Qualitative Histologic Assessment vs. Geomorphometric Analysis of Nerve Fiber Shape after the Intraneural Application of Liposomal Bupivacaine / Evaluación Histológica Cualitativa Versus Análisis Geomorfométrico de la Forma de la Fibra Nerviosa Después de la Aplicación Intraneural de Bupivacaína Liposomal

SUMMARY: The preserved form of all components of the nerve fiber is a prerequisite for the proper conduction of the nerve impulse. various factors can change the shape of nerve fibers. In everyday practice, qualitative histological analysis is the gold standard for detecting changes in shape. Geometric morphometry is an innovative method that objectively enables the assessment of changes in nerve fibers' shape after local anesthetics action. A total of sixty sciatic nerves were used as material, which was intraneural injected with saline solution in the control group (n=30), and a solution of 1.33 % liposomal bupivacaine (n=30) in the test group. After the animals were sacrificed, nerve samples were taken and histological preparations were made. The preparations were first described and examined using a qualitative histological method, after which digital images were made. The images were entered into the MorphoJ program and processed using the method of geometric morphometry. Qualitative histological examination revealed no differences in nerve fibers after intraneurally applied physiological solution and liposomal bupivacaine. Using the method of geometric morphometry, a statistically significant change in the shape of axons was found after intraneurally applied saline solution and liposomal bupivacaine (p=0.0059). No significant differences in histological changes were found after the qualitative histological analysis of nerve fiber cross-section preparations. A statistically significant change in the shape of nerve fiber axons was observed after geometric morphometric analysis of digital images after intraneural application of saline and liposomal bupivacaine.

La forma conservada de todos los componentes de la fibra nerviosa es un requisito previo para la conducción correcta del impulso nervioso. Varios factores pueden cambiar la forma de las fibras nerviosas. En la práctica diaria, el análisis histológico cualitativo es el estándar de oro para detectar cambios de forma. La morfometría geométrica es un método innovador que permite evaluar objetivamente los cambios en la forma de las fibras nerviosas después de la acción de los anestésicos locales. Se utilizó como material un total de sesenta nervios ciáticos, que se inyectaron intraneuralmente con solución salina en el grupo control (n=30), y una solución de bupivacaína liposomal al 1,33 % (n=30) en el grupo de prueba. Después de sacrificados los animales, se tomaron muestras de nervios y se realizaron preparaciones histológicas. Primero se describieron y examinaron las preparaciones utilizando un método histológico cualitativo, después de lo cual se tomaron imágenes digitales. Las imágenes fueron ingresadas al programa MorphoJ y procesadas mediante el método de morfometría geométrica. El examen histológico cualitativo no reveló diferencias en las fibras nerviosas después de la aplicación intraneural de solución fisiológica y bupivacaína liposomal. Usando el método de morfometría geométrica, se encontró un cambio estadísticamente significativo en la forma de los axones después de la aplicación intraneural de solución salina y bupivacaína liposomal (p = 0,0059). No se encontraron diferencias significativas en los cambios histológicos después del análisis histológico cualitativo de las preparaciones de secciones transversales de fibras nerviosas. Se observó un cambio estadísticamente significativo en la forma de los axones de las fibras nerviosas después del análisis de morfometría geométrica de imágenes digitales después de la aplicación intraneural de solución salina y bupivacaína liposomal.

Inhalation of dimethyl fumarate-encapsulated solid lipid nanoparticles attenuate clinical signs of experimental autoimmune encephalomyelitis and pulmonary inflammatory dysfunction in mice.

