A novel approach to oral iron delivery using ferrous sulphate loaded solid lipid nanoparticles

Iron (Fe) loaded solid lipid nanoparticles (SLN’s) were formulated using stearic acid and iron absorp-tion was evaluated in vitro using the cell line Caco-2 with intracellular ferritin formation as a marker ofiron absorption. Iron loading was optimised at 1% Fe (w/w) lipid since an inverse relation was observedbetween initial iron concentration and SLN iron incorporation efficiency. Chitosan (Chi) was included toprepare chitosan coated SLN’s. Particle size analysis revealed a sub-micron size range (300.3 ± 31.75 nmto 495.1 ± 80.42 nm), with chitosan containing particles having the largest dimensions. As expected,chitosan (0.1%, 0.2% and 0.4% w/v) conferred a net positive charge on the particle surface in a concen-tration dependent manner. For iron absorption experiments equal doses of Fe (20 �M) from selectedformulations (SLN-FeA and SLN-Fe-ChiB) were added to Caco-2 cells and intracellular ferritin proteinconcentrations determined. Caco-2 iron absorption from SLN-FeA (583.98 ± 40.83 ng/mg cell protein)and chitosan containing SLN-Fe-ChiB (642.77 ± 29.37 ng/mg cell protein) were 13.42% and 24.9% greaterthan that from ferrous sulphate (FeSO4) reference (514.66 ± 20.43 ng/mg cell protein) (p ≤ 0.05). Wedemonstrate for the first time preparation, characterisation and superior iron absorption in vitro fromSLN’s, suggesting the potential of these formulations as a novel system for oral iron delivery

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PLGA microparticles entrapping chitosan-based nanoparticles for the ocular delivery of ranibizumab

Age-related macular degeneration (AMD) is the leading cause of certified vision loss worldwide. The standard treatment for neovascular AMD involves repeated intravitreal injections of therapeutic proteins directed against vascular endothelial growth factor, such as ranibizumab. Biodegradable polymers, such as poly(lactic-co-glycolic acid) (PLGA), form delivery vehicles which can be used to treat posterior segment eye diseases, but suffer from poor protein loading and release. This work describes a “system-within-system”, PLGA microparticles incorporating chitosan-based nanoparticles, for improved loading and sustained intravitreal delivery of ranibizumab. Chitosan-N-acetyl-l-cysteine (CNAC) was synthesized and its synthesis confirmed using FT-IR and 1H NMR. Chitosan-based nanoparticles composed of CNAC, CNAC/tripolyphosphate (CNAC/TPP), chitosan, chitosan/TPP (chit/TPP), or chit/TPP-hyaluronic acid (chit/TPP-HA) were incorporated in PLGA microparticles using a modified w/o/w double emulsion method. Nanoparticles and final nanoparticles-within-microparticles were characterized for their protein–nanoparticle interaction, size, zeta potential, morphology, protein loading, stability, in vitro release, in vivo antiangiogenic activity, and effects on cell viability. The prepared nanoparticles were 17–350 nm in size and had zeta potentials of −1.4 to +12 mV. Microscopic imaging revealed spherical nanoparticles on the surface of PLGA microparticles for preparations containing chit/TPP, CNAC, and CNAC/TPP. Ranibizumab entrapment efficiency in the preparations varied between 13 and 69% and was highest for the PLGA microparticles containing CNAC nanoparticles. This preparation also showed the slowest release with no initial burst release compared to all other preparations. Incorporation of TPP to this formulation increased the rate of protein release and reduced entrapment efficiency. PLGA microparticles containing chit/TPP-HA showed the fastest and near-complete release of ranibizumab. All of the prepared empty particles showed no effect on cell viability up to a concentration of 12.5 mg/mL. Ranibizumab released from all preparations maintained its structural integrity and in vitro activity. The chit/TPP-HA preparation enhanced antiangiogenic activity and may provide a potential biocompatible platform for enhanced antiangiogenic activity in combination with ranibizumab. In conclusion, the PLGA microparticles containing CNAC nanoparticles showed significantly improved ranibizumab loading and release profile. This novel drug delivery system may have potential for improved intravitreal delivery of therapeutic proteins, thereby reducing the frequency, risk, and cost of burdensome intravitreal injections

Cholesterol-poly(ethylene) glycol nanocarriers for the transscleral delivery of sirolimus

