Lactoferrin (Lf), which has a molecular weight of 77-80 kDa, is an iron-binding glycoprotein that has multiple functions in the body; it is involved in the apoptosis of cancer cells and can regulate various immune responses (1). Lf was first discovered in 1939 and is a 'red protein' found in milk; it can be separated and purified from human milk and cow's milk. The isolated protein structure of Lf is similar to that of serum transferrin, with a 60% sequence homology, and it reversibly binds to iron (Fe³⁺) ions (2). Therefore, Lf is classified as a member of the transferrin family, together with serum transferrin, melanotransferrin and ovotransferrin (3). This multifunctional protein is present in mucosal secretions, including tears, saliva, vaginal secretions, semen, nasal secretions, bronchial secretions, bile, gastrointestinal secretions, urine, cow's milk and human milk (4). Lf is also present on the mucosal surface and granules of white blood cells. Human milk and cow's milk are the most abundant sources of Lf (5). Lf is very similar between different species. In fact, the homology between the Lf of humans and cattle is 77% (6).

Lf promotes the absorption of iron by the body; it regulates cell growth, removes harmful free radicals and inhibits the formation of toxic compounds. In the regulation of immune responses, Lf exerts antibacterial, antiviral, anti-oxidation, anticancer and anti-inflammatory activities (5). Accordingly, Lf is added to a number of commercial products, including infant formula, fermented milk, cosmetics, therapeutic drinks, toothpaste and other products used in daily life (7).

In human endometrial stromal cells (8) and human embryonic kidney cells, Lf can enhance DNA synthesis in normal cells in a dose-dependent manner (9). In addition, another form of Lf, ∆Lf, can be expressed in glandular epithelium cells such as those of the prostate and salivary glands. ∆Lf is a protein subtype of Lf that lacks the leader sequence and the first 25 residues of the original protein (10). The truncated protein mRNA is detected in all normal tissues, but not in some tumor-derived cell lines. Several studies have shown that the chromosomal region encoding Lf is deleted in various tumors, which is a spontaneous process that may occur during cancer (11). ∆Lf plays a role as a transcription factor in cells, participates in the regulation of specific gene expression and plays a role in cancer (12). A number of in vivo studies have indicated the potential antitumor effect of Lf, suggesting that the oral bovine Lf (bLf) administration could decrease chemically induced carcinogenesis in rodents, along with marked cytotoxic and anti-metastatic activity against numerous cancer cell lines (13,14). The antitumor effect of Lf acts via a number of different mechanisms, including the induction of apoptosis in tumor tissues (7).

Owing to the overexpression of a number of cell surface receptors, Lf has a positive targeting effect; therefore, it is considered to be an ideal nanocarrier for certain hydrophobic therapeutic agents. In addition, as Lf can cross the blood-brain barrier (BBB), it has proven to be a good candidate for manufacturing nanocarriers to specifically deliver drugs for brain tumors. Therefore, Lf appears as a promising molecule with multiple applications in the fields of cancer treatment and nanomedicine. Lf has numerous advantages in terms of its ability to actively participate in the manufacture of nanocarriers. Furthermore, it is one of the few proteins that have a net positive charge under physiological conditions [isoelectric point (pI) 8.0-8.5]. Owing to its high pI value, Lf is positively charged over a wide range of pH values (15), is fairly stable in the gastrointestinal tract and possesses a number of intestinal receptors that facilitate the oral absorption and bioavailability of Lf-based nanocarriers within the circulation.

