In vitro and in vivo assessment of delivery of hydrophobic molecules and plasmid DNAs with PEO-PPO-PEO polymeric micelles on cornea.

The stability and bio-distribution of genes or drug complexes with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO, Pluronic F-68) polymeric micelles (PM) are essential for an effective nanosized PM delivery system. We used Förster resonance energy transfer (FRET) pairs with PM and measured the FRET ratio to assess the stability of PM in vitro and in vivo on the cornea. The FRET ratio reached a plateau at 0.8 with 3% PM. Differential scanning calorimetry measurement confirmed the complex formation of FRET pairs with PM. Confocal imaging with the fluorophores fluorescein isothiocyanate isomer I (FITC) and rhodamine B base (RhB) also showed the occurrence of FRET pairs in vitro. The fluorophores were mixed with 3% PM solution or the FITC-labeled PEO-PPO-PEO polymers (FITC-P) were mixed with RhB-labeled plasmids (RhB-DNA). In addition, the in vitro corneal permeation of FRET pair complexes with PM reached a 0.8 FRET ratio. One hour after eye drop administration, FRET pairs colocalized in the cytoplasm, and surrounded and entered the nuclei of cells in the cornea, and the polymers were located in the corneal epithelial layers, as detected through anti-PEG immunohistochemistry. Furthermore, fluorescence colocalization in the cytoplasm and cell nucleus of the corneal epithelium was confirmed in tissues where RhB or RhB-DNA complexed with FITC-P was found to accumulate. We demonstrate that at a concentration of 3%, PM can encapsulate FRET pairs or RhB-DNA and retain their integrity within the cornea 1 h after administration, suggesting the feasibility and stability of PEO-PPO-PEO polymers as a vehicle for drug delivery.

Nanosponges encapsulating dexamethasone for ocular delivery: formulation design, physicochemical characterization, safety and corneal permeability assessment.

Nanosponges (NS) were synthesized by crosslinking of beta cyclodextrins with diphenyl carbonate. It is a hyper-branched polymer with an ultra high encapsulation efficiency leading to formation of colloidal systems. Dexamethasone (DEX) on the other hand is a poorly soluble drug with a poor corneal permeability. Excessive instillation of ocular suspension of dexamethasone leads to various complications thereby necessitating a novel nanotherapeutic system with greater ocular retention and permeation. This study aimed at formulating complexes of DEX with three types of beta-cyclodextrin NS obtained with different cross-linking ratio (viz. 1:2, 1:4 and 1:8 on molar basis with the cross-linker) for ocular applications. Nano-encapsulation was done by incubation-lyophilization technique to yield various formulations of Nanosponges (viz. F1:2, FI:4 and F1:8). From drug loading studies it was found that DEX was loaded in the highest amount in (NS 1:4), as much as 10% w/w as compared to 3% w/w and 5% w/w in 1:2 and 1:8 types of NS respectively. In vitro release studies showed that release of DEX was in a controlled manner for around 300 minutes. The particle sizes of the loaded NS formulations were between 350 and 660 nm with low polydispersity indices. The zeta potentials were sufficiently high (-20 to -27 mV) to obtain a stable colloidal nanosuspension. X-ray powder diffraction, differential scanning calorimetry and Fourier transform infra-red attenuated transmittance reflectance spectroscopy studies confirmed the interactions and encapsulation of DEX with NS. Transmission electron microscopy and atomic force microscopy studies confirmed its spherical colloidal nature. Ex vivo safety assessment done on bovine cornea showed no adverse reactions proving the safety of the system. Adhesion and retention properties of NS formulations were confirmed by use of 6-Coumarin as a model fluorescent marker. Corneal permeability of DEX from optimized formulations done on excised bovine cornea in corneal holders showed that the NS formulation showed higher permeability than the marketed formulation.

Assessment of corneal hydration sensing in the terahertz band: in vivo results at 100 GHz.

Terahertz corneal hydration sensing has shown promise in ophthalmology applications and was recently shown to be capable of detecting water concentration changes of about two parts in a thousand in ex vivo corneal tissues. This technology may be effective in patient monitoring during refractive surgery and for early diagnosis and treatment monitoring in diseases of the cornea. In this work, Fuchs dystrophy, cornea transplant rejection, and keratoconus are discussed, and a hydration sensitivity of about one part in a hundred is predicted to be needed to successfully distinguish between diseased and healthy tissues in these applications. Stratified models of corneal tissue reflectivity are developed and validated using ex vivo spectroscopy of harvested porcine corneas that are hydrated using polyethylene glycol solutions. Simulation of the cornea's depth-dependent hydration profile, from 0.01 to 100 THz, identifies a peak in intrinsic reflectivity contrast for sensing at 100 GHz. A 100 GHz hydration sensing system is evaluated alongside the current standard ultrasound pachymetry technique to measure corneal hydration in vivo in four rabbits. A hydration sensitivity, of three parts per thousand or better, was measured in all four rabbits under study. This work presents the first in vivo demonstration of remote corneal hydration sensing.