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.

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.

DoE development of ionic gradient liposomes: A successful approach to improve encapsulation, prolong anesthesia and decrease the toxicity of etidocaine.

Etidocaine (EDC) is a long-acting local anesthetic of the aminoamide family whose use was discontinued in 2008 for alleged toxicity issues. Ionic gradient liposomes (IGL) are nanostructured carriers for which an inner/outer gradient of ions increases drug upload. This work describes IGLEDC, a formulation optimized by Design of Experiments, composed of hydrogenated soy phosphatidylcholine:cholesterol:EDC, and characterized by DLS, NTA, TEM/Cryo-TEM, DSC and 1H NMR. The optimized IGL showed significant encapsulation efficiency (41 %), good shelf stability (180 days) and evidence of EDC interaction with the lipid bilayer (as seen by DSC and 1H NMR results) that confirms its membrane permeation. In vitro (release kinetics and cytotoxicity) tests showed that the encapsulation of EDC into the IGL promoted sustained release for 24 h and decreased by 50 % the intrinsic toxicity of EDC to Schwann cells. In vivo IGLEDC decreased the toxicity of EDC to Caenorhabditis elegans by 25 % and extended its anesthetic effect by one hour, after infiltrative administration, at clinically used (0.5 %) concentration, in rats. Thus, this novel drug delivery system is a promise for the possible reintroduction of EDC in clinics, aiming at the control of operative and postoperative pain.

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.

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.

Hybrid Pectin-Liposome Formulation against Multi-Resistant Bacterial Strains.

This work describes the development of a gastroresistant antimicrobial formulation composed of two carriers, pectin and liposomes, intended to improve the efficiency of norfloxacin (NOR) against multi-resistant bacterial strains. The formulations showed physicochemical stability for 180 days (4 °C) in terms of size, polydispersity, and zeta potential of the vesicles, prolonging the in vitro release of NOR for 11 h. The hybrid nanocarriers improved the in vitro antimicrobial activity against different multidrug-resistant bacterial strains, such as Salmonella sp., Pseudomonasaeruginosa, E. coli and Campylobacterjejuni, in comparison to commercial NOR and liposomal suspensions. The in vivo toxicity assay in chicken embryos revealed that the hybrid systems were not toxic in any of the different parameters analyzed, a result also corroborated by the analyses of biochemical biomarkers of the chicken-embryos liver function.

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes.

BACKGROUND: Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. METHODS: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. RESULTS: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. CONCLUSIONS: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.

Nanocarriers From Natural Lipids With In Vitro Activity Against Campylobacter jejuni.

Campylobacter jejuni (CJ) is the most prevalent zoonotic pathogen of chicken meat and related products, which may lead to gastroenteritis and autoimmune diseases in humans. Although controlling this bacterium is important, CJ strains resistance against traditional antibiotic therapy has been increased. Vegetable oils and fats are natural biomaterials explored since the Ancient times, due to their therapeutic properties. Nanotechnology has promoted the miniaturization of materials, improving bioavailability and efficacy, while reducing the toxicity of loaded active molecules. In this work, a screening of 28 vegetable oils was firstly performed, in order to select anti-CJ candidates by the disc diffusion test. Thus, the selected liquid lipids were used as active molecules in nanostructured lipid carriers (NLC) formulations. The three resultant systems were characterized in terms of particle size (~200 nm), polydispersity index (~0.15), and zeta potential (~-35mV), and its physicochemical stability was confirmed for a year, at 25°C. The structural properties of NLC were assessed by infrared (FTIR-ATR) and differential scanning calorimetry (DSC) analyses. The spherical nanoparticle morphology and narrow size distribution was observed by transmission electron microscopy (TEM) and field emission scanning electron (FE-SEM) analyses, respectively. Then, the in vitro antimicrobial activity test determined the minimum inhibitory concentration (MIC) of each formulation against CJ strains, in both free (1-3 mg/ml-1) and sessile (0.78 mg/ml-1) forms. Finally, the in vitro biocompatibility of NLC was demonstrated through cell viability using VERO cell line, in which F6 was found twice less cytotoxic than pure olibanum oil. Considering the abovementioned achieved, F6 formulation is able to be evaluated in the in vivo anti-CJ efficacy assays.

