Assessment of the Conformation Stability and Glycosylation Heterogeneity of Lactoferrin by Native Mass Spectrometry.

Lactoferrin (LTF) has diverse biological activities and is widely used in functional foods and active additives. Nevertheless, evaluating the proteoform heterogeneity, conformational stability, and activity of LTF remains challenging during its production and storage processes. In this study, we describe the implementation of native mass spectrometry (nMS), glycoproteomics, and an antimicrobial activity assay to assess the quality of LTF. We systematically characterize the purity, glycosylation heterogeneity, conformation, and thermal stability of LTF samples from different sources and transient high-temperature treatments by using nMS and glycoproteomics. Meanwhile, the nMS peak intensity and antimicrobial activity of LTF samples after heat treatment decreased significantly, and the two values were positively correlated. The nMS results provide essential molecular insights into the conformational stability and glycosylation heterogeneity of different LTF samples. Our results underscore the great potential of nMS for LTF quality control and activity evaluation in industrial production.

Targeting Intracellular Bacteria with Dual Drug-loaded Lactoferrin Nanoparticles.

Treatment of microbial infections is becoming daunting because of widespread antimicrobial resistance. The treatment challenge is further exacerbated by the fact that certain infectious bacteria invade and localize within host cells, protecting the bacteria from antimicrobial treatments and the host's immune response. To survive in the intracellular niche, such bacteria deploy surface receptors similar to host cell receptors to sequester iron, an essential nutrient for their virulence, from host iron-binding proteins, in particular lactoferrin and transferrin. In this context, we aimed to target lactoferrin receptors expressed by macrophages and bacteria; as such, we prepared and characterized lactoferrin nanoparticles (Lf-NPs) loaded with a dual drug combination of antimicrobial natural alkaloids, berberine or sanguinarine, with vancomycin or imipenem. We observed increased uptake of drug-loaded Lf-NPs by differentiated THP-1 cells with up to 90% proportion of fluorescent cells, which decreased to about 60% in the presence of free lactoferrin, demonstrating the targeting ability of Lf-NPs. The encapsulated antibiotic drug cocktail efficiently cleared intracellular Staphylococcus aureus (Newman strain) compared to the free drug combinations. However, the encapsulated drugs and the free drugs alike exhibited a bacteriostatic effect against the hard-to-treat Mycobacterium abscessus (smooth variant). In conclusion, the results of this study demonstrate the potential of lactoferrin nanoparticles for the targeted delivery of antibiotic drug cocktails for the treatment of intracellular bacteria.

A high-throughput differential scanning fluorimetry method for rapid detection of thermal stability and iron saturation in lactoferrin.

Thermal stability and iron saturation of lactoferrin (LF) are of great significance not only for the evaluation of the biological activities of LF but also for the optimization of the isolation and drying process parameters. Differential scanning calorimetry (DSC) is a well-established and efficient method for thermal stability and iron saturation detection in LF. However, multiple DSC measurements are typically performed sequentially, thus time-consuming and low throughput. Herein, we introduced the differential scanning fluorimetry (DSF) approach to overcome such limitations. The DSF can monitor LF thermal unfolding with a commonly available real-time PCR instrument and a fluorescent dye (SYPRO orange or Glomelt), and the measured melting temperature of LF is consistent with that determined by DSC. On the basis of that, a new quantification method was established for determination of iron saturation levels using the linear correlation of the degree of ion saturation of LF with DSF measurements. Such DSF method is simple, inexpensive, rapid (<15 min), and high throughput (>96 samples per experiment), and provides a valuable alternative tool for thermal stability detection of LF and other whey proteins.

Thermal stability and in vitro biological fate of lactoferrin-polysaccharide complexes.

Lactoferrin (LF) is a thermally sensitive iron-binding globular glycoprotein. Heat treatment can induce its denaturation and aggregation and thus affect its functional activity. In this study, carrageenan (CG), xanthan gum (XG) and locust bean gum (LBG), allowed to apply in infant food, were used to form protein-polysaccharide complexes to improve the thermal stability of LF. Meanwhile, in vitro simulated infant digestion and absorption properties of LF were also estimated. The results showed that the complexes formed by CG and XG with LF (LF-CG and LF-XG) could significantly inhibit the loss of α-helix structure of LF against heating. LF-CG and LF-LBG could protect LF from digestion in simulated infant gastric fluid and slow down the degradation of LF under the simulated intestinal conditions. Besides, LF, LF-CG and LF-XG showed no adverse effects on the growth of Caco-2 cells in the LF concentration range of 10-300 µg/mL, and LF-XG exhibited better beneficial to improve the cell uptake of the digestive product than the other protein-polysaccharides at the LF concentration of 100 µg/mL. This study may provide a reference for the enhancement of thermal processing stability of LF and development infant food ingredient with high nutrients absorption efficiency in the gastrointestinal environment in the future.

Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Lactoferricin B Combined with Antibiotics Exhibits Leukemic Selectivity and Antimicrobial Activity.

The fusion of penetrating peptides (PPs), e.g., cell penetration peptides (CPPs) or antimicrobial peptides (AMPs), together with antimicrobial agents is an expanding research field. Specific AMPs, such as lactoferricin B (LfcinB), have demonstrated strong antibacterial, antifungal, and antiparasitic activity, as well as valuable anticancer activity, proving beneficial in the development of anticancer conjugates. The resulting conjugates offer potential dual functionality, acting as both an anticancer and an antimicrobial agent. This is especially necessary in cancer treatment, where microbial infections pose a critical risk. Leukemic cells frequently exhibit altered outer lipid membranes compared to healthy cells, making them more sensitive to compounds that interfere with their membrane. In this study, we revisited and reanalyzed our earlier research on LfcinB and its conjugates. Furthermore, we carried out new experiments with a specific focus on cell proliferation, changes in membrane asymmetric phosphatidylserine location, intracellular reactive oxygen species (ROS) generation, mitochondrial functions, and in vitro bacterial topoisomerase inhibition.

Site-specific glycoproteomic analysis of purified lactoferrin from human and animal milk.

Lactoferrin is a highly glycosylated protein, which have important biological functions in the growth and development of neonates. However, the glycoforms and glycosylation sites differed between species. The aim of the study was to identify the glycosylation profile (including glycosites, glycan structures, and glycoforms) of purified lactoferrin from human and animal (cow, goat, sheep) milk by using site-specific glycoproteomics technique. In total, a number of 89 N-glycans were identified in human and animal milk lactoferrin. We identified three N-glycosites with 23 different compositions of N-glycans in cow lactoferrin (CLF), four distinctive N-glycosites with 34 dissimilar N-glycan compositions in goat lactoferrin (GLF), five N-glycosites with 57 different N-glycan compositions in sheep lactoferrin (SLF), while five unique N-glycosites with 50 different N-glycan compositions were ascertained in human lactoferrin (HLF). HLF had the most complex glycan, while animal lactoferrin had the most high-mannose glycoforms. The results of this study further our understanding of lactoferrin differences between human and animal milk, which can provide a perspective on the analysis of differences in functional characteristics.

Study of pH and Thermodynamic Parameters via Circular Dichroism Spectroscopy of a Recombinant Human Lactoferrin.

The production of human recombinant proteins to be used for therapeutic or nutritional purposes must focus on obtaining a molecule that is as close as possible to the native human protein. This biotechnological tool has been documented in various studies published in recent decades, with lactoferrin being one of those that has generated the most interest, being a promising option for recombinant technology. However, stability studies including thermodynamic parameters have not been reported for recombinant lactoferrin (Lf). The objective of this work was to obtain the human recombinant protein using the yeast Komagataella phaffii to study structural changes modifying pH and temperature using circular dichroism spectroscopy (CD). Thermodynamic parameters such as ΔH, ΔS and Tm were calculated and compared with commercial human lactoferrin. We propose the potential use of CD and thermodynamic parameters as a criterion in the production of recombinant proteins to be used in the production of specialized recombinant proteins.

Immunoregulation of bovine lactoferrin together with osteopontin promotes immune system development and maturation.

The immune system of infants is partly weak and immature, and supplementation of infant formula can be of vital importance to boost the development of the immune system. Lactoferrin (LF) and osteopontin (OPN) are essential proteins in human milk with immunoregulation function. An increasing number of studies indicate that proteins have interactions with each other in milk, and our previous study found that a ratio of LF : OPN at 1 : 5 (w/w, denoted as LOP) had a synergistic effect on intestinal barrier protection. It remains unknown whether LOP can also exert a stronger effect on immunoregulation. Hence, we used an in vitro model of LPS-induced macrophage inflammation and in vivo models of LPS-induced intestinal inflammation and early life development. We showed that LOP increased the secretion of the granulocyte-macrophage colony-stimulating factor (132%), stem cell factor (167%) and interleukin-3 (176%) in bone marrow cells, as well as thymosin (155%) and interleukin-10 (161%) in the thymus, more than LF or OPN alone during development, and inhibited changes in immune cells and cytokines during the LPS challenge. In addition, analysis of the components of digested proteins in vitro revealed that differentially expressed peptides may provide immunoregulation. Lastly, LOP increased the abundance of Rikenellaceae, Muribaculum, Faecalibaculum, and Elisenbergiella in the cecum content. These results imply that LOP is a potential immunomodifier for infants and offers a new theoretical basis for infant formula innovation.

