Hyaluronic Acid Coated Nanoemulsions for CD44 Mediated Targeted Antifungal Therapy : Mechanistic Insights, Formulation Strategies & Considerations

Invasive fungal infections (IFIs) constitute a major and more and more recognized health concern at the global levels. Researchers invariably report about life-threatening fungal infections particularly those caused by Candida, Aspergillus, and Cryptococcus remain a serious health hazard on human health in the world. These organisms negatively affect individuals with weakened immunity due to conditions such as cancer, organ transplantation or critical illness. Although antifungal drugs exist, there are some shortcomings like its ability to combat these infections is hindered by resistance, toxicity and limited ability to penetrate into infected tissues^[1].

Shortcomings of the existing Azole antifungal agents wherein the most commonly used agents include voriconazole that works by inhibiting one of the enzymes of the cell membrane synthesis used by fungi. Although they are wide-ranging, they experience fluctuating blood levels, interactions with drugs and toxicity, particularly with chronic treatment. The possibility of improving delivery of azole via nanotechnology has been put forward to eliminate side effects by enhancing solubility and controlled release.^[2]

Nanoemulsions are tiny droplets of oil suspended in water which are capable of accommodating drugs that are poorly soluble in water, in a form that make it easier to absorb from the body. By encapsulating antifungal agents within the internal oil phase, nanoemulsions can effectively bypass the dissolution barrier that limits conventional solid dosage forms. Moreover, the contact of lipid droplets with the bile salts and digestive enzymes can bypass  hepatic first-pass metabolism and increase the extent of lymphatic transport and improving  systemic exposure.

Review on nanoparticle platforms (such as nanoemulsions and other systems) highlight that difficulties such as low bioavailability, tissue penetration issues and drug resistance can be overcome by these formulations. Nano-based approaches can increase uptake into infected tissues and can sustain release of drugs at places of infection ^[3].

Although enhancing solubility and pharmacokinetics is a must, it is necessary to deliver the drug to the target area such as inflamed and infected tissues is also another therapeutic frontier. Inflammatory responses stimulated by fungal infections are characterized by the increase in adhesion molecules, cytokines and cell surface receptors within the affected tissues.one such receptor, CD44,  is a transmembrane glycoprotein abundantly expressed on epithelial cells and macrophages, and activated immune cells. Although a large portion of the literature on CD44 is on cancer, a common theme is that CD44 is a receptor which is increased in inflamed tissues and interacts well with hyaluronic acid (HA) a naturally occurring sugar molecule in the body^[4]. This interaction has been utilized by researchers to obtain HA-coated nanoparticles which are absorbed more efficiently into cells which overexpress CD44 and enhance targeted delivery.

Nanocarriers functionalized with hyaluronic acid have been documented to be used extensively in oncology and  CD44 mediated models of targeted delivery using inflammatory disease. These systems are exploitative of the natural interaction of HA and CD44 to facilitate receptor-mediated endocytosis, and thus, improve uptake of drugs in the CD44-enriched tissues^[5]. Although its outcomes have been promising in other fields of therapy, the use of CD44-targeted nanocarriers in antifungal treatment is still very limited. Most current antifungal nanoformulations have their core centered on passive improvement of solubility rather than  active targeting of infected tissues. Given the inflammatory microenvironment characteristic of fungal infections and the documented overexpression of CD44 in such settings, integrating CD44-targeting strategies with nanoemulsion platforms represents a rational and innovative approach.

This review aims to critically examine current advances in nanoemulsion-based antifungal delivery, evaluate the rationale for CD44-mediated targeting, and identify existing gaps that must be addressed to translate these strategies into clinically viable therapy.

Table 1 :  Comparision of Conventional Nanoemulsions VS  CD44 Targeted Nanoemulsions

2. Pathophysiology of Fungal Infections

2.1 Mechanisms of Fungal Pathogenesis

Pathophysiology of fungal infections is divided into various categories that include: Aspergillus and Candida.

Pathogenesis: The mechanisms of pathogenesis are not well understood but it is believed that the process is complex and multi-faceted, involving enzymatic and non-enzymatic processes.

The development of the fungal infections follows a series of events that is well-coordinated to enable the normally harmless or environmental fungi to develop invasive pathogenicity, particularly in those people with a weakened immune defenses. This usually entails the initial colonization, attack of the host tissues, avoidance of immune responses, as well as, in most instances, biofilm formation. All of these adaptive strategies establish the severity and persistence of infection^[6].

