Pharmacokinetics and pharmacodynamics of dextroketamine alone or combined with midazolam in Caiman crocodilus.

Pharmacokinetics studies of anesthetic agents are important for understanding of the pharmacology and metabolism of anesthetic agents in reptilians. This study was designed to examine the pharmacokinetic and pharmacodynamic properties of intravenous dextroketamine alone or combined with midazolam in Caiman crocodilus. Eight caimans were anesthetized with dextroketamine (10 mg/kg; group D) or dextroketamine and midazolam (10 and 0.5 mg/kg respectively; group DM) into the occipital venous sinus. The pharmacokinetic parameters were calculated by HPLC using a non-compartmental modeling. Serial blood samples were collected at baseline and within 15 and 30 min, and 11.5, 2, 4, 8, 12, 24 and 48 h of drug administration. Sedation status over time differed between groups. All animals in group D (8/8; 100%) showed signs of light sedation at t10. Half (4/8; 50%) of these caimans did not progress to deeper levels of sedation. In spite of light sedation at t10, animals in group DM were deeply sedated within 13.13 ± 7.04 min of anesthetic agent injection. The area under the plasma concentration-time curve (AUC0-48) and half-life of dextroketamine changed significantly after combination with midazolam. Even without significant changes in clearance, the almost two-fold increase in the half-life of dextroketamine suggests a slower rate of elimination.

Extracellular vesicles from the mycoparasitic fungus Trichoderma harzianum.

Trichoderma harzianum is a filamentous fungus that can act as a mycoparasite, saprophyte, or a plant symbiotic. It is widely used as a biological control agent against phytopathogenic fungi and can also be used for plant growth promotion and biofortification. Interaction between T. harzianum and phytopathogenic fungi involves mycoparasitism, competition, and antibiosis. Extracellular vesicles (EVs) have been described as presenting a central role in mechanisms of communication and interaction among fungus and their hosts. In this study, we characterized extracellular vesicles of T. harzianum produced during growth in the presence of glucose or S. sclerotiorum mycelia. A set of vesicular proteins was identified using proteomic approach, mainly presenting predicted signal peptides.

Photothermal Properties of IR-780-Based Nanoparticles Depend on Nanocarrier Design: A Comparative Study on Synthetic Liposomes and Cell Membrane and Hybrid Biomimetic Vesicles.

Biomimetic nanoparticles hold great promise for photonic-mediated nanomedicine due to the association of the biological functionality of the membrane with the physical/chemical goals of organic/inorganic structures, but studies involving fluorescent biomimetic vesicles are still scarce. The purpose of this article is to determine how photothermal therapy (PTT) with theranostic IR-780-based nanoparticles depends on the dye content, cholesterol content, lipid bilayer phase and cell membrane type. The photophysical responses of synthetic liposomes, cell membrane vesicles and hybrid nanoparticles are compared. The samples were characterized by nanoparticle tracking analysis, photoluminescence, electron spin resonance, and photothermal- and heat-mediated drug release experiments, among other techniques. The photothermal conversion efficiency (PCE) was determined using Roper's method. All samples excited at 804 nm showed three fluorescence bands, two of them independent of the IR-780 content. Samples with a fluorescence band at around 850 nm showed photobleaching (PBL). Quenching was higher in cell membrane vesicles, while cholesterol inhibited quenching in synthetic liposomes with low dye content. PTT depended on the cell membrane and was more efficient for melanoma than erythrocyte vesicles. Synthetic liposomes containing cholesterol and a high amount of IR-780 presented superior performance in PTT experiments, with a 2.4-fold PCE increase in comparison with free IR-780, no PBL and the ability to heat-trigger doxorubicin release.

Interplay between mucus mobility and alveolar macrophage targeting of surface-modified liposomes.

