Simultaneous amelioration of diabetic ocular complications in lens and retinal tissues using a non-invasive drug delivery system.

Topically administered delivery systems for ophthalmic applications have been studied for the treatment of anterior or posterior eye diseases. However, simultaneous treatment of both anterior and posterior eye diseases has not been explored. In this study, we fabricated a topically administrable polymeric nanoparticle (NP)- based delivery system consisting of pluronic®F-68 shell and polycaprolactone core for the simultaneous treatment of both anterior and posterior eye diseases. These NPs were loaded with pyrrolidine dithiocarbamate (PDTC) or triamcinolone acetonide (TA) separately. The drug loading in NPs was optimized to initially achieve a moderate burst release of PDTC followed by slow and sustained release of both PDTC and TA. The resultant delivery system was studied for its in vivo efficacy in a diabetic retinopathy (DR) and cataract rat model. The results demonstrated that administration of PDTC NPs + TA NPs minimized oxidative stress in lens as evidenced by reduced levels of protein carbonyls and malondialdehyde, and, ameliorated DR complications in retina as evidenced by reduced expression of hypoxia inducible factor-1α along with a reduction in number of neovascular tufts and acellular capillaries. Therefore, delivery of PDTC and TA using PCL-PF68 NPs could be a useful approach for simultaneous treatment of diabetic cataract and DR.

A non-invasive nanoparticle mediated delivery of triamcinolone acetonide ameliorates diabetic retinopathy in rats.

Diabetic retinopathy (DR) is a multifactorial manifestation associated with microvascular complications and is the fourth leading cause of visual impairment and blindness world-wide. Current day treatment of DR relies heavily on invasive techniques such as intravitreal injections of therapeutic agents. Unfortunately, intravitreal injections are associated with various complications such as intraocular bleeding, endophthalmitis, pain and discomfort resulting in poor patient compliance. To date, there has been no non-invasive drug delivery system reported for DR treatment. To address this, we developed a core-shell nanoparticle-based delivery system consisting of a hydrophobic polycaprolactone core and a hydrophilic Pluronic® F68 shell, loaded with triamcinolone acetonide and evaluated its efficacy in a DR rat model. After being administered as eye drops, the drug loaded nanoparticles significantly improved structural (retinal thickness and vascular health) and functional activity (rod and cone function) of retina as compared to DR controls that were treated with the drug alone or placebo nanoparticles. Furthermore, drug loaded nanoparticles reduced retinal inflammation as evidenced by a decrease in NF-κB, ICAM-1 and TNFα expression after 20 days of treatment. Similarly, a reduction in glial cell hyperplasia as evidenced by reduced GFAP expression, and a decrease in microvascular complications as evidenced by a decrease in VEGF secretion and microvascular tuft formation were observed in rat retinas after 40 days of treatment. The combined reduction in retinal inflammation and vascular abnormalities, both hallmarks of DR, demonstrates the potential of the nanoparticulate delivery system for use as a topical formulation for treating DR.

Deleterious effects of combination of lead and ß-amyloid peptides in inducing apoptosis and altering cell cycle in human neuroblastoma cells.

Lead (Pb) is a toxic pollutant known to cause several abnormalities related to the brain, including cognitive dysfunction, and it is ubiquitous in nature. ß-amyloid peptides (AP) are crucially involved in Alzheimer's disease (AD). It has been reported that there is a connection between lead and amyloid peptides in exerting similar kinds of altered functions in the brain and long-term exposure to lead leads ultimately to increased beta amyloid formation in the brain, lethal to human brain cells. There is still a lack of information on the mechanism by which Pb affects AP formation, exerting combined toxicity in AD patients. To fill the gap, we have systematically analyzed the toxicity individually and in combination of Pb and AP in human brain cells. We found that the combination of Pb and AP exerted a higher toxicity than individual exposures in human neuroblastoma cells. The lower inhibitory concentration values were determined by both time and concentration dependent manner on using MTT assay. The data resulted in the development of enhanced toxicity on exposure to Pb with both the combinations of AP(1-40) or (25-35) and with all combinations in human brain cells compared to individual exposures to Pb (1-40) or AP(25-35). The severe apoptotic effect and alteration in cell cycle by arresting at the S-phase evidenced the increased toxicity of combinational exposure to Pb and AP on human neuroblastoma cells. Furthermore, the quantitative determination of LDH and caspase-3 activity indicated the induction of severe toxicity. We conclude that both are synergistically associated with effects such as arresting the cell cycle and triggering apoptosis during the progression of Alzheimer's disease.

Ellagic acid inhibits non-enzymatic glycation and prevents proteinuria in diabetic rats.

The formation of advanced glycation end products (AGEs) is a characteristic feature of diabetic tissues and accumulation of these products has been implicated in the pathogenesis of micro- and macrovascular complications of diabetes including diabetic nephropathy (DN). Compelling evidence suggests that AGEs mediate progressive alteration in the renal architecture and loss of renal function whereas inhibitors of AGEs prevent the progression of experimental DN. We have investigated the potential of ellagic acid (EA), a polyphenol present abundantly in fruits and vegetables, to prevent in vivo accumulation of AGE and to ameliorate renal changes in diabetic rats. Streptozotocin-induced diabetic rats were fed with either 0.2% or 2% of EA in the diet for 12 weeks. Dietary supplementation of EA to diabetic rats prevented the glycation mediated RBC-IgG-cross-links and HbA1c accumulation. EA inhibited the accumulation of N-carboxymethyl lysine (CML), a predominant AGE in the diabetic kidney. Further, EA also prevented the AGE-mediated loss of expression of podocyte slit diaphragm proteins: nephrin and podocin. By inhibiting CML formation, EA improved renal function in rats as evidenced by urinary albumin and creatinine levels. In conclusion, EA inhibited AGE accumulation in the diabetic rat kidney and ameliorated AGE-mediated pathogenesis of DN.

