New RP-HPLC method for the simultaneous determination of process yield and percentage of encapsulation of Gallein in albumin nanoparticles.

A study was performed for the development and validation of a method of High Performance Liquid Chromatography (HPLC) for the identification and simultaneous quantification of Gallein and Human Serum Albumin (HSA). In addition, this work presents the development and physicochemical characterization of this new pharmaceutical formulation of HSA nanoparticles loaded with Gallein for potential use in the treatment of Alzheimer's disease. The method was developed with the purpose of determining the performance of the synthesis process of nanoparticles and the efficiency of encapsulation of the drug in the nanosystem. The HPLC mobile phase consisted of ACN:H2O:TEA:H3PO4 (50:49.8:0.1:0.1 v/v/v) pumped at a flow rate of 0.8 mL/min, isocratic mode, and the measurement were carried out at 220 nm. Chromatographic runs were performed on a C18 column (150 × 4.60 mm; 5 µm size particles). The HPLC-method was validated following the International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines and was used to simultaneously quantify the two components of the nanoformulation. Thus, the values obtained through the validated method were 43 % for drug encapsulation efficiency (% EE) and the synthesis performance (% yield) was 96 %. Moreover, the nanoformulation was characterized by DLS, the results showed that the average particle size was 217 nm, with a PDI of (0.085 ± 0.005) and a potential Z of -29.7 mV. Therefore, the developed method has proven useful in providing accurate simultaneous measurements of HSA and Gallein from albumin nanoparticles. It is advantageous since it is able to reduce the time and facilitate the determination of Gallein encapsulation efficiency and yield of albumin nanoparticles.

Evidence of the protective role of Carvacrol in a retinal degeneration animal model.

Neurodegenerative pathologies affecting the posterior segment of the eye, are characterized by being devastating and responsible for the majority of visual dysfunctions worldwide. These diseases are primarily degenerative, progressing chronically, and can inflict gradual harm to the optic nerve, retinal ganglion cells (RGC), photoreceptors, and other retinal cells. This retinal damage leads to a progressive loss of vision, marking these conditions as a significant health concern worldwide. The intravitreal administration of the phytochemical Carvacrol (CAR) is expected to demonstrate a neuroprotective and antiapoptotic effect on retinal cells, with a specific focus on RGC. This effect will be observed in a retinal degeneration model (RDM) in rabbits induced by cytotoxic and oxidative agents, namely glutamate (GLUT) and L-buthionine-S, R-sulfoximine (BSO). An in vivo study was conducted using New Zealand rabbits in which retinal damage was created to evaluate the effectiveness of CAR. The effectiveness of CAR on the functionality of retinal neuronal cells in RDM was evaluated using pupillary light reflection (PLR). Furthermore, the phytotherapeutic's influence on cell viability was determined through flow cytometry analysis. Finally, the neuroprotective and antiapoptotic capabilities of CAR were specifically scrutinized in RGC through histological studies, quantifying cell survival, and employing immunohistochemical assays to detect the apoptotic index (%) using the TUNEL technique. Our results demonstrated that CAR promoted the recovery of the pupillary contraction profile over time, maintaining the functionality of retinal cells as healthy controls. Additionally, it showed increased cell viability under oxidative and cytotoxic conditions given by GLUT-BSO agents. Finally, we found that CAR protects the survival of RGC and decreases the percentage of apoptotic cells when compared to RDM. CAR demonstrated to have positive effects on the functionality of photoreceptive nerve cells by restoring pupillary contraction. Likewise, it was shown to have neuroprotective and antiapoptotic effects when evaluated in a general and specific way on retinal nerve cells.

Improved biodistribution and enhanced immune response of subunit vaccine using a nanostructure formed by self-assembly of ascorbyl palmitate.

