Strategies to enhance bioavailability of Lf

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As presented in Chapter 2, bLf is fragile due to its susceptibility to enzymatic degradation in the GI tract, and such conformational changes may reduce its therapeutic efficiency. Moreover, the absorption barrier presented by the mucus layer covering GI epithelial membranes also limits the drug transfer to systemic sites of action due to the large hydrophilic nature of the drug molecule (60). Consequently, the absolute oral bioavailability levels of native bLf may be less than 1% (51).
Recently, increasing attention has been focused on SLPs as an efficient drug carrier. Due to small particle size (< 1000 nm), SLPs may exhibit bioadhesion to the GI tract wall by lodging in the intervillous space, thus increasing their residence time leading to enhanced bioavailability (157, 158). Orally administered SLPs incorporating proteins, such as calcitonin (161) and insulin (403), have showed prolonged plasma levels and increased therapeutic effects. However, there have been no reported studies on SLPs to encapsulate bLf.
The oral administration of liposomal bLf has exhibited enhanced bioavailability due to the capacity of liposomes to increase protein uptake and its accumulation into cells as well as protecting bLf from enzymatic degradation (187-190). However, liposomes are prone to the combined degrading effects of the acidic pH of the stomach, bile salts, and pancreatic lipases. To overcome these drawbacks, crosslinking the liposomes with biopolymers such as pectin, alginate or chitosan could potentially prolong drug release and improve the pharmacological action by increasing mucoadhesiveness (192, 429).
The physicochemical characterizations of these lipid based particles, including EE, drug loading, surface charge and particle size show variation according to formulation components, such as lipid concentration, surfactant concentration and process conditions (430), which can influence drug release, in vivo stability and biodistribution (431). A strategy for evaluation and optimization of the formulation parameters in an efficient approach is necessary. Experimental factorial design is considered to be the effective method for estimating the correlations between factors and performances as well as identifying interactions between different factors (432).
A full factorial experiment consists of two or more factors, each with discrete possible levels, and the whole experimental units take on all combinations of the experimental factor levels. A k factor, n level, full experimental design involves nk experiments. Two-level (2k) factorial design is the most popular design in pharmaceutical development due to its saving of experimental time and materials (433). The use of variance analysis and factorial design of experiments allows us to express relationship of factors and responses as a polynomial model. By using the generated mathematical polynomial model, each experimental response can be predicted in relation to changing independent variables levels. An interaction is the failure of a factor to produce the same response at the different levels of the other factor (434). A significant interaction effect shows the influence of an individual factor on the response is highly dependent on the other factors.
In this chapter, development and optimization of bLf loaded liposomes and SLPs modified by pectin and chitosan were attempted. Firstly, a 24 full factorial design was employed to evaluate the main factors which affect liposomes and SLPs particle size and EE. Optimized liposomes and SLPs formulations were then prepared on the basis of the predicted optimum levels of the factors from the factorial design. Secondly, different proportions of pectin or chitosan were used to further modify the optimized liposomes and SLPs. In vitro drug release profiles were evaluated to determine the optimized proportions of polymers added. Finally, morphological examination, FTIR and DSC were performed to characterize the properties of the selected pectin and chitosan modified liposomes and SLPs.


bLf from bovine colostrums was provided by Fonterra (Palmerston North, NZ). TFA (puriss. p.a., for HPLC, ≥ 99% GC), L-α-phosphatidylcholine from egg yolk, cholesterol (Sigma Grade, ≥ 99%), stearic acid (Grade I, ≥ 99% GC) and mix high and low methoxyl pectin from apple were purchased from Sigma Aldrich (St. Louis, USA). HPLC grade acetonitrile and Tween® 80 were purchased from Merck (Whitehouse Station, USA). Soybean lecithin was purchased from BDH (Poole, UK). Poloxamer 188 was purchased from BASF (Ludwigshafen, Germany). Chitosan low viscosity was purchased from Fluka (Park Rabin Rehovot, Israel). All other reagents and chemicals were of analytical grade.