Alveolar macrophages play a crucial role in the initiation and resolution of the immune response in the lungs. Pro-inflammatory M1 alveolar macrophages are an interesting target for treating inflammatory and infectious pulmonary diseases. One commune targeting strategy is to use nanoparticles conjugated with hyaluronic acid, which interact with CD44 overexpressed on the membrane of those cells. Unfortunately, this coating strategy may be countered by the presence on the surface of the nanoparticles of a poly(ethylene glycol) corona employed to improve nanoparticles' diffusion in the lung mucus. This study aims to measure this phenomenon by comparing the behavior in a murine lung inflammation model of three liposomal platforms designed to represent different poly(ethylene glycol) and hyaluronic acid densities (Liposome-PEG, Liposome-PEG-HA and Liposome-HA). In this work, the liposomes were obtained by a one-step ethanol injection method. Their interaction with mucin and targeting ability toward pro-inflammatory macrophages were then investigated in vitro and in vivo in a LPS model of lung inflammation. In vitro, poly(ethylene glycol) free HA-liposomes display a superior targeting efficiency toward M1 macrophages, while the addition of poly(ethylene glycol) induces better mucus mobility. Interestingly in vivo studies revealed that the three liposomes showed distinct cell specificity with alveolar macrophages demonstrating an avidity for poly(ethylene glycol) free HA-liposomes, while neutrophils favored PEGylated liposomes exempt of HA. Those results could be explained by the presence of two forces exercising a balance between mucus penetration and receptor targeting. This study corroborates the importance of considering the site of action and the targeted cells when designing nanoparticles to treat lung diseases.

Topical application of melatonin accelerates the maturation of skin wounds and increases collagen deposition in a rat model of diabetes.

AIMS: This study aimed to evaluate the cicatricial potential of melatonin when applied to wounds of diabetic rats. MATHERIALS AND METHODS: The formulation containing melatonin was developed and applied topically to cutaneous wounds of diabetic rats. 48 Wistar rats were used, divided into two groups of 24 diabetic animals each: (i) control group (CG), the animals received topical application of the no-melatonin formulation; (ii) treatment group (TG), the animals received topical application of the melatonin-containing formulation. All animals in each group were treated at four time points: 3, 7, 14, and 21 days. Each subgroup consisted of six animals. RESULTS: The treatment with melatonin improved wound healing by promoting wound closure earlier than the control group evaluated. Also improved a better resolution of the inflammatory phase observed mainly at 7 days, higher tissue maturation and expressive collagen deposition. CONCLUSION: The observed data reveal that the use of melatonin topically could be a promising strategy for the healing of wounds in diabetes. The results of this study elucidate the effects of previously described pathways in which it is proposed that melatonin acts promoting wound healing in diabetes.

Plasma concentration, cardiorespiratory and analgesic effects of ketamine-fentanyl infusion in dogs submitted to mastectomy.

BACKGROUND: The analgesic and cardiorespiratory effects of ketamine, fentanyl, or ketamine-fentanyl constant rate infusion (CRI) in dogs undergoing mastectomy were evaluated. Seventeen female dogs received CRI of ketamine (GK [n = 6]: bolus 0.5 mg/kg; CRI 20 µg/kg/min in intra- and postoperative periods], fentanyl (GF [n = 5]: bolus 20 µg/kg; intraoperative CRI 5 20 µg/kg/hour and postoperative CRI 2 20 µg/kg/hour), or combination of ketamine-fentanyl (GKF [n = 6]: aforementioned doses) for 8 h. Cardiorespiratory, blood gas analyses, plasma drug concentrations, sedation score (SS), Pain Scores were evaluated. RESULTS: The heart rate decreased in the GF and GKF (p < 0.04); the mean arterial pressure was lower in the GKF than in the GK at 35 min (p < 0.001). Maximum plasma concentrations were observed 5 min after bolus in the GK (2847.06 ± 2903.03 ng/mL) and GKF (2811.20 ± 1931.76 ng/mL). Plasma concentration in intraoperative period of ketamine was of > 100 ng/mL in 5/5 and 2/5 animals in the GKF and GK, respectively; and > 1.1 ng/mL of fentanyl in 4/5 and 3/5 in GKF and GF, respectively. CONCLUSION: Ketamine with/without fentanyl provided analgesia without significant cardiorespiratory and guaranteed the minimal plasma levels with analgesic potential during the 8 h.

