Evaluation of nanoformulated therapeutics in an ex-vivo bovine corneal irritation model

Aim : To determine the internalization and protective effects of potential ophthalmic formulations and nanoformulated natural proteins in ex-vivo bovine corneal alkali burn model.

Methods : The bovine cornea obtained were subjected to the 0.5 N NaOH insult that induced alkali burn and inflammation as observed in the in vivo situation. The toxic effects of the nanoformulation were evaluated in the normal and insult induced cornea using histological analysis. Internalization studies were carried out using in vivo imaging and analysis (IVIS, PerkinElmer, USA).

Results : The nanoformulations employed in this study showed no obvious changes in the integrity of the cornea. Further, improvements in the light transmittance and reduced inflammation were observed. The IVIS showed a dose dependant increase in the uptake of the nanoformulations with time.

Conclusion : The nanoformulated bovine lactoferrin and SurR9-C84A (SR9) proteins evaluated in the ex vivo bovine corneal irritation model is the first of its kind, and we report here the non-toxic and therapeutic potential of these formulations for topical applications.

A novel nanoplatform for oral delivery of anti-cancer biomacromolecules

Oral administration of bio–macromolecules is an uphill task and the challenges from varying pH and enzymatic activity are difficult to overcome. In this regard, nanotechnology promises the new hope and offers advantages such as controlled release, target specific delivery, combinatorial therapy and many more. In this study, we demonstrate the formulation of a novel alginate enclosed, chitosan coated ceramic, anti cancer nano carrier (ACSC NC). These NC were loaded with multi functional anti cancer bovine lactoferrin (Lf), a natural milk based protein, for improvement of intestinal absorption, in order to develop a novel platform to carry anti cancer protein and/or peptides for oral therapy. Here we demonstrate the size, morphology, internalisation and release profiles of the nanoparticles (NC) under varying pH as perceived in human digestive system. We further determine the uptake of these particles by colon cancer cell lines by measuring the endocytosis and transcytosis of the NC. These NC can be used for future targeted protein/peptide or nucleic acid based drug delivery to treat difficult diseases including cancer.

SurR9-C84A mutant nanocarrier and milk protein therapeutics for neurological disorders

 The current project revealed that the novel nanoformulation of SurR9-C84A was able to rescue the neurons following the Alzheimer’s related ß-amyloid toxicity and inflammation. In addition, bovine lactoferrin was found to have potential differentiating effect in the tumor cells and hence is a valuable therapeutic for strengthening the degenerating neurons.

Upregulation of sodium iodide symporter (NIS) protein expression by an innate immunity component: promising potential for targeting radiosensitive retinoblastoma

Retinoblastoma (RB), a malignant tumour of the eye arising from developing retina, is the most frequent primary intraocular malignancy of childhood. Its primary management with chemotherapy involves combination regimen of etoposide, vincristine and carboplatin and intra vitreal chemotherapy using melphalan when vitreous seeds develop. Radiotherapy is another effective mode in treating RB. We recently explored the notion if radiotherapy in RB can be mediated via Sodium Iodide Symporter (NIS), an intrinsic membrane glycoprotein which is a key regulator of iodide access to thyroid gland. Its expression has been exploited successfully for diagnostic imaging and molecular radionuclide-based therapy of thyroid cancer. We determined that NIS is expressed endogenously in RB tumour tissues, and in retinoblastoma cell lines Y79 and Weri-Rb-1, and therefore made an attempt to enhance the endogenously low expression of NIS protein in both Y79 and Weri-Rb-1 cells. Here we report about the potential of bovine lactoferrin (bLf) which is a known chemo preventive and emerging safe anti-cancer bio drug, as well as a natural transcriptional activator of genes, to enhance the endogenous expression of NIS in Y79 and Weri-Rb-1 cells. Real time PCR revealed that both cell lines express mRNA of lactoferrin receptors while flow cytometry and confocal microscopy showed the cells efficiently internalize bLf which upregulates NIS expression. These findings highlight an important step that could be taken towards the development of less harmful approaches for the treatment of RB by employing natural supplement bLf (with its clinically proven safe profile), and warrants further studies in future, focussing on enhancing NIS expression in RB cells and NIS functional assays in these cells.

