Delivery of fluorescent-labeled cyclodextrin by liposomes: role of transferrin modification and phosphatidylcholine composition.

Drug-in-CD-in-liposome (DCL) systems which encapsulate the drug/CD inclusion complexes into inner aqueous phase of liposomes have been applied as a novel strategy to improve efficacy of lipophilic antitumor drugs. The aim of this work was to assess the role of transferrin (Tf) modification and phosphatidylcholine (PC) composition on the properties of liposomes containing hydroxypropyl-β-cyclodextrin (HP-β-CD). Fluorescence dye, FITC, was conjugated with HP-β-CD to facilitate the analysis. The resulting FITC-HP-β-CD was further encapsulated into liposomes and then the liposomes were modified with Tf. The FITC-HP-β-CD-loaded liposomes with different PC compositions were compared in terms of particle size, zeta potential, FITC content, FITC-HP-β-CD leakage, phase transition temperature (Tm) and cellular uptake. The apparent partition coefficient values of different PCs were also determined. Compared to PEGylated liposomes, FITC-HP-β-CD-loaded liposomes modified with Tf had been proved to significantly increase vesicle stability and specific cellular uptake. Moreover, PC composition affected the properties of liposomes. Soybean phosphatidylcholine (SPC) liposomes modified with Tf were found to be more easily internalized into tumor cells than 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and hydrogenated soybean phosphatidylcholine (HSPC) while Tf density on the liposomal surface was similar. And the lipophilicity of SPC was found to be much higher than DPPC and HSPC. Collectively, by the optimization of PC composition, the development of DCL modified with Tf might represent a potential strategy for the antitumor application of lipophilic drugs.