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes

Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. Methods: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. Results: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. Conclusions: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.(AU)

Fusogenic Liposomes Increase the Antimicrobial Activity of Vancomycin Against Staphylococcus aureus Biofilm.

Objective: The aim of the present study was to encapsulate vancomycin in different liposomal formulations and compare the in vitro antimicrobial activity against Staphylococcus aureus biofilms. Methods: Large unilamellar vesicles of conventional (LUV VAN), fusogenic (LUVfuso VAN), and cationic (LUVcat VAN) liposomes encapsulating VAN were characterized in terms of size, polydispersity index, zeta potential, morphology, encapsulation efficiency (%EE) and in vitro release kinetics. The formulations were tested for their Minimum Inhibitory Concentration (MIC) and inhibitory activity on biofilm formation and viability, using methicillin-susceptible S. aureus ATCC 29213 and methicillin-resistant S. aureus ATCC 43300 strains. Key Findings: LUV VAN showed better %EE (32.5%) and sustained release than LUVfuso VAN, LUVcat VAN, and free VAN. The formulations were stable over 180 days at 4°C, except for LUV VAN, which was stable up to 120 days. The MIC values for liposomal formulations and free VAN ranged from 0.78 to 1.56 µg/ml against both tested strains, with no difference in the inhibition of biofilm formation as compared to free VAN. However, when treating mature biofilm, encapsulated LUVfuso VAN increased the antimicrobial efficacy as compared to the other liposomal formulations and to free VAN, demonstrating a better ability to penetrate the biofilm. Conclusion: Vancomycin encapsulated in fusogenic liposomes demonstrated enhanced antimicrobial activity against mature S. aureus biofilms.

Lipid-based carriers for the delivery of local anesthetics.

INTRODUCTION: There is a clinical need for pharmaceutical dosage forms devised to prolong the acting time of local anesthetic (LA) agents or to reduce their toxicity. Encapsulation of LA in drug delivery systems (DDSs) can provide long-term anesthesia for inpatients (e.g. in immediate postsurgical pain control, avoiding the side effects from systemic analgesia) and diminished systemic toxicity for outpatients (in ambulatory/dentistry procedures). The lipid-based formulations described here, such as liposomes, microemulsions, and lipid nanoparticles, have provided several nanotechnological advances and therapeutic alternatives despite some inherent limitations associated with the fabrication processes, costs, and preclinical evaluation models. AREAS COVERED: A description of the currently promising lipid-based carriers, including liposomes, microemulsions, and nanostructured lipid carriers, followed by a systematic review of the existing lipid-based formulations proposed for LA. Trends in the research of these LA-in-DDS are then exposed, from the point of view of administration route and alternatives for non-traditionally administered LA molecules. EXPERT OPINION: Considering the current state and potential future developments in the field, we discuss the reasons for why dozens of formulations published every year fail to reach clinical trials; only one lipid-based formulation for the delivery of local anesthetic (Exparel®) has been approved so far.

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.

Rational design of polymer-lipid nanoparticles for docetaxel delivery.

In this work, a stable nanocarrier for the anti-cancer drug docetaxel was rational designed. The nanocarrier was developed based on the solid lipid nanoparticle preparation process aiming to minimize the total amount of excipients used in the final formulations. A particular interest was put on the effects of the polymers in the final composition. In this direction, two poloxoamers -Pluronic F127 and F68- were selected. Some poloxamers are well known to be inhibitors of the P-glycoprotein efflux pump. Additionally, their poly-ethylene-oxide blocks can help them to escape the immune system, making the poloxamers appealing to be present in a nanoparticle designed for the treatment of cancer. Within this context, a factorial experiment design was used to achieve the most suitable formulations, and also to identify the effects of each component on the final (optimized) systems. Two final formulations were chosen with sizes < 250 nm and PDI < 0.2. Then, using dynamic light scattering and nanotracking techniques, the stability of the formulations was assessed during six months. Structural studies were carried on trough different techniques: DSC, x-ray diffraction, FTIR-AR and Molecular Dynamics. The encapsulation efficiency of the anticancer drug docetaxel (> 90%) and its release dynamics from formulations were measured, showing that the polymer-lipid nanoparticle is suitable as a drug delivery system for the treatment of cancer.