RATIONALE: The FDA-approved Dimethyl Fumarate (DMF) as an oral drug for Multiple Sclerosis (MS) treatment based on its immunomodulatory activities. However, it also caused severe adverse effects mainly related to the gastrointestinal system. OBJECTIVE: Investigated the potential effects of solid lipid nanoparticles (SLNs) containing DMF, administered by inhalation on the clinical signs, central nervous system (CNS) inflammatory response, and lung function changes in mice with experimental autoimmune encephalomyelitis (EAE). MATERIALS AND METHODS: EAE was induced using MOG35-55 peptide in female C57BL/6J mice and the mice were treated via inhalation with DMF-encapsulated SLN (CTRL/SLN/DMF and EAE/SLN/DMF), empty SLN (CTRL/SLN and EAE/SLN), or saline solution (CTRL/saline and EAE/saline), every 72 h during 21 days. RESULTS: After 21 days post-induction, EAE mice treated with DMF-loaded SLN, when compared with EAE/saline and EAE/SLN, showed decreased clinical score and weight loss, reduction in brain and spinal cord injury and inflammation, also related to the increased influx of Foxp3+ cells into the spinal cord and lung tissues. Moreover, our data revealed that EAE mice showed signs of respiratory disease, marked by increased vascular permeability, leukocyte influx, production of TNF-α and IL-17, perivascular and peribronchial inflammation, with pulmonary mechanical dysfunction associated with loss of respiratory volumes and elasticity, which DMF-encapsulated reverted in SLN nebulization. CONCLUSION: Our study suggests that inhalation of DMF-encapsulated SLN is an effective therapeutic protocol that reduces not only the CNS inflammatory process and disability progression, characteristic of EAE disease, but also protects mice from lung inflammation and pulmonary dysfunction.

Lipid-Based Nanostructures for the Delivery of Natural Antimicrobials.

Encapsulation can be a suitable strategy to protect natural antimicrobial substances against some harsh conditions of processing and storage and to provide efficient formulations for antimicrobial delivery. Lipid-based nanostructures, including liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid nanocarriers (NLCs), are valuable systems for the delivery and controlled release of natural antimicrobial substances. These nanostructures have been used as carriers for bacteriocins and other antimicrobial peptides, antimicrobial enzymes, essential oils, and antimicrobial phytochemicals. Most studies are conducted with liposomes, although the potential of SLNs and NLCs as antimicrobial nanocarriers is not yet fully established. Some studies reveal that lipid-based formulations can be used for co-encapsulation of natural antimicrobials, improving their potential to control microbial pathogens.

Gelatin-based membrane containing usnic acid-loaded liposomes: A new treatment strategy for corneal healing.

PURPOSE: To evaluate the safety and potential healing efficacy of the topical ocular administration of a gelatin membrane containing usnic acid/liposomes (UALs) for corneal cicatrization. UALs have shown healing activity in animal models of dermal burn lesions. We evaluated the safety of topical ocular administration of UAL and its potential healing efficacy as an ophthalmic treatment on chemical lesions in rabbit eyes. METHOD: The Draize test was used to check for ocular toxicity and the score was zero at each observation, indicating the ocular safety of a gelatin membrane containing usnic acid/liposome. Its potential healing efficacy as an ophthalmic treatment on chemical lesions in rabbit eyes was also assessed. RESULTS: After epithelial removal and treatment with UAL, there was a 49.4 % reduction in injury under in vivo conditions compared with a 36.6 % reduction in the control, a gelatin membrane containing liposome without usnic acid. Histological analysis of ocular surface chemical injury-tissue sections after treatment with UAL supported these observations. The corneal expression of VEGF and TGF-ß1increased by 70 % and 50 % respectively following treatment with UAL gelatin membrane. CONCLUSION: These results indicate the potential therapeutic application of UAL gelatin membranes as an ophthalmic treatment that may be used for corneal cicatrization.

Topical Triamcinolone Acetonide-Loaded Liposomes as Primary Therapy for Macular Edema Secondary to Branch Retinal Vein Occlusion: A Pilot Study.

