The effect of oral administration of iron saturated-bovine lactoferrin encapsulated chitosan-nanocarriers on osteoarthritis

In this study, the therapeutic potentials of 100% iron saturated-bovine lactoferrin encapsulated in alginate-chitosan polymeric nanocarriers (AEC-CP-Fe-bLf-NCs) were examined in in vitro inflammatory OA model and in collagen-induced arthritis (CIA) mice. Oral administration of nanocarriers in mice were non-toxic and significantly induced disease modifying activity by reducing joint inflammation and downregulating the expression of catabolic genes, IL-1β, NO, JNK and MAPK. In addition, up-regulation of type II collagen, aggrecan and inflammation depleted iron and calcium metabolisms via inhibition of miRNA of iron transporting receptors was shown in AEC-CP-Fe-bLf-NCs treated mice.

Identification of unprecedented anticancer properties of high molecular weight biomacromolecular complex containing bovine lactoferrin (HMW-bLf)

With the successful clinical trials, multifunctional glycoprotein bovine lactoferrin is gaining attention as a safe nutraceutical and biologic drug targeting cancer, chronic-inflammatory, viral and microbial diseases. Interestingly, recent findings that human lactoferrin oligomerizes under simulated physiological conditions signify the possible role of oligomerization in the multifunctional activities of lactoferrin molecule during infections and in disease targeting signaling pathways. Here we report the purification and physicochemical characterization of high molecular weight biomacromolecular complex containing bovine lactoferrin (≥250 kDa), from bovine colostrum, a naturally enriched source of lactoferrin. It showed structural similarities to native monomeric iron free (Apo) lactoferrin (&sim;78-80 kDa), retained anti-bovine lactoferrin antibody specific binding and displayed potential receptor binding properties when tested for cellular internalization. It further displayed higher thermal stability and better resistance to gut enzyme digestion than native bLf monomer. High molecular weight bovine lactoferrin was functionally bioactive and inhibited significantly the cell proliferation (p<0.01) of human breast and colon carcinoma derived cells. It induced significantly higher cancer cell death (apoptosis) and cytotoxicity in a dose-dependent manner in cancer cells than the normal intestinal cells. Upon cellular internalization, it led to the up-regulation of caspase-3 expression and degradation of actin. In order to identify the cutting edge future potential of this bio-macromolecule in medicine over the monomer, its in-depth structural and functional properties need to be investigated further.

Drying and denaturation characteristics of α-lactalbumin, β-lactoglobulin, and bovine serum albumin in a convective drying process

Drying and denaturation kinetics of aqueous droplets of α-lactalbumin (α-lac), β-lactoglobulin (β-lg), and bovine serum albumin (BSA) were measured in a convective drying environment. Single droplets having an initial droplet diameter of 2 ± 0.1 mm and containing 10% (w/v) protein concentration were dried using conditioned air (65 and 80 °C, 2-3% RH, 0.5 m/s velocity) for 600 s. The denaturation of these proteins was measured by using reversed-phase HPLC. At the end of 600 s of drying 13.3 and 19.4% α-lac was found to be lost due to denaturation at 65 and 80 °C, respectively. Up to 31.0% of β-lg was found to be denatured, whereas BSA was not found to be significantly (p > 0.05) denatured in these drying conditions. The formation and strength of skin and the associated morphological features were found to be linked with the degree of denaturation of these proteins. The secondary structure of these proteins was significantly (p < 0.05) affected and altered by the drying stresses. The β-sheet and random coil contents were increased in α-lac by 6.5 and 4.0%, respectively, whereas the α-helix and β-turn contents decreased by 5.5 and 5.0%, respectively. The β-sheet and random coil contents in β-lg were increased by 7.5 and 2.0%, respectively, whereas the α-helix and β-turn contents decreased by 3.5 and 6.0%, respectively. In the case of BSA the β-sheet, α-helix, and random coil contents were found to increase, whereas the β-turn content decreased.

Nanocapsules loaded with iron-saturated bovine lactoferrin have antimicrobial therapeutic potential and maintain calcium, zinc and iron metabolism

Aim: This study aimed to evaluate the potential antimicrobial efficacy of alginate gel-encapsulated ceramic nanocarriers loaded with iron-saturated bovine lactoferrin (Fe-bLf) nanocarriers/nanocapsules (AEC-CP-Fe-bLf NCs). Materials & methods: The antimicrobial activities of non-nanoformulated apo (iron free), Fe-bLf and native forms of Australian bLf against pathogenic Salmonella typhimurium (wild strain) were studied in vitro. The efficacy of AEC-CP-Fe-bLf NCs were checked in vivo using Balb/c mice model. Results: The study revealed that native bLf is more effective in combating infection than the conventional drug ciprofloxacin (0.4 mg/ml). The efficacy of the drug was also revealed in vivo when BALB/c mice that, after being challenged with S. typhimurium (200 μl of 10(8) CFU/ml suspension), were fed orally with a nanoformulated bLf diet and the infection was observed to be eliminated. However, chronic infection developed in the group of infected mice that did not receive any drug treatment, as well as the mice treated with ciprofloxacin. The immune response to bacterial infection and to various drug treatments thereafter was studied in the mice. Conclusion: The study concludes that bLf and nanoformulated Fe-bLf are more effective in the treatment of Salmonella-infected mice than ciprofloxacin.

