Biomedical Applications of Lactoferrin on the Ocular Surface

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Francisco Otero-Espinar	--	4323	2023-03-14 16:18:46	\|
2	format	Jason Zhu	Meta information modification	4323	2023-03-15 02:52:57	\|

This entry is adapted from the peer-reviewed paper 10.3390/pharmaceutics15030865

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.

drug delivery systems dry eye keratoconus lactoferrin

1. Introduction

Lactoferrin (LF), also known as lactotransferrin, is a mammalian iron-binding glycoprotein belonging to the transferrin (TF) family. For the first time, LF was discovered in bovine milk ^[1], and its similarity to the structure of TF focused further studies on understanding its properties in iron absorption and microbial growth regulation by iron derivation. In addition to iron, LF has a high affinity for other metals, such as zinc, copper, or manganese ions, being an important regulator of metal homeostasis in exocrine fluids ^[2]^[3]^[4]. Related to their iron-binding properties, TF and LF have been recognized as a powerful team in controlling free iron levels in body fluids, providing a high antioxidant capacity that contributes to mitigating damaging events such as oxidative stress or inflammation. Additionally, the wide array of LF biological functions as an anti-inflammatory, immunomodulatory, antiviral, antimicrobial, and antitumoral protein brings out its potential as a preventive and therapeutic target in different diseases.

2. Lactoferrin Structure

Human LF has a molecular weight of approximately 80 kDa, with an amino acid sequence of 691 amino acids, quite similar to different mammalian species ^[5]^[6]^[7]. Its structure includes two homologous globular lobes (N-lobe and C-lobe) with two domains per lobe (N1, N2, C1, and C2) ^[8]. Both lobes have specific ligands with ideal chemical and geometrical properties for high-affinity binding to ferric ions (Fe³⁺) ^[9]. These ligands contain two tyrosine residues, one histidine, and one aspartic acid. During the iron-binding process, interaction of the four amino acid residues is required, so Asp60, Tyr92, Tyr192, and His253 covalently bind to the Fe³⁺ atom in the presence of one carbonate ion (CO₃²⁻). Moreover, the two oxygen atoms of the carbonate ion also bind to Fe³⁺. Iron binding by the LF has been described as a cooperative binding process in which the N lobe binds the iron ion first and then promotes the C lobe to bind another Fe³⁺ ion.

Two conformational states of this protein can be characterized: holo-LF, as an iron-free protein (the two lobes, C and N lobes, are free of iron), and apo-LF in a fully iron-loaded protein mode (when the two lobes have one iron atom bonded), the last one stable and resistant to protease degradation ^[10]^[11]. The helical bonding of the LF lobes allows maintaining the binding with ferric ions, even at pH as low as 3.5, making LF a powerful iron scavenger that prevents the precipitation of the ions into insoluble hydroxides ^[9]. Changes in the structure of the LF iron domains promote the release of the ions. The main events that give rise to the structural changes responsible for iron release are: (I) the reduction of the ferric iron to the ferrous form (LF has a low affinity for the ferrous ions), (II) specific LF receptors (such as the LF receptor, mainly expressed in the intestinal epithelial cells), and (III) a pH decrease (which can induce the protonation of the carbonate ion, tyrosine, and histidine ligands, weakening the binding to iron and leading to its release) ^[12].

3. Lactoferrin Biological Functions

LF’s multifaceted nature has aroused great interest in its potential as a therapeutic and pharmacological target. In this sense, its iron-binding ability turns it into an essential regulator of toxic-free iron levels in exocrine fluids. In addition, LF has shown anti-inflammatory, immunomodulatory, antioxidant, antimicrobial, antiviral, and anticancerogenic properties, playing a fundamental role in the host defense.

3.1. Lactoferrin and Iron Homeostasis: A Crucial Protein in Antioxidant Protection

Iron is an essential element required for a wide range of cellular functions and pathways. However, iron homeostasis must be strictly controlled, because its dysregulation can lead to potentially cell-damaging events. Increased free iron levels catalyze some reactions (such as Fenton or Haber/Weiss reactions) that produce free radicals highly toxic and harmful to cells through their ability to damage lipids, proteins, and other cellular components ^[13].

LF also participates in the regulation of iron homeostasis through several pathways due to its iron-binding ability. Firstly, LF binds to Fe³⁺ ions, reducing the quantity of free or unbound iron available in the extracellular space. Moreover, LF can interact with ceruloplasmin (Cp), a ferroxidase that converts ferrous into ferric ions. In this regard, the direct transfer of ferric ions from Cp to LF avoids free iron circulation and, consequently, the activation of damaging reactions ^[14]^[15]. Therefore, LF is a key factor in regulating free iron and protecting underlying cells and tissues against free radicals and oxidative stress in the extracellular space.

Cellular iron uptake, storage, and export are controlled by strict mechanisms responsible for the proper balance of intracellular iron levels. There is evidence that LF can protect against intracellular iron overload by its anti-inflammatory action over the proinflammatory cytokine interleukin-6 (IL-6) ^[16]^[17]^[18]. The protection against iron overload derives from the effects of IL-6 on the expression of molecules involved in regulating the intracellular iron levels, such as ferroportin (Fpn), the main protein responsible for the export of ferrous iron from an intracellular to extracellular space. IL-6 induces an under-expression of Fpn, reducing the iron release and, consequently, promoting iron overload inside the cell. The anti-inflammatory activity of LF on IL-6 involves the downregulation of this cytokine expression, which stimulates Fpn activity and shields the cell from the iron overload ^[19].

3.2. Lactoferrin in the Immune and Inflammatory Response

LF is secreted by innate immune cells, epithelial cells, and some glands, supporting the presence of significant concentrations of LF in biological fluids and exocrine secretions that cover mucosal sites as gateways for pathogens. The active participation of LF in the modulation of innate immune responses has been confirmed in several ways through its ability to: (I) regulate T- and B-cell maturation, (II) increase natural killer (NK) cell activity, (III) block or inhibit the complement pathway, (IV) induce macrophage function from cytokine production, and (V) inhibit intracellular pathogen proliferation ^[20]^[21]^[22]^[23].

