Ophthalmic Nanoemulsions: From Composition to Technological Processes and Quality Control.

Nanoemulsions are considered as the most promising solution to improve the delivery of ophthalmic drugs. The design of ophthalmic nanoemulsions requires an extensive understanding of pharmaceutical as well as technological aspects related to the selection of excipients and formulation processes. This Review aims at providing the readers with a comprehensive summary of possible compositions of nanoemulsions, methods for their formulation (both laboratory and industrial), and differences between technological approaches, along with an extensive outline of the research methods enabling the confirmation of in vitro properties, pharmaceutical performance, and biological activity of the obtained product. The composition of the formulation has a major influence on the properties of the final product obtained with low-energy emulsification methods. Increasing interest in high-energy emulsification methods is a consequence of their scalability important from the industrial perspective. Considering the high-energy emulsification methods, both the composition and conditions of the process (e.g., device power level, pressure, temperature, homogenization time, or number of cycles) are important for the properties and stability of nanoemulsions. It is advisible to determine the effect of each parameter on the quality of the product to establish the optimal process parameters' range which, in turn, results in a more reproducible and efficient production.

Thymol and eugenol microemulsion for Rhiphicephalus sanguineus sensu lato control: Formulation development, field efficacy, and safety on dogs.

The present study aimed to develop a microemulsion formulation containing thymol and eugenol for field control of Rhipicephalus sanguineus sensu lato on dogs, as well to evaluate its safety and the physical characteristics of the formulation. The microemulsion using thymol and eugenol (5.0 + 5.0 mg/mL) had as vehicles water, propylene glycol, polysorbate 80 and canola oil. On the next day the preparation (formulation freshly prepared) and after 24 months, the size of the microemulsion droplets, polydispersion index (PdI), organoleptic properties (color, viscosity), and presence of precipitate in the microemulsion were evaluated. For the field assay, on day -1, 10 English Cocker Spaniel dogs were experimentally infested with 200 larvae, 100 nymphs and 30 adults of R. sanguineus s.l. On day 0, after tick counts, the animals were divided into two groups: treated with the freshly prepared microemulsion (10 mL/kg), and control, which received the vehicle (10 mL/kg). Tick counts on dogs were performed daily for three more days. Engorged females were recovered from the dogs and their biological and reproductive parameters were monitored. The dogs' clinical parameters (temperature, mucosa color, and general physical condition) were evaluated daily. In addition, blood samples were collected before infestation to verify hematological (packed cell volume) and biochemical parameters (total serum protein, albumin, globulins, creatinine, urea, alanine transaminase, aspartate aminotransferase, and alkaline phosphatase). Freshly prepared and 24-month aged microemulsions had droplets with mean sizes of 30.94 nm and 27.93 nm, and PdI values of 0.214 and 0.161, respectively. In addition, no difference in the organoleptic properties and no precipitation formation were observed, indicating physical stability. Treatment with the microemulsion resulted in reduction of larvae (p < 0.05) parasitizing the dogs on day 1 while the number of nymphs and adults was not reduced (P> 0.05). In the evaluation of the reproductive biology of engorged females, the larval hatchability (%) was compromised (p < 0.05), and the microemulsion had control rate of 85.5 %. The microemulsion and its vehicles did not change the clinical, hematological and biochemical parameters of the dogs. We concluded that the microemulsion was efficient against R. sanguineus s.l. by reducing the number of larvae and affecting the reproductive parameters of engorged females, safe for dogs, and stable (physical stability) during a two-year interval.

Assembled protein nanoparticles in food or nutrition applications.

Proteins are one of the essential components of nutritional food materials and an excellent source for food-grade nanomaterials. This review focuses on select examples of nanoparticles assembled naturally, found in food-relevant materials, major approaches in assembling nanoscale structure from proteins, and general applications of protein nanoparticles in food or nutrition. Animal-sourced casein and non-animal grain storage proteins and legume storage proteins are discussed in terms of their structural assemblies. Protein solubility is a key factor in assembling protein nanoparticles with desired functional properties. Desolvation is the most common technique to prepare protein nanoparticles for insoluble proteins. Well-hydrated protein assemblies have been extensively studied through electrostatic complexes, assembled with fatty acid and starch, reassembled protein structure, and nanogels. These protein-based nanoparticles have been utilized for filler materials of films, encapsulation of bioactive molecules, and stabilization of emulsions. Most studies exploiting protein-based nanoparticles have focused on developing technologies in extraction of proteins from sources and assembly of nanoparticles in different environmental conditions.