Regulation of the colon-targeted release rate of lactoferrin by constructing hydrophobic ethyl cellulose/pectin composite nanofibrous carrier and its effect on anti-colon cancer activity.

In order to modify colonic release behavior of lactoferrin (Lf), a hydrophobic composite nanofibrous carrier (CNC) was constructed by emulsion coaxial electrospinning. Ethylcellulose/pectin based water-in-oil emulsion and Lf-contained polyvinyl alcohol solution were used as shell and core fluids, respectively. An electrospinning diagram was first constructed to screen out suitable viscosity (51-82 cP) and conductivity (960-1300 µS/cm) of the dispersed phase of pectin solution for successful electrospinning of shell emulsion. Varying mass fraction of pectin solution (5 %-20 %) of shell emulsion during emulsion coaxial electrospinning obtained CNCs with different micro-structures, labeled as 5&95 CNC, 10&90 CNC, 15&85 CNC, 20&80 CNC. These CNCs all achieved colonic delivery of Lf (>95 %), and the time for complete release of Lf in simulated colon fermentation process were 10, 7, 5 and 3 h, respectively. That is, the greater the pectin content in CNC, the faster the release rate of stabilized Lf in colon. Lf release in simulated colon fermentation fluid involved complex mechanisms, in which diffusion release of Lf was dominant. Increasing colonic release rate of Lf enhanced its regulation effect on the expression levels of cell cycle arrest and apoptosis-related protein and promote its effective inhibition on the proliferation of HCT116 cell.

The novel lactoferrin and DHA-codelivered liposomes with different membrane structures: Fabrication, in vitro infant digestion, and suckling pig intestinal organoid absorption.

Inspired by membrane structure of breast milk and infant formula fat globules, four liposomes with different particle size (large and small) and compositions (Single phospholipids contained phosphatidylcholine, complex phospholipids contained phosphatidylcholine, phosphatidylethanolamine and sphingomyelin) were fabricated to deliver lactoferrin and DHA. In vitro infant semi-dynamic digestive behavior and absorption in intestinal organoids of liposomes were investigated. Liposomal structures were negligible changed during semi-dynamic gastric digestion while damaged in intestine. Liposomal degradation rate was primarily influenced by particle size, and complex phospholipids accelerated DHA hydrolysis. The release rate of DHA (91.7 ± 1.3 %) in small-sized liposomes (0.181 ± 0.001 µm) was higher than free DHA (unencapsulated, 64.6 ± 3.4 %). Complex phospholipids liposomal digesta exhibited higher transport efficiency (3.4-fold for fatty acids and 2.0-fold for amino acids) and better organoid growth than digesta of bare nutrients. This study provided new insights into membrane structure-functionality relationship of liposomes and may aid in the development of novel infant nutrient carriers.

New Horizons of Covalent Complex of Plant-Derived Recombinant Human Lactoferrin (OsrhLF) Combined with Different Polyphenols: Formation, Physicochemical Properties, and Gastrointestinal Fate.

Four typical dietary polyphenols ((-)-epigallocatechin gallate (EGCG), quinic acid (QA), caffeic acid (CA), and ferulic acid (FA)) were covalently prepared with rice recombinant human lactoferrin (OsrhLF) and bovine lactoferrin (bLF), and their structure and physicochemical properties were investigated, different lycopene emulsions were made by ultrasonic emulsification to analyze gastrointestinal fate. The results indicated that the covalent modification polyphenols changed the secondary/tertiary structure of LF, significantly improving the surface hydrophilicity, thermal stability, and antioxidant activity of LF. Compared with the bLF group, the OsrhLF group was more hydrophilic and the thermal denaturation temperature of the OsrhLF-CA reached 104.4 °C. LF-polyphenol emulsions significantly enhanced the photochemical stability and bioavailability of lycopene and achieved effective encapsulation and protection of lycopene compared to free lycopene, and the OsrhLF-EGCG reached 58.94% lycopene bioavailability. In short, OsrhLF does not differ much from bLF in terms of physicochemical properties and has a strong potential in the field of dietary supplements.

