Three different methods were carried out to prepare Lf nanoliposomes.

Lf nanoliposomes were prepared by reverse-phase evaporation method 19 . Briefly, a certain amount of PC and CH were dissolved in chloroform-diethyl ether and Lf was dissolved in phosphate buffer solution (pH7.4). The organic phase was mixed with the aqueous phase using probe sonication for 5 min. The mixture was placed in a round-bottom flask and a gel was formed by evaporating the organic solvent under reduced pressure at 35°C using a rotary evaporator. Then 30 mL phosphate buffer solution (0.20 M, pH 7.4, PBS) containing tween 80 was added and evaporated for another 20 min.

The method of preparing Lf nanoliposomes was described by Bangham and Lea 20 . Lipids were dissolved in chloroform-diethyl ether forming a mixture. The organic solvent was then removed by rotary evaporation under reduced pressure at 35°C using a rotary evaporator. The dry lipid film was hydrated with a solution of Lf dissolved in phosphate buffer solution (0.20 M, pH 7.4, PBS).

The method of preparing Lf liposomes was described by Dream and Bangham 21 . PC and Chol were dissolved in a certain volume of diethyl ether and the Lf was dissolved in amount of phosphate buffer (0.02 M, pH7.4). The organic solution was injected into the aqueous solution. The mixture was placed into a glass bottle fitted with a silicone rubber injection cap and this bottle was placed in a water jacket connected to a circulating water bath maintained at 35°C with rapid mixing until diethyl ether removed.

The particle size was measured by Mastersizer 2000 instrument (Malvern), equipped with HydroMu dispersing unit (Malvern). Measurements were taken in the range between 0.1 and 1000 μm, under the following conditions: water refractive index 1.33, and general calculation model for irregular particles. The data obtained were averaged by software (Mastersizer 2000).

The encapsulation efficiency was determined by centrifuge-UV method. Take nanolipsomes suspension (500 μL) by spinning at 10000 rpm for 30 min using centrifuge, the protein content of the supernatant was measured by Bradford. The same suspension was ruptured using sufficient volume of ethanol, and the total amount of Lf was determined spectrophotometrically.

Encapsulation efficiency was calculated using Eq.1.

EE % = Q t − Q f Q t ∗ 100

Where Q_f is the amount of free Lf and Qt is the total amount of Lf present in 500 μL of nanoliposomes.

Lf nanoliposomes were stored in a refrigeratory at 4°C. The MDA value was determined as an index of the PL peroxidation. The MDA value was detected spectrophotometrtically by the thiobarbituric acid (TBA) reaction following the method of Weng and Chen 22 . Taking 5 mL of a mixture of 25 mmol/L TBA, 0.9 mol/L TCA and 50 mmol/L HC1 in a test-tube and 1 mL Lf nanoliposomes was heated to 100°C for 30 min and After reaching the room temperature, the absorbance of the solutions were measured at 535 nm.

Lf nanoliposomes (10 ml) were put into a 30 mL beaker and were ultrasonicated with a probe sonicator (VCX400, Sonics & Material, Inc., USA) in an ice bath with 1 s ON, 1 s OFF intervals. Samples of 0.2 mL were taken at predetermine intervals. Encapsulation efficiency of withdrawn samples was determined. The release ratios were calculated.

The controlled release was examined in simulated gastric juice of pH 1.3 and intestinal juice of pH 7.5. The solution of pH 1.3 consisted of HCl(0.10 M), pepsin and deionized water, while the solution of pH 7.5 was made up of KH₂PO₄ (6.8 mg/mL), NaOH (0.10 M, adjusted to pH 7.5), Trypsin (10 mg/mL) and deionized water. Five milliliters of Lf nanoliposome suspension was mixed with the equal volume of simulated gastrointestinal juice in a 50 mL beaker. The beaker was placed on a magnetic stirrer adjusted to a constant speed of 150 rpm at 37°C. Aliquots of 0.2 mL were sampled from the beaker at predetermine intervals. The release of Lf from nanoliposomes was evaluated by release ratio. The release ratio was calculated using the Eq. (2).

