The effect of charges on the corneal penetration of solid lipid nanoparticles loaded Econazole after topical administration in rabbits.

Fungal keratitis is an infectious disease caused by pathogenic fungi with a high blindness rate. Econazole (ECZ) is an imidazole antifungal drug with insoluble ability. Econazole-loaded solid lipid nanoparticles (E-SLNs) were prepared by microemulsion method, then modified with positive and negative charge. The mean diameter of cationic E-SLNs, nearly neutral E-SLNs and anionic E-SLNs were 18.73±0.14, 19.05±0.28, 18.54±0.10 nm respectively. The Zeta potential of these different charged SLNs formulations were 19.13±0.89, -2.20±0.10, -27.40±0.67 mV respectively. The Polydispersity Index (PDI) of these three kinds of nanoparticles were all about 0.2. The Transmission Electron Microscopy (TEM) and Differential Scanning Calorimetry (DSC) analysis showed that the nanoparticles were a homogeneous system. Compared with Econazole suspension (E-Susp), SLNs exhibited sustained release capability, stronger corneal penetration and enhanced inhibition of pathogenic fungi without irritation. The antifungal ability was further improved after cationic charge modification compared with E-SLNs. Studies on pharmacokinetics showed that the order of the AUC and t1/2 of different preparations was cationic E-SLNs > nearly neutral E-SLNs > anionic E-SLNs > E-Susp in cornea and aqueous humor. It was shown that SLNs could increase corneal penetrability and ocular bioavailability while these capabilities were further enhanced with positive charge modification compared with negative charge ones.

In Vitro and Ex Vivo Models for Assessing Drug Permeation across the Cornea.

Drug permeation across the cornea remains a major challenge due to its unique and complex anatomy and physiology. Static barriers such as the different layers of the cornea, as well as dynamic aspects such as the constant renewal of the tear film and the presence of the mucin layer together with efflux pumps, all present unique challenges for effective ophthalmic drug delivery. To overcome some of the current ophthalmic drug limitations, the identification and testing of novel drug formulations such as liposomes, nanoemulsions, and nanoparticles began to be considered and widely explored. In the early stages of corneal drug development reliable in vitro and ex vivo alternatives, are required, to be in line with the principles of the 3Rs (Replacement, Reduction, and Refinement), with such methods being in addition faster and more ethical alternatives to in vivo studies. The ocular field remains limited to a handful of predictive models for ophthalmic drug permeation. In vitro cell culture models are increasingly used when it comes to transcorneal permeation studies. Ex vivo models using excised animal tissue such as porcine eyes are the model of choice to study corneal permeation and promising advancements have been reported over the years. Interspecies characteristics must be considered in detail when using such models. This review updates the current knowledge about in vitro and ex vivo corneal permeability models and evaluates their advantages and limitations.

Pronounced capping effect of olaminosomes as nanostructured platforms in ocular candidiasis management.

The aim of this study was to formulate and boost ocular targeting of Fenticonazole Nitrate (FTN)-loaded olaminosomes in order to improve drug corneal permeation and candidiasis treatment. Olaminosomes were formulated by ethanol injection technique applying a central composite design. The independent variables were: span 80 amount (mg) (A), oleylamine concentration (mg%) (B) and oleic acid: drug ratio (C). The dependent responses were: percent entrapment efficiency (EE %), particle size (PS), poly-dispersity index (PDI), zeta potential (ZP) and in vitro drug release after 10 hours (Q10h). Numerical optimization by Design-Expert® software was adopted to select the optimum formula. This formula was chosen based on highest EE %, ZP (as absolute value) and Q10h and lowest PS and PDI. The optimum formula was subjected to further in vitro characterization via Differential scanning calorimetry, Transmission electron microscopy, Fourier transform infrared spectroscopy, pH determination, effect of storage, influence of terminal sterilization, detection of Minimal Inhibitory Concentration and ex vivo corneal penetration analysis. Safety and antifungal activity of the optimum formula were tested through various in vivo studies like ocular irritancy, corneal tolerance, corneal uptake and susceptibility test. The optimum formula with the maximum desirability value (0.972) revealed EE% (84.24%), PS (117.55 nm), ZP (-74.85 mV) and Q10h (91.26%) respectively. The optimum formula demonstrated ocular tolerance with enhanced corneal penetration behavior (428.66 µg/cm2) and boosted antifungal activity (56.13%) compared to FTN suspension (174.66 µg/cm2 and 30.83%). The previous results ensured the ability of olaminosomes to enhance the corneal penetration and antifungal efficacy of Fenticonazole Nitrate.

