Harnessing Nanotechnology and Natural Antifungals: Essential Oil Nanoemulsions for Improved Vaginal Candidiasis Therapy

Up to 75% of women have at least one episode of VVC, which is mostly caused by Candida albicans, and many of them have repeated occurrences. Every year, almost 138 million women worldwide suffer from VVC.(1) Candida albicans is the primary cause of vulvovaginal candidiasis (VVC), a chronic and common multifactor an infectious condition. After bacterial vaginosis, it is one of the most common mucosal infections, accounting for 50–72% of cases. C. albicans develops in the vaginal mucosa under ideal circumstances, causing symptomatic VVC.The synthetic antifungal drugs that are now on the market have a number of drawbacks, including rapid development, a significant degree of resistance, systemic and local adverse effects, and high cost. According to recent research, C. albicans has acquired azole resistance as a result of a genetic alteration, increasing the likelihood of VVC recurrence. Antifungal drugs now on the market have limited the spectrum of Candida and are unsuccessful at reducing future recurrence rates.(2)

Natural products stand out in the quest for alternative therapies, particularly essential oils, which are thought to be promising antifungal agents. Essential oils, which come from the secondary metabolism of plants, have a number of medicinal uses.(3)In addition to being extremely volatile and having limited water solubility, essential oils are typically unstable in the presence of light, heat, oxygen, and humidity. In this instance, the nano essential oil formulations are seen to be an effective means to get over these stability-related restrictions, with the goals of higher therapeutic efficacy, safeguards against instability and decomposition, controlled release, increased bioavailability, and decreased toxicity.(3)

Essential oils can suppress or eradicate Candida albicans due to their antifungal characteristics. As a natural product, essential oil is one of the most promising antibiotic substitutes due to its safety, effectiveness, lack of residue, lack of resistance, good tolerance within the animal body, and fewer negative side effects. The antibacterial properties of essential oils, in contrast to some chemical treatments, are the product of several interrelated components. This intricate mix of components lowers the risk by making it more difficult for microorganisms to become resistant to essential oils.(4)Since essential oil faces limitations of solubility and stability , novel approaches are being investigated.

In recent years, there has been a notable surge in the discovery and application of alternative medicines, such as nano emulsions, which have gained popularity in the treatment of fungal infections, including VVC. Because of a number of benefits, including making lipophilic compounds more soluble, protecting against degradation and instability mechanisms, boosting the bioavailability of bioactive substances, promoting controlled delivery, lowering side effects, and improving therapeutic efficacy, nanoemulsions have been used extensively in therapy. Nano emulsions, which give superior drug solubilization for poorly water-soluble azoles, are thermodynamically or kinetically stable emulsions with droplet sizes in the 20–500 nm range. Increased cell wall penetration and better drug-membrane interaction are responsible for the improved antifungal activity. Because surfactant components have antibacterial capabilities, nano emulsions also have self-preserving qualities.(5)

Vulvovaginal candidiasis (VVC)

Candida albicans, an opportunistic fungal pathogen, is the primary cause of vulvovaginal candidiasis (VVC), a mucosal infection of the vulva and vaginal tissue of the lower female reproductive tract. Candida albicans accounts for more than 90% of VVC cases, with non-albicans Candida (NAC) species such Candida glabrata, Candida krusei, Candida tropicalis, and Candida parapsilosis accounting for the remaining 10%. VVC is a global health concern because to rising genital tract infections and their correlation with both direct and indirect economic implications. According to reports, this vaginal yeast infection affects 75% of women at least once in their lifetime .(6) The second most common underlying cause of vaginal inflammation, after anaerobic bacterial vaginosis, is candidiasis. It can cause symptoms like itching, irritation, dysuria, dyspareunia, cottage cheese-like vaginal discharges, or vulvar soreness that can last for days or even weeks and be made worse by sexual activity. Furthermore, compared to other mammals (pH of 5.4–7.8), the normal human vaginal microenvironment is more acidic (pH of 3.8–4.5), and Lactobacilli are the predominant organisms in the human vaginal microbiota—more than 70% of it in certain women. Direct microscopy from vaginal swabs or smears, which show yeast cells, pseudo hyphae, and hyphae, is used to diagnose VVC. Common symptoms of VVC include vulval oedema, erythema of the vaginal mucosa, an elevation in vaginal pH of roughly 4.5, and a thick white discharge. (7)