Molecular targets in arthritis and recent trends in nanotherapy

Due to its severity and increasing epidemiology, arthritis needs no description. There are various forms of arthritis most of which are disabling, very painful, and common. In spite of breakthroughs in the field of drug discovery, there is no cure for arthritis that can eliminate the disease permanently and ease the pain. The present review focuses on some of the most successful drugs in arthritis therapy and their side effects. Potential new targets in arthritis therapy such as interleukin-1β, interleukin-17A, tumor necrosis factor alpha, osteopontin, and several others have been discussed here, which can lead to refinement of current therapeutic modalities. Mechanisms for different forms of arthritis have been discussed along with the molecules that act as potential biomarkers for arthritis. Due to the difficulty in monitoring the disease progression to detect the advanced manifestations of the diseases, drug-induced cytotoxicity, and problems with drug delivery; nanoparticle therapy has gained the attention of the researchers. The unique properties of nanoparticles make them highly attractive for the design of novel therapeutics or diagnostic agents for arthritis. The review also focuses on the recent trends in nanoformulation development used for arthritis therapy. This review is, therefore, important because it describes the relevance and need for more arthritis research, it brings forth a critical discussion of successful drugs in arthritis and analyses the key molecular targets. The review also identifies several knowledge gaps in the published research so far along with the proposal of new ideas and future directions in arthritis therapy.

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: Reversal by holotransferrin not by apotransferrin

The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.

Determination of carbohydrate-deficient transferrin in human serum by capillary zone electrophoresis: evaluation of assay performance and quality assurance over a 10-year period in the routine arena.

The performance of high-resolution CZE for determination of carbohydrate-deficient transferrin (CDT) in human serum based on internal and external quality data gathered over a 10-year period is reported. The assay comprises mixing of serum with a Fe(III) ion-containing solution prior to analysis of the iron saturated mixture in a dynamically double-coated capillary using a commercial buffer at alkaline pH. CDT values obtained with a human serum of a healthy individual and commercial quality control sera are shown to vary less than 10%. Values of a control from a specific lot were found to slowly decrease as function of time (less than 10% per year). Furthermore, due to unknown reasons, gradual changes in the monitored pattern around pentasialo-transferrin were detected, which limit the use of commercial control sera of the same lot to less than 2 years. Analysis of external quality control sera revealed correct classification of the samples over the entire 10-year period. Data obtained compare well with those of HPLC and CZE assays of other laboratories. The data gathered over a 10-year period demonstrate the robustness of the high-resolution CZE assay. This is the first account of a CZE-based CDT assay with complete internal and external quality assessment over an extended time period.

Determination of genetic transferrin variants in human serum by high-resolution capillary zone electrophoresis(†)

High-resolution capillary zone electrophoresis in the routine arena with stringent quality assurance is employed for the determination of carbohydrate-deficient transferrin in human serum. The assay comprises mixing of human serum with a Fe(III) -containing solution prior to analysis of the iron-saturated mixture in a dynamically double-coated capillary using a commercial buffer at alkaline pH. In contrast to other assays, it provides sufficient resolution for proper recognition of genetic transferrin variants. Analysis of 7290 patient sera revealed 166 isoform patterns that could be assigned to genetic variants, namely, 109 BC, 53 CD, one BD and three CC variants. Several subtypes of transferrin D can be distinguished as they have large enough differences in pI values. Subtypes of transferrin C and B cannot be resolved. However, analysis of the detection time ratios of tetrasialo isoforms of transferrin BC and transferrin CD variants revealed multimodal frequency histograms, indicating the presence of subtypes of transferrin C, B and D. The data gathered over 11 years demonstrate the robustness of the high-resolution capillary zone electrophoresis assay. This is the first account of a capillary zone electrophoresis based carbohydrate-deficient transferrin assay with a broad overview on transferrin isoform patterns associated with genetic transferrin variants.

