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.

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.

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.

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.

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.

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.

Albendazole nanocrystals in experimental alveolar echinococcosis: Enhanced chemoprophylactic and clinical efficacy in infected mice.

Human alveolar echinococcosis is caused by the fox tapeworm Echinococcus multilocularis and is usually fatal if left untreated. Medical treatment with albendazole (ABZ) remains an effective option. However, due to its low aqueous solubility, ABZ is poorly and erratically absorbed following oral administration resulting in low drug levels in plasma and liver distribution. Thus, there arises the need to find a simple, efficient and scalable method to produce new ABZ formulations with increased bioavailability. Bearing this in mind, ABZ nanocrystals (ABZ-NCs) appears to be a useful tool to achieve this goal. The aim of the current study was to investigate the chemoprophylactic and clinical efficacy of an ABZ-NC formulation on mice infected with E. multilocularis. In the chemoprophylactic efficacy study, mean weight of the cysts recovered from the ABZ-NC group was 50% lower than that recorded from untreated mice, whereas the treatment with ABZ suspension did not show preventive effect. The viability of protoscoleces isolated from ABZ-NC treated mice was significantly lower than control groups. In the clinical efficacy studies, both ABZ formulations resulted in a reduction in the mean weight of the cysts obtained from mice, however only the treatment with the nanosuspension revealed significant differences (P < 0.05) compared to the control groups. Treatment with ABZ-NCs reduced the weight of the cysts by 77% and the viability of their protoscoleces to 34%. All these results coincided with the tissue damage determined at the ultrastructural level. The enhanced chemoprophylactic and clinical efficacy of ABZ-NCs observed in this study could be attributed to an increase in the oral bioavailability of the drug. In a next step, we will characterize the cyst concentration profile after the administration of ABZ-NCs in mice infected with E. multilocularis.

Antioxidant Stability Study of Oregano Essential Oil Microcapsules Prepared by Spray-Drying.

Release kinetics of the volatile compounds of oregano EO microcapsules and the relation with the antioxidant activity were studied. Different wall material (WM) to core (C) ratios (1:1 and 2:1; WM:C), addition of colloidal silicon dioxide (CSD); and different storage conditions: 23 °C (room temperature; R) and 4 °C (fridge temperature; F) were evaluated for 90 d. Volatile compounds, total phenolic content (TPC), free radical scavenging activity (FRSA), and Trolox equivalent antioxidant capacity (TEAC) were measured. The formulas 2:1 (WM:C) (R and F) without CSD behaved differently from the rest, exhibited a higher antioxidant activity, and released less amount of volatile compounds after 90 d. These treatments grouped together in the cluster analysis, showing the highest TPC (81.54 mg gallic ac/g), FRSA (8.66%), and TEAC (12.35 µg Trolox/g). The addition of CSD facilitated the released of volatile compounds through storage time and promoted losses in the antioxidant activity. The temperature had a significant effect in most of the evaluated variables. However, this effect was more noticeable in F2 (1:1, CSD). PRACTICAL APPLICATION: Oregano essential oil has antioxidant, antimicrobial, and sensory preserving properties. However, it is susceptible to volatilization and is degraded by external factors. Its addition into food matrices is restricted due to low solubility and hydrophobicity. The antioxidant activity of oregano EO is preserved after the process of microencapsulation by spray-drying that extends its stability during storage. Oregano EO microcapsules are an alternative of delivery which protects and extends the shelf life of this essential oil, overcomes stability related limitations and preserves its desirable characteristics allowing these kind of microcapsules to be later incorporated into food products. These microcapsules could be used as a natural additive/flavouring with antioxidant properties.

Evaluation of the Performance of an Ophthalmic Thermosensitive Hydrogel Containing Combination of Suramin and Bevacizumab.