Immunological functions of LF are associated with its cationic charge, allowing its interaction and binding to negatively charged cells of the immune system, triggering signaling pathways and modulating cellular processes such as differentiation, migration, and proliferation. Beyond its functions from the extracellular space, LF can be internalized into the cell and transported to the nucleus, where it binds to the DNA and triggers different signaling cascades ^[24]. Different LF receptors have been in vitro characterized, including intelectin-1 (ITLN1), low-density lipoprotein receptor-related protein 1 (LRP1), toll-like receptors 2 (TLR2) and 4 (TLR4), cluster of differentiation 14 (CD14), asialoglycoprotein receptor (ASGPR), nucleolin, and cytokine receptor 4 ^[25]^[26]^[27]^[28]^[29]^[30]. ITLN1 is the receptor with the highest affinity for LF ^[31]. It is expressed by intestinal and biliary epithelial cells and promotes LF uptake and internalization, probably contributing to iron absorption. Moreover, ITLN1 recognizes microbial carbohydrate chains in a calcium-dependent manner, playing a defensive role against microorganisms. LRP1 is a low-specific LF transmembrane receptor expressed by neurons, hepatocytes, smooth muscle cells, skin keratinocytes, and fibroblasts. LRP1 is involved in multiple processes such as the endocytosis and phagocytosis of apoptotic cells, lipid homeostasis, kinase-dependent intracellular signaling, and β-amyloid precursor protein (APP) metabolism, as well as neuronal calcium signaling and neurotransmission. LF binding to LRP1 promotes and modulates several LRP1 functions and induces the activation of processes such as mitogenesis in osteoblasts or keratinocyte proliferation and migration ^[31]. In this regard, LF’s target molecules, cells, and receptors directly control certain biological functions. Likewise, by the TLRs and NF-kB pathway, LF acquires the ability to modulate the inflammatory response, preventing and reducing the release of some proinflammatory proteins (including IL-6, IL-1B, and IL-8); promoting the expression of anti-inflammatory cytokines such as IL-4 or IL-10; and regulating the activity of the complement. Its anti-inflammatory power is highlighted as one of its most interesting pharmacological properties ^[23]^[32]^[33]^[34].

3.3. Antibacterial Activity

The Fe³⁺ uptake capacity of LF provides a strong antimicrobial potential by limiting the availability of Fe³⁺ required for bacterial growth. LF bacteriostatic function has demonstrated its effectiveness in vivo and in vitro against a wide range of both Gram-positive and Gram-negative bacteria ^[35]^[36]^[37]^[38]. In addition, LF shows bactericidal properties related to its interaction with the bacterial surface, inducing alterations in the osmotic function of membranes. As a result of the LF’s highly cationic charge, it can interact with negatively charged molecules on the bacterial surface (such as lipoteichoic acid in Gram-positive bacteria or lipopolysaccharides (LPS) in Gram-negative bacteria), inducing damage to the lipid bilayer of the microbial membrane. Lipid bilayer disruption increases the membrane permeability, damaging or even leading to bacterial death ^[39]^[40]^[41]. In this line, other crucial functions of LF in the defense against pathogens include the inhibition of biofilm production (biofilms as a crucial component of bacterial virulence) and the promotion of the actions of other natural antimicrobials (such as lysozyme) by reducing the negative charge on the bacterial surface during its interaction with LPS or lipoteichoic acid ^[42]^[43]^[44].

3.4. Antiviral and Antifungal Activity

The antiviral capacity of LF has been reported through extensive studies on a wide range of viral infections ^[45]. Binding to host cell surface glycosaminoglycans (especially heparin sulfate (HSPG)) has been postulated as the central mechanism underlying protection against viral infections, inhibiting the interaction between the virus and the host cell ^[46]. The benefit of LF in the prevention of viral infection and replication (mainly through the activation of interferon α/β) has been demonstrated in vitro for numerous viruses affecting humans, such as human immunodeficiency virus (HIV), respiratory syncytial virus, herpesvirus, cytomegalovirus, hepatitis C virus, and SARS-CoV-2 ^[47]^[48]^[49]^[50]; nevertheless, its clinical usefulness in this field is currently limited. Likewise, LF also protects against fungal infections ^[51]^[52]^[53]. The ability of LF to damage the cell membranes of pathogens, as well as its function as an iron scavenger, have been shown to play a decisive role in this context.

3.5. Anticarcinogenic Properties

The interest in LF as an anticarcinogenic strategy is supported by its capacity to bind free iron, prevent the formation of toxic species, modulate cytokine secretion, regulate the immune–inflammatory response, participate in cell growth, promote apoptosis, and activate NK cells ^[54]^[55]^[56]^[57]. Previous studies have shown that iron chelators prevent the progression of estrogen-dependent neoplasms by limiting the bioavailability of free iron, which can damage cellular DNA and promote tumor cycling, linking the antitumor properties of LF to its action as an iron scavenger ^[58]. Moreover, LF inhibits the growth of cancer cells by blocking the tumoral circle, induces the expression of antitumoral cytokines (such as IL-18), and regulates NK cell cytotoxicity and CD8+ T-lymphocytes ^[59]^[60]. In this regard, exogenous treatment with LF has been effective in inhibiting tumor growth, but the mechanisms underlying this effect are still relatively unknown.

4. Drug Delivery Alternatives for the LF Topical Administration

The eye is one of the best-isolated organs, as it has effective protection mechanisms against external agents and exogenous substances clearance. The average volume of the precorneal tear film is 7–10 μL. This volume may increase at 30 μL after the instillation of the eye drop, decreasing again to 7 μL with the first blink, causing an approximate decrease in the concentration of the instilled drug by 80%. Moreover, it is estimated that the renewal of the tear film is 1.2 μL per minute, which suggests that hardly any drug remains on the ocular surface five minutes after the instillation of the eye drops. Thus, the permanence and bioavailability of drugs administered by conventional eye drops on the eye surface are very low. Typically, less than 5% of the administered dose by classic eye drops can be absorbed by ocular tissues ^[61]^[62].

In the last decades, different strategies have been proposed to ameliorate topical ophthalmic therapeutic effects and minimize the non-desirable adverse effects. Different alternatives based on the improvement of the drug permanence on the ocular surface, drug bioavailability, and penetration into the ocular tissues have been proposed ^[63]. Several hydrogel-based ophthalmic formulations, as well as different hydrophobic colloidal systems, have been recently reported as alternatives to improve the drug permanence on the ocular surface, drug absorption into the cornea and controlled drug release ^[61]^[62].

