Innovative HPMC/PVP K90 Dissolving Microneedles Incorporating Tacrolimus-Loaded Cubosomes: A Novel Strategy for Managing Allergic Conjunctivitis

Abstract

Background/Objectives: Allergic conjunctivitis (AC) is the most common inflammatory disease affecting the ocular conjunctiva. Tacrolimus (TCR), a potent calcineurin inhibitor, is limited by poor aqueous solubility and low ocular bioavailability. This study aimed to develop TCR-loaded cubosomes (TCR-Cubs) incorporated into HPMC/PVP K90 dissolving microneedles (MNs) to enhance their therapeutic efficacy.

Methods: TCR-Cubs were prepared using a modified top-down fragmentation method with glyceryl monooleate and poloxamer 407, optimized via Box–Behnken design, and incorporated into dissolving MNs. The system was evaluated in vitro, ex vivo, and in vivo using a rabbit model of allergic conjunctivitis.

Results: The optimized formulation exhibited the smallest particle size (210 ± 0.91 nm), polydispersity index (0.29 ± 0.03), zeta potential (−21 ± 0.87 mV), and the highest entrapment efficiency (% 93.3 ± 0.45). The optimized formulation was incorporated into MNs via micro molding. Scanning electron microscopy (SEM) confirmed well-defined, sharp microneedles, with low height reduction (<10%) by mechanical testing and high penetration efficiency (>85–90%). In vitro release studies revealed sustained drug release of (~75–80%) over 24 h, compared to (~40%) from the TCR suspension, following diffusion-controlled kinetics. Ex vivo permeation studies showed a (~2–3-fold) enhancement in corneal drug flux. In vivo pharmacodynamic evaluation using an ovalbumin-induced allergic conjunctivitis model demonstrated significant reductions in inflammatory mediators, including inflammatory markers (TNF-α, IL-1β, IL-6, NLRP3), which were reduced by (~50–75%), with modulation of CPA3, BCL2, and TGF-β1 by qRT-PCR. Histopathology and TLR4 analysis confirmed reduced inflammation without irritation.

Conclusions: This dual-delivery system offers a promising, non-invasive platform for enhanced ocular delivery of tacrolimus with superior anti-inflammatory efficacy in allergic conjunctivitis.

Introduction

Allergic conjunctivitis (AC) exemplifies an IgE-mediated hypersensitivity reaction. In response to an environmental allergen, the body triggers mast cell degranulation, leading to the release of histamine and subsequent conjunctival hyperemia, pruritus, lacrimation, and, ultimately, chronic inflammatory changes [1,2]. Over six million Americans of all ages and walks of life have conjunctivitis, the leading cause of red eye. Frontline therapy for allergic conjunctivitis consists of both topical antihistamine and mast cell-stabilizing eye drops [3,4]. Because combined antihistamine formulations can relieve pruritus and redness while inhibiting mast cell degranulation, they are the preferred choice for ocular therapy. Pure antihistaminic drops (e.g., amaprine, emedastine) are relatively ineffective for providing anti-pruritic and anti-redness relief, which is why they are used in conjunction with prophylactic mast cell stabilizers (e.g., sodium cromolyn, ketotifen fumarate, or nedocromil), which are used for longer-term treatment and act much more slowly; and are used more as a treatment to prevent allergic conjunctivitis [1,5]. In more severe or refractory cases (such as vernal and atopic keratoconjunctivitis), a short course of topical corticosteroids, such as prednisolone acetate, dexamethasone, loteprednol, or fluocinolone, may be required. However, because prolonged use of these medications may result in serious adverse effects, patients must be evaluated regularly to identify early signs of complications (such as ocular hypertension, cataracts, and infections) [6,7]. For patients with chronic, severe, steroid-dependent allergic eye disease, calcineurin inhibitors have been shown to reduce the need for steroids and, therefore, type II and type IV closed (cavity) skin through free immunomodulation [8,9].