Assessment of Broad-Spectrum Antimicrobial, Antibiofilm, and Anticancer Potential of Lactoferrin Extracted from Camel Milk.

Lactoferrin is a multifunctional glycoprotein present in mammalian milk. It possesses antimicrobial, antioxidant, immunomodulatory, and several biological functions. Owing to the current trend of increasing antibiotic resistance, our study was designed to purify lactoferrin from camel milk colostrum using cation exchange chromatography on the SP-Sepharose high-performance column. The purity and molecular weight of lactoferrin were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The chromatogram of the purification procedure illustrated a single peak corresponding to lactoferrin, while the SDS-PAGE revealed 78 kDa molecular weight protein. Furthermore, lactoferrin protein and its hydrolysate form were assessed for its antimicrobial potential. The highest inhibitory effect of whole lactoferrin at the concentration (4 mg/ml) was observed against methicillin-resistant S. aureus (MRSA) and S. aureus, while 10 mg/ml concentration was effective against K. pneumonia, and 27 mg/ml was potent against multidrug-resistant (MDR) bacteria, P. aeruginosa. Likewise, MRSA was more sensitive toward iron-free lactoferrin (2 mg/ml) and hydrolyzed lactoferrin (6 mg/ml). The tested lactoferrin forms showed variability in minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) among tested bacteria. The scanning electron microscopy (SEM) analysis images revealed distortions of the bacterial cells exposed to lactoferrin. The antibiofilm effect differed depending on the concentration and the type of the bacteria; biofilm inhibition ranged from 12.5 to 91.3% in the tested pathogenic bacteria. Moreover, the anticancer activity of lactoferrin forms exhibited a dose-dependent cytotoxicity against human lung cancer cell line (A549).

Design of lactoferrin functionalized carboxymethyl dextran coated egg albumin nanoconjugate for targeted delivery of capsaicin: Spectroscopic and cytotoxicity studies.

The increased mortality rates associated with colorectal cancer highlight the pressing need for improving treatment approaches. While capsaicin (CAP) has shown promising anticancer activity, its efficacy is hampered due to low solubility, rapid metabolism, suboptimal bioavailability, and a short half-life. Therefore, this study aimed to prepare a lactoferrin-functionalized carboxymethyl dextran-coated egg albumin nanoconjugate (LF-CMD@CAP-EGA-NCs) for the targeted CAP delivery to enhance its potential for colorectal cancer therapy. Briefly, LF-CMD was synthesized through an esterification reaction involving LF as a receptor and CMD as a shell. Concurrently, CAP was incorporated into an EGA carrier using gelation and hydrophobic interactions. The subsequent production of LF-CMD@CAP-EGA-NCs was achieved through the Maillard reaction. Spectral characterizations confirmed the successful synthesis of smooth and spherical-shaped LF-CMD@CAP-EGA-NCs using LF-CMD and EGA-CAP nanoparticles, with high entrapment efficiency and satisfactory drug content. Furthermore, LF-CMD@CAP-EGA-NCs demonstrated a sustained release of CAP (76.52 ± 1.01 % in 24 h, R2 = 0.9966) in pH 5.8 buffer with anomalous transport (n = 0.68) owing to the shell of the CMD and EGA matrix. The nanoconjugate exhibited enhanced cytotoxicity in HCT116 and LoVo cell lines, which is attributed to the overexpression of LF receptors in colorectal HCT116 cells. Additionally, LF-CMD@CAP-EGA-NCs demonstrated excellent biocompatibility, as observed in the FHC-CRL-1831 cell line. In conclusion, LF-CMD@CAP-EGA-NCs can be considered as a promising approach for targeted delivery of CAP and other anticancer agents in colorectal cancer treatment.

The Multifaceted Roles of Bovine Lactoferrin: Molecular Structure, Isolation Methods, Analytical Characteristics, and Biological Properties.