Introduction
The first closed bilayer phospholipid systems, called liposomes, were described in 1965 and soon were proposed as a biodegradable, low toxic and solubilized drug delivery system (Allen & Cullis, 2013). Liposomes are microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane. They can encapsulate hydrophilic molecules in the aqueous core, whereas lipophilic drugs are entrapped in the lipid bilayer membrane. Many hundreds of drugs, including anticancer and antimicrobial agents, chelating agents, peptide hormones, enzymes, proteins, vaccines and genetic materials, have been incorporated into the aqueous or lipid phases of liposomes, with various sizes, compositions and other characteristics, to provide selective delivery to the target site for in vivo application (Chang & Yeh, 2012). Following intravenous administration, liposomes show improved pharmacokinetics and biodistribution of therapeutic agents and thus minimize toxicity by their accumulation at the target tissue Yan et al., 2012). Since the first liposomal pharmaceutical product, Doxil, received FDA approval in 1995, liposomes have been widely applied as drug carriers in clinic. Until now, several important types of liposomes, such as PEGylated liposomes (Doxil and Lipodox), temperature sensitive liposomes (ThermoDox), cationic liposomes (EndoTAG1-1) and virosomes (Expal and Inflexal V) have been investigated for clinical use (Chang & Yeh, 2012).
Many of clinically effective drugs are hydrophobic. However, to be pharmacologically active, all drugs must possess some degree of water solubility, especially for intravenous administration. One well-known method to increase solubility of lipophilic drugs is by incorporation in liposomal lipid bilayer membranes. Lipophilic drugs can distribute at high percentages in lipid bilayers, increasing their solubility in aqueous media in the form of liposomal dispersions (Nii & Ishii, 2005). Such incorporation in liposomal membranes may also increase its stability and consequently its circulation period in the blood circulation (Chen et al., 2008). However, when liposomes are administered in vivo, due to the high dilution factor in the blood, lipophilic molecules incorporated within the liposome membranes will most possibly leak out from the membranes, at a degree which depends on the log P value of the particular compound, its aqueous solubility at physiological pH and its affinity to plasma proteins (Matloob et al., 2014). Furthermore, the entrapment efficiency (EE) of a lipophilic drug by the lipid bilayer usually relies on the drug-lipid mass ratio. It is hard to get high EE in the lipid bilayers because the space offered by lipid bilayer membranes is limited, and a large amount of lipophilic drug molecules can destabilize the structural integrity of liposomal bilayers.
Another well-known method to increase solubility of lipophilic drugs is by complexation using cyclodextrins (CDs) (Zhang & Ma, 2013). CDs are hydrophilic water-soluble oligosaccharides that contain hydrophobic cavities in which they can accommodate lipophilic drugs, while their outer surface permits aqueous miscibility. However, complex formation is a dimensional fit between host cavity and guest drug molecule. No covalent bonds are formed during formation of the inclusion complex. Therefore, the binding force between CD host molecule and guest drug molecule is relatively weak. Following administration, rapid dissociation of drug/CD inclusion complexes takes place either because of dilution by the plasma and extracellular fluids or because blood components displace the included drug.
With the aim to avoid such high degrees of lipophiliccompound leakage from liposomes or CD inclusion complexes in vivo, drug-in-CD-in-liposome (DCL) systems to encapsulate the drug/CD inclusion complexes into inner aqueous phase of liposomes was proposed (McCormack & Gregoriadis, 1994). DCL combines the advantages offered by these two carriers. CDs increase drug solubility and preserving the bilayer integrity from the perturbation due to the drug insertion into the liposomal membrane, while liposomes prevent CD complex dissociation due to dilution into the plasma, favoring a proper targeted delivery of lipophilic drugs, especially the lipophilic antitumor drugs (Chen et al., 2014;Gharib et al., 2015;McCormack & Gregoriadis, 1996). Among various CDs, hydroxypropylb-cyclodextrin (HP-b-CD) is usually applied for the preparation of DCL formulations (Chen et al., 2014).
The effectiveness of DCL for targeted delivery of antitumor drugs has been investigated but remains a question. Sometimes the efficacy of drug-loaded DCL was even lower than that of free drug. For curcumin, the median effective dose (EC 50 ) of the formulations on colon cancer cells were calculated to be 0.96 mM for curcumin-entrapped liposomes, 1.9 mM for curcumin, 2.95 mM for curcumin b-CD complexes and 3.25 mM for curcumin-loaded DCL. The EC 50 of the formulations on lung cancer cells followed the same pattern, being 0.90 mM for curcumin-entrapped liposomes, 1.5 mM for curcumin, 2.4 mM for curcumin b-CD complexes and 2.9 mM for curcumin-loaded DCL (Rahman et al., 2012). Moreover, for honokiol, the results of cytotoxicity revealed that the bioactivity of honokiol-in-HP-b-CD-in liposome was even a bit weaker compared with free honokiol (Wang et al., 2011).
To improve the targeted efficiency and antitumor efficacy, it is recognized that a means of increasing the selectivity of the interaction of liposomes with tumor cells was desirable. This interaction is expected to trigger receptor-medicated endocytosis of the liposomes and its cargo into the desired cellular target. After the development of long-circulating (PEGylated) liposomes, new coupling methods were developed to attach the specific ligands to the terminus of PEG molecules engrafted to the liposome surface. By using the ligand coupled to the surface of liposomes, the liposomal antitumor drugs can be actively taken up by receptormedicated endocytosis or electrostatic uptake.
The over-expression of transferrin receptor (TfR) on tumor cells makes this glycoprotein an attractive and effective target for site-specific delivery of antitumor drugs into proliferating tumor cells. In our previous studies, Tf was coupled to the surface of 9-nitrocamptothecin (9-NC)-loaded DCL to achieve active targeting to tumor cells. The antitumor efficacy was thus significantly enhanced in vitro and in vivo (Chen et al., 2015).
Besides ligand modification, it is also well known that the phosphatidylcholine (PC) used to prepare DCL has significant effect on the targeted properties of resulting liposomes (Zhou et al., 2012). PC is an important consideration with liposome stability and targeted ability, and the state of liposomes at physiological temperature can be altered by inclusion of PC with different T m s (Anderson & Omri, 2004;Dadashzadeh et al., 2010). Different PCs, such as soybean phosphatidylcholine (SPC) (Yuan et al., 2014), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) (Doi et al., 2008) and hydrogenated soybean phosphatidylcholine (HSPC) (Gandhi et al., 2015) were applied to prepare Tf-modified liposomes. However, the effect of PC composition on the selective uptake by tumor cells is still unclear.
In DCL, lipophilic drugs were encapsulated into the hydrophobic cavities of CD molecules and the drug-CD inclusion complexes were further entrapped into the liposomal inner aqueous phase. In the present paper, HP-b-CD was conjugated with fluorescent dye to facilitate its analysis. Then the fluorescent-labeled HP-b-CD was further encapsulated into liposomes and Tf was conjugated to the liposomal surface by the ''post-insertion'' technique. The effect of PC composition on the targeted delivery of fluorescent-labeled HP-b-CD by Tf-modified liposomes was intensively investigated in vitro.