Curcumin-rich extract from Central Brazil Curcuma longa protects human umbilical vein endothelial cells under oxidative stress / Extrato rico em Curcumina do Brasil Central Curcuma longa protege células endoteliais de veias umbilicais humanas sob estresse oxidativo

INTRODUCTION: In vascular diseases, the interruption of the local blood flow and the subsequent reperfusion of oxygen can cause deleterious oxidative effects on the cells. Turmeric (Curcuma longa L.) presents the capacity to neutralize free radicals along with preventive and therapeutic effects for several diseases. OBJECTIVE: To analyze the bioactive compounds and the antioxidant capacity of the ethanolic extract of Curcuma (EEC), to evaluate its effect on human umbilical vein endothelial cells, and to analyze its effect on cellular signaling pathways. METHODS: Cells were exposed to different concentrations of EEC for 24, 48, and 72 h. Folin-Ciocalteau test, HPLC-Fluorescence analysis, and DPPH method were used to determine the phenolic compounds, curcumin content, and antioxidant action, respectively; the tetrazolium salt reduction to obtain cell viability, cytotoxicity, and the concentration that inhibits 50% of cell viability; and the immunocytochemistry technique to analyze the expression of caspase3, SIRT1, and mTOR. RESULTS: We found the presence of polyphenols in the classes of phenolic acids and curcuminoids in EEC, with 16.7% curcumin content. The number of antioxidants needed to reduce the initial DPPH concentration by 50% was 18.1 µmol/g. The extract mitigated cell damage at a dosage of 100 µg/ml, decreased the immunoexpression of caspase3, and promoted the signaling of the SIRT1 and mTOR survival pathways. CONCLUSION: EEC had a protective effect on human umbilical vein endothelial cells, subjected to oxidative stress, with decreased apoptosis (caspase3) at lower concentrations, cytoprotection by maintaining essential cell functions (mTOR), and signaling of the survival pathway (SIRT1).

INTRODUÇÃO: Em doenças vasculares, a interrupção do fluxo sanguíneo locale subsequente reperfusão de oxigênio pode causar efeitos deletérios e danos irreparáveis às células. Curcuma (Curcuma longa L.) neutraliza radicais livres além de apresentar efeitos preventivos e terapêuticos. OBJETIVO: Caracterizar os compostos bioativos e a capacidade antioxidante do extrato etanólico de cúrcuma (EEC); avaliar seu efeito nas células endoteliais da veia umbilical humana, e analisar a expressão de vias de sinalização celular. MÉTODOS: As células foram expostas a diferentes concentrações de EEC por 24, 48 e 72 horas. Utilizamos o teste de Folin-Ciocalteau, análise por HPLC-Fluorescência e método DPPH para determinar os compostos fenólicos, conteúdo de curcumina e ação antioxidante, respectivamente; o método de redução de tetrazólio para viabilidade celular, a citotoxicidade e a concentração que inibe 50% da viabilidade celular; e a técnica de imunocitoquímica para analisar a expressão de caspase3, SIRT1 e mTOR. RESULTADOS: Observou-se presença de polifenóis nas classes de ácidos fenólicos e curcuminóides no EEC, com teor de curcumina de 16,7%. A quantidade de antioxidante necessária para reduzir a concentração inicial de DPPH em 50% foi de 18,1 µmol/g. O extrato mitigou o dano celular na dosagem de 100 µg/ml, diminuiu a imunoexpressão da caspase3 e promoveu a sinalização das vias de sobrevivência SIRT1 e mTOR. CONCLUSÃO: O EEC teve efeito protetor nas células endoteliais de veia umbilical humana, submetidas ao estresse oxidativo, com diminuição da apoptose (caspase3) em concentrações mais baixas, citoproteção pela manutenção das funções celulares essenciais (mTOR) e sinalização da via de sobrevivência (SIRT1).

Safety and efficacy of a mucoadhesive phytomedication containing curcuminoids and Bidens pilosa L. extract in the prevention and treatment of radiochemotherapy-induced oral mucositis: Triple-blind, randomized, placebo-controlled, clinical trial.

BACKGROUND: Oral mucositis (OM) is the significant complication of radio/chemotherapy treatment. This study evaluated the safety and efficacy of a mucoadhesive phytomedication containing curcuminoids and Bidens pilosa L. (FITOPROT) in the prevention/treatment of OM. METHODS: Sixty-two patients were randomized into the group's intervention and placebo. Adverse effect assessment, OM grading, pain, and saliva collection were carried at the 1st, 15th, 21st, and final of radiotherapy (RT). Inflammatory salivary mediators were measured. RESULTS: FITOPROT decreased the severity of OM from the 15th to the final RT, while the placebo showed an increase in the severity (p < 0.05). Intervention group had a lower number of patients with ulcerated OM at the final RT (p < 0.05). Phytomedication prevented increases of IL-8 levels and reduced the salivary nitrite during RT. CONCLUSIONS: FITOPROT does not promote adverse effects, it appears to be effective at reducing the severity of OM, and it controls the concentration of pro-inflammatory mediators.

