Development of a Novel Drug Delivery System to Enhance the Oral Bioavailability of Lactoferrin

Abstract

Background and objectives: Bovine lactoferrin (bLf) is a therapeutic protein that occurs naturally in cow’s milk. In recent years, there has been considerable interest to develop a bLf nutraceutical product for improvement of general wellbeing, especially with respect to improving immune responses due to its superior source of iron and antibacterial properties. However, the bioavailability of orally administered bLf is extremely low due to biochemical and physical barriers, including gastrointestinal proteases, the epithelial barrier of the small intestine and efflux pumps. Even after bLf is absorbed, another potential ‘barrier’ to take into account is that of avoiding intracellular lysosome degradation in order to reach blood supply. To achieve its therapeutic effects, encapsulation of bLf via liposomes or solid lipid particles (SLPs) has many advantages to accomplish the requirements in oral delivery. This study aims to design these lipid based systems and to evaluate their influence on the effective delivery of bLf. Methods: bLf loaded liposomes and SLPs were prepared by reverse phase evaporation (REV) method and emulsion/solvent evaporation method, respectively. Surface modification and biopolymer coating by chitosan and pectin were further applied to develop these particles with enhanced mucoadhesive properties and sustained release profile. The physio-chemical properties of mucoadhesive polymer coated liposomes and SLPs including particle size, encapsulation efficiency (EE), zeta potential, polydispersity index (PDI), drug-lipid-polymer interactions and stability against enzymatic degradation were measured as a comparison. The cytotoxicity, the cellular uptake and transport mechanisms were investigated for the optimum formulation using Colon adenocarcinoma (Caco-2) cells. Pharmacokinetic parameters associated with different formulations were determined using an in vivo rat tail vein model. Results and discussion: Particle size of optimized polymer coated liposome and SLPs were determined as from 429.6 to 740.2 nm or from 459.5 to 518.5 nm, respectively, depending on the polymer type. The drug EE of mucoadhesive SLPs was 92.02% higher than that of liposomes. SEM and TEM confirmed that hydrophilic polymer chains not only gathered around the globular surface of these lipid particles forming the coating layer, but also attached on particles forming self-assembling polymer-particle networks. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) revealed that bLf was presented in SLPs in an amorphous state and no interactions among drug-lipid-polymer were observed, whereas bLf may insert into liposomal membrane. Compared with liposomes, polymer coated SLPs exhibited better physical stability against aggregation induced by pH change, high temperature and high electrolyte concentration. Up to 83.67% of bLf remained in polymer coated SLPs after 6 hours (hr) digestion in rat luminal extract. ~ 30-40% of bLf can be retained in SLPs formulations over long term storage of 180 days. Moreover, SLPs formulations were less susceptible to lipolysis than liposomes, as cleaving into low amounts (~ 12%) of free fatty acids (FFA) in simulated intestinal fluids (SIF). Owing to the benefits above, SLPs were further studied at the level of the cell. Polymer coated SLPs showed biocompatibility towards Caco-2 cells up to 300 μg/mL for 8 hr. In vitro uptake of SLPs was time -, temperature - and concentration - dependent, suggesting the particles were taken up by endocytosis. Polymer coated SLPs, particularly chitosan, increased cellular uptake (~ 19%) and transport rate (~ 136.9%) of bLf across the Caco-2 cells. In vivo studies, chitosan coated SLPs also proved to be the most effective carrier in ehancing the oral bioavailability of bLf as relative bioavailability (Fbio) increased 2.69-fold in comparison to liposomes (up to 2.24-fold) and free bLf as the control. Conclusion: The project has demonstrated that the developed mucoadhesive polymer coated liposomes and SLPs were able to improve the physio-chemical stability of bLf; impart an absorption ehnacing effect in the Caco-2 cells, hence increase the oral bioavailability of bLf in rat models. The research also highlighted mucoadhesive SLPs were superior to mucoadhesive liposomes, and possess promising features for its future applications as a potential oral delivery system for therapeutic proteins and peptides.