A dual-functional chitosan derivative platform for fungal keratitis.

Fungal keratitis remains a serious infectious ocular disease, and the traditional administration of eye drops is limited by ocular intrinsic barriers and drug shortages. Herein, we fabricated a chitosan-based dual-functional platform for ocular topical delivery of econazole. The platform can prolong the residence time on the ocular surface due to its strong interaction with the mucin layer by physical adhesion and covalent bonding, and also open corneal epithelial tight junctions for being positively charged, thereby enhancing corneal penetration of drug. Using these strategies, dosing concentration was reduced from 0.3 wt% to 0.1 wt%, dosing frequency was reduced from once-an-hour to twice-daily, in vitro and in vivo antifungal therapeutic effects were achieved and patient compliance could be improved. Given its high structural adaptability, many other ocular anterior segment-related diseases would benefit from this platform.

Assessment to the Antifungal Effects in vitro and the Ocular Pharmacokinetics of Solid-Lipid Nanoparticle in Rabbits.

INTRODUCTION: Fungal keratitis (FK) remains a severe sight-threatening disease, and case management is difficult due to ocular intrinsic barriers and drug shortages. Econazole (ECZ), a broad-spectrum antifungal agent, is limited in ocular applications due to the poor water solubility and strong irritant property. METHODS: We successfully prepared solid-lipid nanoparticle-based ECZ eye drops (E-SLNs) by microemulsion method, and the physicochemical properties of E-SLNs were investigated. Corneal permeability, antifungal ability against Fusarium spp., irritation and bioavailability compared to ECZ Suspension (E-Susp) were evaluated in vitro and in vivo. RESULTS: E-SLNs were a uniform and stable system which had an average particle size of 19 nm and a spherical morphology. E-SLNs also exhibited controlled release, enhanced antifungal activity without irritation. The pharmacokinetic analysis in vivo confirmed that E-SLNs showed an improved ocular bioavailability and the drug concentration in the cornea were above minimum inhibitory concentration (MIC) for 3 h after single administration. CONCLUSION: The E-SLNs colloid system is a promising therapeutic approach for fungal keratitis and could serve as a candidate strategy for other ocular diseases.

Celastrol-based nanomedicine promotes corneal allograft survival.

Effectively promoting corneal allograft survival remains a challenge in corneal transplantation. The emerging therapeutic agents with high pharmacological activities and their appropriate administration routes provide attractive solutions. In the present study, a celastrol-loaded positive nanomedicine (CPNM) was developed to enhance corneal penetration and to promote corneal allograft survival. The in vitro, in vivo and ex vivo results demonstrated the good performance of CPNM prolonging the retention time on ocular surface and opening the tight junction in cornea, which resulted in enhanced corneal permeability of celastrol. Both in vitro and in vivo results demonstrated that celastrol inhibited the recruitment of M1 macrophage and the expression of TLR4 in corneal allografts through the TLR4/MyD88/NF-κB pathway, thereby significantly decreasing secretion of multiple pro-inflammatory cytokines to promote corneal allograft survival. This is the first celastrol-based topical instillation against corneal allograft rejection to provide treatment more potent than conventional eye drops for ocular anterior segment diseases.