Biological effects of formulation containing curcuminoids and Bidens Pilosa L. in oral carcinoma cell line.

FITOPROT, which contains curcuminoids and Bidens pilosa L. extract, is an innovative mucoadhesive formulation indicated for the topical treatment of chemoradiotherapy-induced oral mucositis (OM) in patients with advanced and visible oral squamous cell carcinoma. The formulation is used as a mouthwash directly on tumor tissue of patients with advanced neoplasms, without triggering cancer cell proliferation or tumor invasiveness. Thus, the aim of this study was to evaluate the biological effects of FITOPROT on an oral squamous cell carcinoma cell line (SCC-4). The viability of SCC-4 cells was assessed after exposure to FITOPROT using MTT reduction assay. The effects of the mucoadhesive formulation on cell cycle progression and cell death parameters were evaluated using flow cytometry. In addition, the inflammatory profile of the tumor cells was evaluated using the cytometric bead array (CBA) assay. FITOPROT promoted a concentration-dependent decrease in cell viability and cell cycle arrest at the G2/M phase (p < 0.05). Mitochondrial membrane potential was also altered after exposure to the formulation (p < 0.05), in parallel with a reduction in VEGF and IL-8 production (p = 0.01 and p = 0.05, respectively). In summary, the results indicate that FITOPROT reduces SCC-4 cell viability, promotes cell cycle arrest, modulates mitochondrial membrane potential, and exhibits antiangiogenic and anti-inflammatory properties, thus indicating its potential for topical use in patients with OM and visible tumors in the mouth.

Nasal biocompatible powder of Geraniol oil complexed with cyclodextrins for neurodegenerative diseases: physicochemical characterization and in vivo evidences of nose to brain delivery.

Recently, many studies have shown that plant metabolites, such as geraniol (GER), may exert anti-inflammatory effects in neurodegenerative diseases and, in particular, Parkinson's disease (PD) models. Unfortunately, delivering GER to the CNS via nose-to-brain is not feasible due to its irritant effects on the mucosae. Therefore, in the present study ß-cyclodextrin (ßCD) and its hydrophilic derivative hydroxypropyl-beta-cyclodextrin (HPßCD) were selected as potential carriers for GER nose-to-brain delivery. Inclusion complexes were formulated and the biocompatibility with nasal mucosae and drug bioavailability into cerebrospinal fluid (CSF) were studied in rats. It has been demonstrated by DTA, FT-IR and NMR analyses that both the CDs were able to form 1:1 GER-CD complexes, arising long-term stable powders after the freeze-drying process. GER-HPßCD-5 and GER-ßCD-2 complexes exhibited comparable results, except for morphology and solubility, as demonstrated by SEM analysis and phase solubility study, respectively. Even though both complexes were able to directly and safely deliver GER to CNS, GER-ßCD-2 displayed higher ability in releasing GER in the CSF. In conclusion, ßCD complexes can be considered a very promising tool in delivering GER into the CNS via nose-to-brain route, preventing GER release into the bloodstream and ensuring the integrity of the nasal mucosa.

Innate and adaptive immune responses toward nanomedicines.

Since the commercialization of the first liposomes used for drug delivery, Doxil/Caelyx® and Myocet®, tremendous progress has been made in understanding interactions between nanomedicines and biological systems. Fundamental work at the interface of engineering and medicine has allowed nanomedicines to deliver therapeutic small molecules and nucleic acids more efficiently. While nanomedicines are used in oncology for immunotherapy or to deliver combinations of cytotoxics, the clinical successes of gene silencing approaches like patisiran lipid complexes (Onpattro®) have paved the way for a variety of therapies beyond cancer. In parallel, the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the potential of mRNA vaccines to develop immunization strategies at unprecedented speed. To rationally design therapeutic and vaccines, chemists, materials scientists, and drug delivery experts need to better understand how nanotechnologies interact with the immune system. This review presents a comprehensive overview of the innate and adaptative immune systems and emphasizes the intricate mechanisms through which nanomedicines interact with these biological functions.

Development and validation of a rapid RP-HPLC method for simultaneous quantification of paclitaxel and cetuximab in immunoliposomes.

