In Vitro and in Vivo Evaluation of Lactoferrin-Conjugated Liposomes as a Novel Carrier to Improve the Brain Delivery

In this study, lactoferrin-conjugated PEGylated liposomes (PL), a potential drug carrier for brain delivery, was loaded with radioisotope complex, 99mTc labeled N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine (99mTc-BMEDA) for in vitro and in vivo evaluations. The hydrophilicity of liposomes was enhanced by PEGylation which was not an ideal brain delivery system for crossing the blood brain barrier (BBB). With the modification of a brain-targeting ligand, lactoferrin (Lf), the PEGylated liposome (PL) might become a potential brain delivery vehicle. In order to test the hypothesis in vitro and in vivo, 99mTc-BMEDA was loaded into the liposomes as a reporter with or without Lf-conjugation. The mouse brain endothelia cell line, bEnd.3 cells, was cultured to investigate the potential uptake of liposomes in vitro. The in vivo uptake by the mouse brain of the liposomes was detected by tissue biodistribution study. The results indicated that Lf-conjugated PEGylated liposome showed more than three times better uptake efficiency in vitro and two-fold higher of brain uptake in vivo than PEGlyated liposome. With the success of loading the potential Single Photon Emission Tomography (SPECT) imaging probe, 99mTc-BMEDA, Lf-PL might serve as a promising brain delivery system for loading diagnostics or therapeutics of various brain disorders.


Introduction
The blood-brain barrier (BBB) is a unique structure which is formed by the brain endothelial cells lined with cerebral capillaries, together closely with perivascular neurons, astrocytic end-foot, and pericytes [1]. The main role of the BBB is to protect the central nervous system from exogenous toxicants, but on the other aspect, this structure is the most redoubtable obstacle for drug delivery for the treatment of brain disorders [2]. One prospective modality for delivering drugs for brain tumor treatment and for transporting the nuclear imaging probe for neurodegenerative diseases diagnosis are via the receptor-mediated targeting on luminal side of the BBB [3]. Lactoferrin (Lf, MW ~80 kDa), a single-chain iron-binding glycoprotein containing 690 amino acids folded into two globular lobes, is part of transferrin (Tf) family [4] which can penetrate the BBB via receptor-mediated transcytosis [5]. Due to the less concentration of endogenous Lf than Tf, Lf was observed to exhibit better BBB uptake than Tf resulting by the less competitive inhibition [6]. As such, Lf has been used to enhance the polymer-based drug delivery system and superparamagnetic iron oxide (SPIO)-based MRI contrast agent to brain [7][8][9]. Study further showed the applicability of using Lf-conjugated polymersome as drug carrier for the treatment of glioma in rodent model [10]. Moreover, the lactoferrin receptor density had been found increase in the mesencephalon region of patient with Parkinson disease [11]. Studies also showed the accumulation of lactoferrin in the lesions from different neurodegenerative diseases such as Alzheimer's disease, Down syndrome, and Pick's disease [12,13].
Liposomes, a cell membrane-like phospholipid-bilayer structure, contain a hydrophilic phase inside the core and a lipophilic phase between the bilayers. The biocompatible liposome nanoparticles can be loaded with hydrophilic drugs in the internal water compartment and hydrophobic drugs into the membrane [14]. The size, charge and surface properties of the liposome nanoparticles can be easily changed or modified by adding new ingredients to the phospholipid mixture during fabrication. Most recently, Chen et al. [15,16] developed Lf-modified procationic liposomes as a drug carrier for brain delivery. In this study, we used the radioisotope 99m Tc as a reporter of the PEGylated liposomes (PL) by loading 99m Tc-N,N-bis(2-mercaptoethyl)-N',N'-diethylethylenediamine ( 99m Tc-BMEDA) into the liposome core. Cell-based in vitro brain delivery efficacy and in vivo tissue biodistribution of the PEGylated liposomes with or without Lf-conjugation will be discussed in detail.