Recurrent VVC (RVVC)

If a woman has more than three episodes in a 12-month period, it is known as recurrent VVC (RVVC). C. albicans is the primary source of infections in women with RVVC. It has been determined that predisposing genetic variables or hypersensitivity to Candida make women with RVVC more vulnerable to this infection.(8)

Pathophysiology

C. albicans is a frequent vaginal colonizer that is found in the lumen and typically produces no symptoms as part of the normal human microbiota. When Candida spp. pierce the vaginal mucosal linings, an inflammatory reaction is triggered by its pseudo hyphae and hyphae, which activate the NLRP3 (NOD-like receptor protein 3) inflammasome in epithelial cells, resulting in vulvovaginal candidiasis. The inflammatory reaction results in a dense discharge, irritation, itching, burning, excoriations, dysuria, dyspareunia, and even edema. The main inflammatory cells are often polymorphonuclear cells and macrophages. Neutrophil infiltration caused by Candida albicans is known, however the underlying mechanism is yet unknown.(9)

Stages of Candida albicans Infection: From Mycobiome Dysbiosis to Symptomatic Disease

Figure 1. Schematic illustration of the stepwise pathogenesis of vulvovaginal candidiasis, highlighting mycobiome dysbiosis, yeast-to-hypha transition, adhesion, biofilm formation, immune evasion, and tissue invasion leading to inflammation and clinical symptoms.

Figure 2. Constructed pseudo ternary phase diagram showing the nanoemulsion region and the influence of oil, Smix, and aqueous phase concentrations on nanoemulsion formation and stability.

Physical Characterization Techniques of nanoemulsions

The physical aspects of the nanoemulsion characterisation procedures involves the nanoemulsions' size, size distribution, zeta potential, refractive index, viscosity, conductance, stability, etc.

Transmission Electron Microscopy (TEM)

For the morphological analysis of nanoemulsions, Transmission Electron Microscopy (TEM) is frequently used because it allows for the direct imaging of droplet size, shape, and dispersion at the nanoscale. To achieve an appropriate concentration and avoid droplet aggregation, the nanoemulsion is usually diluted with distilled water for TEM investigation. A copper grid coated with carbon is covered with a little amount of the diluted sample, which is then left to adsorb for a brief amount of time. After carefully removing any excess liquid with filter paper, the sample is negatively stained with an electron-dense contrast agent, such as uranyl acetate or phosphotungstic acid, to make the droplets more visible.(49)

After staining, the grid is allowed to air dry at room temperature before being viewed using a transmission electron microscope running at the proper accelerating voltage, which is typically between 80 and 200 kV. Droplet shape, size distribution, structural integrity, and aggregation behaviour can all be evaluated using TEM images taken at different magnifications. For a thorough grasp of nanoemulsion properties, dynamic light scattering (DLS) data is frequently contrasted with the particle size determined from TEM micrographs. TEM is still one of the most dependable methods for verifying the nanoscale dimensions and homogeneity of nanoemulsion formulations because of its excellent resolution and imaging capability.(50)

Dynamic Light Scattering (DLS)

The size distribution profile of tiny particles in suspension or polymers in solution is frequently determined using DLS. DLS is also known as photon correlation spectroscopy or quasi-elastic light scattering since this method often uses the intensity or photon auto-correlation function to study the temporal variations. DLS analysis is now the fundamental but required method for determining the hydrodynamic size of nanoemulsion droplets. This is made possible by the one-step DLS size measurement procedure and accurate hydrodynamic size range estimation of nanoemulsion droplets.(51)