The development of rational therapies against complex diseases, such as cancer, has increased in the past few years due to the advances of 'omics' technologies. Concomitantly, several efforts have been made to design sophisticated drug delivery systems in order to increase specificity and drug accumulation in tumor sites. The complexity of these drug delivery systems highlights the need for suitable analytical methods to determine encapsulation/conjugation efficiency of drugs and molecules responsible for the targeted delivery. Therefore, this study focuses on the development and validation of a RP-HPLC-DAD methodology for concurrent quantification of paclitaxel (PTX) and cetuximab (CTX) in immunoliposomes. Chromatographic separation was achieved using a wide pore C8 column, and a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in Milli-Q water/acetonitrile/isopropanol with a flow rate of 1 mL min-1. Drug peaks were fully separated and detected at 280 nm using UV detector. The method was validated according to ICH and FDA guidelines in terms of specificity and forced degradation studies, system suitability, linearity, limit of detection, limit of quantification, repeatability, intermediate precision, accuracy, robustness, and short-term stability. The developed method was linear over the concentration range of 37.5-150 µg mL-1 of PTX and 75-300 µg mL-1 of CTX. All parameters evaluated satisfied the acceptance criteria, according to both FDA and ICH guidelines. The applicability of the analytical method was assessed following the development of PTX-loaded immunoliposomes conjugated with CTX. Thus, the present study shows a novel, simple, stability-indicating and suitable method to quantify simultaneously PTX and CTX in immunoliposomes.

Role of the complement cascade in the biological fate of liposomes in rodents.

Nanomedicines, including liposomes, have been used to improve the clinical efficacy and safety of drugs. In some liposomal formulations, a hydrophilic polymer coating of poly(ethylene glycol) (PEG) is used to increase the circulation time. Understanding the biological mechanisms responsible for the clearance of PEGylated and non-PEGylated nanomedicines is necessary to develop better-performing materials. The purpose of this work is to explore the role of complement in the elimination of intravenously administered liposomes (PEGylated and non-PEGylated) in mice and rats. Here, the complement cascade was depleted by intraperitoneal injections of cobra venom factor (CVF) 12 and 24 hours before the intravenous injection of radiolabeled liposomes. In both mice and rats, non-PEGylated liposomes showed faster elimination than PEGylated liposomes. At a lipid dose of 20 mg kg-1, the abrogation of the complement cascade (in CVF group) did not alter the circulation time of either PEGylated or non-PEGylated liposomes. In contrast, at lower doses (2 mg kg-1), animals treated with CVF had slightly higher levels of circulating liposomes, especially during the 24 hours pharmacokinetic studies. The complement cascade seems to govern the uptake of non-PEGylated liposomes by splenic B cells. Altogether, these results suggest that although PEGylated and non-PEGylated liposomes can activate complement, the impact of this cascade on their circulation time is minor and mostly perceivable at later phases of distribution. This work enlightens biological pathways responsible for in vivo clearance of liposomes and will help in orienting future research in elucidating the nano-bio interface.

Nose-to-brain drug delivery mediated by polymeric nanoparticles: influence of PEG surface coating.

Intranasal administration of mucus-penetrating nanoparticles is an emerging trend to increase drug delivery to the brain. In order to overcome rapid nasal mucociliary clearance, low epithelial permeation, and local enzymatic degradation, we investigated the influence of PEGylation on nose-to-brain delivery of polycaprolactone (PCL) nanoparticles (PCL-NPs) encapsulating bexarotene, a potential neuroprotective compound. PEGylation with 1, 3, 5, and 10% PCL-PEG did not affect particle diameter or morphology. Upon incubation with artificial nasal mucus, only 5 and 10% of PCL-PEG coating were able to ensure NP stability and homogeneity in mucus. Rapid mucus-penetrating ability was observed for 98.8% of PCL-PEG5% NPs and for 99.5% of PCL-PEG10% NPs. Conversely, the motion of non-modified PCL-NPs was markedly slower. Fluorescence microscopy showed that the presence of PEG on NP surface did not reduce their uptake by RMPI 2650 cells. Fluorescence tomography images evidenced higher translocation into the brain for PCL-PEG5% NPs. Bexarotene loaded into PCL-PEG5% NPs resulted in area under the curve in the brain (AUCbrain) 3 and 2-fold higher than that for the drug dispersion and for non-PEGylated NPs (p < 0.05), indicating that approximately 4% of the dose was directly delivered to the brain. Combined, these results indicate that PEGylation of PCL-NPs with PCL-PEG5% is able to reduce NP interactions with the mucus, leading to a more efficient drug delivery to the brain following intranasal administration. Graphical abstract.