A. Tissue Invasion

Attachment to host surfaces is the initial stage of the pathogenesis of the fungi. Most of the pathogenic fungi express surface proteins, which are specific to them and allow them to target host components of the extracellular matrix like collagen, laminin and fibronectin. This binding process is fundamental in colonization and it comes before deeper invasion.

In Candida albicans, which is one of the most extensively studied fungal pathogens, the progress of infection occurs enormously associated with its morphological transformation capacity. Under favourable conditions,the organism shifts from a round yeast form to elongated filamentous hyphae.This transition improves its ability to enter epithelial barriers. The enzymes secreted by the fungus during the invasion, which include proteases, phospholipases and lipases, rupture the host cell membranes, structural proteins, and leads to damage of tissues and systemic spread.

On the same note, inhalation of Aspergillus fumigatus spores may extend to the lower respiratory system.These spores are normally eliminated by immunocompetent individuals. However,in immunocompromised hosts, they germinate into filamentous hyphae that invade lung tissue.A hallmark of invasive aspergillosis is angioinvasion, where hyphae infiltrate blood vessels, potentially causing thrombosis, hemorrhage and tissue necrosis.

B. Immune Evasion

Pathogenic fungi avoid or manipulate the host immune defences in order to cause an infection. One relevant strategy involves in masking structural cell wall components like as β-glucans, which would otherwise get recognized by host pattern recognition receptors such as Dectin-1. By concealing these molecules, fungi suppress early immune response and slow down inflammatory responses.

Another example of immune evasion is encapsulated fungi. The carbohydrate covering of Cryptococcus neoformans has a key part in its virulence. The capsule disrupts phagocytosis through macrophages and suppressing inflammatory signaling which enables the survival of the organism and spread especially into the central nervous system.

Candida albicans species also exhibit immune adaptation by surviving within macrophages and subsequently escaping by the way of filaments. This intracellular persistence adds to invasion of bloodstream and systemic candidiasis.

C. Biofilm Formation

The other significant determinant of fungal virulence is biofilm formation particularly in Candida infection related to medical devices. A biofilm is an organized group of fungal cells imbedded within a self-generated extracellular matrix that is made of polysaccharides, proteins, lipids, and extracellular DNA^.[7]

These biofilms are common on the indwelling devices including catheters, prosthetic valves and implants. Under this structured environment, fungal cells take on altered metabolic conditions that make them resistant to antifungal agents. The surrounding matrix restricts penetration of drugs whereas biofilm-associated cells usually enhance efflux pumps and stress-response mechanisms that enhance resistance to Antifungal drugs, such as azoles and others.

Figure 1: Schematic illustration of Tissue invasion and Immune evasion

Figure 2: Schematic illustration of Biofilm formation

3. Hyaluronic Acid-Coated Nanoemulsions for CD44 targeting in Antifungal therapy

3.1 Hyaluronic Acid: Structural characteristics & molecular weight dependent behaviour

Structural Characteristics of  Hyaluronic acid

Hyaluronic acid (HA) is a naturally occurring polysaccharide that forms the basic constituent of the connective, epithelial and neural tissue extracellular matrix. Unlike many other glycosaminoglycans, HA is not sulfated and it is not attached to a core protein. Instead, it exists as a free, linear polymer made up of repeating disaccharide units of the D-glucuronic acid and N-acetyl-D-glucosamine.The sugars are also interchangeably connected by  β-(1→3) and β-(1→4) glycosidic bonds, and link themselves together into long chains which may be vary widely in size. This linear polymer can reach extremely high molecular weights of between a few kilodaltons up to several million daltons according to its biological source and processing.^[8]

Unlike sulfated glycosaminoglycans such as heparin or chondritin sulfate, HA is synthesized at the plasma membrane and extruded directly into the extracellular space without covalent attachment to a core protein. Its single repeating structure, and lack of sulfation make it less immunogenic, which enhances its great biocompatibility and biodegradability. Enzyme degradation takes place through hyaluronidases, producing smaller fragments, which are capable of having specific biological effects.

From a drug delivery perspective, several structural features make HA highly attractive:

• Hydrophilicity, enabling surface hydration and stealth properties
• Mucoadhesive behavior, due to hydrogen bonding and electrostatic interactions
• Receptor specificity, particularly toward CD44
• Chemical modifiability, allowing conjugation or surface coating of nanoparticles

These structural attributes form the basis for HA’s dual role as both a biological ligand and a functional polymer in nanocarrier systems.

