrent diabetic macular edema (DME). Methods: Retrospective, observational, single-center study of patients with recurrent or persistent DME. All patients received 0.2 µg/day of fluocinolone acetonide 0.19mg, and data were collected at baseline and months 1, 3, 6, 12, 24 and 36 after implantation. Outcomes measured included best-corrected visual acuity (BCVA), central macular thickness (CMT), intraocular pressure (IOP), and safety outcomes. Results: A total of 28 eyes from 28 patients were included. The mean age was 66.5 years (95% CI 62.8-70.2) with a mean duration of DME of 8.8 years (95% CI 7.7-10.0). Only two eyes were phakic. Mean follow-up was 25.4 months (95% CI 21.2-29.6). Mean BCVA at baseline was 48.6 ETDRS letters (95% CI 41.3-55.8) and improved as early as month 1 of follow-up with a mean gain in BCVA of 7.8 (95% CI 4.3-11.3) ETDRS letters (p<0.001). Statistically significant improvements in BCVA were also observed at months 6, 12 and 24. At baseline, patients had a mean CMT of 530.5µm (95% CI 463.0-598.0), and a decrease in CMT was observed, starting at the first month of follow-up (mean CMT reduction of -170.5µm, 95% CI -223.8- -117.1; p<0.001). Statistically significant decreases in CMT were also observed at months 6, 12, 24, and 36, with the maximum decrease observed at month 12 (p<0.001). Mean IOP at baseline was 16.4mmHg (95% CI 15.3-17.5) and nine eyes (32.1%) had an IOP ≥21mmHg during follow-up. Conclusion: Our results support the effectiveness and safety profile of fluocinolone acetonide. Although additional long-term real-world evidence is required, fluocinolone acetonide may represent a safe strategy for daily, low-dose, sustained and localized release to the posterior segment of the eye, providing both functional and anatomical benefits in DME.

Analysis of Key Factors for Evaluating Mucosal Adhesion Using Swine Buccal Tissue.

The assessment of the mucoadhesive properties peak mucoadhesive force (Fmax) and work of mucoadhesion (Wmuc) with texture analyzers is a common in vitro method for analyzing formulation capabilities. Challenges arise in selecting and standardizing experimental conditions due to various variables influencing mucoadhesion. This complexity hampers direct product performance comparisons. In our study, we explored factors (contact force and time, probe speed and mucin in artificial saliva) impacting a model formulation's mucoadhesive capacity. Using Omcilon-A®Orabase on porcine buccal mucosa, we systematically varied experimental conditions, employing a statistical approach (Central Composite Design - CCD). Three variables (contact force, contact time, probe speed) and their interactions were assessed for their impact on Fmax and Wmuc. Results showed that contact time and force positively affected Fmax, while only contact time influenced Wmuc. In the mucin artificial saliva test, a force of 0.5 N, time of 600 s, and speed of 1 mm/s yielded optimal Fmax (0.587 N) and Wmuc (0.468 N.s). These conditions serve as a reference for comparing mucoadhesive properties of formulations for topical oral use.

Antitumor activity of essential oils-based nanostructured lipid carriers on prostate cancer cells.

Prostate cancer (PCa) is the second most frequent malignancy in men worldwide. Essential oils (EOs) are natural products which can act in cancer suppression by several mechanisms. In this work, a nanotechnological approach was used to develop and evaluate the antineoplastic effects of EOs loaded by nanostructured lipid carriers (NLCs). Three different NLC systems composed of cinnamon, sage or thyme EOs were optimized using factorial design (23). The optimal formulations were characterized in terms of biophysical parameters, structure, stability, in vivo safety and efficacy. All optimized NLC formulations exhibited excellent structural properties and stability over a year (25 °C). They proved to be in vitro and in vivo biocompatible on PNT2 normal prostate cells and on chicken embryos (CE), respectively. In PC3 PCa cells, optimized NLCs inhibited cell proliferation and migration and changed its morphology. In CE xenograft tumor, NLCs have inhibited tumor growth and angiogenesis. The results from this work suggested that all developed EO-based NLC formulations had their stability improved while the biological activity remains unchanged.

Interference with Bacterial Conjugation and Natural Alternatives to Antibiotics: Bridging a Gap.

Horizontal gene transfer (HGT) in food matrices has been investigated under conditions that favor gene exchange. However, the major challenge lies in determining the specific conditions pertaining to the adapted microbial pairs associated with the food matrix. HGT is primarily responsible for enhancing the microbial repertoire for the evolution and spread of antimicrobial resistance and is a major target for controlling pathogens of public health concern in food ecosystems. In this study, we investigated Salmonella Heidelberg (SH) and Escherichia coli (EC) regarding gene exchange under conditions mimicking the industrial environment, with the coproducts whey (SL) and chicken juice (CJ). The S. Heidelberg strain was characterized by antibiotic susceptibility standards and PCR to detect the blaTEM gene. A concentration of 0.39 mg/mL was determined to evaluate the anti-conjugation activity of nanostructured lipid nanocarriers (NLCs) of essential oils to mitigate ß-lactam resistance gene transfer. The results showed that the addition of these coproducts promoted an increase of more than 3.5 (whey) and 2.5 (chicken juice) orders of magnitude in the conjugation process (p < 0.01), and NLCs of sage essential oil significantly reduced the conjugation frequency (CF) by 74.90, 90.6, and 124.4 times when compared to the transfers in the absence of coproducts and the presence of SL and CJ, respectively. For NLCs from olibanum essential oil, the decrease was 4.46-fold for conjugations without inhibitors and 3.12- and 11.3-fold in the presence of SL and CJ. NLCs associated with sage and olibanum essential oils effectively control the transfer of antibiotic resistance genes and are a promising alternative for use at industrial levels.

Lipid nanoparticles based on natural matrices with activity against multidrug resistant bacterial species.

Lately, the bacterial multidrug resistance has been a reason to public health concerning around world. The development of new pharmacology therapies against infections caused by multidrug-resistant bacteria is urgent. In this work, we developed 10 NLC formulations composed of essential oils (EO), vegetable butter and surfactant. The formulations were evaluated for long-term and thermal cycling stability studies in terms of (particle size, polydispersion index and Zeta potential). In vitro antimicrobial assays were performed using disk diffusion test and by the determination of the minimum inhibitory concentration (MIC) performed with fresh and a year-old NLC. The most promising system and its excipients were structurally characterized through experimental methodologies (FTIR-ATR, DSC and FE-SEM). Finally, this same formulation was studied through nanotoxicity assays on the chicken embryo model, analyzing different parameters, as viability and weight changes of embryos and annexes. All the developed formulations presented long-term physicochemical and thermal stability. The formulation based on cinnamon EO presented in vitro activity against strains of Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from humans and in vivo biocompatibility. Considering these promising results, such system is able to be further tested on in vivo efficacy assays.