Kinetics of reduction of FeO from slag by graphite and coal chars

The rates of reduction of FeO from iron-saturated FeO-CaO-Al2O3-SiO2 slags by graphite, coke, bituminous coal and anthracitic coal chars at temperatures in the range 1 673-1873 K have been measured using a sessile drop technique. The extents of reaction were determined using EPMA analysis of quenched samples, and on line gas analysis using a quadrupole mass spectrometer. The reaction rates have been shown to be dependent critically on carbon type. For the reaction geometry used in this investigation the reduction rates of graphite and coke are observed to be faster than with coal chars. This unexpected finding is shown to be associated with differences in the dominant chemical and mass transfer mechanisms occurring at the reaction interface. High reaction rates are observed to occur with the formation of liquid Fe-C alloy product and the associated gasification of carbon from the alloy. The rates of reduction by coal chars are determined principally by the chemical reaction at the carbon/gas interface and slag phase mass transfer.

Observations of the reduction of FeO from slag by graphite, coke and coal char

The reduction of FeO from iron-saturated FeO-CaO-Al2O3-SiO2 slags by graphite, coke and coal char at 1 673 K has been investigated using a sessile drop technique. Metallographic analysis of samples quenched from the reaction temperature, and in situ observations of the reaction interface, reveal significant differences in the slag/carbon contact, and in the morphologies of the product iron and its composition; these differences were found to depend on the carbon type used in the reduction. In particular it has been shown that, in the case of graphite and coke, liquid Fe-C droplets were rapidly formed at the slag/C interface. Reactions of the slag with coal chars, in contrast, result predominantly in the formation of solid iron. These observations indicate that the reaction pathways, and hence reaction kinetics, are dependent on carbon type.

Engineered atherosclerosis-specific zinc ferrite nanocomplex-based MRI contrast agents

BACKGROUND: Cardiovascular diseases are the most prevalent cause of morbidity and mortality affecting millions of people globally. The most effective way to counter cardiovascular complications is early diagnosis and the safest non-invasive diagnostic approach is magnetic resonance imaging (MRI). In this study, superparamagnetic ferrite nanoparticles doped with zinc, exhibiting highly enhanced saturation magnetization and T2 and computed tomography (CT) contrast were synthesized. These nanoparticles have been strategically engineered using bovine lactoferrin (Lf), polyethylene glycol (PEG), and heat shock protein (Hsp)-70 antibody specifically targeting atherosclerosis with potential therapeutic value. The nanocomplexes were further validated in vitro to assess their cytotoxicity, internalization efficiency, effects on cellular proliferation and were assessed for MRI as well as X-ray CT in ex vivo Psammomys obesus rat model.

RESULTS: Optimized zinc doped ferrite nanoparticles (Zn0.4Fe2.6O4) with enhanced value of maximum saturation magnetization value on 108.4 emu/g and an average diameter of 24 ± 2 nm were successfully synthesized. Successfully incorporation with bovine lactoferrin, PEG and Hsp-70 (70 kDa) antibody led to synthesis of spherical nanocomplexes (size 224.8 nm, PDI 0.398). A significantly higher enhancement in T2 (p < 0.05, 1.22-fold) and slightly higher T1 (1.09-fold) and CT (1.08-fold) contrast compared to commercial ferrite nanoparticles was observed. The nanocomplexes exhibited effective cellular internalization within 2 h in both THP-1 and Jurkat cells. MRI scans of contrast agent injected animal revealed significant arterial narrowing and a significantly higher T2 (p < 0.05, 1.71-fold) contrast in adult animals when compared to juvenile and control animals. The excised heart and aorta agar phantoms exhibited weak MRI contrast enhancement in juvenile animal but significant contrast enhancement in adult animal specifically at the aortic arch, descending thoracic aorta and iliac bifurcation region with X-ray CT scan. Histological investigation of the contrast agent injected aorta and heart confirmed site target-specific accumulation at the atherosclerotic aortic arch and descending thoracic aorta of the adult animal with severely damaged intima full of ruptured microatheromas.