Purpose: To explore safety and therapeutic efficacy of a topical ophthalmic triamcinolone acetonide-loaded liposome formulation (TA-LF) as primary therapy in patients with macular edema (ME) secondary to branch retinal vein occlusion (BRVO). Methods: Twelve eyes of 12 patients with ME secondary to BRVO were exposed to a topical instillation of 1 drop of TA-LF (TA 0.2%) 6 times a day for 12 weeks to evaluate safety and efficacy. Best corrected visual acuity (BCVA) intraocular pressure (IOP), slit lamp examination, and central foveal thickness (CFT) were analyzed at every visit. In addition, the morphology of TA-LF was analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results: Patients presented a significant improvement of BCVA and CFT without significant IOP modification (P = 0.94). Treated eyes showed BCVA improvement from 40 ± 12.05 to 64.83 ± 15.97 letters and CFT reduction from 682.91 ± 278.60 to 271.58 ± 57.66 µm after 12 weeks of TA-LF therapy (P < 0.001). No adverse events, including IOP rising, were registered. SEM analysis of liposomal formulations showed that liposome (LP) size depends on its concentration. As the concentration of TA increased, the average size of LPs and the number of larger particles increased as well. TEM study displayed that LP formulation efficiently solubilizes TA crystals in nanoparticles and encapsulates them. Conclusion: LPs can function as nanocarriers of TA and they could be used as topical ophthalmic primary therapy instead of intravitreal drugs in patients with ME secondary to BRVO.

Brain Targeted Gold Liposomes Improve RNAi Delivery for Glioblastoma.

INTRODUCTION: Glioblastoma (GBM) is the most common and lethal of the central nervous system (CNS) malignancies. The initiation, progression, and infiltration ability of GBMs are attributed in part to the dysregulation of microRNAs (miRNAs). Thus, targeting dysregulated miRNAs with RNA oligonucleotides (RNA interference, RNAi) has been proposed for GBM treatment. Despite promising results in the laboratory, RNA oligonucleotides have clinical limitations that include poor RNA stability and off-target effects. RNAi therapies against GBM confront an additional obstacle, as they need to cross the blood-brain barrier (BBB). METHODS: Here, we developed gold-liposome nanoparticles conjugated with the brain targeting peptides apolipoprotein E (ApoE) and rabies virus glycoprotein (RVG). First, we functionalized gold nanoparticles with oligonucleotide miRNA inhibitors (OMIs), creating spherical nucleic acids (SNAs). Next, we encapsulated SNAs into ApoE, or RVG-conjugated liposomes, to obtain SNA-Liposome-ApoE and SNA-Liposome-RVG, respectively. We characterized each nanoparticle in terms of their size, charge, encapsulation efficiency, and delivery efficiency into U87 GBM cells in vitro. Then, they were administered intravenously (iv) in GBM syngeneic mice to evaluate their delivery efficiency to brain tumor tissue. RESULTS: SNA-Liposomes of about 30-50 nm in diameter internalized U87 GBM cells and inhibited the expression of miRNA-92b, an aberrantly overexpressed miRNA in GBM cell lines and GBM tumors. Conjugating SNA-Liposomes with ApoE or RVG peptides increased their systemic delivery to the brain tumors of GBM syngeneic mice. SNA-Liposome-ApoE demonstrated to accumulate at higher extension in brain tumor tissues, when compared with non-treated controls, SNA-Liposomes, or SNA-Liposome-RVG. DISCUSSION: SNA-Liposome-ApoE has the potential to advance the translation of miRNA-based therapies for GBM as well as other CNS disorders.

Novel Triamcinolone Acetonide-Loaded Liposomal Topical Formulation Improves Contrast Sensitivity Outcome After Femtosecond Laser-Assisted Cataract Surgery.