LF has demonstrated an active contribution, not only on the ocular surface but also in the corneal epithelium and stroma. Considering the high molecular weight of LF (80 kDa), its corneal mean residence time (MRT) and its diffusion ability into the epithelium are essential parameters to be considered to achieve significant LF concentrations. According to Subrizi et al. ^[64] large molecules with molecular weights up to 5 kDa can permeate across the conjunctiva and macromolecules as LF can permeate across the sclera. Consequently, if it is necessary to improve LF uptake in the corneal epithelium and stroma to increase its efficacy, LF delivery systems that promote an increase in LF residence time on the ocular surface may be a good alternative.

Despite the availability of several systems concerning the release of LF for different uses previously described in the literature, in the specific case of topical ophthalmic administration cyclodextrin, nanotechnology, and mucoadhesion have been proposed as the main technological approaches ^[61]^[64]. Different researchers have studied polysaccharide nanoparticles, biodegradable polymeric nanoparticles, lipid systems based on liposomes, and nanostructured lipid systems or medicate soft contact lens to increase the LF ocular permanence and diffusion. Nonetheless, there are hardly any studies of its behavior on the ocular surface and cornea at present.

In this line, Varela et al., in 2021 ^[65], proposed the preparation of two different types of LF-loaded chitosan mucoadhesive nanospheres, crosslinked with sodium tripolyphosphate (TPP) and with sulfobutylether-β-cyclodextrin (SBE-β-CD), respectively, for the KC treatment. Taking advantage of the presence of sialic acid and negative charges in the ocular mucosa, they proposed nanoparticles with a positive surface charge to promote mucoadhesive interactions. The researchers developed both types of nanospheres using an ionotropic gelation technique. With this methodology, the researchers obtained nanoparticles with a mean diameter of less than 300 nm, ζ-potential values ranging from +17.13 to +19.89 mV, and an LF immobilization yield of around 50%. The polysaccharide nanoparticles showed excellent stability over a three-month storage period under biological conditions of pH and ionic strength, as well as an LF-controlled release for more than 24 h in an artificial tear medium. The in vitro, ex vivo, and in vivo bioadhesion studies showed good mucoadhesive properties of both types of nanospheres with a prolonged residence time on the ocular surface. The in vivo ocular surface permanence studies developed by means of a computerized PET/CT (Positron Emission Tomography/Computerized Tomography) image analysis showed t_1/2 and MRT values in the cornea of 114 min and 127.3 min for CS/TPP, and 60.5 and 89.9 min for CS/SBE-β-CD, respectively, vs. the 17.7 min and 59.1 min of the free radiolabeling molecule. Both nanospheres exhibited higher ocular permanence compared to the control, with apparently low permanence of CS/SBE-β-CD vs. CS/TPP. The authors suggested that the increased ionic strength and osmolarity values caused by the high sodium concentration of the SBE-β-CD derivative may lead to the aggregation of the CS/SBE-β-CD nanoparticles, and an increase in the blinking after instillation may reduce its surface ocular permanence.

Recently, López-Machado et al. (2021) ^[66] and Varela et al. (2022) ^[67] have also proposed the use of biodegradable polymeric nanoparticles to increase the concentration of LF in the ocular tissues improving the ocular surface permanence time. Varela et al. ^[67] developed non-toxic LF-loaded nanospheres and nanocapsules for KC treatment by a one-step and a two-step nanoprecipitation method, respectively, using a variety of 50:50 polyacrylic-polyglycolic acid (PLGA) copolymer of different molecular weights (10,000, 17,000, 24,000, and 38,000 Da). All nanospheres showed an average diameter between 100 and 150 nm regardless of the PLGA composition. Nanocapsules were larger in size, with diameters between 150 and 300 nm, depending on the molecular weight of the PLGA. In contrast, the ζ-potential values were slightly higher in the nanospheres than in the nanocapsules. The authors also demonstrated the low loading capacity of the nanospheres compared to the nanopcapsules to immobilize. Nanospheres showed an LF loading capacity of less than 10% in comparison to the nanocapsules obtained with the lower molecular weight PLGA, with dosage values of 60%, as well as production yield and encapsulation efficiency values above 80%. PLGA nanocapsules were stable during storage under biological conditions of pH and ionic strength. The in vitro release studies showed a LF-controlled release of almost 24 h with a release kinetic dependent on the molecular weight and the PLGA variety used in the manufacture of the nanocapsules. The in vivo ocular permanence studies by computerized PET/CT showed a t_1/2 higher than the reference (protein solution), with values of 93.31 min and 51.32 min for PLGA nanospheres and nanocapsules, respectively. The t_1/2 values were in the same order as those obtained in previous studies with chitosan nanospheres.

López-Machado et al. ^[66] prepared PLGA nanocapsules (50:50; 38,000 Da molecular weight) for the treatment of inflammatory processes of the anterior segment of the eye. The researchers used a central composite experimental design to obtain the best composition and production conditions. Optimized nanoparticles showed a monodisperse population in terms of size, with an average diameter below 250 nm and high negative ζ-potential values. In vitro cellular studies showed that the nanoparticles were not cytotoxic, had the ability to incorporate through the LRP1 pathway, and were able to inhibit the LPS-induced inflammatory response in an HCE-2 cell line. Ex vivo permeation studies using isolated corneas from New Zealand rabbits show that LF immobilized into nanoparticles can permeate slightly faster than free LF through the cornea. The authors found significant differences in the values of flux, permeability, and amount permeated at 24 h between LF-loaded nanocapsules and free LF. However, no differences were found in the amount of LF retained in the cornea at the end of the experiment. In vivo ocular studies of tolerance (Draize test) and anti-inflammatory efficacy carried out in a rabbit model pointed to the ability of the nanoparticles to prevent and treat ocular inflammation. The authors used a model of inflammation induced by arachidonic acid and studied the protective and curative effects of the LF-loaded PLGA nanoparticles, finding effectiveness in both treatments.