Preparation and characterization of novel, mucoadhesive ofloxacin nanoparticles for ocular drug delivery

The efficacy of conventional ocular formulations is limited by poor corneal retention and permeation, resulting in low ocular bioavailability. Mucoadhesive chitosan (CS)/ tripolyphosphatesodium (TPP) and chitosan (CS)/ tripolyphosphatesodium (TPP)-alginate (ALG) nanoparticles were investigated for the prolonged topical ophthalmic delivery of ofloxacin. A modified ionotropic gelation method was used to produce ofloxacin-loaded nanoreservoir systems. The ofloxacin-loaded CS/TPP and CS/TPP-ALG nanoparticles were characterized for particle size, morphology, zeta potential, encapsulation efficiency, subsequent release and corneal penetration study. The designed nanoparticles have a particle size from 113.8 nm to 509 nm and zeta potential from 16.2 mV to 40.3 mV and encapsulation efficiency values ranging from 19.7% to 33.1%. Nanoparticles revealed a release during the first hours, followed by a more gradual drug release. The ofloxacin-loading CS/TPP or CS/TPP-ALG NPs developed are pronounced penetration enhancing effect as compared to OFX solution (5-6.5 times). Thus, these nanoparticles have a strong potential for ocular drug delivery.

Gel in core carbosomes as novel ophthalmic vehicles with enhanced corneal permeation and residence.

Carbopol is a good bio-adhesive polymer that increases the residence time in the eye. However, the effect of blinking and lacrimation still reduce the amount of polymer and the incorporated drug available for bioadhesion. Gel-core liposomes are advanced systems offering benefits making it a good tool for improved ocular drug delivery and residence time. Incorporation of carbopol in gel-core liposomes and their potential in ocular delivery have not so far been investigated. Fluconazole (FLZ) was selected as a challenging important ocular antifungal suffering from poor corneal permeation and short residence time. In this study, gel-core carbosomes have been elaborated as novel carbopol-based ophthalmic vehicles to solve ocular delivery obstacles of FLZ and to sustain its effect. Full in vitro appraisal was performed considering gel-core structure, entrapment efficiency, particle size and stability of the vesicles as quality attributes. Structure elucidation of the nanocarrier was performed using optical, polarizing and transmission electron microscopy before and after Triton-X100 addition. Ex-vivo ocular permeation and in vivo performance were investigated on male albino rabbits. Optimized formulation (CBS5) showed gel-core structure, nanosize (339.00 ±â€¯5.50 nm) and not defined before (62.00% ±â€¯1.73) entrapment efficiency. Cumulative amount of CBS5 permeated ex-vivo after 6 h, was 2.43 and 3.43 folds higher than that of conventional liposomes and FLZ suspension, respectively. In-vivo corneal permeation of CBS5 showed significantly higher AUC0-24 h (487.12 ±â€¯74.80) compared to that of FLZ suspension (204.34 ±â€¯7.46) with longer residence time in the eye lasts for more than 18 h. In conclusion, novel gel-core carbosomes could successfully be used as a promising delivery system for chronic ocular diseases.

Fabrication of a drug delivery system that enhances antifungal drug corneal penetration.

Fungal keratitis (FK) remains a severe eye disease, and effective therapies are limited by drug shortages and critical ocular barriers. Despite the high antifungal potency and broad spectrum of econazole, its strong irritant and insolubility in water hinder its ocular application. We designed and fabricated a new drug delivery system based on a polymeric vector for the ocular antifungal application of econazole. This novel system integrates the advantages of its constituent units and exhibits superior comprehensive performance. Using the new system, drug content was significantly increased more than 600 folds. The results of in vivo and in vitro experiments demonstrated that the econazole-loaded formulation exhibited significantly enhanced corneal penetration after a single topical ocular administration, excellent antifungal activity, and good tolerance in rabbits. Drug concentrations and ocular relative bioavailability in the cornea were 59- and 29-time greater than those in the control group, respectively. Following the topical administration of one eye drop (50 µL of 0.3% w/v econazole) in fungus-infected rabbits, a high concentration of antimycotic drugs in the cornea and aqueous humor was sustained and effective for 4 h. The mechanism of corneal penetration was also explored using dual fluorescent labeling. This novel drug delivery system is a promising therapeutic approach for oculomycosis and could serve as a candidate strategy for use with various hydrophobic drugs to overcome barriers in the treatment of many other ocular diseases.

Semifluorinated alkane based systems for enhanced corneal penetration of poorly soluble drugs.