Lf is a natural immune modulator that plays roles in the innate and acquired immune systems, which regulate antibody formation, T- and B-cell maturation, and increase the percentage of natural killer cells in the lymphocyte population (16). The ability of Lf to regulate the activity of the immune response may be due to its ability to bind endotoxins [lipopolysaccharides (LPSs)] (17,18). In a previous study, Lf was found to alleviate the cellular inflammation induced by LPS by attenuating the nuclear factor-κB/mitogen-activated protein kinase pathways, mitigating oxidative stress and maintaining cellular barrier integrity. Such a finding implies that Lf plays an important role in immune regulation (19). When gram-negative bacteria try to invade the human host, the bacteria come into contact with various proteins of the innate immune system. Part of the bacterial outer membrane contains LPS. When this 'pathogen-related molecular pattern' is recognized by Toll-like receptor 4, it triggers a number of immune responses in various white blood cells and platelets (20,21). The combination of Lf and endotoxins released by bacteria can decrease the degree of stimulation of the immune system. This process can prevent overstimulation, which sometimes occurs in diseases such as sepsis. The hLf1-11 peptide derived from human lactoferrin (hLf) can inhibit myeloperoxidase, which is a major host defense enzyme found in a variety of white blood cells, which may further decrease the innate immune response (22). Furthermore, hLf has been shown to stimulate the maturation of dendritic cells and recruit various white blood cells (23). Therefore, Lf plays an activating role in innate and adaptive immune responses.

Lactoferrin is an allosteric enhancer of the proteolytic activity of cathepsin G, thereby affecting the function of adaptive immune cells (24). Lf has a positive charge that enables it to bind to the negatively charged surface molecules of various immune system cells, and this connection is believed to trigger signaling pathways that result in cellular responses such as activation, differentiation and proliferation. Lf can be transported to the nucleus in order to bind DNA and activate various signaling pathways (25). Lactoferrin can bind to DNA, and through its highly positively charged N-terminal region, which remains associated with the extruded DNA in the neutrophil extracellular traps, can still contribute to the bacterial killing in this process. As the granules also secrete a variety of proteolytic enzymes, Lf or other polypeptides may also be locally released from intact Lf (26).

As well as the induction of systemic immunity, skin immunity is promoted and allergic reactions are suppressed by Lf (27). The immune system is activated against skin allergens, resulting in the dose-dependent inhibition of Langerhans cell migration and dendritic cell accumulation in lymph nodes. The exposure of leukocyte Lf to cytokines, pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β may be adjusted to increase and decrease. The production of these factors depends on the type of signal that is recognized by the immune system. At the cellular level, Lf can increase the number of CD4⁺ and CD8⁺ cells in natural killer (NK) cells and T cells (28), promote the recruitment of leukocytes in the blood, induce phagocytosis and regulate the process of bone marrow formation (29). Lf also increases the expression of hyaluronic acid, which is required for the formation of granulation tissue, upregulates platelet-derived growth factors and promotes the proliferation and migration of keratinocytes; this is a necessary condition for the re-epithelialization of wounds. Lf also protects cells from apoptosis (30).

Lf is considered as a key component of the first line of defense for the human body (15) and has a variety of biological effects, including regulation of the immune response, iron absorption, and anti-inflammatory and antioxidant activities. Lf exerts antitumor effects through a variety of mechanisms. Oral bLf can decrease chemically induced carcinogenesis in rodents and has significant cytotoxicity and anti-metastatic activity in numerous cancer cell lines, such as breast cancer and stomach cancer cell lines (31,32). Table I summarizes some of the anti-carcinogenic mechanisms of Lf.

Lf is a survival factor of rheumatoid synovial neutrophils, an iron-binding protein that is released from activated neutrophils in inflammatory sites, and has anti-inflammatory and antibacterial properties (33). Although the isolation of iron by Lf and the direct effect on reactive oxygen intermediates are major factors in decreasing excessive inflammatory response damage by directly controlling the development of higher-order immune functions, Lf can regulate injury and pathology caused by injury. This ultimately leads to a decrease in the pathological damage during inflammation (34). The mechanism of action of Lf involves a component that differentially regulates the cellular immune response in sepsis models in vivo. Apoptotic cells can release Lf and combine with neutrophils to inhibit the chemotaxis of neutrophils, enabling macrophages to swallow apoptotic cells, thereby exerting anti-inflammatory effects (Fig. 1) (35).