Development of a novel oil-in-water emulsion and evaluation of its potential adjuvant function in a swine influenza vaccine in mice.

BACKGROUND: Vaccination is the principal strategy for prevention and control of diseases, and adjuvant use is an effective strategy to enhance vaccine efficacy. Traditional mineral oil-based adjuvants have been reported with post-immunization reactions. Developing new adjuvant formulations with improved potency and safety will be of great value. RESULTS: In the study reported herein, a novel oil-in-water (O/W) Emulsion Adjuvant containing Squalane (termed EAS) was developed, characterized and investigated for swine influenza virus immunization. The data show that EAS is a homogeneous nanoemulsion with small particle size (~ 105 nm), low viscosity (2.04 ± 0.24 cP at 20 °C), excellent stability (at least 24 months at 4 °C) and low toxicity. EAS-adjuvanted H3N2 swine influenza vaccine was administrated in mice subcutaneously to assess the adjuvant potency of EAS. The results demonstrated that in mice EAS-adjuvanted vaccine induced significantly higher titers of hemagglutination inhibition (HI) and IgG antibodies than water-in-oil (W/O) vaccines or antigen alone, respectively, at day 42 post vaccination (dpv) (P < 0.05). EAS-adjuvanted vaccine elicited significantly stronger IgG1 and IgG2a antibodies and higher concentrations of Th1 (IFN-γ and IL-2) cytokines compared to the W/O vaccine or antigen alone. Mice immunized with EAS-adjuvanted influenza vaccine conferred potent protection after homologous challenge. CONCLUSION: The O/W emulsion EAS developed in the present work induced potent humoral and cellular immune responses against inactivated swine influenza virus, conferred effective protection after homologous virus challenge and showed low toxicity in mice, indicating that EAS is as good as the commercial adjuvant MF59. The superiority of EAS to the conventional W/O formulation in adjuvant activity, safety and stability will make it a potential veterinary adjuvant.

Application of Check-All-That-Apply (CATA) Questions for Sensory Characterization of Cosmetic Emulsions by Untrained Consumers.

The sales potential of cosmetic products is greatly determined by skin feel and skin sensory performance. To please the target audience, it is important to gather information about consumers' perception of products' sensory characteristics. In this study, six different emulsions were formulated. Samples represented three different types of emulsions, including steric-stabilized oil-in-water (O/W), liquid crystal-stabilized O/W, and water-in-oil emulsions, providing different skin feel and aesthetics. Emulsions within the same group differed in the emollients, providing similar sensory attributes. The aim was to have 50 consumers evaluate the emulsions' sensory characteristics. Using a check-all-that-apply (CATA) survey, consumers provided information about their perception of appearance, rub-out, pick-up, and afterfeel. Consumers effectively discriminated between the emulsions. Statistical analysis showed significant differences for 15 sensory attributes in the before, during, and after phases. Our findings suggest that emulsifiers, and not emollients, have the dominant role in determining the aesthetics of a skin care emulsion, similar to previous findings. The fact that untrained consumers provided similar results as trained panelists suggests the validity of the CATA survey and its reliability as a screening tool in the product development process. CATA questions may serve as a viable complimentary to descriptive sensory analysis performed by trained panelists.

Preparation of Aqueous Core-Poly(d,l-Lactide-co-Glycolide) Shell Microcapsules With Mononuclear Cores by Internal Phase Separation: Optimization of Formulation Parameters.