In Vivo Biodistribution of Respirable Solid Lipid Nanoparticles Surface-Decorated with a Mannose-Based Surfactant: A Promising Tool for Pulmonary Tuberculosis Treatment?

The active targeting to alveolar macrophages (AM) is an attractive strategy to improve the therapeutic efficacy of 'old' drugs currently used in clinical practice for the treatment of pulmonary tuberculosis. Previous studies highlighted the ability of respirable solid lipid nanoparticle assemblies (SLNas), loaded with rifampicin (RIF) and functionalized with a novel synthesized mannose-based surfactant (MS), both alone and in a blend with sodium taurocholate, to efficiently target the AM via mannose receptor-mediated mechanism. Here, we present the in vivo biodistribution of these mannosylated SLNas, in comparison with the behavior of both non-functionalized SLNas and bare RIF. SLNas biodistribution was assessed, after intratracheal instillation in mice, by whole-body real-time fluorescence imaging in living animals and RIF quantification in excised organs and plasma. Additionally, SLNas cell uptake was determined by using fluorescence microscopy on AM from bronchoalveolar lavage fluid and alveolar epithelium from lung dissections. Finally, histopathological evaluation was performed on lungs 24 h after administration. SLNas functionalized with MS alone generated the highest retention in lungs associated with a poor spreading in extra-pulmonary regions. This effect could be probably due to a greater AM phagocytosis with respect to SLNas devoid of mannose on their surface. The results obtained pointed out the unique ability of the nanoparticle surface decoration to provide a potential more efficient treatment restricted to the lungs where the primary tuberculosis infection is located.

Nasal administration of nanoencapsulated geraniol/ursodeoxycholic acid conjugate: Towards a new approach for the management of Parkinson's disease.

The combined use of different therapeutic agents in the treatment of neurodegenerative disorders is a promising strategy to halt the disease progression. In this context, we aimed to combine the anti-inflammatory properties of geraniol (GER) with the mitochondrial rescue effects of ursodeoxycholic acid (UDCA) in a newly-synthesized prodrug, GER-UDCA, a potential candidate against Parkinson's disease (PD). GER-UDCA was successfully synthetized and characterized in vitro for its ability to release the active compounds in physiological environments. Because of its very poor solubility, GER-UDCA was entrapped into both lipid (SLNs) and polymeric (NPs) nanoparticles in order to explore nose-to-brain pathway towards brain targeting. Both GER-UDCA nanocarriers displayed size below 200 nm, negative zeta potential and the ability to increase the aqueous dissolution rate of the prodrug. As SLNs exhibited the higher GER-UDCA dissolution rate, this formulation was selected for the in vivo GER-UDCA brain targeting experiments. The nasal administration of GER-UDCA-SLNs (1 mg/kg of GER-UDCA) allowed to detect the prodrug in rat cerebrospinal fluid (concentration range = 1.1 to 4.65 µg/mL, 30-150 min after the administration), but not in the bloodstream, thus suggesting the direct nose to brain delivery of the prodrug. Finally, histopathological evaluation demonstrated that, in contrast to the pure GER, nasal administration of GER-UDCA-SLNs did not damage the structural integrity of the nasal mucosa. In conclusion, the present data suggest that GER-UDCA-SLNs could provide an effective and non-invasive approach to boost the access of GER and UDCA to the brain with low dosages.

In vivo and in vitro toxicity evaluation of liposome-encapsulated sirolimus.