Purpose: To assess visual results, macular modifications, and the incidence of clinically significant macular edema (CSME) in patients using a topical triamcinolone acetonide-loaded liposomal formulation (TA-LF) after femtosecond laser-assisted cataract surgery (FLACS). Methods: Fifty-six eyes after FLACS were selected. Twenty-eight eyes in the combined therapy group (P + N) were treated with prednisolone 1% and nepafenac 0.1% for 21 days postoperatively, whereas 28 eyes in the TA-LF group received a liposomal formulation containing 2 mg/mL of TA (0.2%) for the same period of time. Follow-up visits at 1 day, 6 weeks, and 12 weeks after surgery consisted of visual acuity, contrast sensitivity (CS), central foveal thickness (CFT), total macular volume (TMV) measurements, and the detection of CSME. Results: CS improved in the TA-LF group (basal value: 1.087 ± 0.339 vs. 1.276 ± 0.147 at week 12, P = 0.0346), whereas in the P + N group, CS was not different from the baseline (basal value: 1.130 ± 0.331 vs. 1.274 ± 0.133 at week 12, P = 0.1276). There were similar increases in postoperative CFT and TMV in both groups. CFT and TMV significantly correlate with CS only in the TA-LF group. The r2 for CFT and CS was 0.1963 (P = 0.0206), whereas the r2 for TMV and CS was 0.3615 (P = 0.0007) at 12 weeks. No difference was observed in the incidence of CSME between the groups. Conclusion: TA-LF is associated with better CS outcomes compared to combined therapy after FLACS.

The Efficacy of Liposomal Bupivacaine Over Traditional Local Anesthetics in Periarticular Infiltration and Regional Anesthesia During Total Knee Arthroplasty: A Systematic Review and Meta-Analysis.

BACKGROUND: Since its Food and Drug Administration approval in 2011 as a local anesthetic for postsurgical analgesia, liposomal bupivacaine (LB) has been incorporated into the periarticular injection (PAI) of many knee surgeons. The slow release of this medication from vesicles should significantly extend the duration of its analgesic effect, but current evidence has not clearly demonstrated this benefit. METHODS: We systematically searched electronic databases including PubMed, MEDLINE, Cochrane Library, EMBASE, ScienceDirect, and Scopus, as well as the Journal of Arthroplasty web page for relevant articles. All calculations were made using Review Manager 5.3. RESULTS: We identified 42 studies that compared LB to an alternate analgesic modality. Seventeen of these studies were controlled trials that were included in meta-analysis. Significant differences were seen in pain scores with LB over a peripheral nerve block (mean difference = 0.45, P = .02) and LB over a traditional PAI (standard mean difference = -0.08, P = .004). CONCLUSION: While LB may offer a statistically significant benefit over a traditional PAI, the increase in pain control may not be clinically significant and it does not appear to offer a benefit in reducing opioid consumption. However, there is no standardization among current studies, as they vary greatly in design, infiltration technique, and outcome measurement, which precludes any reliable summarization of their results. Future independent studies using a standardized protocol are needed to provide clear unbiased evidence.

Enhanced Immunogenicity and Protective Efficacy of a Campylobacter jejuni Conjugate Vaccine Coadministered with Liposomes Containing Monophosphoryl Lipid A and QS-21.