Suramab (SUM) is a new pharmaceutical combination made up of suramine (SUR) and bevacizumab (BVM), which showed a high synergistic effect when administered jointly. As the pharmaceutical vehicle, poloxamer aqueous dispersions were used since this system is able to maintain their fluidity at low temperatures (<15ºC) but which become gel in the corporal environment (>35ºC). In the present study we aimed at evaluating the effect of Poloxamer to prolong the effect of SUM. These formulations were characterized using rheological, biopharmaceutical (drug release) and morphological (SEM) technique. Corneal NV was induced in Sprague Dawley rats Corneal. At 15 days of follow up animals were sacrificed and perfused with black drawing ink. Digital photographs were taken and the area of neovascularisation (ANV) was calculated using the image programmed. The rheological behavior was influenced by the addition of drugs, resulting in a decrease in the gelation temperature (Tsol/gel). Both drugs were released from poloxamer gels by means of an anomalous mechanism. However, BVM was released faster than SUR, with their combination (SUM) to appearing to reduce delivery, probably due to interactions between the drugs or with the polymeric matrix. The in vivo studies showed that SUM-poloxamer gel was able to increase the corneal antiangiogenic effect compared to the SUM solution and BVM alone at 15 days of follow-up. Furthermore no injurious effects were observed in the histological tissue examination after drug administration. The presence of Poloxamer, known to modulate control release of biological agents, seems to have a favorable effect on SUM subconjunctival administered.

Cystic echinococcosis therapy: Albendazole-loaded lipid nanocapsules enhance the oral bioavailability and efficacy in experimentally infected mice.

Therapeutic failures attributed to medical management of cystic echinococcosis (CE) with albendazole (ABZ) have been primarily linked to the poor drug absorption rate resulting in low drug level in plasma and hydatid cysts. Lipid nanocapsules (LNCs) represent nanocarriers designed to encapsulate lipophilic drugs, such as ABZ. The goals of the current work were: (i) to characterize the plasma and cyst drug exposure after the administration of ABZ as ABZ-LNCs or ABZ suspension (ABZ-SUSP) in mice infected with Echinococcus granulosus, and ii) to compare the clinical efficacies of both ABZ formulations. Enhanced ABZ sulphoxide (ABZ-SO) concentration profiles were obtained in plasma and cysts from ABZ-LNC treated animals. ABZSO exposure (AUC0-LOQ) was significantly higher in plasma and cyst after the ABZ-LNC treatments, both orally and subcutaneously, compared to that observed after oral administration of ABZ-SUSP. Additionally, ABZSO concentrations measured in cysts from ABZ-LNC treated mice were 1.7-fold higher than those detected in plasma. This enhanced drug availability correlated with an increased efficacy against secondary CE in mice observed for the ABZ-LNCs, while ABZ-SUSP did not reach differences with the untreated control group. This new pharmacotechnically-based strategy could be a potential alternative to improve the treatment of human CE.

Enhanced chemoprophylactic and clinical efficacy of albendazole formulated as solid dispersions in experimental cystic echinococcosis.

Cystic echinococcosis is a chronic, complex, and still neglected disease. Although albendazole has demonstrated efficacy, only about one-third of patients experience complete remission or cure and 30-50% of treated patients develop some evidence of a therapeutic response. Different strategies have been developed in order to improve the albendazole water solubility and dissolution rate. The aim of the current work was to investigate the chemoprophylactic and clinical efficacy of an albendazole:poloxamer 188 solid dispersion formulation on mice infected with Echinococcus granulosus metacestodes. Albendazole formulated as solid dispersion had greater chemoprophylactic and clinical efficacy than albendazole alone. The improved in therapeutic efficacy could be a consequence of the increase in the systemic availability of albendazole sulfoxide. The work reported here demonstrates that in vivo treatment with albendazole:poloxamer 188 impairs the development of the hydatid cysts. This new pharmacotechnically based strategy could be a suitable alternative for treating cystic echinococcosis in humans.

Improved albendazole dissolution rate in pluronic 188 solid dispersions.

Solids dispersions (SDs) have been proposed as an alternative to improve the dissolution rate of low solubility drugs. SDs containing albendazole (ABZ; 5, 10, 25, and 50% w/w) and Pluronic 188 (P 188) as hydrophilic carrier were formulated. The obtained SDs were assessed in comparison to physical mixtures (PMs). Drug-polymer interactions in solid state were investigated using Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction analysis. No chemical interaction was found between ABZ and poloxamer. The dissolution profiles indicated that ABZ incorporated in SDs and PMs was rapidly released, reaching rapidly the steady state. Increased dissolution rates are usually observed at the highest polymer proportions. However, an opposite effect for SDs as well as for PMs was observed in the assays described here. The systems with the lowest P 188 percentages (SD4, SD3; PM4, PM3) tended to be more effective in increasing the ABZ dissolution rate. Such a result can be attributed to the fact that concentrated aqueous solutions of Poloxamer may form thermo-reversible gels. The physical-mechanical properties indicated that SDs possess improved flow and compacting properties compared to PMs. Thus, ABZ SDs would be more convenient for solid dosage form design and manufacture.