New adjuvant strategies are needed to improve protein-based subunit vaccine immunogenicity. We examined the potential to use nanostructure of 6-O-ascorbyl palmitate to formulate ovalbumin (OVA) protein and an oligodeoxynucleotide (CpG-ODN) (OCC). In mice immunized with a single dose, OCC elicited an OVA-specific immune response superior to OVA/CpG-ODN solution (OC). Rheological studies demonstrated OCC's self-assembling viscoelastic properties. Biodistribution studies indicated that OCC prolonged OVA and CpG-ODN retention at injection site and lymph nodes, reducing systemic spread. Flow-cytometry assays demonstrated that OCC promoted OVA and CpG-ODN co-uptake by Ly6ChiCD11bhiCD11c+ monocytes. OCC and OC induced early IFN-γ in lymph nodes, but OCC led to higher concentration. Conversely, mice immunized with OC showed higher serum IFN-γ concentration compared to those immunized with OCC. In mice immunized with OCC, NK1.1+ cells were the IFN-γ major producers, and IFN-γ was essential for OVA-specific IgG2c switching. These findings illustrate how this nanostructure improves vaccine's response.

Significant progress in improving Atorvastatin dissolution rate: Physicochemical characterization and stability assessment of self-dispersible Atorvastatin/Tween 80® nanocrystals formulated through wet milling and freeze-drying.

Atorvastatin (ATV) is a first-line drug for the treatment of hyperlipidemia. This drug presents biopharmaceutical problems, partly due to its low solubility and dissolution rate. In this work, nanocrystals of ATV stabilized with Tween 80® were designed by wet milling. A full factorial design was applied to optimize the process. Additionally, a cryoprotectant agent (maltodextrin, MTX) was identified, which allowed maintaining the properties of the nanocrystals after lyophilization. The storage stability of the nanocrystals was demonstrated for six months in different conditions. The obtained nanocrystal powder was characterized using SEM, EDXS, TEM, DSC, TGA, FT-IR, and XRD, showing the presence of irregular crystals with semi-amorphous characteristics, likely due to the particle collision process. Based on the reduction in particle size and the decrease in drug crystallinity, a significant increase in water and phosphate buffer (pH 6.8) solubility by 4 and 6 times, respectively, was observed. On the other hand, a noticeable increase in the dissolution rate was observed, with 90 % of the drug dissolved within 60 min of study, compared to 30 % of the drug dissolved within 12 h in the case of the untreated drug or the physical mixture of components. Based on these results, it can be concluded that the nano-milling of Atorvastatin stabilized with Tween 80® is a promising strategy for developing new formulations with improved biopharmaceutical properties of this widely used drug.

Development of melatonin-loaded, human-serum-albumin nanoparticles formulations using different methods of preparation for ophthalmic administration.

The use of proteins such as human serum albumin (HSA) to form nanometric systems seems very promising since they are non-toxic, biodegradable and have no antigenic activity. This molecule is ideal to transport insoluble drugs such as melatonin (Mel), which has antiapoptotic and antioxidant properties and appears promising for the treatment of neurodegenerative eye diseases. The objective of this study was to obtain nanoparticulate systems loaded with Mel, improving the conventional desolvation method. Systems were stabilised using two different strategies: one through the use of Eudragit S100 as a cross-linking agent and the other through thermal stabilisation. The systems thus obtained (Np-HSA-Eu-Mel and Np-HSA-Mel, respectively) were characterised and compared in terms of physicochemical and pharmacotechnical parameters. Whitish colloidal dispersions of nanometric size (≈170 nm), spherical shape, and monodisperse population were obtained. Besides, the pH was close to neutrality reaching 20 % drug encapsulation whereas the process performance was higher than 80 %. In FT-IR studies, thermal analysis and X-ray diffraction (XRD), the incorporation of the drug in the cavities of the nanoparticles could be evidenced. Regarding the physical stability of nanoparticles, for Np-HSA-Eu-Mel instability was observed at pH > 7. However, Np-HSA-Mel was able to remain stable at different pH levels. Mel release from these systems was consequently affected, where the former released faster than the active compared to the last. On the other hand, it was observed that the drying process (lyophilization in this case) applied to the nanoparticles suspensions does not affect their original properties after redispersion over a three months period. Likewise, the formulation did not produce irritation when administered topically, whereas when administered subconjunctivally, only slight irritation was observed 24 h after administration. According to the result of this study, the Np-HSA-Mel formulation could achieve advantageous properties as a vehicle for the transport of insoluble drugs for the treatment of neurodegenerative diseases at the ocular level.

