Biomedical Applications of Lactoferrin on the Ocular Surface.

Lactoferrin (LF) is a first-line defense protein with a pleiotropic functional pattern that includes anti-inflammatory, immunomodulatory, antiviral, antibacterial, and antitumoral properties. Remarkably, this iron-binding glycoprotein promotes iron retention, restricting free radical production and avoiding oxidative damage and inflammation. On the ocular surface, LF is released from corneal epithelial cells and lacrimal glands, representing a significant percentage of the total tear fluid proteins. Due to its multifunctionality, the availability of LF may be limited in several ocular disorders. Consequently, to reinforce the action of this highly beneficial glycoprotein on the ocular surface, LF has been proposed for the treatment of different conditions such as dry eye, keratoconus, conjunctivitis, and viral or bacterial ocular infections, among others. In this review, we outline the structure and the biological functions of LF, its relevant role at the ocular surface, its implication in LF-related ocular surface disorders, and its potential for biomedical applications.

Immunomodulatory Effect of Human Lactoferrin on Toll-like Receptors 2 Expression as Therapeutic Approach for Keratoconus.

Keratoconus (KC) is a corneal disorder whose etiology shares a close relationship with Lactoferrin (LTF) dysregulation and Toll-like Receptors 2 (TLR2) overexpression. This study shows how these two important biomarkers are clinically and molecularly interrelated, increasing knowledge about KC pathophysiology, and opening the door to future therapies. In this prospective clinical study, serum and tear LTF concentrations were quantified in 90 KC patients and 60 controls. A correlation analysis with multiple blood and tear immunoinflammatory mediators, and KC-associated tomographic parameters, was performed. An in vitro study using HEK-BlueTMhTLR2 cell cultures was also conducted to determine the expression and functionality of TLR2 under the influence of LTF treatment. As a result, a LTF decreased was observed in KC patients compared to controls (p < 0.0001), evidencing the strong correlation with TLR2 overexpression at systemic and ocular surface level, with inflammatory mediator upregulation and with KC severity. In stimulated cell cultures, TLR2 expression was decreased using 2 mg/mL of LTF. The levels of secreted embryonic alkaline phosphatase (SEAP) and interleukin-8 (IL-8) were also reduced in supernatants after LTF treatment. As conclusions, the dysregulation of LTF and TLR2 in the ocular surface of KC patients contributes to KC severity by maintaining a detrimental chronic immune−inflammatory state. The immunomodulatory properties of LTF on TLR2 expression suggest its potential as a therapeutic approach for KC.

Shotgun Proteomics for the Identification and Profiling of the Tear Proteome of Keratoconus Patients.

Purpose: The qualitative approach followed in this study aims to obtain an extensive view of the keratoconus (KC) tear proteome, which could highlight proteins previously undetected and enlarge our knowledge of the disease's pathophysiology. Methods: Twenty-five patients diagnosed with KC and 25 control subjects were studied in a prospective, cross-sectional study. KC screening examinations, including clinical and tomographic examinations, were performed on all participants. Tear samples were collected using Schirmer strips and analyzed by liquid chromatography-tandem mass spectrometry in a data-dependent workflow. A spectral count was used as a semiquantification tool. The tear proteomes of both groups were identified and profiled, and the functional interactions and biological characterization of differential proteins were analyzed using in silico tools. Results: We identified a total of 232 proteins, of whom 133 were expressed in both groups' samples; 41 were observed only in control samples and 58 were identified just in tears of patients with KC. A semiquantitative analysis showed the dysregulation of 17 proteins in the KC samples. An in silico analysis linked proteins only expressed in KC samples to oxidative stress, skin development, and apoptosis. The dysregulation of proteins involved in iron transport, inflammation, oxidative stress, and protease inhibition was observed in the semiquantitative results. Conclusions: A shotgun analysis showed that the tear proteome of patients with KC differed from controls by more than one-third of the total proteins identified, highlighting the relationship of the proteins only expressed in KC tears with processes of cell death, oxidative damage, and inflammation. The underexpression of proteins involved in iron pathways might support the iron imbalance as a contributing factor to cellular damage and death in KC disease.

Mucoadhesive PLGA Nanospheres and Nanocapsules for Lactoferrin Controlled Ocular Delivery.