Preparation of fluorescent-labeled HP--CD
For labeling HP-b-CD with fluorescence dye (Chen et al., 2015;Cheng et al., 2012), 30 mg of FITC was dissolved in 1 mL of DMSO, and then introduced dropwise into 10% HPb-CD solution. The mixture was magnetically stirred at room temperature for 24 h in the dark. The resultant fluorescentlabeled HP-b-CD (FITC-HP-b-CD) was precipitated with 100 mL of ethanol and the obtained sediment was re-dissolved in 10 mL of deionized water and dialyzed with a dialysis bag (2 kDa cut-off) against deionized water in the dark for 72 h to remove any free FITC. The fluorescence of the sample was measured with a LS55 Fluorescence Spectrophotometer (PerkinElmer, Waltham, MA) using 495 nm excitation wavelength and 525 nm emission wavelengths.

Fourier transform infrared spectroscopy analysis
FITC, HP-b-CD and FITC-HP-b-CD were pressed into thin KBr tablets. The tablets were scanned on a Tensor 37 Spectrometer (Bruker Corp., Karlsruhe, Germany) from 400 to 4000 cm À1 .

Preparation of liposomes
Liposomes containing FITC-HP-b-CD were prepared using ethanol injection method which is easy to scale up (Chen et al., 2015). Briefly, phospholipids (PL, composed of SPC:DSPE-PEG2000-COOH ¼ 20:1, molar ratio) were dissolved in 5 mL of anhydrous ethanol at the lipid concentration of 50 mM. FITC-HP-b-CD was weighed and dissolved in 6 mL water to obtain the concentration of 20%. The lipid solution was then added to FITC-HP-b-CD solution and the mixture was magnetically stirred at 60 C. The ethanol was evaporated to no odor. Then the liposomes were ultrasonically treated for 10 min, using a JY92-II probe ultrasonicator (Xinzhi Biotechnology Co., Ltd, Ningbo, China) equipped with a tapered microtip. The resultant unilamellar liposomes were filtered through a 0.22 mm micropore filter to remove the titanium fragments. Non-entrapped FITC-HP-b-CD was removed from liposomes by four consecutive dialysis (10 kDa cut-off) exchanges (0.5 h at a time) against 30 vols. of 5% glucose solution under room temperature.

Transferrin conjugation
Transferrin (Tf) was coupled to terminal carboxyl group of PEG according to the previously described methodology (Li et al., 2009) with minor modifications. The pH value of liposome suspension was adjusted to 5.2 with citric acid. Then both EDC (0.5 mol/L) and NHS (0.5 mol/L) were added at the ratio of 360 mL/10 mmoL PL. After stirring under room temperature for 10 min, excess EDC and NHS were removed by dialysis. After adjusting to pH 7.5 with 1 M NaOH, Tf was added at the ratio of 125 mg Tf/mmoL PL and gently stirred for 8 h at 4 C. Unbound protein was removed by four consecutive dialysis (300 kDa cut-off) exchanges (0.5 h at a time) against 100 vols. of 5% glucose solution under room temperature. Then the Tf-modified liposomes containing FITC-HP-b-CD (Tf-C-L-s) were lyophilized.
Tf-modified liposomes containing FITC-HP-b-CD composed of DPPC or HSPC (Tf-C-L-d or Tf-C-L-h) were also prepared using DPPC or HSPC instead of SPC, respectively. PEGylated (P-C-L-s) was prepared using DSPE-PEG2000 instead of DSPE-PEG2000-COOH according to the preparation of SPC liposomes described above.