Conjugation of Lactoferrin (Lf) to PEGylated liposome (PL)
The neutral PEGylated liposomes (PL) was fabricated with zeta potential as −0.60 ± 0.51 mV and particle size as 91.23 ± 17.88 nm with polydispersity index (PDI) of 0.12 ± 0.02 (n = 3). The thiolated Lf (Lf-SH) was validated by Ellman's assay with the content of the terminal sulfhydryl group (-SH) on Lf-SH at 0.0243 ± 0.001 mM (n = 3). The Lf-PL was purified by a Sepharose TM 4B gel-filtration column eluted with normal saline with the chromatogram as shown in Figure 1. The zeta potential of the final product of Lf-PL was slightly negative as −25.4 mV and its particle size measured to be 96.68 ± 17.84 nm with PDI of 0.14 ± 0.03 (n = 3).
The particle size is an important factor that affects the liposome endocytosis in the brain capillary cells. In our study, the size of the prepared PL[ 99m Tc] and Lf-PL[ 99m Tc] were between 75 and 120 nm, indicating a favorable condition for brain drug delivery by liposomes [17,18]. The particle size of Lf-PL[ 99m Tc] slightly increased about 5 nm in comparison with that of PL[ 99m Tc].

Characterization of PL[ 99m Tc] and Lf-PL[ 99m Tc]
The original concentration of phospholipid in the prepared PL solution was 16.7 ± 0.01 μmole/mL and the concentration of phospholipid in the final working Lf-PL solution was determined to be 4.17 ± 0.013 μmole/mL. From Bradford assay, the concentration of Lf in the Lf-PL solution was found to be 0.238 ± 0.0058 mg/mL. Based on these values, the number of Lf molecule in each Lf-PL particle was calculated to be 63.3 ± 2.17. Accordingly, the coupling efficiency of Lf to Lf-PL was estimated to be 74%.
The achievable radiochemical yield of 99m Tc-BMEDA was greater than 98%. For loading of the radioisotope complex, 99m Tc-BMEDA, into PL and Lf-PL liposomes, the loading yield of PL[ 99m Tc] (75%) was better than Lf-PL[ 99m Tc] (26%) that might be due to the steric obstruction of the additional Lf ligand. After purification by PD-10 column, the radiochemical purity of either PL[ 99m Tc] or Lf-PL[ 99m Tc] was around 96%.

Stability Study
The stability of PL[ 99m Tc] and Lf-PL[ 99m Tc] during incubation in normal saline at room temperature and rat plasma at 37 °C is shown in Figure 2. Either PL[ 99m Tc] or Lf-PL[ 99m Tc] showed high stability with over 87% intact after 48 h of incubation in both conditions which was suitable for in vitro and in vivo studies.

In Vitro Cell Uptake Study for Evaluation of BBB Penetration Potential
Current most common methods for modeling the BBB penetration include in situ perfusion model in animal and in vitro culture of endothelial cells. In this study, we used the rat brain endothelia cells, bEnd.3, to mimic the endogenous microvascular endothelial cells due to its expression of tight junction proteins which had been reported to a suitable BBB model for in vitro brain delivery study [19].
The in vitro cell uptake index of Lf-PL[ 99m Tc], PL[ 99m Tc], and 99m Tc-BMEDA was evaluated in a mouse brain endothelia cell line, bENd.3 cells, with incubation at 37 °C for 0.5, 1, and 2 h ( Figure 3). The incubation time did not affect the uptake level in all three groups. However, Lf-PL[ 99m Tc] showed significant higher uptake compared to PL[ 99m Tc] or 99m Tc-BMEDA. The significant higher uptake values (p < 0.0001) between Lf-PL[ 99m Tc] and PL[ 99m Tc] in bEnd.3 cells indicated that the enhanced uptake efficacy was mediated by Lf receptor. This receptor was also found high level of expression in brain endothelial capillary cells (BCECs) [20]. Hence, further examination of in vivo animal study with Lf-conjugated liposome is needed to validate more precisely the relationship with affinity between Lf and Lf receptor in the BBB.