Inhibition of advanced glycation end-product formation on eye lens protein by rutin.

Formation of advanced glycation end products (AGE) plays a key role in the several pathophysiologies associated with ageing and diabetes, such as arthritis, atherosclerosis, chronic renal insufficiency, Alzheimer's disease, nephropathy, neuropathy and cataract. This raises the possibility of inhibition of AGE formation as one of the approaches to prevent or arrest the progression of diabetic complications. Previously, we have reported that some common dietary sources such as fruits, vegetables, herbs and spices have the potential to inhibit AGE formation. Flavonoids are abundantly found in fruits, vegetables, herbs and spices, and rutin is one of the commonly found dietary flavonols. In the present study, we have demonstrated the antiglycating potential and mechanism of action of rutin using goat eye lens proteins as model proteins. Under in vitro conditions, rutin inhibited glycation as assessed by SDS-PAGE, AGE-fluorescence, boronate affinity chromatography and immunodetection of specific AGE. Further, we provided insight into the mechanism of inhibition of protein glycation that rutin not only scavenges free-radicals directly but also chelates the metal ions by forming complexes with them and thereby partly inhibiting post-Amadori formation. These findings indicate the potential of rutin to prevent and/or inhibit protein glycation and the prospects for controlling AGE-mediated diabetic pathological conditions in vivo.

A novel mutation (F71L) in alphaA-crystallin with defective chaperone-like function associated with age-related cataract.

Age-related cataract (ARC) is a multifactorial disease and the leading cause of blindness worldwide. Genetic predisposition in association with other etiological factors may contribute to ARC. However, gene mutation studies on ARC are scanty. In the present work, we identified a genetic variation (F71L) in the exon-2 of CRYAA (alphaA-crystallin) gene in three unrelated female sporadic cases among 711 ARC patients but not in 265 normal non-cataractous controls by SSCP and RFLP analysis. By comparing human recombinant wild-type and F71L-alphaA-crystallin, we characterized the functional significance of this missense mutation. Chromatography, fluorescence and far- and near-UV CD studies indicated that F71L missense mutation did not significantly affect the apparent molecular mass, secondary and tertiary structures and hydrophobicity of alphaA-crystallin. While the mutant alphaA-crystallin displayed significant (35-90%) loss of chaperone-like activity (CLA) in thermal aggregation of carbonic anhydrase, betaL- and gamma-crystallins, it showed moderate (10-50%) loss in CLA in DTT-induced aggregation of insulin and lysozyme. This is the first report of an alphaA-F71L mutation being associated with ARC and suggests that ARC in individuals carrying this mutation (F71L) might be due to the overall loss of in vivo chaperone activity due to interaction with other environmental factors.

Enhanced degradation and decreased stability of eye lens alpha-crystallin upon methylglyoxal modification.

Methylglyoxal (MGO), a potent glycating agent, forms advanced glycation end products (AGEs) with proteins. Several diabetic complications including cataract are thought to be the result of accumulation of these protein-AGEs. alpha-Crystallin, molecular chaperone of the eye lens, plays an important role in maintaining the transparency of the lens by preventing the aggregation/inactivation of several proteins/enzymes in addition to its structural role. Binding of adenosine-5-triphosphate (ATP) to alpha-crystallin has been shown to enhance its chaperone-like function and protection against proteolytic degradation. In the earlier study, we have shown that modification of alpha-crystallin by MGO caused altered chaperone-like activity along with structural changes, cross-linking, coloration and subsequent insolubilization leading to scattering of light [Biochem. J. 379 (2004) 273]. In the present study, we have investigated ATP binding, stability and degradation of MGO-modified alpha-crystallin. Proteolytic digestion with trypsin and chymotrypsin showed that MGO-modified alpha-crystallin is more susceptible to degradation compared to native alpha-crystallin. Furthermore, ATP was able to protect native alpha-crystallin against proteolytic cleavage but not MGO-modified alpha-crystallin. Interestingly, binding studies indicate decreased ATP binding to MGO-modified alpha-crystallin and support the decreased protection by ATP against proteolysis. In addition, differential scanning calorimetric and denaturant-induced unfolding studies indicate that modification of alpha-crystallin by MGO leads to decreased stability. These results indicate that MGO-modification of alpha-crystallin causes partial unfolding and decreased stability leading to enhanced proteolysis. Cross-linking of these degraded products could result in aggregation and subsequent insolubilization as observed in senile and diabetic cataract lenses.

A predominantly hydrophobic recognition of H-antigenic sugars by winged bean acidic lectin: a thermodynamic study.

The thermodynamics of binding of winged bean (Psophocarpus tetragonolobus) acidic agglutinin to the H-antigenic oligosaccharide (Fucalpha1-2Galbeta1-4GlcNAc-oMe) and its deoxy and methoxy congeners were determined by isothermal titration calorimetry. We report a relatively hydrophobically driven binding of winged bean acidic agglutinin to the congeners of the above sugar. This conclusion is arrived, from the binding parameters of the fucosyl congeners, the nature of the enthalpy-entropy compensation plots and the temperature dependence of binding enthalpies of some of the congeners. Thus, the binding site of winged bean acidic agglutinin must be quite extended to accommodate the trisaccharide, with non-polar loci that recognize the fucosyl moiety of the H-antigenic determinant.