Campylobacter jejuni is among the most common causes of diarrheal disease worldwide and efforts to develop protective measures against the pathogen are ongoing. One of the few defined virulence factors targeted for vaccine development is the capsule polysaccharide (CPS). We have developed a capsule conjugate vaccine against C. jejuni strain 81-176 (CPS-CRM) that is immunogenic in mice and nonhuman primates (NHPs) but only moderately immunogenic in humans when delivered alone or with aluminum hydroxide. To enhance immunogenicity, two novel liposome-based adjuvant systems, the Army Liposome Formulation (ALF), containing synthetic monophosphoryl lipid A, and ALF plus QS-21 (ALFQ), were evaluated with CPS-CRM in this study. In mice, ALF and ALFQ induced similar amounts of CPS-specific IgG that was significantly higher than levels induced by CPS-CRM alone. Qualitative differences in antibody responses were observed where CPS-CRM alone induced Th2-biased IgG1, whereas ALF and ALFQ enhanced Th1-mediated anti-CPS IgG2b and IgG2c and generated functional bactericidal antibody titers. CPS-CRM + ALFQ was superior to vaccine alone or CPS-CRM + ALF in augmenting antigen-specific Th1, Th2, and Th17 cytokine responses and a significantly higher proportion of CD4+ IFN-γ+ IL-2+ TNF-α+ and CD4+ IL-4+ IL-10+ T cells. ALFQ also significantly enhanced anti-CPS responses in NHPs when delivered with CPS-CRM compared to alum- or ALF-adjuvanted groups and showed the highest protective efficacy against diarrhea following orogastric challenge with C. jejuni This study provides evidence that the ALF adjuvants may provide enhanced immunogenicity of this and other novel C. jejuni capsule conjugate vaccines in humans.IMPORTANCECampylobacter jejuni is a leading cause of diarrheal disease worldwide, and currently no preventative interventions are available. C. jejuni is an invasive mucosal pathogen that has a variety of polysaccharide structures on its surface, including a capsule. In phase 1 studies, a C. jejuni capsule conjugate vaccine was safe but poorly immunogenic when delivered alone or with aluminum hydroxide. Here, we report enhanced immunogenicity of the conjugate vaccine delivered with liposome adjuvants containing monophosphoryl lipid A without or with QS-21, known as ALF and ALFQ, respectively, in preclinical studies. Both liposome adjuvants significantly enhanced immunity in mice and nonhuman primates and improved protective efficacy of the vaccine compared to alum in a nonhuman primate C. jejuni diarrhea model, providing promising evidence that these potent adjuvant formulations may enhance immunogenicity in upcoming human studies with this C. jejuni conjugate and other malaria and HIV vaccine platforms.

Liposomes Co- encapsulating Anticancer Drugs in Synergistic Ratios as an Approach to Promote Increased Efficacy and Greater Safety.

The era of chemotherapy began in the 1940s, but it was in the 1960s that it was seen as really promising when the first patients with childhood acute lymphoblastic leukemia were cured with combination chemotherapy. Today, it is known that due to resistance to single agents, combination therapy is essential for tumor eradication and cure. In the last decade, studies have shown that anticancer drug combinations can act synergistically or antagonistically against tumor cells in vitro, depending on the ratios of the individual drugs forming the combination. From this observation and facing the possibility of maintaining the in vivo synergistic ratio of combinations came the idea of co-encapsulating anticancer agents in nanosystems. In vivo studies validated this idea by showing that the co-encapsulation of anticancer agents in liposomes allows the maintenance of drug ratios in the plasma and the delivery of fixed drug ratios directly to tumor tissue, leading to a better efficacy compared to the administration of the free drugs combination. Liposomes co-encapsulating irinotecan/floxuridine are now in Phase II trial, and liposomes co-encapsulating cytarabine/daunorubicin were recently approved by the FDA for treatment of patients with acute myeloid leukemia.

[99mTc-HYNIC-N-dodecylamide]: a new hydrophobic tracer for labelling reconstituted high-density lipoproteins (rHDL) for radioimaging.

Despite the widespread use of nanotechnology in radio-imaging applications, lipoprotein based delivery systems have received only limited attention so far. These studies involve the synthesis of a novel hydrophobic radio-imaging tracer consisting of a hydrazinonicotinic acid (HYNIC)-N-dodecylamide and 99mTc conjugate that can be encapsulated into rHDL nanoparticles (NPs). These rHDL NPs can selectively target the Scavenger Receptor type B1 (SR-B1) that is overexpressed on most cancer cells due to excess demand for cholesterol for membrane biogenesis and thus can target tumors in vivo. We provide details of the tracer synthesis, characterization of the rHDL/tracer complex, in vitro uptake, stability studies and in vivo application of this new radio-imaging approach.

Toward biomedical application of amino-functionalized silicon nanoparticles.