Previously, our group employed the internal phase separation method to produce aqueous core-PLGA [poly(d,l-lactide-co-glycolide)] shell microcapsules with polynuclear core morphologies. This report describes the preparation of the more desired and challenging architecture with mononuclear cores. Optimization of formulation parameters including (1) varying the composition of the internal phase and (2) incorporating selected organic solvents (dichloromethane, chloroform, methanol, and acetonitrile) into the internal phase was systematically evaluated. Varying the composition of the internal phase (i.e., PLGA and water levels) failed to produce mononuclear microcapsules. However, incorporating methanol or acetonitrile into the internal phase produced microcapsules with mononuclear cores as confirmed by phase-contrast microscopy, transmission electron microscopy, and scanning electron microscopy. Stability of the prepared emulsions (internal phase of PLGA, acetone, acetonitrile, and water) was optimized by evaluating different types of surfactants with varying concentrations. Among them, lecithin in the range of 0.5%-5% wt/wt provided the best emulsion stability. Interestingly, increasing lecithin concentrations led to the production of microcapsules with smaller sizes (from 2.4 ± 1.6 to 1.1 ± 0.8 µm) and higher percentage of mononuclear cores. The resulting aqueous core-PLGA shell microcapsules are expected to have interesting applications in drug delivery systems with controlled release for hydrophilic drugs and proteins.

Light backscatter fiber optic sensor: a new tool for predicting the stability of pork emulsions containing antioxidative potato protein hydrolysate.

The objective of this study was to determine whether light backscatter response from fresh pork meat emulsions is correlated to final product stability indices. A specially designed fiber optic measurement system was used in combination with a miniature fiber optic spectrometer to determine the intensity of light backscatter within the wavelength range 300-1100 nm (UV/VIS/NIR) at different radial distances (2, 2.5 and 3mm) with respect to the light source in pork meat emulsions with two fat levels (15%, 30%) and two levels (0, 2.5%) of the natural antioxidant hydrolyzed potato protein (HPP). Textural parameters (hardness, deformability, cohesiveness and breaking force), cooking loss, TBARS (1, 2, 3, and 7 days of storage at 4 °C) and CIELAB color coordinates of cooked emulsions were measured. The light backscatter was directly correlated with cooking losses, color, breaking force and TBARS. The optical configuration proposed would compensate for the emulsion heterogeneity, maximizing the existing correlation between the optical signal and the emulsion quality metrics.

In line monitoring of the preparation of water-in-oil-in-water (W/O/W) type multiple emulsions via dielectric spectroscopy.

Multiple emulsions offer various applications in a wide range of fields such as pharmaceutical, cosmetics and food technology. Two features are known to yield a great influence on multiple emulsion quality and utility as encapsulation efficiency and prolonged stability. To achieve a prolonged stability, the production of the emulsions has to be observed and controlled, preferably in line. In line measurements provide available parameters in a short time frame without the need for the sample to be removed from the process stream, thereby enabling continuous process control. In this study, information about the physical state of multiple emulsions obtained from dielectric spectroscopy (DS) is evaluated for this purpose. Results from dielectric measurements performed in line during the production cycle are compared to theoretically expected results and to well established off line measurements. Thus, a first step to include the production of multiple emulsions into the process analytical technology (PAT) guidelines of the Food and Drug Administration (FDA) is achieved. DS proved to be beneficial in determining the crucial stopping criterion, which is essential in the production of multiple emulsions. The stopping of the process at a less-than-ideal point can severely lower the encapsulation efficiency and the stability, thereby lowering the quality of the emulsion. DS is also expected to provide further information about the multiple emulsion like encapsulation efficiency.

Technology transfer of oil-in-water emulsion adjuvant manufacturing for pandemic influenza vaccine production in Romania.

Many developing countries lack or have inadequate pandemic influenza vaccine manufacturing capacity. In the 2009 H1N1 pandemic, this led to delayed and inadequate vaccine coverage in the developing world. Thus, bolstering developing country influenza vaccine manufacturing capacity is urgently needed. The Cantacuzino Institute in Bucharest, Romania has been producing seasonal influenza vaccine since the 1970s, and has the capacity to produce ∼5 million doses of monovalent vaccine in the event of an influenza pandemic. Inclusion of an adjuvant in the vaccine could enable antigen dose sparing, expanding vaccine coverage and potentially allowing universal vaccination of the Romanian population and possibly neighboring countries. However, adjuvant formulation and manufacturing know-how are difficult to access. This manuscript describes the successful transfer of oil-in-water emulsion adjuvant manufacturing and quality control technologies from the Infectious Disease Research Institute in Seattle, USA to the Cantacuzino Institute. By describing the challenges and accomplishments of the project, it is hoped that the knowledge and experience gained will benefit other institutes involved in similar technology transfer projects designed to facilitate increased vaccine manufacturing capacity in developing countries.

