Innovative Microneedles for Enhanced Amphotericin B Delivery

Introduction to Chitosan-Based Microneedles

Fungal keratitis (FK) remains a significant global health concern, with an alarming incidence rate leading to blindness due to inadequate treatment methods. The primary challenge in treating FK is the limited penetration of antifungal agents like Amphotericin B (AmB) into the corneal tissue, compounded by the increasing resistance of fungal pathogens. This issue necessitates innovative drug delivery systems that can enhance therapeutic efficacy while minimizing toxicity.

Microneedles (MNs) represent a promising solution for the ocular delivery of drugs, particularly in the context of FK treatment. These devices, typically measuring between 60 and 1000 μm in height, create microchannels in the epithelial layer of the cornea, thereby facilitating improved drug absorption. Chitosan, a biocompatible polysaccharide derived from chitin, is an ideal material for constructing MNs due to its favorable properties such as biodegradability, non-toxicity, and ability to enhance drug solubility and stability.

Recent advancements have leveraged chitosan-based MNs for the delivery of AmB encapsulated in oleosomes—lipid-based carriers that enhance the solubility and stability of hydrophobic drugs. Oleosomes consist of a lipid bilayer that can encapsulate the drug, allowing for a controlled release profile and prolonged contact time with ocular tissues. The incorporation of oleosome-loaded MNs into therapeutic protocols could significantly alter the management of fungal keratitis, providing a robust platform for sustained drug delivery directly to the site of infection.

Advantages of Amphotericin B in Fungal Keratitis Treatment

AmB is a polyene antifungal agent known for its broad-spectrum activity against various fungal pathogens, making it a cornerstone in treating severe mycoses. Its mechanism of action involves binding to ergosterol in fungal cell membranes, leading to the formation of pores that disrupt membrane integrity and ultimately cause cell death. However, the clinical utility of AmB is hindered by its poor solubility in aqueous environments and significant renal toxicity associated with intravenous formulations.

Despite these limitations, AmB remains a potent option for treating FK, particularly against pathogens such as Candida albicans and Aspergillus species. The emergence of antifungal resistance has further accentuated the need for effective formulations of AmB to ensure therapeutic success. The use of novel delivery systems such as chitosan-based MNs loaded with oleosomes can enhance the corneal permeability of AmB, allowing for higher concentrations at the infection site while mitigating systemic side effects.

Additionally, studies have highlighted the anti-inflammatory properties of AmB, which can play a vital role in alleviating keratitis-related inflammation. Recent advancements in formulation techniques aim to utilize AmB’s antifungal and anti-inflammatory properties synergistically, enhancing overall therapeutic outcomes in FK management.

Formulation and Characterization of Oleosome-Loaded Microneedles

The formulation of oleosome-loaded microneedles involves several critical steps, including the preparation of oleosomes and their subsequent incorporation into chitosan-based MNs. Oleosomes are typically prepared using the ethanol injection method, which involves combining phosphatidylcholine and sodium oleate to produce nanosized spherical globules that encapsulate AmB.

Oleosome Preparation

1. Materials:

   • Phosphatidylcholine (Lipoid S100): Serves as the primary lipid component, providing structural integrity and stability to the oleosomes.
   • Sodium oleate: Enhances the emulsifying properties of the oleosomes.
   • Amphotericin B: The active pharmaceutical ingredient.

2. Method:

   • The oleosomes are formed by dissolving the lipid components in ethanol, followed by rapid injection into an aqueous solution, leading to the spontaneous formation of lipid bilayers around the AmB.
   • Characterization of oleosomes includes assessing particle size, polydispersity index (PDI), zeta potential, and entrapment efficiency.

Characterization Techniques

• Dynamic Light Scattering (DLS): Used to measure the size and PDI of oleosomes, ensuring uniformity and stability.
• Zeta Potential Analysis: Evaluates the surface charge of oleosomes, influencing stability and interaction with biological membranes.
• Entrapment Efficiency (EE): Calculated to determine the proportion of AmB successfully encapsulated within the oleosomes.

These optimized oleosomes are then incorporated into MNs fabricated from chitosan and other excipients, facilitating a dual mechanism of action: enhancing the solubility of AmB and providing a minimally invasive delivery method through microneedles.

Antifungal Efficacy and Release Profile of AmB-Ole/MNs

The efficacy of AmB-Ole/MNs has been evaluated through in vitro and in vivo studies, demonstrating significant improvements in antifungal activity against common ocular pathogens:

In Vitro Studies

1. Antifungal Activity:

   • The AmB-Ole/MNs system exhibited potent biofilm disruption effects (>90%) against Candida albicans and Aspergillus niger, with inhibition zones measuring 27 mm and 32 mm, respectively.
   • Comparative studies showed that the oleosome formulation significantly enhanced the antifungal efficacy of AmB compared to conventional formulations.