First, this kind of immunomodulation is highly effective because it targets the T-cell response. Furthermore, the topical administration of tacrolimus ointment (0.03%) to the eyelid margins or conjunctival sac is intended to reduce T-cell inflammation and therefore reduce the need for corticosteroids and control the symptoms of vernal and atopic keratoconjunctivitis [8,10,11]. Additionally, the long-term use of cyclosporine A emulsion or drop formulations has been shown to improve ocular surface inflammation and the stability of the tear film. An iron-binding glycoprotein, Lactoferrin, exhibits anti-inflammatory, antimicrobial, and immunomodulatory effects, and lower levels of lactoferrin are related to ocular surface disease, making adjunct or alternative therapy possible. While it is too early to reach any conclusions, initial exploratory research suggests that the lactoferrin component could alleviate and reduce inflammation in dry and allergic eye disease, as well as in the tear film. This is not to imply that her contribution is fully developed compared to the options offered by antihistamines and calcineurin inhibitors [12,13].

Tacrolimus (TCR) (FK506) is a 23-membered cyclic macrolide lactone with immunomodulatory function derived from the bacteria Streptomyces tsukubensis in 1984 [14,15]. TCR is a topical calcineurin inhibitor that acts as a steroid-sparing immunomodulator for serious or refractory cases of allergic and immune-mediated ocular surface disorders, particularly vernal and atopic keratoconjunctivitis. It inhibits T cell calcineurin, binds to FKBP-12, thereby blocking NF-AT-dependent transcription of cytokines such as IL-2, and reduces T cell, mast cell, and inflammatory mediator release from the eye [16,17]. TCR is classified as a Biopharmaceutics Classification System (BCS) class II drug, with low solubility and high permeability [18]. However, TCR’s physicochemical characteristics (high molecular weight (822.95 g/mol); higher lipophilicity (partition coefficient log P = 3.96 ± 0.83)) indicate that it is unable to permeate the dermal layers of the skin beyond the stratum corneum [19]. Previous studies showed that dermal application of TCR resulted in most of the drug remaining in the stratum corneum, with limited dermal penetration. There is hence a need for a new delivery system for TCR to facilitate penetration into the epidermal and dermal target layers beyond the stratum corneum [20,21].

Currently under investigation are several delivery mechanisms, including polymeric micelle nanocarriers, Solid Lipid Nanoparticles (SLNs), and other nanocarriers. However, the absence of reference products has prevented addressing issues such as poor solubility and high lipophilicity. When treating conjunctivitis, anti-allergic medications are often administered topically because of factors such as ease of administration, non-invasiveness, rapid onset, cost-efficiency, minimal systemic side effects, and high patient compliance [22,23,24]. Topically administered medications can diffuse into various ocular tissues, with the main pathway being the transcorneal route. The nature of the cornea and the poor physicochemical properties of the active drug substances present a challenge to achieving the desired antifungal bioavailability. In response to such challenges, a few non-conventional drug delivery systems have been developed, including lipid nanoparticles, microemulsions, Spanlastics, micelles, and in situ gels [19,25,26].

Recently, MO-based nanosystems, like cubosomes (Cubs), have been gaining attention in ocular drug delivery [27,28]. These cubosomes attain stability after forming a polymeric outer corona, which drives them towards a polymeric corona structure. Due to their large membrane and surface area and ability to achieve optimal drug loading compared to liposomes, cubosomes present promising biocompatibility and drug stability while providing controlled release, maintaining a high level of bioadhesion, and transcorneal permeability with prolonged corneal retention [29,30]. Cubosomes stabilized by nonionic block copolymers, such as Pluronic F127, form a polymeric corona that enhances colloidal stability and inhibits aggregation, surpassing conventional surfactants [31,32,33]. This field has experienced a significant increase in popularity due to recent developments in polymer cubosomes utilizing polystyrene scaffolds, poly (ionic liquid) block copolymers, and photocleavable variations for adjustable release and enhanced loading of hydrophobic pharmaceuticals. These designs provide bicontinuous channels for prolonged administration, suitable for BCS Class II medicines such as tacrolimus [34,35,36].