BACKGROUND: To evaluate the in vivo and in vitro toxicity of a new formulation of liposome-encapsulated sirolimus (LES). METHODS: In vitro experiments were done using ARPE-19 and HRP cells. An MTT assay was used to determine cell metabolic activity and a TUNEL assay for detecting DNA fragmentation. In vivo experiments were conducted on New Zealand albino rabbits that received intravitreal injections of empty liposomes (EL) or different concentrations of LES. Histopathological and immunohistochemical analyses were performed on the rabbit's eyes following injection. RESULTS: Eighteen eyes of nine rabbits were used. MTT assay cell viability was 95.04% in group 1 (12.5 µL/mL LES). 92.95% in group 2 (25 µL/mL LES), 91.59% in group 3 (50 µL/mL LES), 98.09% in group 4 (12.5 µL/mL EL), 95.20% on group 5 (50 µL/mL EL), 98.53% in group 6 (50 µL/mL EL), and 2.84% on group 8 (50 µL/mL DMSO). There was no statistically significant difference among groups 1 to 7 in cell viability (p = 1.0), but the comparison of all groups with group 8 was significant (p < 0.0001). The TUNEL assay comparing two groups was not statistically significant from groups 1 to 7 (p = 1.0). The difference between groups 1 to 7 and group 8 (p < 0.0001) was significant. Histopathological changes were not found in any group. No activation of Müller cells was detected. CONCLUSION: A novel formulation of LES delivered intravitreally did not cause in vitro toxicity, as evaluated by MTT and TUNEL assays, nor in vivo toxicity as evaluated by histopathology and immunohistochemistry in rabbit eyes.

Impact of drug loading in mesoporous silica-amorphous formulations on the physical stability of drugs with high recrystallization tendency.

In this study, a method is described to determine the monolayer loading capacity (MLC) of the drugs naproxen and ibuprofen, both having high recrystallization tendencies, in mesoporous silica (MS), a well known carrier that is able to stabilize the amorphous form of a drug. The stabilization has been suggested to be due to direct absorption of the drug molecules onto the MS surface, i.e. the drug monolayer. In addition, drug that is not in direct contact with MS surface can fill the pores up to its pore filling capacity (PFC) and is potentially stabilized by confinement due to the pore size being smaller than a crystal nuclei. For drugs with high recrystallization tendencies, any drug outside the pores crystallizes due to its poor physical stability. The drug monolayer does not contribute to the glass transition temperature (Tg ) in the DSC, however, the confined amorphous drug above MLC has a Tg and the heat capacity (ΔC p) over the Tg increases with an increasing fraction of confined amorphous drug. Hence, several drug loading values above the MLC were investigated towards the presence of a Tg and ΔC p using differential scanning calorimetry (DSC). A linear correlation between the amount of confined amorphous drug and its ΔC p was identified for the mixtures between the MLC and PFC. By subsequent extrapolation to zero ΔC p the experimental MLC could be determined. Using theoretical density functional theory (DFT) and ab initio Molecular Dynamics (AIMD), the binding energies for the monolayer suggested that the monolayer in fact is thermodynamically more favorable than the crystalline form, whereas the confined amorphous form is thermodynamically less favorable. Consequently, a physical stability study showed that the confined amorphous drugs above the MLC were thermodynamically unstable and consequently flowing out of the pores in order to crystallize, whereas the monolayer remained physically stable.

Increased Nose-to-Brain Delivery of Melatonin Mediated by Polycaprolactone Nanoparticles for the Treatment of Glioblastoma.

PURPOSE: Intranasal administration has been extensively applied to deliver drugs to the brain. In spite of its unfavorable biopharmaceutic properties, melatonin (MLT) has demonstrated anticancer effects against glioblastoma. This study describes the nose-to-brain delivery of MLT-loaded polycaprolactone nanoparticles (MLT-NP) for the treatment of glioblastoma. METHODS: MLT-NP were prepared by nanoprecipitation. Following intranasal administration in rats, brain targeting of the formulation was demonstrated by fluorescence tomography. Brain and plasma pharmacokinetic profiles were analyzed. Cytotoxicity against U87MG glioblastoma cells and MRC-5 non-tumor cells was evaluated. RESULTS: MLT-NP increased the drug apparent water solubility ~35 fold. The formulation demonstrated strong activity against U87MG cells, resulting in IC50 ~2500 fold lower than that of the free drug. No cytotoxic effect was observed against non-tumor cells. Fluorescence tomography images evidenced the direct translocation of nanoparticles from nasal cavity to the brain. Intranasal administration of MLT-NP resulted in higher AUCbrain and drug targeting index compared to the free drug by either intranasal or oral route. CONCLUSIONS: Nanoencapsulation of MLT was crucial for the selective antitumoral activity against U87MG. In vivo evaluation confirmed nose-to-brain delivery of MLT mediated by nanoparticles, highlighting the formulation as a suitable approach to improve glioblastoma therapy.