Silicon blue-emitting nanoparticles (NPs) are promising effectors for photodynamic therapy and radiotherapy, because of their production of reactive oxygen species (ROS) upon irradiation. RESULTS: Amino-functionalized silicon NPs (NH2SiNP) were intrinsically nontoxic below 100 µg/ml in vitro (on two tumor cell lines) and in vivo (zebrafish larvae and embryos). NH2SiNP showed a moderate effect as a photosensitizer for photodynamic therapy and reduced ROS generation in radiotherapy, which could be indicative of a ROS scavenging effect. Encapsulation of NH2SiNP into ultradeformable liposomes improved their skin penetration after topical application, reaching the viable epidermis where neoplastic events occur. CONCLUSION: Subsequent derivatizations after amino-functionalization and incorporation to nanodrug delivery systems could expand the spectrum of the biomedical application of these kind of silicon NPs.

Nanoparticle vaccines against viral infections.

Despite numerous efforts, we still do not have prophylactic vaccines for many clinically relevant viruses, such as HIV, hepatitis C virus, Zika virus, and respiratory syncytial virus. Several factors have contributed to the current lack of effective vaccines, including the high rate of viral mutation, low immunogenicity of recombinant viral antigens, instability of viral antigenic proteins administered in vivo, sophisticated mechanisms of viral immune evasion, and inefficient induction of mucosal immunity by vaccine models studied to date. Some of these obstacles could be partially overcome by the use of vaccine adjuvants. Nanoparticles have been intensively investigated as vaccine adjuvants because they possess chemical and structural properties that improve immunogenicity. The use of nanotechnology in the construction of immunization systems has developed into the field of viral nanovaccinology. The purpose of this paper is to review and correlate recent discoveries concerning nanoparticles and specific properties that contribute to the immunogenicity of viral nanoparticle vaccines, bio-nano interaction, design of nanoparticle vaccines for clinically relevant viruses, and future prospects for viral nanoparticle vaccination.

Nanotechnology-based drug delivery systems for control of microbial biofilms: a review.

Since the dawn of civilization, it has been understood that pathogenic microorganisms cause infectious conditions in humans, which at times, may prove fatal. Among the different virulent properties of microorganisms is their ability to form biofilms, which has been directly related to the development of chronic infections with increased disease severity. A problem in the elimination of such complex structures (biofilms) is resistance to the drugs that are currently used in clinical practice, and therefore, it becomes imperative to search for new compounds that have anti-biofilm activity. In this context, nanotechnology provides secure platforms for targeted delivery of drugs to treat numerous microbial infections that are caused by biofilms. Among the many applications of such nanotechnology-based drug delivery systems is their ability to enhance the bioactive potential of therapeutic agents. The present study reports the use of important nanoparticles, such as liposomes, microemulsions, cyclodextrins, solid lipid nanoparticles, polymeric nanoparticles, and metallic nanoparticles, in controlling microbial biofilms by targeted drug delivery. Such utilization of these nanosystems has led to a better understanding of their applications and their role in combating biofilms.

Hepatic fibropoiesis in dogs naturally infected with Leishmania (Leishmania) infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol.

Hepatic fibropoiesis in canine visceral leishmaniasis (CVL) were evaluated by histological (morphometrical collagen deposition) and immunohistochemical assays characterizing alpha-actin (α-SMA), vimentin, calprotectin (L1 antigen), and TGF-ß in 46 naturally infected dogs with Leishmania infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol separately and in combination. Six treatment groups were defined: meglumine antimoniate encapsulated in nanometric liposomes (LMA), allopurinol (ALLOP); liposome-encapsulated meglumine antomoniate combined with allopurinol (LMA+ALLOP); empty liposomes (LEMP); empty liposomes combined with allopurinol (LEMP+ALLOP) and saline. Relative liver weight was lower in LMA, LMA+ALLOP, and ALLOP groups compared to the LEMP control. Significantly lower granulomatous chronic inflammatory reaction was seen in the ALLOP group compared to a control group. Calprotectin was lowest in liver of those dogs showing lower numbers of intralobular hepatic granulomas. Collagen deposits were significantly higher in LMA compared to ALLOP, LEMP+ALLOP, and Saline groups. LMA+ALLOP group collagen deposition was higher than dogs treated only with allopurinol. Immunohistochemical analysis showed significant higher α-SMA in hepatic stellate cells (HSCs), hepatic perisinusoidal cells, in control groups than LMA+ALLOP and LEMP+ALLOP. Alpha-actin and Vimentin positive cells were diffusely distributed throughout the liver parenchyma in the hepatic lobule, mainly in HSCs. Vimentin expression was significantly higher in the saline group than in the ALLOP group. Our data suggest that allopurinol inhibits HSC and results in lower collagen deposits in liver during CVL progression, as supported by the significantly lower expression of TGF-ß in the ALLOP group compared to other groups. Results demonstrated that treatment with allopurinol inhibited chronic granulomatous inflammatory reaction and hepatic fibrosis in CVL.