Bovine lactoferrin (bLF) is widely known as an iron-binding glycoprotein from the transferrin family. The bLF molecule exhibits a broad spectrum of biological activity, including iron delivery, antimicrobial, antiviral, immunomodulatory, antioxidant, antitumor, and prebiotic functions, thereby making it one of the most valuable representatives for biomedical applications. Remarkably, LF functionality might completely differ in dependence on the iron saturation state and glycosylation patterns. Recently, a violently growing demand for bLF production has been observed, mostly for infant formulas, dietary supplements, and functional food formulations. Unfortunately, one of the reasons that inhibit the development of the bLF market and widespread protein implementation is related to its negligible amount in both major sources─colostrum and mature milk. This study provides a comprehensive overview of the significance of bLF research by delineating the key structural characteristics of the protein and elucidating their impact on its physicochemical and biological properties. Progress in the development of optimal isolation techniques for bLF is critically assessed, alongside the challenges that arise during its production. Furthermore, this paper presents a curated list of the most relevant instrumental techniques for the characterization of bLF. Lastly, it discusses the prospective applications and future directions for bLF-based formulations, highlighting their potential in various fields.

Model based process optimization of an industrial chromatographic process for separation of lactoferrin from bovine milk.

Model based process development using predictive mechanistic models is a powerful tool for in-silico downstream process development. It allows to obtain a thorough understanding of the process reducing experimental effort. While in pharma industry, mechanistic modeling becomes more common in the last years, it is rarely applied in food industry. This case study investigates risk ranking and possible optimization of the industrial process of purifying lactoferrin from bovine milk using SP Sepharose Big Beads with a resin particle diameter of 200 µm, based on a minimal number of lab-scale experiments combining traditional scale-down experiments with mechanistic modeling. Depending on the location and season, process water pH and the composition of raw milk can vary, posing a challenge for highly efficient process development. A predictive model based on the general rate model with steric mass action binding, extended for pH dependence, was calibrated to describe the elution behavior of lactoferrin and main impurities. The gained model was evaluated against changes in flow rate, step elution conditions, and higher loading and showed excellent agreement with the observed experimental data. The model was then used to investigate the critical process parameters, such as water pH, conductivity of elution steps, and flow rate, on process performance and purity. It was found that the elution behavior of lactoferrin is relatively consistent over the pH range of 5.5 to 7.6, while the elution behavior of the main impurities varies greatly with elution pH. As a result, a significant loss in lactoferrin is unavoidable to achieve desired purities at pH levels below pH 6.0. Optimal process parameters were identified to reduce water and salt consumption and increase purity, depending on water pH and raw milk composition. The optimal conductivity for impurity removal in a low conductivity elution step was found to be 43 mS/cm, while a conductivity of 95 mS/cm leads to the lowest overall salt usage during lactoferrin elution. Further increasing the conductivity during lactoferrin elution can only slightly lower the elution volume thus can also lead to higher total salt usage. Low flow rates during elution of 0.2 column volume per minute are beneficial compared to higher flow rates of 1 column volume per minute. The, on lab-scale, calibrated model allows predicting elution volume and impurity removal for large-scale experiments in a commercial plant processing over 106 liters of milk per day. The successful model extrapolation was possible without recalibration or detailed knowledge of the manufacturing plant. This study therefore provides a possible pathway for rapid process development of chromatographic purification in the food industries combining traditional scale-down experiments with mechanistic modeling.

DNA-based molecular recognition system for lactoferrin biosensing.

The paper describes the development of a novel DNA oligonucleotide-based affinity bioreceptor that binds to lactoferrin, a glycoprotein-type immunomodulator. The research was performed using surface plasmon resonance method to investigate affinity of various types of oligonucleotides to the target protein. The 72 base pair-long 5'[(TAGAGGATCAAA)AAA]4TAGAGGATCAAA3' sequence with the highest affinity to lactoferrin was selected for further investigations. Kinetic analysis of the interaction between selected DNA and lactoferrin provided rate and equilibrium constants: ka = (2.49 ± 0.03)∙104 M-1∙s-1, kd = (1.89 ± 0.02)∙10-3 s-1, KA = (0.13 ± 0.05)∙108 M-1, and KD = (7.61 ± 0.18)∙10-8 M. Thermodynamic study conducted to determine the ΔH0, ΔS0, and ΔG0 for van't Hoff characteristic in the temperature range of 291.15-305.15 K, revealed the complex formation as endothermic and entropically driven. The chosen DNA sequence's selectivity towards lactoferrin was confirmed with interferents' response constituting <3 % of the response to lactoferrin. SPR analysis justified utility of the designed DNA oligonucleotide for Lf determination, with LOD of 4.42∙10-9 M. Finally, the interaction between lactoferrin and DNA was confirmed by electrochemical impedance spectroscopy, providing the basis for further quantitative assay of lactoferrin using the developed DNA-based bioreceptor. The interactions were performed under immobilized DNA ligand conditions, thus reflecting the sensor's surface, which facilitates their transfer to other label-free biosensor technologies.