Varela et al. (2022) ^[68] also proposed LF-loaded nanostructured lipid carriers (NLC) to improve LF bioavailability for the treatment of KC. The NLC were prepared by a double emulsion/solvent evaporation technique with a thermosensitive gel core to enhance the LF immobilization into the lipid formulation. The NLCs were monodisperse with an average diameter of 120 nm. The encapsulation efficiency and loading capacity of NLC were high depending on the LF concentration employed in the elaboration procedure. The process was improved when the LF concentration in the manufacturing medium was higher than 1 mg/mL. At this concentration, the encapsulation efficiency and loading capacity were 80% and 70%, respectively. The NLCs proved to be non-toxic and stable in storage under biological conditions. Moreover, the in vivo ocular permanence studies by the PET/CT technique showed NLC with excellent mucoadhesive properties, with values of 107.82 and 141.33 min for t_1/2 and MRT, respectively.

Lopez Machado et al. (2021) ^[69] also proposed the use of hyaluronic acid-coated liposomes as a new lipid formulation for the treatment of dry eye disease and ocular inflammation. The authors produced fat-free soybean phospholipid liposomes with phosphatidylcholine and cholesterol by the lipid film hydration method combined with a high-pressure homogenization process. Hyaluronic acid-coated liposomes showed an average size of 90.5 nm, a positive surface charge with ζ-potential values of +20.5 mV, and an encapsulation efficiency of 50%. In vitro and ex vivo corneal permeation assays (performed on isolated New Zealand rabbit corneas) showed an effective control of the release and an improvement of LF corneal permeation from liposomes. Ex vivo permeation studies showed higher flux values, permeability, and amount permeated at 24 h for LF-loaded liposomes compared to a free LF solution, as well as a decrease in the amount retained in the cornea when liposomes were used. Comparing these results with those obtained in the previous study with nanoparticles ^[66] an increase in the values of all pharmacokinetic parameters for both free LF and immobilized LF into liposomes, and a reduction in the amount of LF retained in the cornea in the case of liposomes were observed. Two different in vivo models using benzalkonium chloride or LPS treatment were developed to study the efficacy of liposomes in the treatment of DED and ocular inflammation. Hyaluronic acid-coated liposomes showed good activity in the Schirmer test in the DED animal model and an improvement in the prevention and treatment of ocular inflammation compared to the control, as well as to the group treated with a solution of free LF, was proved.

Marketed soft contact lens loaded with LF has also been used for the glycoprotein release in the eye for protection against oxidative stress ^[70] and to counteract cytotoxicity caused by keratoconic process ^[71]. Initially, Pastori et al. ^[70] investigated the ability of three types of commercial contact lenses to load and release LF: the silicone-based hydrogel filcon V and filcon IB, and the hydroxyethyl methacrylate-based hydrogel galyfilcon A. Filcon V showed better LF loading and releases behavior with a loaded level of 61 μg of glycoprotein per lens. LF released from the lens maintains its antioxidant activity in human epithelial cell culture, showing a protective effect against oxidative stress. The same authors study the antioxidant activity of LF loaded in the silicone-based hydrogel filcon V on the epithelial cells incubated with keratoconic tears. The incubation of epithelial cells with tears of KC patients produces an increase in cell mortality compared with the incubation with tears of healthy patients. Both works show the in vitro efficacy of LF-loaded contact lenses for protection against oxidative stress.

Finally, LF has also been proposed to treat pathologies of the posterior segment of the eye. An example is the work of Ahmed et al. in 2014 ^[72] that proposed the use of LF-coated nanoparticles as a new strategy to improve cellular uptake in the retinoblastoma treatment.

Therefore, efforts are being made to achieve effective LF delivery systems to the ocular surface for the treatment of inflammatory and degenerative diseases of the eye. Pharmaceutical nanotechnology has demonstrated its efficacy to interact with the ocular mucosa and epithelia, increasing the permanence of the glycoprotein on the ocular surface. The nanoparticles developed have shown good in vitro and in vivo efficacy in animal models in the treatment of inflammatory pathologies or with an important oxidative component. Consequently, these works have laid the groundwork for the future clinical use of LF delivery systems with applicability not only in KC but also in other ocular surface conditions with LF involvement.

An additional problem in the development of new formulations of Lf with the potential to be commercialized is their stability. There are several studies that determine the stability of LF in different media and delivery systems ^[73]^[74]^[75]. Kim and coworkers studied the stability of LF in solution in the presence of various excipients ^[69]. They found that arginine and polysorbate 80 can protect the molecule from physical or chemical destabilization. After the addition of arginine, polysorbate 80, and trehalose, it is possible to obtain a solution that remains stable for more than 150 days in the refrigerator. However, stability declines at room temperature or above.

Yao et al. studied the physicochemical stability of LF-loaded liposomes and solid lipid nanoparticles modified by a chitosan or pectin ^[74]. All the LF-loaded liposomes show a rapid release of the LF overall at 40 °C and a complete degradation after 180 days of storage time, whereas almost 30% of intact Lf still remained after 180 days in solid lipid nanoparticles. However, the instability processes in lipid formulation are related to the premature release and expulsion of LF to the environment than to its degradation within the lipid systems. Finally, there is a complete review of the effects of technological treatments used in the treatment and preservation of food on LF ^[75]. It can be seen how thermal treatments at high temperatures produce the aggregation and denaturation of LF, as well as some high-pressure homogenization processes when very high-pressure values are used. Other processes such as spray-drying or freeze-drying respect the LF structure.

CDs can be a candidate to contribute to the stabilization of the LF in the nanomedicine and hydrogels. CDs are cyclic oligosaccharides that show a good ability to form complexes with drug molecules and to improve their physicochemical properties without molecular modifications, via drug/host interaction ^[76]^[77]. The capacity of CDs to interact with proteins is well known ^[78]^[79]^[80]^[81]^[82]. Different mechanisms have been described by which cyclodextrins interact with proteins improving physical and chemical stability ^[81]. CDs can form inclusion complexes with amino acids, mainly βCD derivatives and hydrophobic and aromatic residues of Phe, Tyr, His, and Trp, modulating the solvent exposure to hydrophobic amino-acidic residues ^[78]^[79]^[80]^[81]. Additionally, the surface activity of some CD-derivative can contribute to protein stabilization by reducing the protein surface adsorption ^[81]. DCs can prevent protein aggregation and adsorption through these mechanisms and improve stability against proteases. Consequently, CDs derivatives are excellent candidates for improving the chemical and physicochemical stability of proteins in the solid and liquid states ^[82].