Pharmacokinetic Study
The pharmacokinetic parameters of the clearance curves of PL[ 99m Tc] and Lf-PL[ 99m Tc] (Figure 4) are summarized in Table 1. Both of the values of the area under the curve (AUC 0→24h ) and the clearance rate (Cl) from Lf-PL[ 99m Tc] showed no significant difference to PL[ 99m Tc] with p-values of 0.89 and 0.31, respectively. From this pharmacokinetic examination, the Lf-conjugated liposomes can provide the similar long-circulation property in vivo as well as Lf-unconjugated liposomes and beneficially improve the drug delivery. For designing a better Lf-PL[ 99m Tc] to target Lf receptor, the number of Lf ligand on the liposomes should have a suitable level. Herewith, we loaded ~63 of Lf ligand on the liposome in this study which was similar to the previous report [8].   Table 2 and Figure 5. The brain uptake of Lf-PL[ 99m Tc] presented 1.47 ± 0.16 fold and 1.34 ± 0.12 fold higher than PL[ 99m Tc] at 1 and 2 h postinjecton, respectively (p < 0.05) ( Figure 5A). The brain-to-blood ratios of Lf-PL[ 99m Tc] were 3-fold and 2-fold higher than PL[ 99m Tc] for 1 and 2 h post-injection, respectively ( Figure 5B). It was noteworthy that the accumulation of Lf-PL[ 99m Tc] in the spleen was obviously higher than that of PL[ 99m Tc], with the uptake values presented as 34.12% ± 4.91%ID/g and 33.00% ± 2.64%ID/g for Lf-PL[ 99m Tc] in comparison with 10.64% ± 0.77%ID/g and 10.91% ± 0.73%ID/g for PL[ 99m Tc], at 1 and 2 h post-injection, respectively. These results indicate that the Lf conjugated PL nanoparticles can significantly enhance the brain uptake in comparison with unmodified PL nanoparticles. Previous report revealed that the Lf receptor of mouse was localized in various regions of brain [21]. As such, the increase of the uptake in the brain for the Lf anchored liposomes may be mediated by the Lf receptors existed. In our biodistribution, spleen was the other organ shown significant difference in uptake levels. The marked increase of the splenic uptake of Lf-PL[ 99m Tc] over PL[ 99m Tc] might be attributed to the existing Lf ligands on the surface of Lf-PL liposomes which are easily bound by opsonins, subsequently recognized by RES system, and terminated in the spleen [14]. Lf receptor mediated function might be also recommended for the Lf-PL[ 99m Tc] increased uptake in the spleen but would not be confirmed with the scanty information of Lf receptors in the organ to date.

Prospective Development of Lf-PL
The related mechanism of Lf receptor in mammalian and/or in some brain diseases has been completely reviewed [22]. Most recently, Lf has been intensively studied for its brain-targeting capacity. A variety of nanoparticles including polyamidoamine dendrimer [7,23,24], poly(ethyleneglycol)-poly(lactide) [8], superparamagnetic iron oxide [9] and procationic liposome [15,16] had been utilized to conjugate with Lf as a vector and constructed as a promising gene or drug delivery system into the brain. In the present study, the PEGylated liposome nanoparticles (PL) was employed and Lf conjugated PL (Lf-PL) was investigated for its brain-targeting capacity. Liposomes are a common-used biocompatible nanoparticle, beneficial for its easily encapsulating hydrophilic or hydrophobic drugs into the internal water compartment or the membrane. We have shown the significant in vitro and in vivo results suggesting Lf-PL as a potential delivery system of the brain. Our radiotracer technique has also provided the biodistribution result in an animal model for the Lf conjugated nanoparticles for the first time. Lf receptor is existing abundantly not only on the cell surface of glioblastomas [9,10,16] but also in the lesion site of neurodegenerative diseases [12,13]. It would be worthy to develop Lf-PL[ 99m Tc] as a single photon emission computed tomography (SPECT) imaging agent for the diagnosis of glioblastomas and neurodegerative diseases or the similar product by 188 Re, a 99m Tc surrogate, Lf-PL[ 188 Re] as a radio-therapeutic agent for glioblastomas treatment.