Hybrid Hydrogel Composed of Polymeric Nanocapsules Co-Loading Lidocaine and Prilocaine for Topical Intraoral Anesthesia.

This study reports the development of nanostructured hydrogels for the sustained release of the eutectic mixture of lidocaine and prilocaine (both at 2.5%) for intraoral topical use. The local anesthetics, free or encapsulated in poly(ε-caprolactone) nanocapsules, were incorporated into CARBOPOL hydrogel. The nanoparticle suspensions were characterized in vitro in terms of particle size, polydispersity, and surface charge, using dynamic light scattering measurements. The nanoparticle concentrations were determined by nanoparticle tracking analysis. Evaluation was made of physicochemical stability, structural features, encapsulation efficiency, and in vitro release kinetics. The CARBOPOL hydrogels were submitted to rheological, accelerated stability, and in vitro release tests, as well as determination of mechanical and mucoadhesive properties, in vitro cytotoxicity towards FGH and HaCaT cells, and in vitro permeation across buccal and palatal mucosa. Anesthetic efficacy was evaluated using Wistar rats. Nanocapsules were successfully developed that presented desirable physicochemical properties and a sustained release profile. The hydrogel formulations were stable for up to 6 months under critical conditions and exhibited non-Newtonian pseudoplastic flows, satisfactory mucoadhesive strength, non-cytotoxicity, and slow permeation across oral mucosa. In vivo assays revealed higher anesthetic efficacy in tail-flick tests, compared to a commercially available product. In conclusion, the proposed hydrogel has potential for provision of effective and longer-lasting superficial anesthesia at oral mucosa during medical and dental procedures. These results open perspectives for future clinical trials.

Nanohybrid hydrogels designed for transbuccal anesthesia.

BACKGROUND: Local anesthesia in dentistry is by far the most terrifying procedure for patients, causing treatment interruption. None of the commercially available topical formulations is effective in eliminating the pain and phobia associated to the needle insertion and injection. MATERIALS AND METHODS: In this work we prepared a nanostructured lipid-biopolymer hydrogel for the sustained delivery of lidocaine-prilocaine (LDC-PLC) for transbuccal pre-anesthesia. The lipid was composed of optimized nanostructured lipid carriers (NLC) loaded with 5% LDC-PLC (NLC/LDC-PLC). The biopolymer counterpart was selected among alginate, xanthan (XAN), and chitosan matrices. The XAN-NLC hydrogel presented the most uniform aspect and pseudoplastic rheological profile, as required for topical use; therefore, it was selected for subsequent analyses. Accelerated stability tests under critical conditions (40°C; 75% relative humidity) were conducted for 6 months, in terms of drug content (mg/g), weight loss (%), and pH. RESULTS: In vitro LDC-PLC release profile through Franz diffusion cells revealed a bimodal kinetics with a burst effect followed by the sustained release of both anesthetics, for 24 hours. Structural analyses (fourier transform infrared spectroscopy, differential scanning calorimetry and scanning electron microscopy) gave details on the molecular organization of the hybrid hydrogel, confirming the synergic interaction between the components. Safety and efficacy were evaluated through in vitro cell viability (3T3, HaCat, and VERO cells) and in vivo antinociceptive (tail-flick, in mice) tests, respectively. In comparison to a control hydrogel and the eutectic mixture of 5% LDC-PLC cream (EMLA®), the XAN-NLC/LDC-PLC hybrid hydrogel doubled and quadrupled the anesthetic effect (8 hours), respectively. CONCLUSION: Considering such exciting results, this multifaceted nanohybrid system is now ready to be further tested in clinical trials.