Importance of bacterial surface properties to control the stability of emulsions.

In colloidal media such as emulsions or food matrixes, the stability results from physicochemical interactions. The same type of interaction is involved in the attachment processes of microorganisms, through their surface properties, to interfaces. When bacteria are present in a food matrix, it is probable that their surface interacts with the other constituents. In this paper, the involvement of bacterial surface properties of Lactococcus lactis subsp lactis biovar diacetylactis (LLD) on the stability of model emulsions has been studied. The hydrophobic and electrostatic cell-surface properties were characterized by the MATH method and by microelectrophoresis, respectively. The oil-in-water emulsions were stabilized by various surface-active compounds, CTAB, SDS or Tween 20, giving differently charged droplets. Two strains with different surface characteristics were added to the emulsion. Contrasting with emulsions made with the non-ionic surfactant, for which the stability was not modified by the addition of bacteria, the emulsions made with ionic surface-active compounds were unstable in the presence of bacteria when the bacterial surface charge was opposite to the one of the emulsion droplets. Moreover, aggregation and flocculation phenomena were observed for emulsions stabilized with the cationic surfactant, particularly for more negatively charged bacteria. The effect of bacteria on the emulsion stability depended on the strain which shows the importance of the choice of the microorganism according to of the characteristics of the colloidal media to obtain a stable system. In addition, these results suggest that the interactions between bacteria and other food components can influence the position of bacteria in food matrixes.

The evaluation of cosmetic and pharmaceutical emulsions aging process using classical techniques and a new method: FTIR.

The purpose of this paper is to show how the utilization of Fourier Transform Infrared (FTIR) spectroscopy can be interesting in stability studying of cosmetic or pharmaceutical "oil in water" (O/W) emulsions. In this study temperature storage tests were performed to accelerate the aging process and evaluate the stability of five emulsions. Emulsions were analyzed by FTIR and classical methods (conductivity, viscosity, pH, texture analysis) in order to determine a method that would enable predicting the emulsion's stability. During the aging process, modifications of chemical functions are measured by FTIR (using spectrometric indices), such modifications included: a decrease of unsaturation index, an increase of carbonyl index and a broadening of the carbonyl band. This band was deconvoluted to evaluate the contribution of different species in the broadening phenomenon, which seems to be caused by the appearance of free fatty acids. Conductimetry seems to be the most sensitive technique to assess physical modifications during emulsion's aging. Concerning the most unstable emulsions, a progressive increasing of conductivity was observed several months before the emulsion destabilizes. Consequently, FTIR and conductimetry are two complementary techniques. Conductimetry is a useful technique to predict emulsion destabilization while FTIR allows the measurement of chemical modifications and helps to understand the chemical mechanisms which occur during the oxidation.

Modelling the effects of (green) antifungals, droplet size distribution and temperature on mould outgrowth in water-in-oil emulsions.

Prevention of fungal spoilage is a key microbiological issue for the shelf life of fat spreads. Our aim was to assess and model the scope of (natural) antimicrobials for extending shelf life of spreads (water-in-oil emulsions). Production conditions were established to make 60% model fat spreads with reproducible droplet size distributions. The mould vulnerabilities ranged from 1 to 20 weeks. The system allowed feasibility testing of lytic enzymes (Novozym 234) and LMW compounds against Penicillium roqueforti, a key-spoilage mould. The action of Novozym 234, carvacrol, undecanol and dihydrocarveol was benchmarked against sorbate and preservative-free controls under ambient and chilled conditions. Novozym 234 was ineffective to prevent outgrowth of P. roqueforti. Carvacrol, undecanol and dihydrocarveol had limited effects on shelf-life extension compared to sorbate. Fungal growth boundaries of (un-)preserved spreads were modelled. The emulsion droplet size distribution (DSD) was first captured in a mechanistic parameter DSD-I (I = Influence). DSD-I was a move away from the mean droplet diameter D3,3 as sole quantitative droplet-size distribution parameter for mould susceptibility of emulsions. DSD-I is a combination of available water droplets and surface area to initiate and sustain fungal outgrowth. Followup experiments showed that modelling D3,3 and distribution width (e(sigma)) instead of DSD-I gave better results for emulsions with high e(sigma). Empirical predictive models were subsequently developed for the effects of D3,3, e(sigma) and undissociated sorbic acid (HSO) on the shelf life of emulsions.