Microneedles (MNs), on the other hand, are more rigid structures with well-defined heights and spacing [1,37]. The use of microneedles (MNs) avoids contact with nerve fibers and blood vessels in the dermis, which can help alleviate pain. These concerns, along with adverse pain reactions, compliance, and treatment efficacy, have led to the development of MNs [38,39]. MNs can be made from various materials, most commonly metals, polymers, and silica. MNs can be classified into five different types depending on the mechanisms of delivery: solid, hollow, coated, dissolving, and hydrogel-forming MNs [40,41]. Dissolving MNs are made from biocompatible and non-toxic polymers that are soluble in water (e.g., hyaluronic acid, maltose, polyvinyl pyrrolidone, sucrose, and hydroxypropyl methylcellulose) [42]. These polymers are safe for use in the human body [43,44]. The active pharmaceutical ingredients (APIs) contained within dissolving MNs are either fully dissolved or uniformly dispersed within the MNs. In order to provide effective drug delivery, the MNs are designed to fully dissolve after insertion into the skin because the MNs are made from materials that will alter their structure, and water or other bodily fluids (e.g., interstitial fluid) will facilitate the MNs to dissolve and the APIs to be released transdermally, thereby leaving the MNs’ original shape behind. In particular, dissolving MNs have a great deal of versatility and have been designed to incorporate a wide variety of APIs and macromolecules (e.g., DNA, RNA, and proteins) as well as even deliver the large molecules donepezil hydrochloride and propranolol hydrochloride [45,46,47]. 3D printing and micro-electromechanical systems/micromachining (MEMS) have both been used to fabricate MN patches. Though the MEMS technique has the possibility for mass production and replication, it is very labor-intensive and requires a lot of training to learn the complicated multi-step process, and is, therefore, not as suitable as a 3D printing method [48,49].

To address these limitations, our study introduces cubosomes, which have previously been used as drug delivery systems, and, when combined with microneedles, provide a non-invasive and patient-friendly method for drug delivery. The dual delivery system, incorporating microneedles and cubosomes, is designed to deliver TCR (targeted cancer therapy) to the conjunctival space to inhibit conjunctivitis-causing inflammation and achieve higher drug concentrations in the conjunctiva, thereby increasing therapeutic effect and reducing adverse effects from systemic circulation. The results from the Box–Behnken optimization of the TCR cubosomes provided a quality statistical distribution of the size parameters, poly dispersity, zeta potential, and entrapment to design a microneedle system with pre-determined parameters for penetration, drug load, dissolution, and structural integrity determined by standard tests of Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC), and Fourier Transform Infrared Spectroscopy (FTIR). The purpose of this study was to evaluate the effectiveness of the new design to deliver TCR in the form of TCR-Cubosomes and TCR-Cub/HPMC/PVP K90- MNs for the first time in vivo to an allergically non-infectious conjunctivitis model (AC) in rabbits. Quantified the gene expression levels of BCL2-like 1/BCL2L1 (BCL2) and carboxypeptidase A3 (CPA3) and transforming growth factor beta 1 (TGF-β1) using qRT-PCR, and the tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) using ELISA. The histopathological evaluation and TLR4 immunohistochemistry of the proposed TCR-Cub/HPMC/PVP K90- MNs TLR4 give us the potential for future clinical use.

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Materials

Tacrolimus (TCR), glyceryl monooleate (GMO; type I, ≥99% purity), poloxamer 407 (Pluronic® F127; pharmaceutical grade), hydroxypropyl methylcellulose (HPMC E50), polyvinylpyrrolidone K-30 (PVP K-30; average MW ≈ 40,000 Da), and polyvinyl alcohol (PVA; average MW ≈160,000 Da, 88–89% hydrolyzed) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ovalbumin (OVA, Grade V) and aluminum hydroxide (AH) were also obtained from Sigma-Aldrich (St. Louis, MO, USA). Spectra/Por® regenerated cellulose dialysis membrane tubing (molecular weight cut-off 12,000–14,000 Da) was obtained from Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA). Chloroform, ethanol, acetonitrile, and methanol (HPLC grade) were supplied by Thermo Fisher Scientific (Waltham, MA, USA).

Elhabal, S.F.; Shoela, M.S.; Hassan, F.E.; Awad AbdelGhany Morsy, S.; Allam, S.; Aldeeb, R.A.E.; Taha, A.A.; Mostafa Abd El Galil, R.; M. Emam, A.; Elzohairy, N.A.; et al. Innovative HPMC/PVP K90 Dissolving Microneedles Incorporating Tacrolimus-Loaded Cubosomes: A Novel Strategy for Managing Allergic Conjunctivitis. Pharmaceutics 2026, 18, 459. https://doi.org/10.3390/pharmaceutics18040459

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