COVID-19: Epidemiological Situation of Argentina and its Neighbor Countries after Three Months of Pandemic.

OBJECTIVE: In this work, in order to establish a better comprehension of the association between Argentina and its neighbor countries' capacity, and COVID-19 burden during the first 3 months, different indicators were evaluated. METHOD: We analyzed the association between GHSI, INFORM index and COVID-19 burden (number of confirmed cases and deaths), also the number of tests, lethality and the stringency of Governmental policies were evaluated. RESULTS: Uruguay, Paraguay, and Bolivia started earlier different prevention measures. The number of tests differs, as Chile is the 1 that makes more. Uruguay and Paraguay register fewer positive cases and deaths from COVID-19. The GHS index is led by Brazil, followed by Argentina, and then Chile. However, the INFORM index is led by Uruguay followed by Argentina, while Chile and Paraguay are on par. CONCLUSION: The countries that took preventive measures earlier and carried out a more tests are the ones that are obtaining the best results against COVID-19.

Eugenol carbonate activity against Plasmodium falciparum, Leishmania braziliensis, and Trypanosoma cruzi.

Neglected tropical diseases are a major health problem throughout the world, and there are few effective and safe drugs. In this study, we report the design and synthesis of a novel series of carbonates of eugenol using different aliphatic alcohols and N,N-carbonyldiimidazole. Spectroscopic techniques, including 1 H nuclear magnetic resonance (NMR), 13 C NMR, Fourier transform infrared, and high-resolution mass spectrometry, were used to confirm the structures of the synthesized compounds. In vitro and in silico studies of prodrugs of eugenol were performed to determine their antiplasmodial, trypanocidal, and leishmanicidal activities, and also their cytotoxicity. Compounds were highly active against Leishmania braziliensis and Plasmodium falciparum, whereas the activity shown for Trypanosoma cruzi was moderate. Molecular docking was used to determine a possible mode of action of eugenol against the dihydroorotate dehydrogenase of the three parasites (TcDHODH, LbDHODH, and PfDHODH). Notably, the docking results showed that eugenol not only has binding energy similar to that of the natural substrate (-7.2 and -7.1, respectively) but also has interactions with relevant biological residues of PfDHODH. This result indicates that eugenol could act as a substrate for PfDHODH in the pyrimidine biosynthesis pathway of P. falciparum. In conclusion, the combination of certain aliphatic alcohols and eugenol through a carbonate bond could significantly increase the antiparasitic activity of this class of compounds, which merits further studies.

Latanoprost-loaded phytantriol cubosomes for the treatment of glaucoma.

Glaucoma is a degenerative optic neuropathy characterized by increased intraocular pressure that if untreated can result in blindness. Ophthalmological drug therapy is a challenge of great clinical importance due to the diversity of ocular biological barriers which commonly causes limited or no effectiveness for drugs delivered through the eye. In this work, we proposed the development of nanosized cubic liquid crystals (cubosomes) as a new drug carrier system for latanoprost, an anti-glaucoma drug. Latanoprost-loaded phytantriol cubosomes (CubLnp) were prepared using a top-down method. Latanoprost concentration in the formulations ranged from 0.00125% to 0.02% w/v. All cubosomes displayed an average size around 200 nm, a low polydispersity index of 0.1 and zeta potential values around -25 mV, with an encapsulation efficiency of about 90%. Structural studies revealed that cubosomes displayed a double-diamond surface, Pn3m cubic-phase structure, and was not affected by drug loading. Calorimetric studies revealed a fast and exothermic interaction between latanoprost and cubosomes. According to in vitro essays, latanoprost release from cubosomes was slow in time, evidencing a sustained release profile. Based on this behavior, the in vivo hypotensive intraocular effect was evaluated by means of the subconjunctival administration of CubLnp in normotensive rabbits. We obtained promising results in comparison with a marketed latanoprost formulation (0.005% w/v).