Molecular insight into binding behavior of caffeine with lactoferrin: Spectroscopic, molecular docking, and simulation study.

The majority of bioactive substances in the human diet come from polyphenols. Here, we use spectroscopy, molecular docking, molecular dynamics simulations, and in vitro digestion to look at the relationship between caffeine (CAF) and bovine lactoferrin (BLF). The correlation analysis of the CAF-BLF fluorescence quenching process revealed that the reaction was spontaneous and that the CAF-BLF fluorescence quenching process may have been static. The predominant intrinsic binding forces were hydrogen bonds and van der Waals forces, which were also supported by molecular docking and molecular dynamics simulations. Through Fourier infrared and circular dichroism spectroscopy experiments, it was found that CAF changed the secondary structure of BLF and might bind to the hydrophobic amino acids of BLF. Compared with BLF, CAF-BLF showed inhibitory effects on digestion in simulated in vitro digestion. It will be helpful to better understand the interaction between CAF and BLF and provide the basis for the development of innovative dairy products.

The Antimicrobial Bifunctional Camel Lactoferrin: In Silico and Molecular Dynamic Perspective.

Lactoferrin (LF) is a major natural antimicrobial agent secreted in body fluids as a natural innate immunity protein. The action and structure of LF are closely related to its iron-binding capacity with structural reporting in open and closed conformations. This study looked at how lactoferrin structures change in camel (cLF), bovine (bLF), and human (hLF) lactoferrin closed forms after iron is removed from their binding sites. Initially, the sequence comparison between cLF and the LFs of marine mammals, bats, and domestic animals was the most intriguing conclusion. Camel LF is revealed to be more closely related to marine animals (~80.36% identity) and bats (~79.3% identity) than to terrestrial mammal species (~75.5% identity). Results indicated that cLF was more dynamic in nature than bLF and hLF by showing higher RMSD values. The cLF is known to be half lactoferrin half transferrin; in this study, we show that there are different MD behavior of both iron-binding sites. While LF contains two lobes (C- and N-lobes), the C-lobe showed high fluctuations as N-lobe was more stable in the absence of ferric ions. The C-lobe and N-lobe of cLF react differently at physiological pH, revealing distinct molecular interactions between these components. In addition, cLF showed higher system flexibility derived from its larger RMSD, RMSF, lower intermolecular hydrogen bonds, and higher solvent accessible surface area (SASA).

Chondroitin/Lactoferrin-dual functionalized pterostilbene-solid lipid nanoparticles as targeted breast cancer therapy.

Breast cancer remains the leading cause of cancer-associated mortality in women. Research investigating novel therapeutic approaches is thus crucial, including phytotherapeutics. Pterostilbene (PTS) is a phytochemical agent with promising efficacy against breast cancer. Poor solubility, low bioavailability and chemical instability are major drawbacks compromising PTS functionality. Herein, novel PTS-loaded solid lipid nanoparticles (PTS-SLNs) were fabricated using the ultrasonication technique. Dual-functionalization with lactoferrin (Lf) and chondroitin-sulfate (CS; CS/Lf/PTS-SLNs) was adopted as active-targeting approach. CS/Lf/PTS-SLNs demonstrated nanoparticle-size (223.42 ± 18.71 nm), low PDI (0.33 ± 0.017), acceptable zeta potential (-11.85 ± 0.07 mV) and controlled release (72.93 ± 2.93% after 24 h). In vitro studies on triple-negative MDA-MB-231 revealed prominent cytotoxicity of CS/Lf/PTS-SLNs (2.63-fold IC50 reduction), higher anti-migratory effect and cellular uptake relative to PTS-solution. The in vivo anti-tumor efficacy in an orthotopic cancer model verified the superiority of CS/Lf/PTS-SLNs; achieving 2.4-fold decrease in tumor growth compared to PTS-solution. On the molecular level, CS/Lf/PTS-SLNs enhanced suppression of VEGF, down-regulated cyclin D1 and upregulated caspase-3 and BAX, compared to PTS-solution. Also, immunohistochemical assay confirmed the higher anti-tumorigenic effect of CS/Lf/PTS-SLNs (5.87-fold decrease in Bcl-2 expression) compared to PTS-solution. Our findings highlight CS/Lf/PTS-SLNs as a promising nanoplatform for phytotherapeutic targeted-breast cancer therapy.