Release ratio % = 1 - EE t EE 0 × 100

Where EE₀ is the encapsulation efficiency of lactoferrin nano-liposomes before incubation and EE_t is the encapsulation efficiency of lactoferrin nanoliposomes after incubation for the time.

In Figure 1 it can be observed size distribution of Lf nanoliposomes prepared by the three different methods. The size distribution is generally used as a characterization tool to evaluate the stability of Lf nanoliposomes. The result showed that the size of nanoliposomes was ranked in the following order, reverse-phase evaporation method< ether injection method <film method.

The effect of three different methods on encapsulation efficiency of Lf nanoliposomes is shown in Figure 2. The encapsulation efficiency prepared with reverse-phase evaporation method, ether injection method and film method was 50.1%, 34.5% and 48.9%, respectively.

Above all, reverse-phase evaporation method is a simple and applicable operation to most of the phospholipid mixture encapsulation volume and has high encapsulation efficiency. This method is suitable for wrapping water soluble drugs and macromolecular biologically active substance.

Phospholipid was used as the major component of liposomal membrane, containing partially polyunsaturated fatty acid residues sensitive to oxidative free radicles 26 . The MDA, which as a final product of fatty acid peroxidation was evaluated in the study. During 30 days of storage at 4°C, the MDA values in Lf nanoliposomes showed no distinct differences in the MDA values were shown in Figure 3. The result showed the Lf nanoliposomes could be stable in a period of time.

Sonication was used to form a w/o emulsion with the REV method, and to control and reduce the size of microvesicles 27 . The stability of Lf nanoliposomes was evaluated by measuring the change of particle size and release ratio after storage at 4°C for 30 days was shown in Figure 4. After 25 min sonication on nanoliposomes, the release ratio was 15.12% which was higher than the release ratio of 20 min sonication. This may be caused by the extension of ultrasonic total time, the crushed particles had been gained new energy, resulting in a change of stability . With time increased, the size of nanoliposomes became smaller. This is due to ultrasound phenomena in liquid media enhance mass transports of their constituents in a non-homogeneous fashion allowing the fast formation of vesicles 28 . After 20 min sonication on Lf nanoliposomes, the particle size did not change so much. Above all, the 20 min sonication time on Lf nanoliposomes may be fit for preparing.

When Lf nanoliposomes could be used as carriers for the oral administration of Lf, they must be able to withstand passage through the stomach and small intestine. In vitro release has been used as a very important surrogate indicator of in vivo performance.

Figure 5 showed the release ratio of Lf from nanoliposomes in simulated gastrointestinal juice. About 23% Lf was released from nanoliposomes within 4 h in the simulated gastric juice. However, because food usually remains in the stomach for more or less 4 h, the liposomal Lf could be effectively protected in the gastric juice 29 . In simulated intestinal juice, bile salts and pancreatic lipase may cause the Lf release from nanoliposomes 30 . This phenomenon may increase the release of nanoliposome.

After cells were incubated with 1, 2.5, 5, 10 mg·mL^-1 of Lf nanoliposomes for 24 h, compared with the Lf in control experiments. Figure 6 showed the MTT results demonstrated a concentration dependent uptake after exposure to Lf nanoliposomes. With the same concentration, the cell activity of Lf nanoliposomes is lower than the cell activity of Lf. It is observed that the cell activity is a statistically significantly different (p<0.01) between the concentration 5 and 10 mg/mL. The MTT results showed that Lf nanoliposomes and Lf activated in the cells in a manner of dose-effect relation and Lf nanoliposomes has a more obvious function to the cells (p<0.01). The possibility of both targeting drugs to specific tissues and cells, and facilitating their uptake and cytoplasmic delivery has rendered liposomes a versatile drug carrier system with numerous potential applications in medicine 31 .