Stephan Reichl

Diclofenac sodium delivery to the eye : In vitro evaluation of novel solid lipid nanoparticle formulation using human cornea construct

Solid lipid nanoparticles (SLNs) were prepared with a combination of homolipid from goat (goat fat) and phospholipid, and evaluated for diclofenac sodium (DNa) delivery to the eye using bio-engineered human cornea, produced from immortalized human corneal endothelial cells (HENC), stromal fibroblasts and epithelial cells CEPI 17 CL 4. Encapsulation efficiency was high and sustained release of DNa and high permeation through the bio-engineered cornea were achieved. Results obtained in this work showed that permeation of DNa through the cornea construct was improved by formulation as SLN modified with phospholipid.

Human corneal equivalent as cell culture model for in vitro drug permeation studies

Aims: For the study of transcorneal in vitro permeation of ophthalmic drugs, excised animal cornea or corneal epithelial cell culture are frequently used as a replacement for the human cornea. The main purposes of this study were to reconstruct a complete human organotypic cornea equivalent, consisting of all three different cell types (epithelial, stromal, and endothelial); to test the barrier function of this bio-engineered human cornea using three different model drugs (pilocarpine hydrochloride (PHCl), befunolol hydrochloride (BHCl), and hydrocortisone (HC)); and to determine its usefulness as an in vitro model for prediction of ocular drug absorption into the human eye. Methods: A multilayer tissue construct was created step by step in Transwell cell culture insert using SV-40 immortalised human endothelial and epithelial cells and native stromal cells (fibroblasts). Morphology was characterised by light microscopy using routine H&E staining. Scanning electron microscopy was used to evaluate ultrastructural features. Ocular permeation of drugs across the human cornea construct was tested using modified Franz cells and compared with data obtained from excised porcine cornea and previously described porcine cornea constructs. Results and conclusion: The cornea construct exhibited typical corneal structures such as a monolayer of hexagonally shaped endothelial cells and a multilayered epithelium consisting of seven to nine cell layers with flat superficial cells. The formation of microplicae and microvilli was also confirmed. The human cornea construct showed similar permeation behaviour for all substances compared with excised porcine cornea. However, permeability (permeation coefficients Kp) of the human cornea equivalent (PHCl 13.4•10−6 (SD 3.01•10−6); BHCl 9.88•10−6 (SD 1.79•10−6); HC 5.41•10−6 (SD 0.40•10−6) cm/s) was about 1.6–1.8 fold higher than excised porcine cornea. Compared with data from the porcine cornea construct the cultivated human equivalent showed a decreased permeability. The reconstructed human cornea could be appropriate to predict drug absorption into the human eye.

Films based on human hair keratin as substrates for cell culture and tissue engineering.

Keratin from hair or wool has been proposed as an appropriate material for producing films or cell cultivation scaffolds. The current study was performed to characterize two different approaches involving substrate coating based on keratin from human hair. Our goal was to evaluate cell growth behavior in these systems in comparison with a standard polystyrene substrate. The coating was made in two different ways: (i) by trichloroacetic acid precipitation or (ii) by casting a keratin nanosuspension. The resulting films were characterized using SDS-PAGE, SEM, and X-ray studies. The growth behaviors of twelve cell lines on the keratin films and on polystyrene were estimated using proliferation studies. Furthermore, we assessed the cell detachment behavior during trypsinization and the seeding efficiency. For epithelial cell lines with tight junction proteins, the transepithelial electrical resistance was measured and compared with values achieved using common coating materials. Both of the keratin coatings exhibited similar protein patterns and X-ray diffraction profiles, but we also detected differences in the transparency and ultrastructural surface morphologies. Culture dishes coated with keratin nanoparticles were used to create a transparent substrate that supports cell adherence and improves cell growth as compared with uncoated polystyrene or coatings that use trichloroacetic acid precipitation. We conclude that this coating method may be a new promising substrate for standard cell cultivation.