The microstructure and physicochemical properties of probiotic buffalo yoghurt during fermentation and storage: a comparison with bovine yoghurt

The physicochemical and rheological properties of yoghurt made from unstandardised unhomogenised buffalo milk were investigated during fermentation and 28 days of storage and compared to the properties of yoghurt made from homogenised fortified bovine milk. A number of differences observed in the gel network can be linked to differences in milk composition. The microstructure of buffalo yoghurt, as assessed by confocal laser scanning microscopy (CLSM) and cryo scanning electron microscopy (cryo-SEM), was interrupted by large fat globules and featured more serum pores. These fat globules have a lower surface area and bind less protein than the homogenised fat globules in bovine milk. These microstructural differences likely lead to the higher syneresis observed for buffalo yoghurt with an increase from 17.4 % (w/w) to 19.7 % (w/w) in the weight of whey generated at days 1 and 28 of the storage. The higher concentration of total calcium in buffalo milk resulted in the release of more ionic calcium during fermentation. Gelation was also slower but the strength of the two gels was similar due to similar protein and total solids concentrations. Buffalo yoghurt was more viscous, less able to recover from deformation and less Newtonian than bovine yoghurt with a thixotropy of 3,035 Pa.s-1 measured for buffalo yoghurt at the end of the storage, at least four times higher than the thixotropy of bovine yoghurt. While the titratable acidity, lactose consumption and changes in organic acid concentrations were similar, differences were recorded in the viability of probiotic bacteria with a lower viability of Lactobacillus acidophilus of 5.17 log (CFU/g) recorded for buffalo yoghurt at day 28 of the storage. Our results show that factors other than the total solids content and protein concentration of milk affect the structural properties of yoghurt. They also illustrate the physicochemical reasons why buffalo and bovine yoghurt are reported to have different sensory properties and provide insight into how compositional changes can be used to alter the microstructure and properties of dairy products. © 2013 Springer Science+Business Media New York.

Oral administration of iron-saturated bovine lactoferrin-loaded ceramic nanocapsules for breast cancer therapy and influence on iron and calcium metabolism

We determined the anticancer efficacy and internalization mechanism of our polymeric-ceramic nanoparticle system (calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate nanocapsules/nanocarriers [ACSC NCs]) loaded with iron-saturated bovine lactoferrin (Fe-bLf) in a breast cancer xenograft model. ACSC-Fe-bLf NCs with an overall size of 322±27.2 nm were synthesized. In vitro internalization and anticancer efficacy were evaluated in the MDA-MB-231 cells using multicellular tumor spheroids, CyQUANT and MTT assays. These NCs were orally delivered in a breast cancer xenograft mice model, and their internalization, cytotoxicity, biodistribution, and anticancer efficacy were evaluated. Chitosan-coated calcium phosphate Fe-bLf NCs effectively (59%, P≤0.005) internalized in a 1-hour period using clathrin-mediated endocytosis (P≤0.05) and energy-mediated pathways (P≤0.05) for internalization; 3.3 mg/mL of ACSC-Fe-bLf NCs completely disintegrated (~130-fold reduction, P≤0.0005) the tumor spheroids in 72 hours and 96 hours. The IC50 values determined for ACSC-Fe-bLf NCs were 1.69 mg/mL at 10 hours and 1.62 mg/mL after 20 hours. We found that Fe-bLf-NCs effectively (P≤0.05) decreased the tumor size (4.8-fold) compared to the void NCs diet and prevented tumor recurrence when compared to intraperitoneal injection of Taxol and Doxorubicin. Receptor gene expression and micro-RNA analysis confirmed upregulation of low-density lipoprotein receptor and transferrin receptor (liver, intestine, and brain). Several micro-RNAs responsible for iron metabolism upregulated with NCs were identified. Taken together, orally delivered Fe-bLf NCs offer enhanced antitumor activity in breast cancer by internalizing via low-density lipoprotein receptor and transferrin receptor and regulating the micro-RNA expression. These NCs also restored the body iron and calcium levels and increased the hematologic counts.