Preparation of bLf loaded liposomes

Liposomal dispersions were prepared by REV method with modification (392). A total of 40 mg L-α-phosphatidylcholine and cholesterol at mass ratios of 4:1 or 7:3 was accurately weighed and adequately dissolved into 10 mL of chloroform/methanol mixture (4:1 v/v) in a round bottom flask. The flask was attached to a rotary evaporator (R215 rotavapor, Buchi, Flawil, Switzerland), and rotated at 260 rpm (8 × g) at 40 °C under vacuum until a thin lipid film was formed in flask. The remaining fumes of solvent mixture were evacuated by nitrogen gas. The lipid film was redissolved in diethyl ether (10 mL), in which the reversed-phase vesicles were formed. Then an aqueous phase of 5 mL PBS buffer (0.1 M, pH 7.4) containing bLf (1 or 3 mg) and Tween 80 (10 or 20 mg) was mixed with the organic phase. The W/O emulsion was sonicated using a probe sonicator (UP200S, Hielscher, Teltow, Germany) for 1 or 5 min at frequency of 0.5 cycles and 50% amplitude in an ice bath before returning to the rotary evaporator again. A gel was obtained during the evaporation processing under atmospheric pressure at 40 °C. The resultant gel was broken to become a semi-transparent liquid indicating the formation of liposomes by further rotary evaporation. Finally, another 5 mL of PBS (0.1 M, pH 7.4) was added with gentle vortex and the remaining fumes of diethyl ether were evacuated by nitrogen gas. A final liposomes suspension ~ 10 mL was stored at 4 °C in a refrigerator until required.

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Preparation of bLf loaded SLPs

SLPs dispersions were prepared by the emulsion/solvent evaporation method (396). The organic phase was formed by dissolving a total of 40 mg stearic acid and lecithin at a mass ratio of 4:1 or 7:3 in 10 mL of acetone/DCM mixture (1:4 v/v) at room temperature. Then an aqueous phase of 5 mL PBS (0.1 M, pH 7.4) containing bLf (1 or 3 mg) was added slowly to the organic phase in a bath sonicator. The mixture was sonicated by a probe sonicator for 1 or 5 min at 0.5 cycles and 50% amplitude in an ice bath. The formed W/O primary emulsion was immediately poured onto 25 mL of PBS buffer (0.1 M, pH 7.4) containing 10 or 20 mg poloxamer 188 and continuously stirred at a speed of 500 rpm (30 × g) to allow solvent evaporation for ~ 6 hr at room temperature. The SLPs suspension ~ 30 mL was collected and stored at 4 °C in a refrigerator until required.

Experimental design: A 24 full factorial design

Four independent variables were analyzed using a 24 full factorial. The four independent factors at two levels were the percentage of cholesterol for liposome preparation (percentage of lecithin for SLPs preparation), initial concentration of bLf, initial concentration of surfactant and sonication time. The normalized factor levels of independent variables were given in Table 3.1. Particle size and drug EE were evaluated as dependent variables (response).
Statistical analysis was performed using Design Expert (Version 7.0.0, State-Ease Inc., USA), for which the interactive statistical first-order computer-generated equation is defined as: Y =α0 + α1X1 + α2X2 + α3X3 + α4X4 + α12X1 X2 + α13X1 X3 + α14X1 X4 + α23X2 X3 + α24X2 X4 + α34X3 X4 + α123X1 X2 X3 + α124X1 X2 X4 + α134X1 X3 X4 + α234X2 X3 X4 (Equation 3-1), where Y is the particle size (nm) or EE (%); X1 to X4 are the coded levels of factors; X1 X2 to X2 X3 X4 represent the interaction terms; α0 is the intercept; α1 to α234 are the regression coefficients of factors and interaction terms. Maximizing the EE and minimizing the particle size were achieved as the objective function in the present study by investigating the relationship between factors and responses.