Characterization of liposomes modified with Tf
Tf-binding efficiency The amount of Tf conjugated to the liposomes containing FITC-HP-b-CD was quantified by a BCA protein assay as previously described (Li et al., 2009;Wei et al., 2012). In brief, 100 mL of liposome was mixed with 400 mL methanol. The mixture was vortexed and centrifuged (10 000 rpm, 10 s). 200 mL of chloroform was added and the mixture was vortexed and centrifuged (10 000 rpm, 10 s) again. For phase separation, 300 mL of water was added and the sample was vortexed and centrifuged for 1 min at 10 000 rpm. The upper phase was carefully removed and discarded. 300 mL of methanol was added to the interphase between chloroform and the precipitated protein. After vigorous mixing and centrifugation (2 min at 10 000 rpm), the supernatant was removed and the protein pellet was dried under a steam of nitrogen gas. The pellet was subsequently dissolved in 100 mL PBS (pH7.4) and the concentration of Tf was determined by a BCA protein assay using pure bovine serum albumin as a standard. The coupling efficiency of Tf was calculated as mg Tf/mmol PL. The PL concentration of liposomes was determined by the Stewart assay (Stewart, 1980).

Liposome size and zeta potential
The particle size, distribution and zeta potential were evaluated in triplicate by dynamic light scattering using a DB-525 Zeta Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY) at 25 C.

FITC content
Liposomes were accurately weighed, solubilized with ethanol-isopropanol (1:4, v/v) and diluted with methanol. Then the FITC content was measured as described in the section ''Preparation of fluorescent-labeled HP-b-CD (FITC-HP-b-CD)''.

DSC analysis
Differential scanning calorimetry (DSC) was conducted to determine the T m of liposomes using a Diamond DSC apparatus (PerkinElmer; Waltham, MA). According to the reported method (Chen et al., 2013), the liposome suspension was transferred into aluminum pans, which were subsequently sealed and analyzed. The samples were scanned from 30 to 60 C at 10 C/min.

Leakage experiments in vitro
The in vitro leakage assays were carried out according to the method described previously (Chen et al., 2010) with minor modification. Briefly, liposomes with different PC compositions were dissolved in isotonic PBS (pH 7.4) to obtain the FITC concentration of 1000 ng/mL. The resulting suspension was incubated at 37 C. At various time points, aliquots were withdrawn for the determination of FITC-HP-b-CD retention. The samples collected at specified time points were passed through a Sephadex G-50 column (1 Â 27 cm) to separate the liposomal FITC-HP-b-CD from the leaked one. The concentration of FITC-HP-b-CD was assayed as described above. The drug retention percentage following incubation was calculated by dividing the liposomal FITC-HP-b-CD content at indicated time points following incubation by the initial liposomal FITC-HP-b-CD content.

Interaction of Tf with HP--CD
Fluorescent spectroscopy is the most convenient method to investigate the interaction of albumin with other compounds. According to the previously described methodology (Zhang et al., 2015), fluorescence quenching spectra were obtained in a 1-cm path length quartz cell using an excitation wavelength of 225 nm and an emission wavelength of 280-380 nm. Slit widths (10 nm) and scan speed (12 000 nm/min) were kept constant within each data set. Excitation and emission bandwidths were 5 nm. The range of synchronous scanning was 225-400 nm and 245-300 nm, with the constant wavelength intervals of 15 and 60 nm, respectively. The fluorescence spectra of Tf with the absence and presence of HP-b-CD in water were measured.

Cell lines
The cell lines, including human ovarian tumor cells (A2780), mice sarcoma 180 cells (S180) and normal human hepatocyte cells (L02) were purchased from cell bank of Shanghai Institute of Cell Biology (Shanghai, China), maintained in RPMI 1640 culture medium plus 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, 75 U/mL streptomycin, in a 37 C incubator supplied with 95% room air and 5% CO 2 . After 60-80% confluency, the cells were trypsinized with 0.25% trypsin (AMRESCO, dissolved in PBS, pH 7.4), counted and placed down at needed density for treatment. Experiments were performed on cells in the logarithmic phase of growth.