Bezault et al (36) confirmed, for the first time, the antitumor activity of Lf in fibrosarcoma and melanoma mouse models. Furthermore, an intraperitoneal injection of hLh could inhibit the growth of solid tumors and lung metastasis, independent of the iron saturation of the protein. Notably, the anticancer ability of LF is related to the presence of NK cells. To further prove the relevance of Lf in anticancer activities, Damiens et al (37) studied the role of Lf in cancer progression under inflammatory conditions. The experimental results showed that Lf regulates NK cell cytotoxicity and the sensitivity of target cells to lysis. Similar results were also obtained in experiments by Shi and Li (38). However, Iyer et al (39) pointed out that the antitumor properties of Lf may partly be due to its iron-binding properties. Free iron may act as a mutagenic promoter via the induction of oxidative damage to the nucleic acid structure (40), thereby decreasing the risk of tumors induced by oxidation (Fig. 2) (41,42).

Numerous studies (43-45) have shown that exogenous treatment with Lf and its derivatives can effectively inhibit tumor growth and decrease tumor susceptibility. Specifically, the downregulation or silencing of Lf and its derivatives can lead to an increased chance of developing a tumor (46). Conversely, the proliferation of cancer cells is prevented after the restoration of the Lf gene (47). However, these studies did not definitively conclude the mechanism underlying the anticancer effect of Lf. This review discusses the potential applications of Lf gene expression in cancer treatment and the association between Lf and cancer. To date, it has been indicated that the cytotoxicity of Lf to several cancers occurs via three methods under different conditions: i) Destruction of cell membranes; ii) induction of cell apoptosis; and iii) cell cycle arrest and cellular immune response.

Tammam et al (66) revealed that the cytotoxicity of Lf to gliomas can be attributed to its cytoplasmic distribution. The nuclear transmission of Lf induces cell proliferation rather than cytotoxicity, suggesting that the mode of action of Lf in glioma is related to cell location.

It is well know that tumor cells overexpress Lf receptors in order to fulfill the increased nutritional demands of these highly proliferative cells (67). Lf is an ideal nanocarrier for certain hydrophobic therapeutics due to its active targeting potential as a result of its receptor being overexpressed on the surface of a number of cells. Moreover, Lf is good potential candidate for fabricating nanocarriers that specifically deliver drugs to brain tumors, as Lf can cross the BBB. Consequently, Lf appears as a promising molecule with multiple applications in the fields of cancer therapy and nanomedicine (68). Song et al (69) demonstrated the potential utility of Lf-conjugated GO@Fe3O4 nanocomposites for therapeutic applications in the treatment of gliomas (69). Lf-conjugated iron oxide nanoparticles can be used as tracers for targeted brain glioma imaging using magnetic particle imaging (70). Lf/phenylboronic acid-functionalized hyaluronic acid (HA) nanogel crosslinked with a disulfide bond crosslinker was generated as a reduction-sensitive dual-targeting glioma therapeutic platform for doxorubicin hydrochloride (DOX) delivery (71). Lf-HA-DOX significantly increased drug delivery to the glioma and may thus serve as a promising anti-glioma therapy (72).

The effect of Lf on gram-positive bacteria is mainly based on the combination of cations on the surface of Lf and anions on the surface of the bacteria, thereby neutralizing the negative charges on the surface of the gram-positive bacteria. For example, lipoteichoic acid decreases the negative charge on the cell wall, thereby facilitating the contact between lysozyme and the peptidoglycan under the cell wall, ultimately exerting an enzymatic effect (15).

Lf functions as an enzyme in certain reactions. Lf is the milk protein with the highest activity of DNase, RNase, ATPase and amylase. However, these activities are not the only enzymatic activities of Lf. bLf binds two HCV envelope proteins (88). Lf has DNA-binding properties (89) and can participate in the transcriptional activation of specific DNA sequences (90) or as a signal transduction mediator (91). Celiac disease has the highest amylase and ATP activities (92). The discovery of the properties of the Lf enzyme helps to clarify a number of its physiological functions.