Figure 3 : Structure & Applications of hyaluronic acid

Molecular Weight-Dependent Biological Behavior

The variability of the molecular weight is one among the typical characteristics of HA. Depending on tissue origin and physiological conditions, HA chains could be a few kilodaltons up to several million daltons. This size difference is biologically significant.HA of high molecular weight is considered to be normally related to anti-inflammatory effects, structural support and hydration. In contrast, smaller-molecular-weight fragments which are frequently produced in case of tissue damage or enzyme activity are capable of taking part in inflammatory communication and immune regulation.^[9]

Table 2 : Molecular weight dependent behaviour of Hyaluronic acid

CD44 is a type I transmembrane glycoprotein and plays the role of a major regulator of cell adhesion, migration, immunodulators, and tissue remodeling. It is widely expressed in numerous cell types, and consists of epithelial and endothelial cells, fibroblasts, macrophages, neutrophils and lymphocytes.

The structure of CD44 consists of three major parts: an extracellular ligand domain responsible for ligand recognition, a single transmembrane segment and a cytoplasmic tail which links to intracellular signaling apparatus and the cytoskeleton. Hyaluronic acid (HA) is the best among its ligands described and most physiologically relevant binding partner.^[14]

CD44 Standard and Variant Isoforms

The gene of the CD44 is highly subject to alternative splicing to produce numerous variants with distinct biological properties. The standard isoform (CD44s) contains the conserved exons encoding the HA-binding extracellular domain, transmembrane region, and cytoplasmic tail. In contrast, variant isoforms (collectively termed CD44v) arise through insertion of additional variant exons into the extracellular portion of the receptor.

These structural variations impact on the interaction of ligands and downstream signaling ability. Variant isoforms are often related to inflammatory, tissue remodelling, and pathological processes. Through communication with cytoskeletal associated proteins and signaling molecules, CD44 isoforms have the power to modulate pathways like MAPK and PI3K/Akt hence affecting cell survival, proliferation and migration.

Figure 4: Schematic protein structure of the CD44 molecule.

CD44 as a molecular target in Fungal Infections

During fungal infections particularly the invasive forms, the host has a strong inflammatory response marked by cytokine release and immune cell recruitment.These proinflammatory agents stimulate a rise in the expression of CD44 on epithelial and immune cells in sites of infection. Studies of pulmonary inflammation has demonstrated that the alveolar epithelial cells and the macrophages increase CD44 in response to inflammatory stimuli. This upregulation has been found, similarly, in mucosal and systemic infections in which tissue damage and immune activation occurs.

CD44 contributes  to the host defense as it helps in the adhesion and migration of leukocytes to inflamed tissues. Its cytoplasmic tail interacts with cytoskeletal components and improving the phagocytic activity. Interaction of CD44 and HA has the potential to stimulate intracellular signaling receptors controlling the production of cytokines, amplification of inflammation and tissue repair.

Therefore, CD44 is not only an adhesive molecule, but also a dynamic regulator of signalling in the course of infection.

HA-CD44 interaction and Receptor-Mediated Internalization.

Hyaluronan-binding domain is a defined domain of CD44 located in its extracellular part and which interacts with HA using a combination of hydrogen bonding and electrostatic forces. Binding of HA can stimulate receptor clustering on the cell surface. Such clustering facilitates rearrangement of cytoskeletons that may induce endocytosis via receptors.^[11]

The internalization mechanism is specifically relevant to drug delivery applications. When nanoemulsions or nanoparticles are coated with HA, they can exploit CD44 recognition to facilitate targeted cellular uptake. Instead of relying soley on passive diffusion, HA-decorated systems may enter the controlled endocytic route of CD44-rich cells to enhances the intracellular drug accumulation.

Some fungal pathogens such as Candida species are able to survive within macrophages, thus delivery of drugs into the cell a significant therapeutic factor. Since the expression of CD44 is elevated in inflamed and infected tissues,HA-coated nanoemulsions can be potentially accumulate in these receptor-rich conditions. Targeting CD44 increases localization to pathological sites and facilitates immune or epithelial cell uptake via receptors.

Such targeted strategy has two possible benefits in antifungal therapy:

• Enhanced drug concentration in the sites of infection.
• Reduced systemic toxicity, which could be a result of reduced exposure of healthy tissues.

The biological properties of CD44 its expression in inflammation, ability to mediate endocytosis, and immune regulation role- make it a rational molecular target of HA-functionalized nanocarrier systems in the management of infectious diseases.