In recent years, 3D printing has emerged as a promising technology for creating complex structures and materials with precise control over their physical properties ^[83]. There has been some research into 3D printing lactoferrin, particularly for its potential use in biomedical applications but not specifically for ophthalmic applications. One example is the 3D printing of lactoferrin-loaded alginate hydrogel scaffolds using a bioprinter ^[84]. The researchers found that the printed scaffolds had good biocompatibility and could support the growth of human mesenchymal stem cells. Another example is the development of a 3D-printed lactoferrin-based hydrogel that could be used as a wound dressing ^[85]. The hydrogel had good mechanical properties and could release lactoferrin in a controlled manner. Overall, 3D printing lactoferrin holds promise for the development of new therapeutic and biomedical applications. However, further research is needed to fully understand this technology’s potential and optimize the printing process of lactoferrin-based hydrogels for ophthalmic applications.

References

Groves, M.L. The Isolation of a Red Protein from Milk. J. Am. Chem. Soc. 1960, 82, 3345–3350.
Smith, C.A.; Anderson, B.F.; Baker, H.M.; Baker, E.N. Metal substitution in transferrins: The crystal structure of human copper-lactoferrin at 2.1-A resolution. Biochemistry 1992, 31, 4527–4533.
Løvstad, R.A. A kinetic study on the distribution of Cu(II)-ions between albumin and transferrin. Biometals 2004, 17, 111–113.
Thompson, M.W. Regulation of zinc-dependent enzymes by metal carrier proteins. Biometals 2022, 35, 187–213.
Metz-Boutigue, M.H.; Jollès, J.; Mazurier, J.; Schoentgen, F.; Legrand, D.; Spik, G.; Montreuil, J.; Jollès, P. Human lactotransferrin: Amino acid sequence and structural comparisons with other transferrins. Eur. J. Biochem. 1984, 145, 659–676.
Baker, E.N.; Baker, H.M. Molecular structure, binding properties and dynamics of lactoferrin. Cell. Mol. Life Sci. 2005, 62, 2531–2539.
Baker, E.N.; Baker, H.M. A structural framework for understanding the multifunctional character of lactoferrin. Biochimie 2009, 91, 3–10.
Wally, J.; Buchanan, S.K. A Structural Comparison of Human Serum Transferrin and Human Lactoferrin. Biometals 2007, 20, 249–262.
Baker, H.M.; Baker, E.N. Lactoferrin and iron: Structural and dynamic aspects of binding and release. Biometals 2004, 17, 209–216.
Andersen, B.F.; Baker, H.M.; Morris, G.E.; Rumball, S.V.; Baker, E.N. Apolactoferrin Structure Demonstrates Ligand-Induced Conformational Change in Transferrins. Nature 1990, 344, 784–787.
Gerstein, M.; Anderson, B.F.; Norris, G.E.; Baker, E.N.; Lesk, A.M.; Chothia, C. Domain closure in lactoferrin. Two hinges produce a see-saw motion between alternative close-packed interfaces. J. Mol. Biol. 1993, 234, 357–372.
Baker, E.N.; Baker, H.M.; Kidd, R.D. Lactoferrin and transferrin: Functional variations on a common structural framework. Biochem. Cell Biol. 2002, 80, 27–34.
Lieu, P.T.; Heiskala, M.; Peterson, P.A.; Yang, Y. The roles of iron in health and disease. Mol. Aspects Med. 2001, 22, 1–87.
Sokolov, A.V.; Pulina, M.O.; Zakharova, E.T.; Shavlovski, M.M.; Vasilyev, V.B. Effect of lactoferrin on the ferroxidase activity of ceruloplasmin. Biochemistry 2005, 70, 1015–1019.
Sokolov, A.V.; Voynova, I.V.; Kostevich, V.A.; Vlasenko, A.Y.; Zakharova, E.T.; Vasilyev, V.B. Comparison of interaction between ceruloplasmin and lactoferrin/transferrin: To Bind or Not to Bind. Biochemistry 2017, 82, 1073–1078.
Berlutti, F.; Pilloni, A.; Pietropaoli, M.; Polimeni, A.; Valenti, P. Lactoferrin and oral diseases: Current status and perspective in periodontitis. Ann. Stomatol. 2011, 2, 10–18.
Sessa, R.; Di Pietro, M.; Filardo, S.; Bressan, A.; Rosa, L.; Cutone, A.; Frioni, A.; Berlutti, F.; Paesano, R.; Valenti, P. Effect of bovine lactoferrin on Chlamydia trachomatis infection and inflammation. Biochem. Cell Biol. 2017, 95, 34–40.
Rosa, L.; Cutone, A.; Lepanto, M.S.; Paesano, R.; Valenti, P. Lactoferrin: A natural glycoprotein involved in iron and inflammatory homeostasis. Int. J. Mol. Sci. 2017, 18, 1985.
Cutone, A.; Frioni, A.; Berlutti, F.; Valenti, P.; Musci, G.; Bonaccorsi di Patti, M.C. Lactoferrin prevents LPS-induced decrease of the iron exporter ferroportin in human monocytes/macrophages. Biometals 2014, 27, 807–813.
Shi, H.; Li, W. Inhibitory Effects of Human Lactoferrin on U14 Cervical Carcinoma through Upregulation of the Immune Response. Oncol. Lett. 2014, 7, 820–826.
Damiens, E.; Mazurier, J.; el Yazidi, I.; Masson, M.; Duthille, I.; Spik, G.; Boilly-Marer, Y. Effects of human lactoferrin on NK cell cytotoxicity against haematopoietic and epithelial tumour cells. Biochim. Biophys. Acta 1998, 1402, 277–287.