A Vaccine Based on Kunitz-Type Molecule Confers Protection Against Fasciola hepatica Challenge by Inducing IFN-γ and Antibody Immune Responses Through IL-17A Production.

Fasciola hepatica is helminth parasite found around the world that causes fasciolosis, a chronic disease affecting mainly cattle, sheep, and occasionally humans. Triclabendazole is the drug of choice to treat this parasite. However, the continuous use of this drug has led to the development of parasite resistance and, consequently, the limitation of its effectiveness. Hence, vaccination appears as an attractive option to develop. In this work, we evaluated the potential of F. hepatica Kunitz-type molecule (FhKTM) as an antigen formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate ester (Coa-ASC16) and the synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) during an experimental model of fasciolosis in mice, and we further dissected the immune response associated with host protection. Our results showed that immunization of mice with FhKTM/CpG-ODN/Coa-ASC16 induces protection against F. hepatica challenge by preventing liver damage and improving survival after F. hepatica infection. FhKTM/CpG-ODN/Coa-ASC16-immunized mice elicited potent IFN-γ and IL-17A with high levels of antigen-specific IgG1, IgG2a, and IgA serum antibodies. Strikingly, IL-17A blockade during infection decreased IgG2a and IgA antibody levels as well as IFN-γ production, leading to an increase in mortality of vaccinated mice. The present study highlights the potential of a new vaccine formulation to improve control and help the eradication of F. hepatica infection, with potential applications for natural hosts such as cattle and sheep.

Neuroprotective effect of melatonin loaded in ethylcellulose nanoparticles applied topically in a retinal degeneration model in rabbits.

We are reporting for the first time the synthesis and application of an innovative nanometric system for the controlled topic release of melatonin in the retina. The ethylcellulose nanocapsules were characterized by diverse physicochemical techniques (scanning electron microscopy, zeta potential, hydrodynamic diameters) and an in vitro release study was done. A complete ex vivo and in vivo trans-corneal permeation and an irritation study were carried out with the new formulations in albino rabbits, to which a retinal degenerative model was induced. The results obtained demonstrate that the in vitro release of melatonin (1 mg/mL and 2 mg/mL) transported by nanocapsules is slower when compared to a solution of melatonin. Greater penetration of melatonin through the cornea was demonstrated by ex vivo and in vivo tests. This can be attributable to an enhanced neuroprotective effect of melatonin on retinal ganglion cells when it is included in ethylcellulose nanocapsules compared to a solution of melatonin. These outstanding findings add promising new perspectives to current knowledge about administrations using nano-technological tools in the treatment of neurodegenerative diseases at the ocular level.

Ricobendazole nanocrystals obtained by media milling and spray drying: Pharmacokinetic comparison with the micronized form of the drug.

Helminthic infections are produced by different types of worms and affect millions of people worldwide. Benzimidazole compounds such as ricobendazole (RBZ) are widely used to treat helminthiasis. However, their low aqueous solubility leads to poor gastrointestinal dissolution, absorption and potential lack of efficacy. The formulation of nanocrystals (NCs) have become the strategy of preference for hydrophobic drugs. In this work, we prepared RBZ NCs (RBZ-NCs) by an optimized combination of bead milling and spray-drying. Following the physicochemical characterization, a comparative pharmacokinetic evaluation of RBZ-NCs was performed in dogs using as controls a micronized powdered form of RBZ (mRBZ) and a physical mixture of drug and stabilizer 1:1 (PM). The particle size of the redispersed RBZ-NCs was 181.30 ± 5.93 nm, whereas DSC, PXRD and FTIR analyses demonstrated that the active ingredient RBZ remained physicochemically unchanged after the manufacture process. RBZ-NCs exhibited improved in vitro biopharmaceutical behaviour when compared to mRBZ. Consequently, the pharmacokinetic trial demonstrated a significant increase in the drug oral absorption, with an AUC0-∞ 1.9-fold higher in comparison to that obtained in animals treated with mRBZ. This novel formulation holds substantial potential for the development of new/alternative treatments for helminth infections both in human and veterinary medicine.