Preparation of Maleimide Functional PEGylated Liposome (PL)
Maleimide (MAL) functional PL was prepared by the lipid film hydration-extrusion method using repeated freeze-thawing as described previously by Huang et al. [25], but with some modifications. Briefly, the mixture of DSPC: cholesterol: DSPE-PEG 2000 : DSPE-PEG 3400 -MAL at the molar ratio of 3:2:0.24:0.06 was dissolved in chloroform followed by removing the solvent by rotary evaporation. The resultant dry lipid film was rehydrated in 250 mM ammonium sulfate (pH 5.0) at 60 °C. After rehydration, the PEGylated liposomes was extruded 3 times through polycarbonate membrane filters with graded pore sizes (0.4, 0.2, 0.1, 0.05, and 0.03 μm) (Costar, Cambridge, MA, USA) via a high-pressure extruder (LIPEX™, Northern Lipids Inc., Burnaby, BC, Canada). Then the extraliposomal buffer was changed to normal saline via elution through a Sephadex G-50 column (Pharmacia, Uppsala, Sweden). The size and zeta potential of the nanoparticles were measured by a dynamic laser scattering (DLS) analyzer (N4 plus; Beckman Coulter). Phospholipid concentration was measured via phosphorus assay with UV-VIS spectrophotometry at λ = 830 nm (JascoV-530, Tokyo, Japan) [26].

Preparation of Lactoferrin Modified PEGylated Liposome (Lf-PL)
The procedure for fabrication of Lf-PL is shown in Scheme 1. In experiment, Lf was thiolated and conjugated to the distal MAL functional groups surrounding on PEGylated liposomes to form the product. Lf was derivated with a terminal sulfhydryl group at the N-termus by adding Traut's Reagent as follows [27]. Traut's Reagent (14.5 mM, 75 μL) and Lf (0.4 mg, 33.2 μL) were added together and dissolved in 20 mM HEPES (150 mM MgCl 2 , 2 mM EDTA, pH 8.0). The mixture was incubated at room temperature for 1 h and then passed through a PD-10 size exclusion column for purification. The resulted thiolated lactoferrin was added with 0.5 mL of PL at pH 6.8 at a molar ratio of 2:1 and reacted at room temperature for 17 h to form a thioether bonding with the MAL functional group at the N-terminus of DSPE-PEG 3400 -MAL chain on PL. L-cysteine (1 mg, 100 μL) was added and reacted for 30 min to block the unreacted MAL functional group. N-ethylmaleimide (8 mM) was then added to stop the above reaction. The resultant Lf-PL was purified via a Sepharose™ 4B column using normal saline as the eluent. The size and zeta potential of Lf-PL were measured via DLS analysis. The phospholipid concentration of Lf-PL was measured by phosphorus assay as precedingly described. The content of the terminal sulfhydryl group on Lf was determined by Ellman's assay. The content of Lf in Lf-PL was measured by Bradford assay. The bioactivity of Lf for Lf-PL was validated by Lf ELISA assay. The number of Lf molecules conjugated on each liposome particle was further calculated based on the assumption of a 100 nm-liposome particle containing about 100,000 molecules of phospholipids [28].

Preparation of 99m Tc-BMEDA Complex, PL[ 99m Tc], and Lf-PL[ 99m Tc]
99m Tc in a form of NaTcO 4 was obtained from a 99 Mo/ 99m Tc generator by elution with normal saline. Labeling BMEDA with 99m Tc was carried out by the procedure reported by Bao et al. [29] and Chen et al. [30], but with some modifications. Briefly, BMEDA (5 mg) was pipetted into a vial. Then, 0.5 mL of 0.17 M sodium gluconate, 0.5 mL of 0.17 M acetate solution, and 120 μL of stannous chloride (10 mg/mL) were added. After flushing the solution with N 2 gas, 1.5-3.7 GBq of Na 99m TcO 4 in saline was added. The mixture was heated at 80 °C for 1 h. The radiochemical yield for 99m Tc-BMEDA was measured by ITLC using silica gel impregnated glass fiber sheet (ITLC SG). The ITLC sheet was sectioned into eight pieces and the radioactivities were measured on an auto gamma counter (2480 WIZARD2 TM , PerKinElmer, Waltham, MA, USA).
For preparation of PL[ 99m Tc] and Lf-PL[ 99m Tc], the 99m Tc-BMEDA solution was adjusted to pH 7.0 beforehand. The 99m Tc-BMEDA solution (0.74-1.85 GBq) was added to PL or Lf-PL (1 mL) and heated at 60 °C for 30 min. The 99m Tc loaded product, PL[ 99m Tc] or Lf-PL[ 99m Tc] was separated from the unreacted free 99m Tc-BMEDA using a PD-10 column eluted with normal saline. Into each tube was 0.5 mL fraction of the eluent collected.