2. Release Profile:

   • The sustained release of AmB from the oleosome-loaded MNs was confirmed, with approximately 70% of the drug permeating through corneal tissue within 80 hours.
   • This extended release profile is critical for providing lasting therapeutic concentrations at the infection site, thereby improving treatment outcomes and reducing the frequency of administration.

In Vivo Studies

In a rabbit model, the ocular tolerance and biocompatibility of the AmB-Ole/MNs system were assessed. Histopathological examinations revealed no significant adverse effects, confirming the safety of the formulation for ocular applications. Importantly, the AmB-Ole/MNs system targeted the TLR4/NLRP3 pathways, enhancing the therapeutic potential through modulation of the inflammatory response associated with fungal keratitis.

Safety and Biocompatibility in Ocular Applications

The safety profile of the AmB-Ole/MNs system is paramount for its clinical application in ocular therapies. The following aspects were evaluated:

1. Cytotoxicity Testing:

   • Cytotoxicity assays demonstrated reduced toxicity levels of AmB when delivered via the oleosome-loaded microneedles, showcasing improved biocompatibility compared to traditional formulations.

2. Histopathological Studies:

   • Comprehensive histopathological evaluations of ocular tissues after the administration of AmB-Ole/MNs indicated minimal inflammatory response and optimal tissue healing.

3. Regulatory Considerations:

   • The formulation strategy adheres to biocompatibility standards set forth by regulatory bodies, ensuring that the materials used in microneedle fabrication do not pose risks when in contact with sensitive ocular tissue.

Conclusion

The innovative formulation of chitosan-based microneedles loaded with oleosomes for delivering Amphotericin B marks a significant advancement in the treatment of fungal keratitis. This system enhances drug solubility, provides sustained release, and increases local drug concentration at the site of infection while minimizing systemic side effects. The promising results from both in vitro and in vivo studies underscore the potential of this delivery system in clinical settings, offering a new avenue for effectively managing ocular fungal infections.

References

1. Elhabal, S. F., Al-Zuhairy, S. A. K. S., Elrefai, M. F. M., El-Nabarawi, M. A., Hababeh, S., Zarif Attalla, K., … & Hamdan, A. M. E. (2025). Chitosan-Based Intelligent Microneedles for Delivery of Amphotericin B Loaded Oleosomes: Antifungal Ocular Patch Targeting for Effective Against Fungal Keratitis Using Rabbit Model via TLR4/NLRP3 Pathway. International Journal of Nanomedicine. Retrieved from https://doi.org/10.2147/IJN.S514798

2. Azzam, F., & Weege, N. (2025). Prooxidant effect of uric acid on human leukocytic DNA: An in vitro and ex vivo study. Turkish Journal of Biology. Retrieved from https://doi.org/10.55730/1300-0152.2735

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4. Smith, M. R., Jarrell, Z. R., Liu, K. H., Lee, C. M., Morgan, E. T., & Go, Y. M. (2025). Redox Metabolomics of Menthol in Children’s Plasma with Second-Hand Cigarette and Electronic Cigarette Exposures. Advances in Redox Research. Retrieved from https://doi.org/10.1016/j.arres.2025.100122

5. Ngamsangiam, P., Wantakan, T., Techa-ay, S., … & Thanee, M. (2025). Distinct chromosome abnormality patterns for differential diagnosis of hepatocellular carcinoma and cholangiocarcinoma. PLOS One. Retrieved from https://doi.org/10.1371/journal.pone.0322408

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FAQ

What are microneedles, and how do they work?

Microneedles are tiny needles, typically measuring between 60 to 1000 μm, that create microchannels in the skin or mucosal surfaces. They facilitate the delivery of drugs directly into the tissues, enhancing absorption and bioavailability.

What is Amphotericin B, and why is it important?

Amphotericin B is a broad-spectrum antifungal medication used to treat severe fungal infections. Its ability to target various pathogens makes it a critical option in managing life-threatening infections, particularly in immunocompromised patients.

How does the AmB-Ole/MNs system enhance treatment for fungal keratitis?

The AmB-Ole/MNs system improves ocular drug delivery by increasing corneal permeability and providing sustained release of Amphotericin B, thus ensuring higher drug concentrations at the site of infection while reducing systemic side effects.

Are there any side effects associated with the use of AmB?

While Amphotericin B is effective, it can have side effects including nephrotoxicity and infusion-related reactions. However, the oleosome-loaded microneedles aim to minimize these effects by localizing treatment and reducing systemic exposure.

What future research directions are being explored for this technology?

Future research will focus on optimizing the formulation parameters, conducting clinical trials to evaluate efficacy and safety in larger patient cohorts, and exploring the applicability of this technology for other ocular diseases.