In vivo efficacy of bevacizumab-loaded albumin nanoparticles in the treatment of colorectal cancer.

Bevacizumab (as other monoclonal antibodies) has now become a mainstay in the treatment of several cancers in spite of some limitations, including poor tumour penetration and the development of resistance mechanisms. Its nanoencapsulation may be an adequate strategy to minimize these problems. The aim of this work was to evaluate the efficacy of bevacizumab-loaded nanoparticles (B-NP-PEG) on a xenograft model of human colorectal cancer. For this purpose, human serum albumin nanoparticles were prepared by coacervation, then coated with poly(ethylene glycol) and freeze-dried. B-NP-PEG displayed a mean size of about 300 nm and a bevacizumab loading of approximately 145 µg/mg. An in vivo study was conducted in the HT-29 xenograft model of colorectal cancer. Both, free and nanoencapsulated bevacizumab, induced a similar reduction in the tumour growth rate of about 50%, when compared to controls. By microPET imaging analysis, B-NP-PEG was found to be a more effective treatment in decreasing the glycolysis and metabolic tumour volume than free bevacizumab, suggesting higher efficacy. These results correlated well with the capability of B-NP-PEG to increase about fourfold the levels of intratumour bevacizumab, compared with the conventional formulation. In parallel, B-NP-PEG displayed six-times lower amounts of bevacizumab in blood than the aqueous formulation of the antibody, suggesting a lower incidence of potential undesirable side effects. In summary, albumin-based nanoparticles may be adequate carriers to promote the delivery of monoclonal antibodies (i.e. bevacizumab) to tumour tissues. Graphical abstract.

Antioxidant status in rabbit aqueous humor after instillation of ascorbyl laurate-based nanostructures.

BACKGROUND: The aim of this work was evaluate the antioxidant effect of ascorbyl laurate (ASC12) based nanostructures applied topically to the cornea of ocular normotensive and hypertensive rabbits. The ASC12 was chosen for its capacity to form liquid lyotropic crystal and keeps its free radical trapping power. METHODS: The hypertension model was performed in six rabbits and was obtained by the application of intracameral injections of alpha-chymotrypsin in the right eye. A single 50 ml dose of ascorbyl laurate coagel 2% w/v (COA-ASC12) was applied topically to the cornea of six normotensive and six hypertensive rabbits. The aqueous humor samples were obtained before and after instillation of COA-ASC12 at different times (2 h and 4 h). Antioxidant capacity was determined via the reduction reaction with iron and tripyridyltriazine (FRAP) and the total proteins were measured using the Bradford reagent. RESULTS: The kinetic antioxidant capacity in the aqueous humor of normotensive and hypertensive rabbits showed a maxim increment at 4 h instillation. Also, the antioxidant capacity in the aqueous humor of hypertensive rabbits was ten times lower than in normotensive rabbits. CONCLUSION: This type of nanostructures has the potential to significantly improve the topical formulation for the prophylaxis and treatment of several eye diseases.

In vivo effect of bevacizumab-loaded albumin nanoparticles in the treatment of corneal neovascularization.

Corneal neovascularization (CNV) is associated with different ocular pathologies, including infectious keratitis, trachoma or corneal trauma. Pharmacological treatments based on the topical application of anti-VEGF therapies have been shown to be effective in the treatment and prevention of CNV. The aim of this work was to evaluate the effect of bevacizumab-loaded albumin nanoparticles in a rat model of CNV. Bevacizumab-loaded nanoparticles, either "naked" (B-NP) or coated with PEG 35,000 (B-NP-PEG), were administered once a day in the eyes of animals (10 µL, 4 mg/mL every 24 h) during 7 days. Bevacizumab and dexamethasone were employed as controls and administered at the same dose every 12 h. At the end of the study, the area of the eye affected by neovascularization was about 2-times lower for animals treated with B-NP than with free bevacizumab. In the study, dexamethasone did not demonstrate an inhibitory effect on CNV at the employed dose. All of these results were confirmed by histopathological analysis, which clearly showed that eyes treated with nanoparticles displayed lower levels of fibrosis, inflammation and edema. In summary, the encapsulation of bevacizumab in human serum albumin nanoparticles improved its efficacy in an animal model of CNV.