Kruzel, M.L.; Zimecki, M.; Actor, J.K. Lactoferrin in a context of inflammation-induced pathology. Front. Immunol. 2017, 8, 1438.
Samuelsen, Ø.; Haukland, H.H.; Ulvatne, H.; Vorland, L.H. Anti-complement effects of lactoferrin-derived peptides. FEMS Immunol. Med. Microbiol. 2004, 41, 141–148.
Legrand, D.; Vigié, K.; Said, E.A.; Elass, E.; Masson, M.; Slomianny, M.C.; Carpentier, M.; Briand, J.P.; Mazurier, J.; Hovanessian, A.G. Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells. Eur. J. Biochem. 2004, 271, 303–317.
Suzuki, Y.A.; Lopez, V.; Lönnerdal, B. Mammalian lactoferrin receptors: Structure and function. Cell. Mol. Life Sci. 2005, 62, 2560–2575.
Shin, K.; Wakabayashi, H.; Yamauchi, K.; Yaeshima, T.; Iwatsuki, K. Recombinant human intelectin binds bovine lactoferrin and its peptides. Biol. Pharm. Bull. 2008, 31, 1605–1608.
Baveye, S.; Elass, E.; Fernig, D.G.; Blanquart, C.; Mazurier, J.; Legrand, D. Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, E-selectin and ICAM-1, induced by the CD14-lipopolysaccharide complex. Infect. Immun. 2000, 68, 6519–6525.
Takayama, Y.; Takezawa, T. Lactoferrin promotes collagen gel contractile activity of fibroblasts mediated by lipoprotein receptors. Biochem. Cell Biol. 2006, 84, 268–274.
Yang, H.G.; Li, H.Y.; Li, P.; Bao, X.Y.; Huang, G.X.; Xing, L.; Zheng, N.; Wang, J.Q. Modulation activity of heat-treated and untreated lactoferrin on the TLR-4 pathway in anoxia cell model and cerebral ischemia reperfusion mouse model. J. Dairy Sci. 2020, 103, 1151–1163.
Regueiro, U.; López-López, M.; Varela-Fernández, R.; Sobrino, T.; Diez-Feijoo, E.; Lema, I. Immunomodulatory effect of human lactoferrin on toll-like receptors 2 expression as therapeutic approach for keratoconus. Int. J. Mol. Sci. 2022, 23, 12350.
Mancinelli, R.; Olivero, F.; Carpino, G.; Overi, D.; Rosa, L.; Lepanto, M.S.; Cutone, A.; Franchitto, A.; Alpini, G.; Onori, P.; et al. Role of lactoferrin and its receptors on biliary epithelium. Biometals. 2018, 31, 369–379.
Håversen, L.; Ohlsson, B.G.; Hahn-Zoric, M.; Hanson, L.A.; Mattsby-Baltzer, I. Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B. Cell. Immunol. 2002, 220, 83–95.
Li, H.Y.; Yang, H.G.; Wu, H.M.; Yao, Q.Q.; Zhang, Z.Y.; Meng, Q.S.; Fan, L.L.; Wang, J.Q.; Zheng, N. Inhibitory Effects of Lactoferrin on Pulmonary Inflammatory Processes Induced by Lipopolysaccharide by Modulating the TLR4-Related Pathway. J. Dairy Sci. 2021, 104, 7383–7392.
He, Y.; Lawlor, N.T.; Newburg, D.S. Human milk components modulate toll-like receptor-mediated inflammation. Adv. Nutr. 2016, 7, 102–111.
Dierick, M.; Ongena, R.; Vanrompay, D.; Devriendt, B.; Cox, E. Lactoferrin Decreases Enterotoxigenic Escherichia Coli-Induced Fluid Secretion and Bacterial Adhesion in the Porcine Small Intestine. Pharmaceutics 2022, 14, 1778.
Hussan, J.R.; Irwin, S.G.; Mathews, B.; Swift, S.; Williams, D.L.; Cornish, J. Optimal Dose of Lactoferrin Reduces the Resilience of in Vitro Staphylococcus Aureus Colonies. PLoS ONE 2022, 17, e0273088.
Wang, X.; Hirmo, S.; Willén, R.; Wadström, T. Inhibition of Helicobacter pylori infection by bovine milk glycoconjugates in a BAlb/cA mouse model. J. Med. Microbiol. 2001, 50, 430–435.
Zarzosa-Moreno, D.; Avalos-Gómez, C.; Ramírez-Texcalco, L.S.; Torres-López, E.; Ramírez-Mondragón, R.; Hernández-Ramírez, J.O.; Serrano-Luna, J.; de la Garza, M. Lactoferrin and its derived peptides: An alternative for combating virulence mechanisms developed by pathogens. Molecules 2020, 25, 5763.
Valenti, P.; Antonini, G. Lactoferrin: An important host defense against microbial and viral attack. Cell. Mol. Life Sci. 2005, 62, 2576–2587.
Ellison, R.T.; Giehl, T.J.; LaForce, F.M. Damage of the outer membrane of enteric gram-negative bacteria by lactoferrin and transferrin. Infect. Immun. 1988, 56, 2774–2781.
Drago-Serrano, M.E.; de la Garza-Amaya, M.; Luna, J.S.; Campos-Rodríguez, R. Lactoferrin-lipopolysaccharide (LPS) binding as key to antibacterial and antiendotoxic effects. Int. Immunopharmacol. 2012, 12, 1–9.
García-Borjas, K.A.; Ceballos-Olvera, I.; Luna-Castro, S.; Peña-Avelino, Y. Bovine lactoferrin can decrease the in vitro biofilm production and show synergy with antibiotics against listeria and escherichia coli isolates. Protein. Pept. Lett. 2021, 28, 101–107.
Parra-Saavedra, K.J.; Macias-Lamas, A.M.; Silva-Jara, J.M.; Solís-Pacheco, J.R.; Ortiz-Lazareno, P.C.; Aguilar-Uscanga, B.R. Human Lactoferrin from Breast Milk: Characterization by HPLC and Its in Vitro Antibiofilm Performance. J. Food Sci. Technol. 2022, 59, 4907–4914.