The aim of this study was to prepare and characterize cholesterol-poly(ethylene) glycol (chol-PEG) nanocarriers of two different molecular weights (1 and 5 kDa) and to determine their effect on the transscleral retention and permeation of a lipophilic multi-therapeutic agent, sirolimus (rapamycin), with potential application in angiogenic and immunogenic ocular diseases. Sirolimus-containing nanocarriers were prepared using the thin-film hydration method and characterized for their physicochemical properties including size, drug entrapment (EE) and loading (DL) efficiencies, stability, surface charge, morphology, critical micelle concentration (CMC) and thermal properties. Ussing chambers were used to determine the retention and permeability of sirolimus-containing nanocarriers in porcine sclera followed by ultrastructural tissue examination. Sirolimus-containing nanocarriers had an average size of 11.7 nm (chol-PEG 1 kDa) and 13.8 nm (chol-PEG 5 kDa) and zeta potentials of 0.41 and -1.05, respectively. Both nanocarriers had similar transscleral permeabilities (chol-PEG 1 kDa 6.44 × 10(-7) and 5 kDa 6.16 × 10(-7) cm2 s(-1)), and very high scleral retention compared with a free solution of sirolimus (chol-PEG 1 kDa 16.9 μg/g; chol-PEG 5 kDa 7.48 μg/g; free sirolimus 0.57 μg/g). The DL (EE) for chol-PEG 1 and 5 kDa were 2.93% (77.4%) and 3.10% (81.6%), respectively. The CMC values for the nanocarriers were similar to those previously reported in literature (3.85 × 10(-7) M for chol-PEG 1 kDa; 4.26 × 10(-7) M for chol-PEG 5 kDa). In conclusion, chol-PEG nanocarriers successfully loaded sirolimus and resulted in scleral permeation and high retention, which shows potential utility for the topical delivery of lipophilic ocular drugs

Determining vitreous viscosity using fluorescence recovery after photobleaching

PURPOSE: Vitreous humor is a complex biofluid whose composition determines its structure and function. Vitreous viscosity will affect the delivery, distribution, and half-life of intraocular drugs, and key physiological molecules. The central pig vitreous is thought to closely match human vitreous viscosity. Diffusion is inversely related to viscosity, and diffusion is of fundamental importance for all biochemical reactions. Fluorescence Recovery After Photobleaching (FRAP) may provide a novel means of measuring intravitreal diffusion that could be applied to drugs and physiological macromolecules. It would also provide information about vitreous viscosity, which is relevant to drug elimination, and delivery. METHODS: Vitreous viscosity and intravitreal macromolecular diffusion of fluorescently labelled macromolecules were investigated in porcine eyes using fluorescence recovery after photobleaching (FRAP). Fluorescein isothiocyanate conjugated (FITC) dextrans and ficolls of varying molecular weights (MWs), and FITC-bovine serum albumin (BSA) were employed using FRAP bleach areas of different diameters. RESULTS: The mean (±standard deviation) viscosity of porcine vitreous using dextran, ficoll and BSA were 3.54 ± 1.40, 2.86 ± 1.13 and 4.54 ± 0.13 cP respectively, with an average of 3.65 ± 0.60 cP. CONCLUSIONS: FRAP is a feasible and practical optical method to quantify the diffusion of macromolecules through vitreous

Influence of molecular shape, conformability, net surface charge, and tissue interaction on transscleral macromolecular diffusion

Purpose: To study the influence of molecular shape, conformability, net surface charge and tissue interaction on transscleral diffusion. Methods: Unfixed, porcine sclera was clamped in an Ussing chamber. Fluorophore labelled, neutral, albumin, dextran, or ficoll were placed in one hemi-chamber and the rate of transscleral diffusion was measured over 24 hours using a spectrophotometer. Experiments were repeated using dextrans and ficoll with positive, or negative, net surface charges. Fluorescence recovery after photobleaching (FRAP) was undertaken to compare transscleral diffusion with diffusion through a solution. All molecules were 70 kDa. Results: Using FRAP, mean ± SD diffusion coefficient (D) was highest for albumin, followed by ficoll, then dextran (p = 0.0005). Positive dextrans diffused fastest, followed by negative, then neutral dextrans (p = 0.0005). Neutral ficoll diffused the fastest, followed by positive then negative ficoll (p = 0.0008). For the neutral molecules, transscleral D was highest for albumin, followed by dextran, then ficoll (p < 0.0001). D was highest for negative ficoll, followed by neutral, then positive ficoll (p < 0.0001). By contrast, D was highest for positive dextran, followed by neutral, then negative dextran (p = 0.0021). Conclusions: Diffusion in free solution does not predict transscleral diffusion and the molecular-tissue interaction is important. Molecular size, shape, and charge may all markedly influence transscleral diffusion, as may conformability to a lesser degree, but all need to be considered when selecting or designing drugs for transscleral delivery