A Natural Peanut Edible Coating Enhances the Chemical and Sensory Stability of Roasted Peanuts.

This study aims to assess the enhancement of the chemical and sensory properties of roasted peanuts during storage, through the application of high-protein defatted peanut flour (DPF) coatings incorporated with and without antioxidants. The control sample without coating, packed in normal atmosphere (control), showed the highest conjugated dienes (CD) increment (from 1.17 on day 0 to 3.60 on day 180). Roasted peanuts without coating, packed in high barrier bags under vacuum, reached the lowest CD at day 180 (1.92). Conjugated trienes and peroxide values were analogous to CD. The control exhibited the greatest decrease in α-tocopherol (from 27.65 mg/100 g on day 0 to 21.32 mg/100 g on day 180) and γ-tocopherol (from 21.91 mg/100 g on day 0 to 14.99 mg/100 g on day 180). 3-Methylpyridine and 2,5-dimethylpyrazine decreased with storage time only for the control, which had the highest increase in oxidized flavor (from 0 on day 0 to 13.30 on day 180), cardboard (from 7.67 on day 0 to 15.23 on day 180), and astringency. The lowest decreases in roasted peanutty scores were seen in coated samples. DPF coatings delayed roasted peanuts oxidation, enhancing their sensory properties and shelf life compared with the control sample. PRACTICAL APPLICATION: Defatted peanut flour (DPF) is a byproduct obtained during peanut oil extraction and is a possible material for edible film preparation. This strategy adds value to the peanut industry by transforming a by-product into a material with the potential to develop biodegradable and economical films. The application of this DPF-based edible coating on the surface of roasted peanuts may have contributed to extent product's shelf life, allowing for coated products to be packaged in lower barrier and less expensive materials. Use of peanut material to coat peanuts avoids the risk of allergen protein cross contamination, which would be highly valuable for the food industry.

Albendazole-lipid nanocapsules: Optimization, characterization and chemoprophylactic efficacy in mice infected with Echinococcus granulosus.

Cystic echinococcosis (CE), which is caused during the metacestode larval stage of Echinococcus granulosus, is a life-threatening disease and is very difficult to treat. At present, the FDA-approved antihelmintic drugs are mebendazole (MBZ), albendazole (ABZ) and its principal metabolite ABZ sulfoxide (ABZSO), but as these have a therapeutic efficacy over 50%, underlining the need for new drug delivery systems. The aim of this work was the optimization and characterization of previously developed ABZ lipid nanocapsules (ABZ-LNCs) and evaluate their efficacy in mice infected with E. granulosus. LNCs were prepared by the phase inversion technique and characterized in terms of size, surface charge, drug loading, and in vitro stability followed by an in vivo proof-of-concept using a murine model infected with E. granulosus. Stable particle dispersions with a narrow size distribution and high efficiency of encapsulation (≥90%) were obtained. ABZ-LNCs showed a greater chemoprophylactic efficacy than ABZ suspension administered by the oral route as 4 out of the 10 ABZ-LNCs treated mice did not develop any cysts, whereas the infection progressed in all mice from the ABZ suspension group. Regarding the ultrastructural studies of cysts, mice treated with ABZ-LNCs or ABZ suspension revealed changes in the germinal layer. However, the extent of the damage appeared to be greater after ABZ-LNC administration compared to the suspension treatment. These results suggest that ABZ-LNCs could be a promising novel candidate for ABZ delivery to treat CE.

Protective role of melatonin on retinal ganglionar cell: In vitro an in vivo evidences.