Khanum, R.; Chung, P.Y.; Clarke, S.C.; Chin, B.Y. Lactoferrin Modulates the Biofilm Formation and Bap Gene Expression of Methicillin-Resistant Staphylococcus Epidermidis. Can. J. Microbiol. 2023, 69, 117–122.
Berlutti, F.; Pantanella, F.; Natalizi, T.; Frioni, A.; Paesano, R.; Polimeni, A.; Valenti, P. Antiviral properties of lactoferrin--a natural immunity molecule. Molecules 2011, 16, 6992–7018.
Lang, J.; Yang, N.; Deng, J.; Liu, K.; Yang, P.; Zhang, G.; Jiang, C. Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans. PLoS ONE 2011, 6, e23710.
Miotto, M.; Di Rienzo, L.; Bò, L.; Boffi, A.; Ruocco, G.; Milanetti, E. Molecular mechanisms behind anti SARS-CoV-2 action of lactoferrin. Front. Mol. Biosci. 2021, 8, 607443.
Mancinelli, R.; Rosa, L.; Cutone, A.; Lepanto, M.S.; Franchitto, A.; Onori, P.; Gaudio, E.; Valenti, P. Viral Hepatitis and Iron Dysregulation: Molecular Pathways and the Role of Lactoferrin. Molecules 2020, 25, 1997.
Picard-Jean, F.; Bouchard, S.; Larivée, G.; Bisaillon, M. The Intracellular Inhibition of HCV Replication Represents a Novel Mechanism of Action by the Innate Immune Lactoferrin Protein. Antivir. Res. 2014, 111, 13–22.
Wakabayashi, H.; Oda, H.; Yamauchi, K.; Abe, F. Lactoferrin for Prevention of Common Viral Infections. J. Infect. Chemother. 2014, 20, 666–671.
Samaranayake, Y.H.; Samaranayake, L.P.; Pow, E.H.; Beena, V.T.; Yeung, K.W. Antifungal effects of lysozyme and lactoferrin against genetically similar, sequential Candida albicans isolates from a human immunodeficiency virus-infected southern Chinese cohort. J. Clin. Microbiol. 2001, 39, 3296–3302.
Machado, R.; da Costa, A.; Silva, D.M.; Gomes, A.C.; Casal, M.; Sencadas, V. Antibacterial and Antifungal Activity of Poly(Lactic Acid)-Bovine Lactoferrin Nanofiber Membranes. Macromol. Biosci. 2018, 18, 1700324.
Pawar, S.; Markowitz, K.; Velliyagounder, K. Effect of Human Lactoferrin on Candida Albicans Infection and Host Response Interactions in Experimental Oral Candidiasis in Mice. Arch. Oral Biol. 2022, 137, 105399.
Rocha, V.P.; Campos, S.P.C.; Barros, C.A.; Trindade, P.; Souza, L.R.Q.; Silva, T.G.; Gimba, E.R.P.; Teodoro, A.J.; Gonçalves, R.B. Bovine Lactoferrin Induces Cell Death in Human Prostate Cancer Cells. Oxid. Med. Cell. Longev. 2022, 2022, 2187696.
Arcella, A.; Oliva, M.A.; Staffieri, S.; Alberti, S.; Grillea, G.; Madonna, M.; Bartolo, M.; Pavone, L.; Giangaspero, F.; Cantore, G.; et al. In Vitro and in Vivo Effect of Human Lactoferrin on Glioblastoma Growth. J. Neurosurg. 2015, 123, 1026–1035.
Ramírez-Sánchez, D.A.; Arredondo-Beltrán, I.G.; Canizalez-Roman, A.; Flores-Villaseñor, H.; Nazmi, K.; Bolscher, J.G.M.; León-Sicairos, N. Bovine Lactoferrin and Lactoferrin Peptides Affect Endometrial and Cervical Cancer Cell Lines. Biochem. Cell Biol. 2021, 99, 149–158.
Nakamura-Bencomo, S.; Gutierrez, D.A.; Robles-Escajeda, E.; Iglesias-Figueroa, B.; Siqueiros-Cendón, T.S.; Espinoza-Sánchez, E.A.; Arévalo-Gallegos, S.; Aguilera, R.J.; Rascón-Cruz, Q.; Varela-Ramirez, A. Recombinant Human Lactoferrin Carrying Humanized Glycosylation Exhibits Antileukemia Selective Cytotoxicity, Microfilament Disruption, Cell Cycle Arrest, and Apoptosis Activities. Investig. New Drugs. 2021, 39, 400–415.
Rodrigues, L.; Teixeira, J.; Schmitt, F.; Paulsson, M.; Månsson, H.L. Lactoferrin and cancer disease prevention. Crit. Rev. Food Sci. Nutr. 2009, 49, 203–217.
Wang, W.P.; Iigo, M.; Sato, J.; Sekine, K.; Adachi, I.; Tsuda, H. Activation of intestinal mucosal immunity in tumor-bearing mice by lactoferrin. Jpn. J. Cancer. Res. 2000, 91, 1022–1027.
González-Chávez, S.A.; Arévalo-Gallegos, S.; Rascón-Cruz, Q. Lactoferrin: Structure, function and applications. Int. J. Antimicrob. Agents. 2009, 33, 301.e1–301.e8.
Varela-Fernández, R.; Díaz-Tomé, V.; Luaces-Rodríguez, A.; Conde-Penedo, A.; García-Otero, X.; Luzardo-Álvarez, A.; Fernández-Ferreiro, A.; Otero-Espinar, F.J. Drug Delivery to the Posterior Segment of the Eye: Biopharmaceutic and Pharmacokinetic Considerations. Pharmaceutics 2020, 12, 269.
Yang, Y.; Lockwood, A. Topical ocular drug delivery systems: Innovations for an unmet need. Exp. Eye Res. 2022, 218, 109006.
Moiseev, R.V.; Morrison, P.W.J.; Steele, F.; Khutoryanskiy, V.V. Penetration Enhancers in Ocular Drug Delivery. Pharmaceutics 2019, 11, 321.
Subrizi, A.; del Amo, E.; Korzhikov-Vlakh, V.; Tennikova, T.; Ruponen, M.; Urtti, A. Design principles of ocular drug delivery systems: Importance of drug payload, release rate, and material properties. Drug Discov. Today 2019, 24, 1446–1457.
Varela-Fernández, R.; García-Otero, X.; Díaz-Tomé, V.; Regueiro, U.; López-López, M.; González-Barcia, M.; Lema, I.; Otero-Espinar, F.J. Design, optimization, and characterization of lactoferrin-loaded chitosan/TPP and chitosan/sulfobutylether-β-cyclodextrin nanoparticles as a pharmacological alternative for keratoconus treatment. ACS Appl. Mater. Interfaces 2021, 13, 3559–3575.