BACKGROUND: the present work describes the preparation, characterization and optimization of eight types of PLGA-based nanosystems (nanospheres and nanocapsules) as innovative mucoadhesive drug delivery systems of lactoferrin, in order to achieve a preclinical consistent base as an alternative pharmacological treatment to different ocular syndromes and diseases. METHODS: All different nanoparticles were prepared via two modified nanoprecipitation techniques, using a three-component mixture of drug/polymer/surfactant (Lf/PLGA/Poloxamer), as a way to overcome the inherent limitations of conventional PLGA NPs. These modified polymeric nanocarriers, intended for topical ophthalmic administration, were subjected to in vitro characterization, surface modification and in vitro and in vivo assessments. RESULTS: An appropriate size range, uniform size distribution and negative ζ potential values were obtained for all types of formulations. Lactoferrin could be effectively included into all types of nanoparticles with appropriate encapsulation efficiency and loading capacity values. A greater, extended, and controlled delivery of Lf from the polymeric matrix was observed through the in vitro release studies. No instability or cytotoxicity was proved for all the formulations by means of organotypic models. Additionally, mucoadhesive in vitro and in vivo experiments show a significant increase in the residence time of the nanoparticles in the eye surface. CONCLUSIONS: all types of prepared PLGA nanoparticles might be a potential alternative for the topical ophthalmic administration of lactoferrin.

Alzheimer's Disease Seen through the Eye: Ocular Alterations and Neurodegeneration.

Alzheimer's Disease (AD) is one of the main neurodegenerative diseases worldwide. Unfortunately, AD shares many similarities with other dementias at early stages, which impedes an accurate premortem diagnosis. Therefore, it is urgent to find biomarkers to allow for early diagnosis of the disease. There is increasing scientific evidence highlighting the similarities between the eye and other structures of the CNS, suggesting that knowledge acquired in eye research could be useful for research and diagnosis of AD. For example, the retina and optic nerve are considered part of the central nervous system, and their damage can result in retrograde and anterograde axon degeneration, as well as abnormal protein aggregation. In the anterior eye segment, the aqueous humor and tear film may be comparable to the cerebrospinal fluid. Both fluids are enriched with molecules that can be potential neurodegenerative biomarkers. Indeed, the pathophysiology of AD, characterized by cerebral deposits of amyloid-beta (Aß) and tau protein, is also present in the eyes of AD patients, besides numerous structural and functional changes observed in the structure of the eyes. Therefore, all this evidence suggests that ocular changes have the potential to be used as either predictive values for AD assessment or as diagnostic tools.

Lactoferrin-loaded nanostructured lipid carriers (NLCs) as a new formulation for optimized ocular drug delivery.

Nanostructured lipid carriers (NLC) are novel lipidic nanosystems that provide significant improvements in terms of high drug loading capacity and controlled drug release. The purpose of the present work was based on the design, development, and physicochemical characterization of lactoferrin-loaded NLCs as a new therapeutic alternative for the keratoconus treatment. Lactoferrin-loaded NLCs were successfully prepared by a double emulsion/solvent evaporation method. The resultant NLC were assessed in terms of particle size, size distribution, surface charge, morphology, encapsulation efficiency (EE), loading capacity (LC), stability, cytotoxicity, in vitro release, and ocular surface retention. Resulting data showed a size of 119.45 ± 11.44 nm, a 0.151 ± 0.045 PDI value and a surface charge of -17.50 ± 2.53 mV. Besides, high EE and LC values were obtained (up to 75%). The in vitro release study demonstrated a lactoferrin controlled release pattern. NLCs were also stable, non-toxic and show mucoadhesive properties. Thus, a consistent preclinical base was obtained, where NLC may be considered as a potential controlled release novel drug delivery system of lactoferrin for the keratoconus treatment.

Tear Proteomics in Keratoconus: A Quantitative SWATH-MS Analysis.