Semifluorinated alkanes (SFAs) are amphiphilic liquids that can dissolve hydrophobic drugs to form clear solutions. This study evaluated the potential of two SFAs to act as vehicle for topical ocular drug delivery. After confirming ocular safety, an ex vivo corneal penetration model was developed to determine drug distribution and corneal bioavailability. Hydrophobic dye distribution in the different corneal layers was visualised under a confocal microscope. Corneal bioavailability of cyclosporine A (CsA) dissolved in perfluorobutylpentane (F4H5) or perfluorohexyloctane (F6H8) was compared to commercially available CsA ophthalmic emulsions, Restasis® and Ikervis®. Precorneal residence of the four test vehicles containing the hydrophobic dye was also compared using an ex vivo corneal tissue model. Preferential accumulation of the hydrophobic dye in the corneal epithelium was observed with higher amounts detectable when delivered via the SFAs compared to Restasis or Ikervis. A significant improvement in corneal CsA penetration was observed after application of a single dose of 0.05% CsA in F4H5 and F6H8 when compared to Restasis with the area under curve over 4 h (AUC(0-4h)) being at least 8-fold greater for both SFAs (p < .0001). Moreover, the AUC(0-4h) of 0.1% CsA in F4H5 was almost 5-fold greater than Ikervis (p < .0001). Finally, the precorneal residence time of both SFA solutions was significantly longer than that of the commercial emulsions with the AUC(0-60min) being 2- to 11-fold greater. This study demonstrated that SFAs can significantly improve the local bioavailability of hydrophobic drugs by increasing corneal penetration as well as prolonging precorneal residence. They therefore offer a promising new platform for topical drug delivery to the eye.

Enhancement in Corneal Permeability of Dissolved Carteolol by Its Combination with Magnesium Hydroxide Nanoparticles.

We prepared magnesium hydroxide (MH) nanoparticles, and investigated their effect when combined with dissolved carteolol on the bioavailability and intraocular pressure (IOP)-reducing effect of carteolol. The carteolol was solved in saline containing additives (0.5% methylcellulose, 0.001% benzalkonium chloride, 0.5% mannitol; CRT-solution). MH nanoparticles were prepared by a bead mill method with additives. Then carteolol/MH microparticle and carteolol/MH nanoparticle fixed combinations (mCMFC and nCMFC) were prepared by mixing the CRT-solution and MH particles. The transcorneal penetration and IOP-reducing effect of carteolol was evaluated in rabbits. The mean particle size of mCMFC was 7.2 µm, and the particle size was reduced to 73.5-113.5 nm by the bead mill treatment. The MH particles in nCMFC remained in the nano size range for 8 days after preparation, and the amounts of lacrimal fluid and corneal damage were unchanged by repetitive instillation of nCMFC (twice a day for 4 weeks). The transcorneal penetration of carteolol was enhanced by the combination with MH nanoparticles, and the IOP-reducing effect of nCMFC was significantly higher than that of CRT-solution or mCMFC. In conclusion, we designed nCMFC, and showed that the high levels of dissolved carteolol can be delivered into the aqueous humor by the instillation of nCMFC. Combination with MH nanoparticles may achieve an enhancement of corneal penetration for water-soluble drugs. These findings provide significant information that can be used to design further studies aimed at developing anti-glaucoma eye drugs.

Corneal delivery of besifloxacin using rapidly dissolving polymeric microneedles.

Penetration of antibiotics into and through the cornea is a major limiting factor in the treatment of ocular infections. Several strategies are in vogue to overcome this limitation such as use of fortified drops, gels, and subconjunctival injections. Here, we present the fabrication of rapidly dissolving polymeric microneedle array to effectively deliver besifloxacin through the cornea. Microneedles were prepared using polyvinyl alcohol and polyvinyl pyrrolidone by the micromolding technique. The model fluoroquinolone antibiotic, besifloxacin, was loaded in 36 microneedles arranged in a 6 × 6 array format within a 1 cm2 area. The average height and base width of microneedles was 961 ± 27 and 366 ± 16 µm, respectively. Each microneedle array contained 103.4 ± 8.5 µg of besifloxacin. Cryosectioning and confocal microscopy of excised human cornea revealed that microneedles penetrated to a depth of up to 200 µm. Microneedles were found to completely dissolve in the cornea within 5 min. Application of microneedles for 5 min significantly (p < 0.05) improved the besifloxacin deposition and permeation through the cornea compared with free besifloxacin solution. Similarly, besifloxacin-loaded microneedles showed greater antibacterial activity in Staphylococcus aureus-infected cornea in comparison to free besifloxacin solution. Taken together, rapidly dissolving microneedles can be developed to effectively deliver besifloxacin to treat bacterial infections in the cornea and eye.