CONCLUSION: Overall, the study demonstrates the strategic development of nanocomplex based bimodal MRI and CT contrast agents and its validation on Psammomys obesus for atherosclerosis diagnostics.

Targeting Survivin and molecular regulation of de-differentiated grade IV Astrocytoma (Glioblastoma)

The study unprecendently provided evidence about the molecular understanding of a dedifferentiation phenotype of glioblastoma-a highly aggressive brain tumour with 12-15 months of patient survival after diagnosis. Further, the efficacies of survivin targeting molecules SurR9-C84A and a natural non-toxic protein bovine lactoferrin as a safe bio-drug to target glioblastoma were evaluated

Bovine Serum Albumin Adsorption to Iron-Oxide Coated Sands Can Change Microsphere Deposition Mechanisms

Particulate colloids often occur together with proteins in sewage-impacted water. Using Bovine Serum Albumin (BSA) as a surrogate for protein in sewage, column experiments investigating the capacity of iron-oxide coated sands to remove latex microspheres from water revealed that microsphere attenuation mechanisms depended on antecedent BSA coverage. Dual pulse experiment (DPE) results suggested that where all BSA was adsorbed, subsequent multiple pore volume microsphere breakthrough curves reflected progressively reduced colloid deposition rates with increasing adsorbed BSA content. Modelling colloid responses suggested adsorption of 1 µg BSA generated the same response as blockage by between 7.1x108 and 2.3x109 deposited microspheres. By contrast, microsphere responses in DPEs where BSA coverage of the deposition sites approached/ reached saturation revealed the coated sand maintained a finite capacity to attenuate microspheres, even when incapable of further BSA adsorption. Subsequent microsphere breakthrough curves demonstrated the matrix’s colloid attenuation capacity progressively increased with continued microsphere deposition. Experimental findings suggested BSA adsorption on the sand surface approaching/ reaching saturation generated attractive deposition sites for colloids, which became progressively more attractive with further colloid deposition (filter ripening). Results demonstrate that adsorption of a single type of protein may either enhance or inhibit colloid mobility in saturated porous media.

Comparing the influence of two different natural organic matter types on colloid deposition in saturated porous medium

Humic acid and protein are two major organic matter types encountered in natural and polluted environment, respectively. This study employed Triple Pulse Experiments (TPEs) to investigate and compare the influence of Suwannee River Humic Acid (SRHA) (model humic acid) and Bovine Serum Albumin (BSA) (model protein) on colloid deposition in a column packed with saturated iron oxide-coated quartz sand. Study results suggest that adsorbed SRHA may inhibit colloid deposition by occupying colloid sites on the porous medium. Conversely, BSA may promote colloid deposition by a 'filter ripening' mechanism. This study provides insight to understand the complex behavior of colloids in organic matter-presented aquifers and sand filters. © (2012) Trans Tech Publications, Switzerland.

Fortified food made by the extrusion of a mixture of chickpea, corn and bovine lung controls iron-deficiency anaemia in preschool children

A fortified food that was rich in protein, vitamins and iron made of chickpea, bovine lung and corn was developed with the aim of controlling iron-deficiency anaemia in children from poorer areas. It was tested in Teresina, State of Piaui, Northeastern Brazil, on a population with high anaemia prevalence. Two local daycare units with similar characteristics were selected and the children at one of them received a 30 g pack three times a week, representing a total iron daily intake of 6.96 mg. The other daycare unit was followed as a control. The capillary haemoglobin concentration was determined for the children at both daycare units, at the beginning of the study and after a two-month intervention period. The mean haemoglobin concentration in the test group at the beginning of the intervention was 11.8 g/dL, which increased to 13.1 g/dL at the end of the intervention. In the control group these figures remained practically constant (11.6-11.8 g/dL). These represented a dramatic and significant drop in anaemia prevalence, from 61.5% to 11.5% in the test group, and an insignificant reduction (63.1-57.7%) in the control group. The acceptance of the fortified snack was excellent and no undesirable effects were observed. (C) 2007 Published by Elsevier Ltd.