Preparation of pectin and chitosan modified liposomes and SLPs

After the optimum formulation of bLf loaded liposomes and SLPs were determined based on 24 full factorial designs, hydrophilic polymer modified liposomes and SLPs were prepared in a two-step process. Firstly, liposomes and SLPs with optimized compositions (F6 and F11) were prepared using the methods mentioned above and centrifuged at the speed of 25,000 rpm (~ 75,000 × g) (Sorvall wx80, Langenselbold, Germany) for 1 hr at 4 °C to remove the supernatant. Secondly, the particle sediments were dispersed into 30 mL of PBS buffer (0.1 M, pH 7.4) followed by pectin and chitosan added into the suspensions, respectively. The mixtures were mixed overnight to facilitate adsorption of the polymer onto the particles. To study the influence of polymer concentrations on particle properties, different polymer/lipid mass ratios of 0, 1:4, 1:2, 1:1 and 1.5:1 were conducted. Lipid content of the particle suspension in the study was kept constant at 40 mg/30mL.

Liposomes and SLPs characterization
Particle size, size distribution and zeta potential

The zeta average mean particle size, PDI and zeta potential of liposomes and SLPs were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire, UK) at 25 °C. All measurements were measured in three replicates. The conditions of measurement were: He/Ne laser (wavelength = 633 nm), scattering angle 90°, refractive index for liposome analysis of 1.34 or 1.33 for SLPs, and the viscosity 0.8872 mPa s. Prior to the measurements, the concentration of liposomes was diluted 200-fold using PBS buffer (0.1 M, pH 7.4), and the concentration of SLPs was diluted 100-fold using PBS buffer (0.1 M, pH 7.4).


of bLf was determined as followed: particle suspensions (4.5-fold dilutions for liposomes but no dilution for SLPs) were centrifuged at the speed of 25,000 rpm (~ 75,000 × g) for 1 hr at 4 °C to separate free bLf from bLf loaded particles. Free bLf in the supernatant was analyzed by the previously established RP-HPLC method in Chapter 2. Briefly, separation was performed on a C18 HPLC column (Jupiter 5u C18 300R, 250 × 4.6 mm, 5 mm, Pheonomenex, North Shore, NZ) fitted with a C18 guard column (10 × 3.0 mm). A constant flow rate of 0.5 mL/min was used. The injection volume was 50 µL and the column temperature was maintained at 37 °C. Mobile phase A consisted of 0.1% TFA in water and acetonitrile (95:5 v/v). Mobile phase B consisted of 0.1% TFA in water and acetonitrile (5:95 v/v). Elution started with an isocratic elution from 35% mobile phase B for 1 min followed by a linear gradient to 60% mobile phase B for 19 min, and a final 5 min (post time) for equilibration. The EE of bLf was calculated using the following equation: EE = (Wt – Wf)/Wt × 100 %, where Wt was the total amount of initially added bLf and Wf is the free amount of bLf in the supernatant.

 In vitro bLf release assessment

The in vitro release of bLf from pectin and chitosan modified liposomes and SLPs were determined as followed: polymer modified liposomes and SLPs dispersions were centrifuged at 25,000 rpm (~ 75,000 × g) for 1 hr at 4 °C to remove free polymer from the sediments. The sediments were dispersed into 10 mL PBS buffer (0.1 M, pH 7.4) in a 30 mL beaker. The beaker was then placed on a shaking water bath (GLS Aqua 18 Plus, Grant, UK) at 100 rpm (1 × g) at 37 °C. 200 µL samples were withdrawn at various times (0, 0.5, 1, 3, 5 and 7 hr) and centrifuged at the speed of 13,000 rpm (~ 20,000 × g) (Minispin, Eppendorf, Germany) for 5 min. The supernatants were analyzed by the previously mentioned RP-HPLC analysis. The sediments dispersed with equal volume (200 µL) of fresh PBS buffer (0.1 M, pH 7.4) were immediately replaced into the beaker to maintain a constant volume. The release of bLf from polymer modified particles was evaluated using the following equation: drug release = Wt / Wi × 100 %, where Wi is the initial amount of encapsulated bLf before incubation and Wt is the free amount of bLf in the supernatant after incubation for time t. Umodified liposomes and SLPs with pectin or chitosan acted as controls.

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The morphology of unmodified, pectin and chitosan modified liposomes and SLPs were observed using SEM (Philips XL30S FEG, Eindhoven, the Netherlands). A small drop of orinigal particle suspension (~ 10 µL) was placed on a piece of cover-glass and then air-dried at room temperature. The samples were mounted on aluminium stubs covered with a carbon tape which was then coated with gold palladium.