Co-instillation of nano-solid magnesium hydroxide enhances corneal permeability of dissolved timolol.

We prepared magnesium hydroxide (MH) nanoparticles by a bead mill method, and investigated whether the co-instillation of MH nanoparticles improves the low transcorneal penetration of water-soluble drugs, such as the anti-glaucoma eye drug timolol maleate (TM). MH particle size was decreased by the bead mill treatment to a mean particle size of 71 nm. In addition, the MH nanoparticles were highly stable. Next, we demonstrated the effect of MH nanoparticles on the corneal surface. MH shows only slight solubility in lacrimal fluid, and the instillation of MH nanoparticles for 14 days did not affect the behavior (balance of secretion and excretion) of the lacrimal fluid in rabbit corneas. Moreover, there was no observable corneal toxicity of MH nanoparticles, and treatment with MH nanoparticles enhanced the intercellular space ratio in the eyes of rats. MH alone did not permeate into the cornea; however, the co-instillation of MH nanoparticles and dissolved TM (nMTFC) enhanced the corneal penetration of TM. In addition, the intraocular pressure (IOP)-reducing effect of nMTFC was significantly higher than those of the TM solution or the co-instillation of MH microparticles and TM. In conclusion, we found that MH nanoparticles enhance the corneal penetration of dissolved TM with no observable corneal stimulation or obstruction of the nasolacrimal duct by the MH nanoparticles. It is possible that the co-instillation of MH nanoparticles may provide a useful way to improve the bioavailability of water-soluble drugs in the ophthalmic field. These findings provide significant information that can be used to design further studies aimed at developing anti-glaucoma eye drugs.

PLGA nanoparticles for ocular delivery of loteprednol etabonate: a corneal penetration study.

The purpose of the present study was to develop loteprednol etabonate (LE) loaded poly(d,l-lactide co-glycolide) (PLGA) nanoparticles (NPs) and study their penetration profile into the excised goat cornea. In the present study, LE loaded PLGA NPs were prepared by solvent evaporation with high speed homogenization method and the penetration profile was studied using confocal laser scanning microscopy (CLSM). Rhodamine (Rd) was used as a fluorescent marker to prepare Rd-LE-PLGA-NPs. The NPs were characterized for particle size, X-ray diffraction (XRD), differential scanning calorimetry (DSC), transmission electron microscopy (TEM), drug entrapment, and permeation profile. Intense fluorescence observed across the depths of goat corneal tissue suggested an improved penetration profile of NPs. The entrapment efficiency and mean diameter of the optimized formulation (F5) were found to be 96.31 ± 1.68% and 167.6 ± 0.37 nm, respectively. These findings indicate that LE loaded PLGA NPs may serve as a potential drug carrier for ocular administration in eye disease.

In vitro and ex vivo corneal penetration and absorption models.

Topical drug administration is the preferred route of drug delivery to the eye despite the poor bioavailability. To develop more efficient drug carriers, reliable in vitro or ex vivo models are required in the early stages of formulation development, with such methods being faster, cheaper, and more ethical alternatives to in vivo studies. In vitro cell culture models are increasingly being used for transcorneal penetration studies, with primary cell cultures and immortalized cell lines now giving way to the development of organotypic corneal constructs for studying ocular drug bioavailability. Artificially cultured human corneal equivalents are still in the early stages of development, but present tremendous potential for corneal penetration studies. Ex vivo models using excised animal tissue are also being used to study corneal penetration with promising results, although significant inter-species variations need to be considered. This review discusses the in vitro and ex vivo models currently being used to study corneal penetration and evaluates their advantages and limitations with a focus on diffusion cell assemblies. In addition to the tissue used, the diffusion cell set-up can significantly influence the penetration profile and should be cautiously adjusted to simulate clinical conditions.

Positively charged micelles based on a triblock copolymer demonstrate enhanced corneal penetration.