Zeta potential

Under particular experimental circumstances, the zeta potential of nanoemulsions can be calculated to assess changes in surface charge. In short, a suitable buffer solution is used to dilute the nanoemulsion formulation, which is then incubated under regulated circumstances, including physiological temperature and constant stirring. The formulation may be subjected to biological fluids, enzymes, or other pertinent agents, depending on the goal of the investigation, while a control sample is kept untreated. Aliquots are taken out and examined using a zeta potential analyser based on electrophoretic light scattering at prearranged intervals. According to the manufacturer's specifications, measurements are usually carried out using a laser source at a set wavelength and suitable scattering angles. In order to track changes in surface charge over time and gather data on nanoemulsion stability, surface modification, and interactions with the environment, the resulting zeta potential values are recorded.(52)

Refractive Index:

The reflectivity of a nanoemulsion is determined using an Abbes refractometer. The refraction describes both the transparency of a nanoemulsion and the way light travels through a substance. The ability to calculate the medium's refractive index is based on the speed of the wave in the reference media (c) in relation to the wave's phase speed in the medium (vp) (n), i.e., n=c/vp. The nanoemulsion is considered transparent if its refractive index is equal to that of water.(53)

Viscosity

A viscometer is a tool used to determine a fluid's viscosity. The rotating viscometer is the primary tool used to characterize nanoemulsions. The concept behind rotational viscometers is that the torque needed to rotate an object in a fluid depends on the fluid's viscosity. They calculate the torque needed to turn a bob or disk in a fluid at a predetermined speed.(51) Evaluation of viscosity is a crucial factor in the physicochemical description of nanoemulsion. The Ostwald viscometer, Hoeppler falling ball viscometer, Stormer viscometer, Brookfield viscometer, and Ferranti-Shirley viscometer are some of the tools used to measure viscosity. The Brookfield viscometer is the best option for determining the viscosity of a nanoemulsion. Viscosity measurements confirm whether the system is an O/W or W/O emulsion. Systems with low viscosity are O/W type, while those with high viscosity are water-in-oil type.(54)

Conductance Measurement

A common technique for describing nanoemulsions and identifying the type of continuous phase they include is conductance measurement. A conductivity meter is used to measure a nanoemulsion's electrical conductivity by submerging an appropriate conductivity probe in the formulation at a regulated temperature. Because water is a good electrical conductor, the presence of water as the exterior continuous phase in oil-in-water (o/w) nanoemulsions typically results in high conductivity values. Water-in-oil (w/o) nanoemulsions, on the other hand, exhibit extremely poor conductivity due to the oil phase surrounding the scattered water droplets, which restricts ion mobility. Therefore, conductance measurements are helpful in determining the type of emulsion, tracking phase inversion occurrences, and evaluating formulation stability. Furthermore, conductivity and dielectric investigations can offer important insights into the dynamic characteristics, droplet interactions, and structural organization of nanoemulsion systems.(55)

Stability studies

One crucial factor to take into account while evaluating nanoemulsions is the stability study. The exceptional stability of nanoemulsions sets them apart from other dispersed systems. Thermodynamic stability studies and accelerated stability studies are two of the many stability investigations carried out for nanoemulsions.

1. Thermodynamic stability studies

To evaluate the physical stability of the chosen formulation, various thermodynamic stability studies were conducted.(56) The procedure comprised three cycles: heating and cooling were done six times at first, then alternatively heated and cooled at 40°C and 4°C, respectively. Centrifugation was then done for 30 minutes at 3500 rpm. Phase separation-related changes in the formulation were monitored for each cycle.

2. Accelerated stability studies

Studies of accelerated stability were conducted on the optimized formulation. Three batches of the nanoemulsions were placed in glass vials and stored under ambient humidity conditions at temperatures of 30°C, 40°C, and 60°C.(57) In accordance with the usual process outlined in the International Conference on Harmonization (ICH) Q1 recommendations, the samples were taken out for drug content analysis. It was determined how much of the medication was broken down at each interval. The graphical method was used to determine the order of the reaction. For every temperature, the degradation rate constant (K) was calculated.(58)

Potential Paths for Future Treatment

New treatment options are critically required due to the increasing resistance to antifungal medications. As a result, many new research projects and preclinical trials have been carried out. The market has recently seen the launch of new antifungal medications.(9)