Keratin films for ocular surface reconstruction

Human amniotic membrane (AM) is frequently used as a substrate for ocular surface reconstruction. Its disadvantages (e.g., reduced transparency and biomechanical strength, heterogeneity depending on donor) create the need for standardized alternatives. Keratin from hair or wool has been proposed as an appropriate material for producing films or cell cultivation scaffolds. The current study was performed to develop transparent, stable and transferable films based on human hair keratin that support cellular adhesion and proliferation. The films were engineered by a multi-step procedure including keratin extraction, neutral and alkaline dialysis, drying and a curing process. Keratin films were investigated by SDS-PAGE, SEM and X-ray analyses. Furthermore, swelling and water absorption of the films were studied, as were tensile strength and light transmission (UV/VIS). Finally, the growth behavior of corneal epithelial cells on the keratin films and AM was estimated in proliferation studies. In addition, we assessed the seeding efficiency and cell detachment behavior during trypsinization. The film-forming process resulted in transparent films composed of nanoparticulate keratin structures. The film characteristics could be varied by changing the protein composition, adding softening agents or varying the curing temperature and duration. Based on these findings, an optimized protocol was developed. The films showed improved light transmission and biomechanical strength in comparison to AM. Furthermore, cell behavior on the films was similar to that found on AM. We conclude that keratin films may represent a new, promising alternative for ocular surface reconstruction.

The use of a porcine organotypic cornea construct for permeation studies from formulations containing befunolol hydrochloride

The purpose of this study was to develop an organotypic cornea equivalent consisting of three different cell types (epithelial, stromal and endothelial cells) and to investigate its usefulness as in vitro model for permeation studies. The different cell types of a porcine cornea were selectively isolated and a multilayer tissue construct was created step-by-step in Transwell cell culture insert. Histology, basement membrane components (laminin, fibronectin) and surfaces of cornea construct were investigated to evaluate the degree of comparability to porcine cornea from slaughtered animals. The cornea construct exhibited similarities to the original cornea. Ocular permeation of befunolol hydrochloride from different formulations across the cornea construct was tested using modified Franz cells and compared with data obtained from excised cornea. The cornea construct showed a similar permeation behavior for befunolol hydrochloride from different formulations compared with excised porcine cornea. However, permeation coefficients K(p) obtained with the construct were about three to fourfold higher for aqueous formulations and same for the w/o-emulsion. The reconstructed cornea could be an alternative to excised animal tissue for drug permeation studies in vitro.

Sustained release and permeation of timolol from surface-modified solid lipid nanoparticles through bioengineered human cornea.

Purpose: The aim of the study was to formulate and evaluate surface-modified solid lipid nanoparticles sustained delivery system of timolol hydrogen maleate, a prototype ocular drug using a human cornea construct. Materials and Methods: Surface-modified solid lipid nanoparticles containing timolol with and without phospholipid were formulated by melt emulsification with high-pressure homogenization and characterized by particle size, wide-angle X-ray diffraction, encapsulation efficiency, and in vitro drug release. Drug transport studies through cornea bioengineered from human donor cornea cells were carried out using a modified Franz diffusion cell and drug concentration analyzed by high-performance liquid chromatography. Results: Results show that surface-modified solid lipid nanoparticles possessed very small particles (42.9 ± 0.3 nm, 47.2 ± 0.3 nm, 42.7 ± 0.7 nm, and 37.7 ± 0.3 nm, respectively for SM-SLN 1, SM-SLN 2, SM-SLN 3, and SM-SLN 4) with low polydispersity indices, increased encapsulation efficiency (> 44%), and sustained in vitro release compared with unmodified lipid nanoparticles whose particles were greater than 160 nm. Permeation of timolol hydrogen maleate from the surface-modified lipid nanoparticles across the cornea construct was sustained compared with timolol hydrogen maleate solution in distilled water. Conclusions: Surface-modified solid lipid nanoparticles could provide an efficient way of improving ocular bioavailability of timolol hydrogen maleate.