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.

Use of nanoparticle concentration as a tool to understand the structural properties of colloids.

Elucidation of the structural properties of colloids is paramount for a successful formulation. However, the intrinsic dynamism of colloidal systems makes their characterization a difficult task and, in particular, there is a lack of physicochemical techniques that can be correlated to their biological performance. Nanoparticle tracking analysis (NTA) allows measurements of size distribution and nanoparticle concentration in real time. Its analysis over time also enables the early detection of physical instability in the systems not assessed by subtle changes in size distribution. Nanoparticle concentration is a parameter with the potential to bridge the gap between in vitro characterization and biological performance of colloids, and therefore should be monitored in stability studies of formulations. To demonstrate this, we have followed two systems: extruded liposomes exposed to increasing CHCl3 concentrations, and solid lipid nanoparticles prepared with decreasing amounts of poloxamer 188. NTA and dynamic light scattering (DLS) were used to monitor changes in nanoparticle number and size, and to estimate the number of lipid components per particle. The results revealed a strong negative correlation between particle size (determined by DLS) and concentration (assessed by NTA) in diluted samples, which should be adopted to monitor nanocolloidal stability, especially in drug delivery.

Encapsulation of ropivacaine in a combined (donor-acceptor, ionic-gradient) liposomal system promotes extended anesthesia time.

Ropivacaine is a local anesthetic with similar potency but lower systemic toxicity than bupivacaine, the most commonly used spinal anesthetic. The present study concerns the development of a combined drug delivery system for ropivacaine, comprised of two types of liposomes: donor multivesicular vesicles containing 250 mM (NH4)2SO4 plus the anesthetic, and acceptor large unilamellar vesicles with internal pH of 5.5. Both kinds of liposomes were composed of hydrogenated soy-phosphatidylcholine:cholesterol (2:1 mol%) and were prepared at pH 7.4. Dynamic light scattering, transmission electron microscopy and electron paramagnetic resonance techniques were used to characterize the average particle size, polydispersity, zeta potential, morphology and fluidity of the liposomes. In vitro dialysis experiments showed that the combined liposomal system provided significantly longer (72 h) release of ropivacaine, compared to conventional liposomes (~45 h), or plain ropivacaine (~4 h) (p <0.05). The pre-formulations tested were significantly less toxic to 3T3 cells, with toxicity increasing in the order: combined system < ropivacaine in donor or acceptor liposomes < ropivacaine in conventional liposomes < plain ropivacaine. The combined formulation, containing 2% ropivacaine, increased the anesthesia duration up to 9 h after subcutaneous infiltration in mice. In conclusion, a promising drug delivery system for ropivacaine was described, which can be loaded with large amounts of the anesthetic (2%), with reduced in vitro cytotoxicity and extended anesthesia time.

Recent advances and perspectives in topical oral anesthesia.

INTRODUCTION: Topical anesthesia is widely used in dentistry to reduce pain caused by needle insertion and injection of the anesthetic. However, successful anesthesia is not always achieved using the formulations that are currently commercially available. As a result, local anesthesia is still one of the procedures that is most feared by dental patients. Drug delivery systems (DDSs) provide ways of improving the efficacy of topical agents. Areas covered: An overview of the structure and permeability of oral mucosa is given, followed by a review of DDSs designed for dental topical anesthesia and their related clinical trials. Chemical approaches to enhance permeation and anesthesia efficacy, or to promote superficial anesthesia, include nanostructured carriers (liposomes, cyclodextrins, polymeric nanoparticle systems, solid lipid nanoparticles, and nanostructured lipid carriers) and different pharmaceutical dosage forms (patches, bio- and mucoadhesive systems, and hydrogels). Physical methods include pre-cooling, vibration, iontophoresis, and microneedle arrays. Expert opinion: The combination of different chemical and physical methods is an attractive option for effective topical anesthesia in oral mucosa. This comprehensive review should provide the readers with the most relevant options currently available to assist pain-free dental anesthesia. The findings should be considered for future clinical trials.