In targeted delivery systems such as hyaluronic acid coated nanoemulsions, HA on the surface of the nanocarrier has a specific binding to receptor of CD44 at sites of infection. The resultant receptor-mediated uptake is able to increase the localization of drugs in diseased tissues and decrease healthy cell exposure. This approach therefore enhances the effectiveness of treatment and reduces the systemic toxicity.

Figure 5 : Hypothetical communication between hyaluronic acid and its receptors.

4. Formulation Development of HA-Coated Nanoemulsions & Safety considerations^[17].

4.1 Components of HA-Coated Nanoemulsion

(A) Drug selection

Poorly water soluble antifungal drugs are best candidates because:

• High lipophilicity
• Low aqueous solubility
• Requirement of better oral bioavailability.

(B) Oil Phase Selection

It should have a high solubilization capacity of the antifungal drug in oil phase and it should maintain acceptable safety profiles. The choice of oil is directed by solubility screening studies.

Table 3 : Safety considerations of Oils

(C) Surfactant and Co-Surfactant Selection

Surfactants with appropriate hydrophilic-lipophilic balance (HLB) values are selected to ensure stable nano-sized droplets. Safety and regulatory acceptability must be considered.

Table 4 : Safety considerations of surfactants & co-surfactants

4.2  Pseudo-Ternary Phase Diagram Construction

The construction of a pseudo-ternary phase diagram is a foundational step in the rational development of nanoemulsion and self-emulsifying systems. It is characterized as pseudo because the surfactant and  co-surfactant blend (Smix) is considered as a single entity and hence makes a four-component system simpler by dividing into three compositional axes of oil, Smix, and water. This is a graphical method that can be used to conduct a systematic approach of phase behaviour mapping as well as determination of areas where spontaneous nanoemulsification takes place.

The recent studies on lipid-based formulations point out that pseudo-ternary phase diagrams reduce empirical trial-and-error experimentation through clearly identifying concentration ranges which yield transparent, isotropic, and thermodynamically stable systems ^[19] .In antifungal nanoemulsion development, this strategy is particularly valuable since there should be enough oil to dissolve highly lipophilic drugs, but the level of surfactants should not be above the safe regulatory ranges.

The process is usually initiated by solubility screening of the drug using desired oils, surfactants and co-surfactants. Surfactant and co-surfactant are then mixed in known proportions (e.g., 1: 1, 2: 1, or 3: 1) to form Smix after the selection of appropriate components. Based on this, oil and Smix are mixed in different proportions ratios, usually 9:1 to 1: 9. Under gentle agitation, water is titrated gradually and a visual assessment of each system is done in terms of clarity, homogeneity, turbidity or phase separation.

Compositions giving clear and low-viscosity systems are plotted on triangular coordinates to trace the nanoemulsion area. The larger the nanoemulsion areas, the higher the emulsification efficiency and flexibility in formulation design. Phase diagram analysis according to Pouton and Porter is still among the reliable methods of maximizing lipid-based drug delivery system, especially SEDDS and nanoemulsions platforms.

Figure 6: Schematic figure of Pseudo-Ternary phase diagram.

4.3 Methods of Nanoemulsion Preparation

Nanoemulsions can be produced through high-energy or low-energy approaches, and the choice depends on formulation requirements, scalability, and desired droplet characteristics.

High-energy methods make use of mechanical forces to rupture coarse emulsions into nanometric droplets. Intense shear and cavitation forces are exerted on the emulsion through the force of high-pressure homogenization via a narrow valve of high pressure. similarly, ultrasonication generates acoustic cavitation bubbles that collapse violently and breaking droplets down to nanoscale dimentions. These approaches are especially effective in the case when a small droplet size distribution and a precise control below 150-200 nm are required and is often desirable to enhance oral absorption of lipophilic antifungal agents ^[19].

Low-energy methods, on the other hand, use the physicochemical nature of surfactants and take advantage of their inherent characteristics where interfacial tension varies instead of extrinsic mechanical energy. Spontaneous emulsification takes place when an oil-surfactant mixture is introduced into the aqueous phase and causes rapid droplet formation driven by interfacial turbulence. Phase inversion techniques make use of temperature or compositional variations to induce the formation of nanoemulsions^[20].

Recent formulation literature suggests that low-energy systems are appealing to oral antifungal delivery due to simplicity, cost-effectiveness, and ease of scale-up, whereas high-energy systems may offer superior control over droplet size distribution and physical stability.

Figure 7 : Schematic representation of high energy and low energy methods for preparing  O/W  nanoemulsions