Purpose: To elucidate dysregulated proteins in keratoconus (KC) to provide a better understanding of the molecular mechanisms that lead to the development of the disease using sequential window acquisition of all theoretical mass spectra (SWATH-MS) as a protein quantification tool of the tear proteomic profile. Methods: Prospective cross-sectional study that includes 25 keratoconic eyes and 25 healthy eyes. All participants underwent a clinical, tomographic, and aberrometric exam. Tear sample was collected using Schirmer strips and analyzed by liquid chromatography with tandem mass spectrometry. SWATH-MS was used as a quantification tool of the tear proteomic profile. The expression of the quantified proteins was compared between groups, and the biological and molecular functions of the dysregulated proteins as well as their functional relationships were studied by in silico analysis. Results: A total of 203 proteins were quantified in tear samples of patients with KC and control participants, of which 18 showed differential expression between groups (P < 0.05). An increase in the expression of 7 proteins and a decrease in the expression of 11 proteins were observed. Protein-protein interactions and gene ontology analysis showed the involvement of these dysregulated proteins in structural, inflammatory-immune, iron homeostasis, oxidative stress, and extracellular matrix proteolysis processes. Conclusions: Tear protein quantification has revealed the dysregulation of proteins involved in biological processes previously associated with KC. Among them, iron homeostasis should be highlighted as a relevant pathway in the KC pathophysiology, and it should be taken into account in the development of therapeutic targets to cope with tissue damage derived from iron accumulation and toxicity.

Design, Optimization, and Characterization of Lactoferrin-Loaded Chitosan/TPP and Chitosan/Sulfobutylether-ß-cyclodextrin Nanoparticles as a Pharmacological Alternative for Keratoconus Treatment.

This research study describes the design, optimization, and characterization of two different types of chitosan-based nanoparticles as novel drug delivery systems of a protein drug, lactoferrin. A preclinical consistent base was obtained for both nanosystems, being considered as the first pharmacological treatment for keratoconus as an alternative to current invasive clinical methods. Both types of nanoparticles were obtained via the ionotropic gelation technique. The size and morphology of the nanoparticles were studied as a function of the preparation conditions. A mean size of 180.73 ± 40.67 nm, a size distribution [polydispersity index (PDI)] of 0.170 ± 0.067, and positive ζ-potential values, ranging from 17.13 to 19.89 mV, were achieved. Lactoferrin was successfully incorporated into both types of nanocarriers. In vitro release profiles showed a lactoferrin enhanced, prolonged, and controlled delivery from the polymeric matrix. These formulations also demonstrated no stability or cytotoxicity problems, as well as appropriate mucoadhesive properties, with a high permanence time in the ocular surface. Thus, both types of nanoparticles may be considered as nanocarriers for the controlled release of lactoferrin as novel topical ophthalmic drug delivery systems.

Corneal and conjunctival alteration of innate immune expression in first-degree relatives of keratoconus patients.

PURPOSE: The innate immune toll-like receptors 2 (TLR2) and 4 (TLR4) may play a key role in the physiopathology of keratoconus (KC). Therefore, the aim of this study was to compare TLR2/TLR4 expression in corneal and conjunctival epithelial cells between healthy first-degree relatives of patients with KC and healthy controls as well as KC patients. METHODS: Case-control study in 72 healthy eyes of 36 control subjects, 53 eyes of 27 first-degree relatives, and 109 eyes with KC (60 patients). All participants were subjected to a clinical, topographic, aberrometric, and tomographic examination with extraction of corneal and conjunctival epithelial cells through scraping. TLR2/TLR4 expression was measured by flow cytometry, and was compared among controls, first-degree relatives, and KC patients. The relationship between TLR expression and epidemiological-clinical variables or topographic-aberrometric-tomographic parameters was also analyzed. RESULTS: Mean TLR2/TLR4 expression showed a significant gradual increase among groups: controls < first-degree relatives < KC patients. Mean expression of TLR2 in corneal epithelial cells and both TLR2/TLR4 in conjunctival epithelial cells were significantly higher in relatives than in controls (p = 0.026, p < 0.001, and p = 0.031, respectively). Sex, age, allergic disease, eye itching, rubbing, and topographic-aberrometric-tomographic parameters were not associated to TLR2/TLR4 expression in relatives. TLR2 conjunctival expression was independently associated to relatives (OR 1.001; CI 95% 1.000-1.002, p = 0.043) after adjustment by sex, age, and rubbing. CONCLUSION: TLR2 and TLR4 are overexpressed in corneal and conjunctival epithelial cells of KC relatives compared with controls. Both biomarkers may monitor early ocular changes in first-degree relatives who not show any abnormal clinical-topographic-aberrometric-tomographic parameters.