Oxidative stress triggers ocular neurodegenerative diseases, such as glaucoma or macular degeneration. The increase of reactive oxygen and nitrogen species in retinal ganglion cells (RGCs) causes damage to the structure and function of the axons that make up the optic nerve, leading to cell death arising from apoptosis, necrosis or autophagy in the RCGs. The use of antioxidants to prevent visual neurodegenerative pathologies is a novel and possibly valuable therapeutic strategy. To investigate in vitro and in vivo neuroprotective efficacy of melatonin (MEL) in RGCs, we used a model of oxidative glutamate (GLUT) toxicity in combination with l-butionin-S, R-sulfoximine (BSO), which induces cell death by apoptosis through cytotoxicity and oxidative stress mechanisms. Histological sectioning and immunohistochemical assays using the TUNEL technique were performed to determine the damage generated in affected cells and to observe the death process of RGCs. Whit BSO-GLUT the results revealed a progressive RGCs death without any significant evidence of a decreased retinal function after 9 days of treatment. In this way, we were able to develop a retinal degeneration model in vivo to carry out treatment with MEL and observed an increase in the survival percentage of RGCs, showing that BSO-GLUT could not exert an oxidant effect on cells to counteract the effect of MEL. These findings reveal that MEL has a neuroprotective and antiapoptotic effect as evidenced by the reduction of oxidative stress damage. MEL demonstrated in this model makes it a promising neuroprotective agent for the treatment of ocular neurodegenerative diseases when administered locally.

Class-B CpG-ODN Formulated With a Nanostructure Induces Type I Interferons-Dependent and CD4+ T Cell-Independent CD8+ T-Cell Response Against Unconjugated Protein Antigen.

There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines.

[Comparison of two experimental models of glaucoma in rabbits] / Comparación de dos modelos experimentales de glaucoma en conejos.

Objective: to compare two models of experimental glaucoma by induction of ocular hypertension in rabbits. Materials and methods: Sixteen New Zealand female rabbits, 2-3 kg were used. Model A (n=6): cauterization of episcleral and perilimbar veins of the right eye (RE) with surgical electrocautery. Model B (n=10): Injection of ?-chymotrypsin in posterior chamber of RE. Intraocular pressure (IOP) was measured before and after the induction of ocular hypertension (OHT), once a week at the same time of day for 40 days, with a manual tonometer. The animals were euthanized by CO2 inhalation. In both models the control was the IOP of the left eye (LE). The mean and standard error (SE) values of IOP, expressed in mmHg, were compared statistically by applying Student's t-test with a significance level of p<0.05. Results: The IOP in LE (control) of model A: was 12.9±1.05 and in model B: 12.9±1.09. There were no significant differences between the models. Model A: The IOP increase in RE was 14.7% (14.8±1.4) with respect to LE. A significant increase in IOP was observed within the first 24 hours: 23.5±1.9 (p<0.05) compared to the control eye. There were no significant differences with subsequent controls. Model B: The increase in IOP in RE was 129.1% (29.6±3.4) with respect to LE. In all cases an increase was observed from Day 1 (p<0.05). The IOP peak in RE was evidenced on Day 25: 35±3.4 (p<0.05). The increase in IOP induced by model B was significantly higher (p<0.01) than in model A. There was loss of ganglion cells of the retina in both models, but the following anatomo-pathological changes were observed only in model B: buphthalmos, subluxation of the lens and increased excavation of the papilla. Conclusion: This study indicates that model B is the most appropriate method to induce a rapid, controlled increase of IOP in rabbits and, more importantly, that this increase may be sustained over extended periods of time. This model could be useful for evaluating the efficacy of new ocular drug delivery systems and for further studies of the physiopathology of glaucoma.