A Salmonella abortusovis inactivated vaccine protects mice from abortion after challenge.

Two types of Salmonella abortusovis vaccines were prepared, one with aluminium hydroxide (vaccine A) and the other with water in oil (vaccine B) adjuvants. They were compared in a pregnant mouse model, aiming at protecting them from abortions after challenge with a virulent strain of S. abortusovis. The protection for vaccine A was from 74% to 77.6% and that for vaccine B from 71% to 79.6%. Abortions occurred 5-10 days post challenge and S. abortusovis was isolated from all aborted fetuses and from the liver and the spleen of their mothers at the end of the experiment (18 days post challenge). The presence of salmonella in the liver and the spleen of vaccinated non-pregnant but challenged mice was studied in a separate experiment. The bacterium was isolated from one out of 12 vaccinated mice 6 days post challenge as well as from the six controls.

A comparison of two quality assessment methods for emulsions.

A common method of assessing the quality of emulsions is to evaluate the size distribution of the globules of the internal phase. The primary aim of this work is to compare the sensitivity of this test to an alternative method. The sizes of the globules of two emulsions, an oral emulsion and a total parenteral nutrition (TPN) emulsion, were determined using a light microscope. Globule size analyses were performed upon preparation and during storage of the emulsions. Using a computer program specially developed for this study, the recorded diameters were placed into size groups and the volumes of each of the measured globules was determined. For each size group, the total volume of all the globules within the group and the volume percentage of the oil phase represented by the group were calculated. The volume distribution of the internal phase across the size groups was found to predict emulsion instability better than the globule number distribution and thus is a better determinant of emulsion quality. This technique may have general application in the evaluation of TPN emulsions and other spheres, such as liposomes.

Physicochemical characterization and acute toxicity evaluation of a positively-charged submicron emulsion vehicle.

Fine, homogeneous, positively-charged emulsions with a mean droplet size of 138 +/- 71 nm and a zeta potential value of 41.06 mV were prepared using a combination of emulsifiers comprising phospholipids, poloxamer 188, and stearylamine. The pH of these emulsions decreased with time. However, the extent of decrease was dependent on the storage temperature. The mean droplet size of the emulsions that had been prepared with 1% poloxamer began to increase slightly after six months' storage, particularly when stored at 23 and 37 degrees C. However, emulsions prepared with 2% poloxamer remained stable for at least 10 months at 4 degrees C, suggesting that the poloxamer 188 concentration is critical for prolonged emulsion stability. The results of the ocular tolerance study in rabbit eye indicate that hourly administration of a positively-charged emulsion vehicle was well tolerated without any toxic or inflammatory response to the ocular surface during the five days of the study. Scanning electron microscopy revealed a normal corneal surface, which was not different from that of the animals treated with physiological saline. No marked acute toxicity was observed when 0.6 mL of positively-charged emulsion was injected intravenously to BALB/c mice. Furthermore, no difference was noted between this group of animals and the group injected with the marketed Intralipid emulsion. These results were further confirmed in a rat study where there were no deaths following intravenous injection of 3.3 mL per rat of the positively-charged emulsion or Intralipid. Neither emulsion elicited any hepatotoxic or nephrotoxic effects. The overall results suggest that the novel positively-charged emulsion is suitable for parenteral use, and for ocular application.

Blood substitutes: where are we?

Plasma volume expanders exist and are available. Balance salt solutions are preferred for initial resuscitation. Albumin should be avoided. Synthetic coagulation factors are not available. They exist because proper component therapy creates them to be stored for selective use. A substitute for the prime function of blood--oxygen delivery by the red cell--does not yet exist. Its clear that one is needed; the form is not yet identified. Fluorocarbon emulsions have a certain chemical cleanliness that makes them appealing, yet there are many unanswered questions, especially as to oxygen-carrying capacity and toxicity. Fluorocarbons have been pushed to clinical testing before their time. Chemically modified stroma-free hemoglobin continues to evolve and develop as a useful blood substitute. Most of the early problems seem to have been resolved and a third generation of molecules, the second set of modifications, is promising.