López-Machado, A.; Díaz, N.; Cano, A.; Espina, M.; Badía, J.; Baldomà, L.; Cristina Calpena, A.; Biancardi, M.; Souto, B.E.; García, M.L.; et al. Development of topical eye-drops of lactoferrin-loaded biodegradable nanoparticles for the treatment of anterior segment inflammatory processes. Int. J. Pharm. 2021, 609, 121188.
Varela-Fernández, R.; García-Otero, X.; Díaz-Tomé, V.; Regueiro, U.; López-López, M.; González-Barcia, M.; Lema, I.; Otero-Espinar, F.J. Mucoadhesive PLGA nanospheres and nanocapsules for lactoferrin controlled ocular delivery. Pharmaceutics 2022, 14, 799.
Varela-Fernández, R.; García-Otero, X.; Díaz-Tomé, V.; Regueiro, U.; López-López, M.; González-Barcia, M.; Lema, I.; Otero-Espinar, F.J. Lactoferrin-loaded nanostructured lipid carriers (NLCs) as a new formulation for optimized ocular drug delivery. Eur. J. Pharm. Biopharm. 2022, 172, 144–156.
López-Machado, A.; Díaz-Garrido, N.; Cano, A.; Espina, M.; Badia, J.; Baldomà, L.; Cristina Calpena, A.; Souto, B.E.; García, M.L.; Sánchez-López, E. Development of Lactoferrin-Loaded Liposomes for the Management of Dry Eye Disease and Ocular Inflammation. Pharmaceutics 2021, 13, 1698–1717.
Pastori, V.; Tavazzi, S.; Lecchi, M. Lactoferrin-loaded contact lenses: Eye protection against oxidative stress. Cornea. 2015, 34, 693–697.
Pastori, V.; Tavazzi, S.; Lecchi, M. Lactoferrin-loaded contact lenses counteract cytotoxicity caused in vitro by keratoconic tears. Contact Lens. Anterior Eye 2019, 42, 253–257.
Ahmed, F.; Ali, M.J.; Kondapi, A.K. Carboplatin loaded protein nanoparticles exhibit improve anti-proliferative activity in retinoblastoma cells. Int. J. Biol. Macromol. 2014, 70, 572–582.
Kim, H.J.; Shin, C.H.; Kim, C.W. Stabilization of Glycoprotein Liquid Formulation Using Arginine: A Study with Lactoferrin as a Model Protein. Biosci. Biotechnol. Biochem. 2009, 73, 61–66.
Yao, X.; Bunt, C.; Cornish, J.; Quek, S.Y.; Wen, J. Oral Delivery of Bovine Lactoferrin Using Pectin- and Chitosan-Modified Liposomes and Solid Lipid Particles: Improvement of Stability of Lactoferrin. Chem. Biol. Drug Des. 2015, 86, 466–475.
Franco, I.; Pérez, M.D.; Conesa, C.; Calvo, M.; Sánchez, L. Effect of Technological Treatments on Bovine Lactoferrin: An Overview. Food Res. Int. 2018, 106, 173–182.
Saokham, P.; Muankaew, C.; Jansook, P.; Loftsson, T. Solubility of Cyclodextrins and Drug/Cyclodextrin Complexes. Molecules 2018, 23, 1161.
Kovacs, T.; Nagy, P.; Panyi, G.; Szente, L.; Varga, Z.; Zakany, F. Cyclodextrins: Only Pharmaceutical Excipients or Full-Fledged Drug Candidates? Pharmaceutics 2022, 14, 2559.
Irie, T.; Uekama, K. Cyclodextrins in peptide and protein delivery. Adv. Drug Deliv. Rev. 1999, 36, 101.
Härtl, E.; Winter, G.; Besheer, A. Influence of hydroxypropyl-Beta-cyclodextrin on the stability of dilute and highly concentrated immunoglobulin g formulations. J. Pharm. Sci. 2013, 102, 4121–4131.
Castañeda Ruiz, A.J.; Shetab Boushehri, M.A.; Phan, T.; Carle, S.; Garidel, P.; Buske, J.; Lamprecht, A. Alternative Excipients for Protein Stabilization in Protein Therapeutics: Overcoming the Limitations of Polysorbates. Pharmaceutics 2022, 14, 2575.
Stolzke, T.; Krieg, F.; Peng, T.; Zhang, H.; Häusler, O.; Brandenbusch, C. Hydroxylpropyl-cyclodextrin as Potential Excipient to Prevent Stress-Induced Aggregation in Liquid Protein Formulations. Molecules 2022, 27, 5094.
Serno, T.; Geidobler, R.; Winter, G. Protein stabilization by cyclodextrins in the liquid and dried state. Adv. Drug Deliv. Rev. 2011, 63, 1086.
Arif, Z.U.; Khalid, M.Y.; Noroozi, R.; Sadeghianmaryan, A.; Jalalvand, M.; Hossain, M. Recent Advances in 3D-Printed Polylactide and Polycaprolactone-Based Biomaterials for Tissue Engineering Applications. Int. J. Biol. Macromol. 2022, 218, 930–968.
Janarthanan, G.; Tran, H.N.; Cha, E.; Lee, C.; Das, D.; Noh, I. 3D Printable and Injectable Lactoferrin-Loaded Carboxymethyl Cellulose-Glycol Chitosan Hydrogels for Tissue Engineering Applications. Mater. Sci. Eng. C Mater. Biol. Appl. 2020, 113, 111008.
Ghosh, S.; Yi, H.G. A Review on Bioinks and Their Application in Plant Bioprinting. Int. J. Bioprinting 2022, 8, 612.

© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.

Upload a video for this entry

Information

Subjects: Ophthalmology

Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :

Uxía Regueiro

Maite López-López

Rubén Varela-Fernández

Francisco Javier Otero-Espinar

Isabel Lema

View Times: 539

Update Date: 15 Mar 2023

Table of Contents

Video Upload Options

Confirm