Objetivo: comparar dos modelos de glaucoma experimental por inducción de hipertensión ocular en conejos y describir cambios anatomo-patológicos. Materiales y métodos: Se utilizaron 16 conejos New Zealand, hembras, de 2-3 kg. Modelo A (n=6): cauterización de venas epiesclerales y perilimbares en ojo derecho (OD) con cauterio eléctrico quirúrgico. Modelo B (n=10): inyección intracamerular en OD de ?-quimotripsina. Se midió la presión intraocular (PIO) antes y después de la inducción de la hipertensión ocular (HTO), una vez por semana, a la misma hora del día, durante 40 días, con tonómetro manual. Los animales fueron sacrificados por inhalación de CO2. En ambos modelos la PIO del ojo izquierdo (OI).fue tomado como valor control. La media y error estándar (EE) de los valores de la PIO, expresada en mmHg, fueron evaluadas y comparadas estadísticamente aplicando Test T de Student considerando un nivel de significación de p < 0.05. Resultados: La PIO en OI (control) del modelo A: fue 12,9±1,05 y en el modelo B: 12,9±1,09. No se observaron diferencias significativas entre ambas. Modelo A: el aumento de la PIO en OD fue 14,7% (14,8±1,4) con respecto a OI. Se observó un incremento significativo de la PIO dentro de las primeras 24 hs: 23,5±1,9 (p<0,05) comparado con el valor del ojo control. No hubo diferencias significativas con los controles posteriores. Modelo B: el aumento de la PIO en OD fue 129,1% (29,6±3,4) con respecto al OI. En todos los casos se observó un incremento desde el día 1 (p<0,05). El pico de PIO en OD se evidenció el día 25: 35±3,4 (p<0,05). El incremento de la PIO inducida en el modelo B fue significativamente mayor (p<0,01) que en el modelo A. En ambos modelos hubo pérdida de células ganglionares de la retina, pero sólo en el modelo B se observaron los siguientes cambios anatomo-patológicos: buftalmus, subluxación del cristalino y aumento de la excavación de la papila. Conclusión: De acuerdo a este estudio, el modelo B aparece como el método más apropiado a los fines de inducir un incremento rápido y controlado de la IOP en conejos y más importante, este incremento sería capaz de mantenerse alto a lo largo de periodos de tiempos extendidos. Este modelo podría ser de gran utilidad para evaluar la eficacia de nuevos sistemas oculares de liberación de fármacos y realizar futuros estudios de la fisiopatología del glaucoma Conclusión: De acuerdo a este estudio, el modelo B aparece como el método más apropiado a los fines de inducir un incremento rápido y controlado de la IOP en conejos y más importante, este incremento sería capaz de mantenerse alto a lo largo de periodos de tiempos extendidos. Este modelo podría ser de gran utilidad para evaluar la eficacia de nuevos sistemas oculares de liberación de fármacos y realizar futuros estudios de la fisiopatología del glaucoma

Human serum albumin nanoparticles for ocular delivery of bevacizumab.

Bevacizumab-loaded nanoparticles (B-NP) were prepared by a desolvation process followed by freeze-drying, without any chemical, physical or enzymatic cross-linkage. Compared with typical HSA nanoparticles cross-linked with glutaraldehyde (B-NP-GLU), B-NP displayed a significantly higher mean size (310 nm vs. 180 nm) and a lower negative zeta potential (-15 mV vs. -36 mV). On the contrary, B-NP displayed a high payload of approximately 13% when measured by a specific ELISA, whereas B-NP-GLU presented a very low bevacizumab loading (0.1 µg/mg). These results could be related to the inactivation of bevacizumab after reacting with glutaraldehyde. From B-NP, bevacizumab was released following an initial burst effect, proceeded by a continuous release of bevacizumab at a rate of 6 µg/h. Cytotoxicity studies in ARPE cells were carried out at a single dose up to 72 h and with repeated doses over a 5-day period. Neither bevacizumab nor B-NP altered cell viability even when repeated doses were used. Finally, B-NP were labeled with 99mTc and administered as eye drops in rats. 99mTc-B-NP remained in the eye for at least 4 h while 99mTc-HSA was rapidly drained from the administration point. In summary, HSA nanoparticles may be an appropriate candidate for ocular delivery of bevacizumab.