FTIR analysis

FTIR analysis was performed on a Bruker Tensor 37 spectrometer using a Miracle Micro ATR attachment with diamond crystal (Bruker, Ettlingen, Germany). Unmodified, pectin and chitosan modified liposomes and SLPs were centrifuged at the speed of 25,000 rpm (~ 75,000
g) for 1 hr at 4 °C to remove the supernatant. The particle sediments were then placed into an oven at 60 °C until dry. After that, a small amount of powder sample was placed on the diamond crystal and compressed gently using the pressure clamp. The crystal was cleaned carefully before each use. Scanning was carried out in the 4000-400 centimetre-1 (cm-1) region with a resolution of 4 cm-1, from 64 parallel scans. The spectra were recorded and analyzed using OPUS software version 6.5 (Bruker, Ettlingen, Germany).

Thermal analysis

Thermograms of unmodified, pectin and chitosan modified liposomes and SLPs were obtained with a Q2000 DSC (TA instruments, New Castle, Delaware, USA). Approximately 3.0 mg of each powder sample was placed on an aluminium hermetic pan which was tightly sealed by an aluminium lid, and an empty pan/lid was used a reference. DSC scans were performed under a nitrogen atmosphere at a heating rate of 10 °C/min in 30-330 °C temperature range.

Data analysis

All data were expressed as mean ± SD. One-way analysis of variance was performed to assess the statistical significant difference between the data using SPSS Statistics Version 22 software (IBM, Chicago, USA). Differences were considered to be statistically significant at *p < 0.05.

Effect of various factors on particle size and EE

As shown in Table 3.2, liposomal size ranged from 292.0 to 656.4 nm, and the percentage of bLf entrapped in the liposomes ranged from 2.05 to 58.14% for the various factor combinations. Mean PDI ranging from 0.38 to 0.60 indicating a narrow size distribution. Negative zeta potentials were observed ranging from -8.21 to -3.30.
Mathematical modelling was carried out using the equation 3-1 (mentioned in Section 3.3.3) to obtain a polynomial equation. Transformed values of independent variables (XL1 to XL4, and XS1 to XS4) and its products as in the equation 3-1 along with dependent variables (YL1, YL2, YS1 and YS2) were subjected to multiple-regression analysis to determine the coefficients (α0 to α234) and P-values of each term of the equation. After neglecting the insignificant terms (P > 0.05), the fitted equations related to particle size, EE and the transformed factors are shown in Table 3.3. R2 value of greater than 0.9 indicated good correlation between the adjusted and predicted values which supports the statistical validity and significance of these equations for optimization.

Table of Contents
List of Figures
List of Tables 
List of Abbreviations 
1.1 An overview of oral delivery of peptides and proteins
1.2 Lactoferrin (Lf)
1.3 Barriers to oral absorption of Lf
1.4 Strategies to enhance bioavailability of Lf
1.5 Bioadhesive lipid based delivery systems selected for this study
1.6 The selection of biodegradable mucoadhesive polymers
1.7 Mechanism of bioadhesive particles interact with mucus layer
1.8 The impact of interactions among polymer, lipid and protein
1.9 Characterisation of bioadhesive liposomes and SLPs
1.10 Preparation of bioadhesive liposomes and SLPs
1.11 Thesis aims and structure
2.1 Introduction
2.2 Materials
2.3 Methods
2.4 Results
2.5 Discussion
2.6 Conclusion
3.1 Introduction
3.2 Materials
3.3 Methods
3.4 Results …
3.5 Discussion
3.6 Conclusion
4.1 Introduction
4.2 Materials
4.3 Methods
4.4 Results
4.5 Discussion
4.6 Conclusion
5.1 Introduction
5.2 Materials
5.3 Methods
5.4 Results
5.5 Discussion
5.6 Conclusions
6.1 Discussion
6.2 Limitations and future directions
6.3 Conclusion
Development of a Novel Drug Delivery System to Enhance the Oral Bioavailability of Lactoferrin

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