Recent Advances and Perspectives in Liposomes for Cutaneous Drug Delivery.

The cutaneous route is attractive for the delivery of drugs in the treatment of a wide variety of diseases. However the stratum corneum (SC) is an effective barrier that hampers skin penetration. Within this context, liposomes emerge as a potential carrier for improving topical delivery of therapeutic agents. In this review, we aimed to discuss key aspects for the topical delivery by drug-loaded liposomes. Phospholipid type and phase transition temperature have been shown to affect liposomal topical delivery. The effect of surface charge is subject to considerable variation depending on drug and composition. In addition, modified vesicles with the presence of components for permeation enhancement, such as surfactants and solvents, have been shown to have a considerable effect. These liposomes include: Transfersomes, Niosomes, Ethosomes, Transethosomes, Invasomes, coated liposomes, penetration enhancer containing vesicles (PEVs), fatty acids vesicles, Archaeosomes and Marinosomes. Furthermore, adding polymeric coating onto liposome surface could influence cutaneous delivery. Mechanisms of delivery include intact vesicular skin penetration, free drug diffusion, permeation enhancement, vesicle adsorption to and/or fusion with the SC, trans-appendageal penetration, among others. Finally, several skin conditions, including acne, melasma, skin aging, fungal infections and skin cancer, have benefited from liposomal topical delivery of drugs, with promising in vitro and in vivo results. However, despite the existence of some clinical trials, more studies are needed to be conducted in order to explore the potential of liposomes in the dermatological field.

Liposomal butamben gel formulations: toxicity assays and topical anesthesia in an animal model.

The aim of this study was to evaluate the in vitro cytotoxicity and the in vivo analgesic effect and local toxicity of the local anesthetic butamben (BTB) encapsulated in conventional or elastic liposomes incorporated in gel formulations. The results showed that both gel formulations of liposomal BTB reduced the cytotoxicity (p < 0.001; one-way ANOVA/Tukey's test) and increased the topical analgesic effect (p < 0.05; one-way ANOVA/Tukey's test) of butamben, compared to plain BTB gel. The gel formulations presented good rheological properties, and stability assays detected no differences in physicochemical stability up to 30 d after preparation. Moreover, histological assessment revealed no morphological changes in rat skin after application of any of the gel formulations tested.

A novel "in-feed" delivery platform applied for oral DNA vaccination against IPNV enables high protection in Atlantic salmon (Salmon salar).