Probiotics

Probiotics are live, helpful bacteria that support the maintenance and restoration of a healthy vaginal microbiota. They are mostly Lactobacillus species. Probiotics are used in conjunction with antifungal medication to restore the Lactobacillus-dominant vaginal environment and lower recurrence rates in vulvovaginal candidiasis (VVC), especially recurrent vulvovaginal candidiasis (RVVC).(59)

Since certain probiotics contain Lactobacillus species that have an antagonistic effect on Candida-induced vulvovaginitis, probiotics are regarded as a natural way to prevent and cure vaginal infections. It is hardly surprising, that they have achieved tremendous achievements in their fight against VVC. Probiotics such Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14, or their combination have been demonstrated to significantly reduce fungal colonization in in-vitro settings. There were no adverse effects, a return to normal microflora from asymptomatic bacterial vaginosis, and a decrease in yeast in a trial comparing the effects of Lactobacillus fermentum RC-14 and Lactobacillus rhamnosus GR-1 versus a placebo. Azoles seem to be controversially replaced by probiotics.(9)

Probiotic-integrated nanotherapeutics

Combining probiotics with cutting-edge nanotechnology-based delivery methods is a promising future strategy for the treatment of vulvovaginal candidiasis. Wei et al. created a probiotic nanozyme hydrogel that combines catalytic nanozymes that can alter the vaginal microenvironment with the helpful bacterium Lactobacillus. This multipurpose platform was created to concurrently restore vaginal microbial equilibrium, control local pH, lower oxidative stress, and inhibit fungal development, in contrast to traditional antifungal treatments that mainly concentrate on getting rid of Candida species. The hydrogel improved therapeutic efficacy by offering a protective matrix for probiotic survival and extended retention in the vaginal cavity.

The study showed that in experimental models, the probiotic nanozyme hydrogel successfully prevented Candida albicans colonization, decreased inflammation, and encouraged the restoration of a healthy vaginal microbiota. Instead of just treating acute fungal overgrowth, this approach addressed the underlying reasons of recurrent infection by combining microbiome restoration with nanotechnology-enabled local delivery. The findings highlight the potential of probiotic-based nano formulations as next-generation therapeutics for recurrent vulvovaginal candidiasis, offering a personalized and microbiome-centered treatment approach with reduced risk of drug resistance and recurrence.(60)

Personalized medicine

The goal of personalized medicine is to tailor therapies to each patient's particular traits, including genetics and lifestyle. This entails tailoring medicine formulations and delivery methods for vaginal drug delivery based on variables such as vaginal pH and flora. Research indicates that hormonal treatments should be customized to maximize patient success and reduce side effects. Implants that are tailored to each patient's anatomy and hormone requirements can be made thanks to 3D printing technology. Regulations are changing to guarantee the effectiveness and safety of these customized devices, especially those made at the point of care. Personalized techniques could greatly improve the management of a variety of gynaecological disorders in women's healthcare, possibly leading to better results and lower costs.(61)

CONCLUSION

Due to its high prevalence, rising antifungal resistance, and frequent recurrence after conventional therapy, vulvovaginal candidiasis (VVC), especially recurrent vulvovaginal candidiasis (RVVC), continues to be a major global health concern. Due to their broad-spectrum efficacy against Candida species and reduced tendency to induce resistance, essential oils have become attractive natural antifungal agents. However, their direct medicinal application is limited by issues such chemical instability, volatility, and low water solubility. By boosting essential oils' solubility, stability, bioavailability, and controlled release while also strengthening their interaction with fungal cells and vaginal mucosa, nanoemulsion-based delivery methods provide a practical solution to these problems.

According to the reviewed studies, essential oil-loaded nanoemulsions had better therapeutic efficiency than traditional formulations, excellent physicochemical properties, and strong antifungal activity against a variety of Candida species. Stable and effective delivery platforms for vaginal administration have been made possible by developments in nanoemulsion preparation and characterization techniques. Additionally, new methods like personalized vaginal medicine and probiotic-integrated nanotherapeutics demonstrate a move toward microbiome-centered and patient-specific therapy techniques. When taken as a whole, these developments present nanoemulsion-based essential oil formulations as prospective next-generation therapeutic approaches for the efficient treatment of VVC and RVVC, necessitating more preclinical and clinical research to enable their transfer into clinical practice.

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