Lf is used as a nanocarrier of DOX, as its receptor is highly expressed on the surface of highly proliferating cells (such as cancer cells). DOX is an effective cytotoxic anticancer drug, but has been reported to exhibit extensive toxicity to the heart and spleen, in addition to its limited oral absorption (93,94). Drug-loaded preparations have shown good physical stability, indicating that damage to the red blood cell membrane is negligible. Drug delivery through nano-formulations not only minimizes the cardiotoxicity of DOX, but also improves the efficacy and bioavailability of the drug in a targeted-specific manner (95).

A study has also shown that Lf nanoparticles are used to encapsulate antiviral drugs. Zidovudine nano-encapsulation into Lf nanoparticles has been achieved through sol-oil chemistry. Zidovudine is an effective antiviral drug with good bioavailability (50-75%); however, it can cause bone marrow suppression, neutropenia and organ toxicity. In the study by Kumar et al (96), the size of the prepared nanoparticles was 50-60 nm, the drug encapsulation efficiency was 67% and good physical stability was observed at room temperature and at 4°C. Furthermore, there was no significant change in particle size or drug content. Oral administration of efavirenz-loaded Lf nanoparticles resulted in anti-HIV-1 effects comparable to those of the free drug. In addition, compared with free efavirenz, drug-loaded nanoparticles showed improved pharmacokinetic characteristics and lower organ toxicity, indicating that this nanoformulation is a safe nanoplatform that can enhance drug delivery (96).

The cationic nature of Lf can be used to form complexes with negatively charged DNA through electrostatic complexation. In this context, in a previous study, plasmid pGFPC1, which encodes the green fluorescent protein, was used as a cargo gene (97). Lf nanoparticles loaded with plasmids were prepared using the sol-oil method. The diameter of the prepared Lf nanoparticles was 60 nm and the PDI was low, indicating the uniformity of the preparation. The prepared Lf nanoparticles also showed enhanced physical stability at a temperature of 4°C for ≤10 weeks without the particle size exhibiting significant changes. Incubation in DMEM containing 10% serum at 37°C for 8 h also did not result in changes to the particle size, which would result in longer plasma levels. This improved stability may be related to the strong electrostatic interactions between the positively charged Lf and negatively charged DNA. According to reports, Lf has a DNA-binding domain, which may help to further promote DNA binding and the formation of a tighter DNA-Lf nanocomplex (98).

The methods used to prepare Lf nanoparticles include nanoparticle albumin-bound (NAB) and thermal denaturation methods. The NAB technology is primarily dependent on the presence of an oily phase, which is slowly added to the aqueous phase containing Lf. As a nanoparticle, Lf forms a gel when exposed to heat treatment, ionic strength or changes in pH. Generally, the thermal gelation of a protein starts with a heating step to denature the protein, followed by the addition of salt to induce protein aggregation (87).

Lf can participate in the production of polyelectrolyte complex nanocarriers. The synthesis of such nanocarriers is based on the use of two oppositely charged molecules, such as a positively charged protein and a negatively charged natural polysaccharide. Nanocarriers based on polyelectrolyte complexes are likely stabilized by strong electrostatic interactions between cationic proteins and anionic polysaccharides. This stabilization can also enhance the stability of the encapsulated active ingredients (99). Table II summarizes representative examples of Lf-based nanocarriers for drug delivery applications.

This review provides an in-depth summary of the biological characteristics of Lf, including the structure of Lf biomolecules, its binding affinity to iron, and the interaction between Lf and the host. Lf plays an important role in the regulation of the immune response, and has immune stimulation, immune activation, anti-inflammatory activity, antibacterial, anti-viral effects and, in particular, anticancer activity. Based on the results of several studies, it is known that Lf-based nanocarriers can be easily prepared by simple methods and have excellent active targeting potential for tumor tissues, particularly brain tumors. The manufacture of Lf-based nanocarriers can broadly enhance the therapeutic potential of encapsulating active molecules. The present review discussed the latest preparation methods for Lf-based nanocarriers prepared by the sol-oil method, NAB technology and thermal denaturation method.