Stability Study
The stability of PL[ 99m Tc] as well as Lf-PL[ 99m Tc] during incubation in normal saline at room temperature and in rat plasma at 37 °C was studied. At each post-incubation time point (1,4,8,20,24,30, and 48 h), a probe of 200 μL was taken and analyzed via a Poly-Prep chromography column (Bio-Red) packed with Sephadex G-50 or Sepharose 4B, by elution with normal saline. The radioactivities were measured on an auto gamma counter. The radiochemical purity was determined by the radioactivity of the separated product fractions divided by the total initial radioactivity of the sample loaded.

Cell Culture and Animals
bEnd.3 cells (BCRC 60515), the immortalized mouse brain endothelial cell line, were cultured in 90% Dulbecco's modified Eagle's medium supplemented with 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose and 10% fetal bovine serum, at 37 °C in a humidified environment with 5% CO 2 .
Normal male BALB/c mice (4~5-week old) were supplied from National Laboratory Animal Center (NLAC), Taiwan. All animal studies were approved by the Institutional Animal Care and Use Committee of National Tsing Hua University.

In Vitro Cell Uptake Study for Evaluation of BBB Penetration Potential
bEnd.3 cells [31] are of an immortalized mouse brain endothelial cell line. The cells are characterized as a model of the BBB by their rapid growth, maintenance of BBB characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions [19].

Biodistribution Study
Twelve normal male BABL/c mice (4-5 week-old) were used for the biodistribution study. Animals were divided into two groups with six mice in each group. Mice were intravenously administrated with either PL[ 99m Tc] (3.7 MBq/100 μL, 4 μmole/mL) or Lf-PL[ 99m Tc] (3.7 MBq/100 μL, 4 μmole/mL). Three mice were sacrificed at 1 and 2 h post-injection at each time point. The organs of interest including brain, blood, skin, kidney, spleen, liver, lung, and heart were dissected, rinsed in saline, blotted dry, weighed and then measured for radioactivities on an auto gamma counter. Aliquots of the injections of PL[ 99m Tc] and Lf-PL[ 99m Tc] were collected beforehand as the standard initial injected doses. The biodistribution results were expressed as percentage of injection dose per gram of organ or tissue (%ID/g).

Statistical Analysis
Data were expressed as mean ± standard deviation (SD). The unpaired t test was used for group comparisons. Values of p < 0.05 were considered significant.

Conclusions
The lactoferrin (Lf) conjugated PEGylated liposome (PL) was constructed in this study as a novel brain delivery system with evaluation of its in vitro and in vivo delivery properties. To evaluate the brain delivery properties of the Lf-PL, 99m Tc was incorporated into it as a radiotracer. The uptake of Lf-PL[ 99m Tc] by bEnd.3 cells was significantly higher than that of Lf-unconjugated PL[ 99m Tc]. The uptake of Lf-PL[ 99m Tc] in the brain was higher than liposome without the Lf targeting ligand in the animal study. Via probing by the radionuclide ( 99m Tc) in place of a commonly used fluorescin, our study showed the comparable results to the Lf conjugated nanoparticles in vitro. With the higher lactoferrin receptor density in various diseases, the conjugation strategy with SPECT radionuclide, 99m Tc, demonstrated the potential of imaging probe development for the diagnosis of neurodegenerative diseases and gliobastoma in the future.