BACKGROUND: DNA vaccination has emerged as a promising tool against infectious diseases of farmed fish. Oral delivery allows stress-free administration that is ideal for mass immunization and of paramount importance for infectious pancreatic necrosis (IPN) and other viral disease that affect young salmonids and cause economic losses in aquaculture worldwide. METHODS: We describe the development and in vivo assessment of an "in-feed" formulation strategy for oral immunization with liposomal DNA vaccines, by delivering a vaccine construct coding for an immunogenic region of the VP2 capsid protein. A challenge against IPNV was carried out to determine the vaccine efficacy, by comparing the mortality of pre-smolt Atlantic salmons immunized and non-immunized with the oral vaccine. The antibody response (ELISA) and hematological parameters after immunization were examined, as well as the vaccine effect on the growth and internal structures of fry salmons (histological analysis). The vaccine distribution in the experimental tank after oral administration was investigated by HPLC and PCR amplification. RESULTS: The oral vaccine induced detectable levels of VP2-specific antibodies and conferred significant protection following IPNV challenge, with relative percent survivals (RPS) of 58.2%, for single dose (1mgpDNA/kgfish⋅d), and 66% for double dose (2mgpDNA/kgfish⋅d). We further provide evidence in favour of the vaccine safety to fish and demonstrated absence of pDNA in the tank water, but presence of vaccine residues in faeces and unconsumed feed sediments (solid wastes). CONCLUSION: The delivery platform for liposomal DNA vaccination via feed was successfully proved against IPNV in Atlantic salmon, showing the oral vaccine to be immunogenic and safe for fish, and providing significant protection after oral administration. The "in-feed" technology for oral DNA vaccination holds potential to be applied against IPNV and other pathogens that currently threaten the aquaculture worldwide.

Fungal diseases: could nanostructured drug delivery systems be a novel paradigm for therapy?

Invasive mycoses are a major problem for immunocompromised individuals and patients in intensive care units. Morbidity and mortality rates of these infections are high because of late diagnosis and delayed treatment. Moreover, the number of available antifungal agents is low, and there are problems with toxicity and resistance. Alternatives for treating invasive fungal infections are necessary. Nanostructured systems could be excellent carriers for antifungal drugs, reducing toxicity and targeting their action. The use of nanostructured systems for antifungal therapy began in the 1990s, with the appearance of lipid formulations of amphotericin B. This review encompasses different antifungal drug delivery systems, such as liposomes, carriers based on solid lipids and nanostructure lipids, polymeric nanoparticles, dendrimers, and others. All these delivery systems have advantages and disadvantages. Main advantages are the improvement in the antifungal properties, such as bioavailability, reduction in toxicity, and target tissue, which facilitates innovative therapeutic techniques. Conversely, a major disadvantage is the high cost of production. In the near future, the use of nanosystems for drug delivery strategies can be used for delivering peptides, including mucoadhesive systems for the treatment of oral and vaginal candidiasis.

Liposome-entrapped GABA modulates the expression of nNOS in NG108-15 cells.

BACKGROUND: Liposomes are concentric lipid vesicles that allow a sustained release of entrapped substances. GABA (γ-aminobutyric acid) is the most prevalent inhibitory neurotransmitter in the central nervous system. NEW METHOD: Using GABA-containing liposomes (GL) prepared by the freeze-thawing method, we determined the effect of sustained release of GABA on expression of neuronal nitric oxide synthase (nNOS) and GABAA receptor (GABAAR) in an in vitro neuronal model. RESULTS: Neuronal cell line NG108-15 treated with different doses of GL during 24h showed an increase in expression of GABAAR (54 and 50% with 10 and 20ng doses, respectively) and nNOS (138, 157 and 165% with 20, 50 and 100ng doses, respectively) compared with cells treated with empty liposomes (EL). Additionally, cells treated with 50ng of GL showed an increase in GABAAR (23%) after 1h followed by an increase in nNOS (55, 46 and 55%) at 8, 12 and 24h time points, respectively. Immunofluorescence experiments confirmed an increase in nNOS (134%) and basal intracellular levels of nitric oxide (84%) after GL treatment. Further, treatment of cells with GL showed a decrease in expression of a protein inhibitor of nNOS (PIN) (26, 66 and 57% with 20, 50 and 100ng doses respectively) compared with control. COMPARISON WITH EXISTING METHODS: This is first demonstration for the development of GL that allows sustained slow release of this neurotransmitter. CONCLUSION: These results suggest that a slow release of GABA can change the expression of nNOS possibly via alteration in PIN levels in neuronal cells.