Cell culture models of the human cornea — a comparative evaluation of their usefulness to determine ocular drug absorption in-vitro

Cell culture models of the cornea are continually developed to replace the isolated animal cornea for transcorneal drug absorption studies. The aim of this study was to determine and compare epithelial tightness and permeability of currently available corneal cell culture models to avoid interlaboratory variability and to assess their usefulness for in‐vitro permeation studies. Pure epithelial cell culture models (CEPI, SIRC and HCE‐T cell lines), primary cultures of human corneal epithelium (HCEpiC) and the two commercially available models (RHC and Epiocular), as well as organotypic human cornea constructs (HCC, HCC‐HCE‐T), were investigated and data were compared with those obtained from the excised bovine cornea. Barrier properties were assessed by measurements of transepithelial electrical resistance (TEER) and permeability of three passively absorbed substances (mannitol, testosterone and timolol maleate) with different physico‐chemical properties. TEER experiments revealed weak barrier functions for all of the investigated epithelial models (≤100–200 Ω cm2), except the HCE‐T cell line. Transport studies confirmed TEER results insofar that models showing low TEER values also had higher permeation rates in comparison with the excised bovine cornea. However, models based on HCE‐T cells demonstrated similar barrier properties to isolated corneal tissue. The corneal models investigated in our laboratory show clear differences in epithelial barrier function. In‐vitro systems comprising the HCE‐T cell line seem to be most appropriate to replace excised animal cornea for assessing corneal permeability.

RPMI 2650 epithelial model and three-dimensional reconstructed human nasal mucosa as in vitro models for nasal permeation studies

The purpose of this study was to investigate the human nasal epithelial cell line RPMI 2650 regarding its usefulness as in vitro model for drug permeation studies. Particularly, the influence of the air-liquid interface in culture and coculture with human nasal fibroblasts (HNF) on the differentiation and permeation barrier properties of the cell layer was examined. In addition to a non-contact coculture, we developed a three-dimensional construct of the human nasal mucosa composed of a collagen matrix with embedded HNF, covered by a RPMI 2650 epithelial cell layer. Microscopic examination as well as measurement of the transepithelial electrical resistance and permeation experiments showed the importance of cultivation at the air-liquid interface. Permeation studies were performed using a paracellular marker (sodium fluorescein), a transcellular marker (propranolol-HCl) and a model substance with high molecular weight (FITC-dextran, MW 4000). The epithelial model showed an organotypic permeation barrier for paracellular, transcellular and high MW permeation. Three-dimensional reconstructed human nasal mucosa showed four- to fivefold higher permeation coefficients. Regardless of the limits of these models, both offer promise to evaluate passive drug permeation through the nasal mucosa.

Review of Alternative Carrier Materials for Ocular Surface Reconstruction

Abstract Severe ocular surface disorders can result in deficiency of limbal stem cells that is potentially associated with chronic inflammation, impaired vision and even blindness. Advanced stem cells deficiency requires reconstruction of the OS with autologous or allogeneic limbal stem cells. To address such deficiency, a limbal tissue biopsy is taken and limbal cells are expanded on a carrier, which then can be used for OS reconstruction. Human amniotic membrane – currently the most common carrier for transplantation of limbal epithelial stem cells – has the downsides of carrying the risk of disease transmission, limited transparency, variable and unstable quality and low mechanical strength. This article reviews the advantages and disadvantages of the established carrier materials for limbal stem cell transplantation, as well as discussing emerging alternatives, including carriers based on collagen, fibrin, siloxane hydrogel contact lenses, poly(ε-caprolactone), gelatin–chitosan, silk fibroin, human anterior lens capsule, keratin, poly(lactide-co-glycolide), polymethacrylate, hydroxyethylmethacrylate and poly(ethylene glycol) for their potential use in the treatment of limbal stem cell deficiency.

Keratin film made of human hair as a nail plate model for studying drug permeation.

The limited source of human nail plate for studying drug permeation inspired us to develop a nail plate model made of human hair keratin. The manufacturing process consisted of keratin extraction, dialysis, molding, solvent evaporation, and curing, producing a water-resistant film. The permeability of the film was examined using three markers: sodium fluorescein, rhodamine B, and fluorescein isothiocyanate-dextran as water-soluble, lipid-soluble, and large molecule models, respectively. Bovine hoof was used for comparison. First investigation showed that keratin films (thickness 120 μm) resembled hooves (thickness 100 μm) except that these films were more permeable to rhodamine B compared with hooves (1.8-fold, p<0.01). Subsequent investigations using ungual penetration enhancers (urea, thioglycolic acid, and papain) showed that keratin films were generally more susceptible than hooves. This study revealed that the produced keratin film offers a possibility as a human nail plate substitute. However, inclusion of the penetration enhancer must be carefully interpreted.