Short communication: bovine-derived proteins activate STAT3 in human skeletal muscle in vitro

Bovine milk contains biologically active peptides that may modulate growth and development within humans. In this study, targeted bovine-derived proteins were evaluated for their effects on signal transducer and activator of transcription-3 (STAT3) phosphorylation in human skeletal muscle cells. Following an acute exposure, bovine-derived acidic fibroblast growth factor-1 (FGF) and leukemia inhibitory factor (LIF) activated STAT3 in differentiating myotubes. Chronic exposure to FGF and LIF during the proliferative phase reduced myoblast proliferation and elevated MyoD and creatine kinase (CKM) mRNA expression without altering apoptotic genes. In mature myotubes, neither FGF nor LIF elicited any action. Together, these data indicate that a reduction in proliferation in the presence of bovine-derived FGF or LIF may stimulate early maturation of myoblasts.

Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer

BACKGROUND: Iron binding, naturally occurring protein bovine lactoferrin (bLf) has attracted attention as a safe anti-cancer agent capable of inducing apoptosis. Naturally, bLf exists partially saturated (15-20%) with Fe(3+) however, it has been demonstrated that manipulating the saturation state can enhance bLf's anti-cancer activities. METHODS: Apo-bLf (Fe(3+) free) and Fe-bLf (>90% Fe(3+) Saturated) were therefore, tested in MDA-MB-231 and MCF-7 human breast cancer cells in terms of cytotoxicity, proliferation, migration and invasion. Annexin-V Fluos staining was also employed in addition to apoptotic protein arrays and Western blotting to determine the specific mechanism of bLf-induced apoptosis with a key focus on p53 and inhibitor of apoptosis proteins (IAP), specifically survivin. RESULTS: Apo-bLf induced significantly greater cytotoxicity and reduction in cell proliferation in both cancer cells showing a time and dose dependent effect. Importantly, no cytotoxicity was detected in normal MCF-10-2A cells. Both forms of bLf significantly reduced cell invasion in cancer cells. Key apoptotic molecules including p53, Bcl-2 family proteins, IAP members and their inhibitors were significantly modulated by both forms of bLf, though differentially in each cell line. Most interestingly, both Apo-bLf and Fe-bLf completely inhibited the expression of survivin protein (key IAP), after 48 h at 30 and 40 nM in cancer cells. CONCLUSIONS: The capacity of these forms of bLf to target survivin expression and modulation of apoptosis demonstrates an exciting potential for bLf as an anti-cancer therapeutic in the existing void of survivin inhibitors, with a lack of successful inhibitors in the clinical management of cancer.

Bovine lactoferrin targets apoptotic and EGFR mechanisms in breast cancer

 This research investigated the anti-cancer effects of milk protein, lactoferrin. It was found that lactoferrin specifically induced cell death in breast cancer cells and was non-toxic to normal mammary gland cells. Key molecular mechanisms targeted by lactoferrin were elucidated in this study which provides important insight into the activity of this protein as an anti-cancer agent.

Biodegradable Eri silk nanoparticles as a delivery vehicle for bovine lactoferrin against MDA-MB-231 and MCF-7 breast cancer cells

This study used the Eri silk nanoparticles (NPs) for delivering apo-bovine lactoferrin (Apo-bLf) (~2% iron saturated) and Fe-bLf (100% iron saturated) in MDA-MB-231 and MCF-7 breast cancer cell lines. Apo-bLf and Fe-bLf-loaded Eri silk NPs with sizes between 200 and 300 nm (±10 nm) showed a significant internalization within 4 hours in MDA-MB-231 cells when compared to MCF-7 cells. The ex vivo loop assay with chitosan-coated Fe-bLf-loaded silk NPs was able to substantiate its future use in oral administration and showed the maximum absorption within 24 hours by ileum. Both Apo-bLf and Fe-bLf induced increase in expression of low-density lipoprotein receptor-related protein 1 and lactoferrin receptor in epidermal growth factor (EGFR)-positive MDA-MB-231 cells, while transferrin receptor (TfR) and TfR2 in MCF-7 cells facilitated the receptor-mediated endocytosis of NPs. Controlled and sustained release of both bLf from silk NPs was shown to induce more cancer-specific cytotoxicity in MDA-MB-231 and MCF-7 cells compared to normal MCF-10A cells. Due to higher degree of internalization, the extent of cytotoxicity and apoptosis was significantly higher in MDA-MB-231 (EGFR+) cells when compared to MCF-7 (EGFR-) cells. The expression of a prominent anticancer target, survivin, was found to be downregulated at both gene and protein levels. Taken together, all the observations suggest the potential use of Eri silk NPs as a delivery vehicle for an anti-cancer milk protein, and indicate bLf for the treatment of breast cancer.

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.