PURPOSE: The cornea is a main barrier to drug penetration after topical application. The aim of this study was to evaluate the abilities of micelles generated from a positively charged triblock copolymer to penetrate the cornea after topical application. METHODS: The triblock copolymer poly(ethylene glycol)-poly(ε-caprolactone)-g-polyethyleneimine was synthesized, and the physicochemical properties of the self-assembled polymeric micelles were investigated, including hydrodynamic size, zeta potential, morphology, drug-loading content, drug-loading efficiency, and in vitro drug release. Using fluorescein diacetate as a model drug, the penetration capabilities of the polymeric micelles were monitored in vivo using a two-photon scanning fluorescence microscopy on murine corneas after topical application. RESULTS: The polymer was successfully synthesized and confirmed using nuclear magnetic resonance and Fourier transform infrared. The polymeric micelles had an average particle size of 28 nm, a zeta potential of approximately +12 mV, and a spherical morphology. The drug-loading efficiency and drug-loading content were 75.37% and 3.47%, respectively, which indicates that the polymeric micelles possess a high drug-loading capacity. The polymeric micelles also exhibited controlled-release behavior in vitro. Compared to the control, the positively charged polymeric micelles significantly penetrated through the cornea. CONCLUSION: Positively charged micelles generated from a triblock copolymer are a promising vehicle for the topical delivery of hydrophobic agents in ocular applications.

Novel in situ gel systems based on P123/TPGS mixed micelles and gellan gum for ophthalmic delivery of curcumin.

Curcumin, a natural polyphenol compound, has been widely reported for diverse pharmacological effects and already been investigated for eye diseases. However, the water-insolubility of curcumin and the inherent penetration barriers in cornea make it difficult for curcumin to enter eye. This work aimed to develop ion-sensitive curcumin-loaded Pluronic P123 (P123)/D-a-tocopheryl polyethylene glycolsuccinate (TPGS) mixed micelle in situ gels (CUR-MM-ISGs) to prolong ocular retention time and improve cornea permeability. Central composite design-response surface methodology was applied for the optimization of curcumin-loaded P123/TPGS mixed micelles (CUR-MMs). Characterization tests showed that CUR-MMs were in spherical shape with small size and low critical micelle concentration. After dispersing the micelles in gellan gum solution (0.2%, w/w) at the ratio of 3:1 and 1:1 (v/v), respectively, CUR-MM-ISGs were formed and presented transparent appearance. Sustained release profile was obtained in vitro for both CUR-MM-ISGs (3:1 or 1:1, v/v). The irritation test proved that CUR-MM-ISGs as ophthalmic formulations were gentle and biocompatible towards ocular tissues. In addition, the ex vivo corneal penetration study indicated that the cumulative drug permeation amount of CUR-MM-ISGs (3:1, v/v) was respectively 1.16-fold and 1.32-fold higher than CUR-MM-ISGs (1:1, v/v) and curcumin solution. It can be concluded from these results that the developed ion-sensitive mixed micelle in situ gel system is a potential ophthalmic delivery carrier for curcumin as a poorly soluble drug.

Clinical utility of voriconazole eye drops in ophthalmic fungal keratitis.

Fungal keratitis is one of the major causes of ophthalmic mycosis and is difficult to treat. The range of common antifungal agents available for fungal keratitis remains inadequate and is generally associated with poor clinical outcomes. Voriconazole is a new generation triazole antifungal agent. Only marketed in systemic formulation and, with broad-spectrum activity and high intraocular penetration, voriconazole has demonstrated effectiveness against fungal keratitis. Systemic voriconazole, however, is not without side effects and is costly. Voriconazole eye drops have been prepared extemporaneously and used for the treatment of ophthalmic fungal keratitis. The current article sought to review the literature for evidence related to the effectiveness and safety of topical voriconazole and its corneal penetration into the aqueous humor of the eye. The voriconazole eye drops used are typically of 1% concentration, well tolerated by the eye, and are stable. Despite existing evidence to suggest that the eye drops are effective in the treatment of fungal keratitis, more studies are needed, especially in relation to using the eye drops as first-line and stand-alone treatment, preparation of higher concentrations, and optimal dosing frequency.