Flow cytometry analysis
Cells (A2780, S180 or L02) were seeded at a density of 1 Â 10 6 cells/well in 6-well plates and incubated at 37 C for 24 h to allow cell attachment. After 24 h, the medium was replaced with liposomes (Tf-C-L-s, Tf-C-L-d, Tf-C-L-h or P-C-L-s) or free FITC-HP-b-CD (the concentration of FITC was 100 ng/mL). After 1 h incubation at 37 C, the cells were washed three times with ice cold PBS (pH 7.4). The cells were then harvested by trypsinization and centrifuged at 1000 rpm for 5 min and resuspended in 500 mL PBS medium and examined by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA).

Confocal microscopy analysis
Following incubation of cells on glass-bottomed dishes containing culture medium at 37 C for 24 h, free FITC-HPb-CD, Tf-FC-L or P-C-L formulations were added to each dish to obtain the final FITC concentration of 100 ng/mL and incubated for another 1 h at 37 C. The medium was then removed and cells were washed with ice cold PBS followed by fixing with 4% paraformaldehyde for 10 min. After fixing, cells were treated with Hoechst 33258 for 5 min. The fluorescent images of the cells were analyzed using a TCS SP5 confocal microscope (Leica, Solms, Germany) at excitation and emission wavelengths of 488 and 520 nm, respectively.

Determination of apparent partition coefficient of PC
The apparent partition coefficients of PC (SPC, DPPC or HSPC) were measured by shake-flask method. PC was dissolved n-octanol (presaturated with water) at the concentration of 10 mg/mL. The resulted solution was mixed with water (presaturated with n-octanol) in equal volume, shaken and incubated in a water bath at 37 C for 4 h. After centrifugation, the concentrations of PC in the aqueous phase and in the octanol phase were determined by HPLC following dilution with methanol. Then the apparent partition coefficient was determined by the ratio of the concentration in octanol phase to that in the aqueous phase.
An Ultimate3000 system (ThermoFisher Scientific, Waltham, MA) equipped with quaternary pump, mobile phase degasser, temperature controlled autosampler and column thermostat was used for HPLC analysis. The separation was carried out on an AcclaimTM120 C18 column (5 mm, 250 mm Â 4.6 mm) at a column temperature of 35 C. Phosphatidylcholines were eluted using binary linear gradients starting from a mixture of 10% A and 90% B in 10 min followed by a 40-min plateau at 100% B and return to 10% A and 90% B in 5 min, where A is 4 mM ammonium acetate in water (pH 4.0) and B is 4 mM ammonium acetate in methanol. The flow rate of the mobile phase was set to 1.0 mL/min. Sample injection volume of 20 mL was used. The separation of PCs was detected using a 2000ES ELSD detector. The ELSD conditions were as follows: the drift tube temperature was set at 45 C, the high-purity nitrogen gas flow rate was set at 1.9 mL/min, and the gain was set to 4.

Characterization of FITC-HP-b-CD
The content of FITC in FITC-HP-b-CD was measured to be 1.18 ± 0.02% (n ¼ 3). The FT-IR spectrograms of FITC and HP-b-CD were compared with that of FITC-HP-b-CD (Figure 1). FITC has the benzene ring while HP-b-CD has no benzene structure. In the planar structure of benzene, each carbon is bonded to two other carbons and the carbon-carbon bonds are alike for all six carbons. There are aromatic CC stretch bands (for the carbon-carbon bonds in the aromatic ring) at 1593 cm À1 . Therefore, the presence of band at 1593 cm À1 in the spectrogram of FITC-HP-b-CD indicated the successful conjugation of FITC with HP-b-CD.

Characterization of Tf-conjugated liposomes
The properties of FITC-HP-b-CD-loaded liposomes are shown in Table 1. With Tf attachment, the particle size of liposomes was obviously increased. The polydispersity index was lower than 0.3, indicating that liposome populations were homogeneous in size. Zeta potential of P-C-L-s showed negative values, and it was demonstrated that the negatively charged DSPE-PEG2000 was successfully inserted into the outer monolayer of the vesicles. Tf-C-L with different PC composition all exhibited more negative zeta potential than P-C-L-s, indicating that negatively charged Tf might have conjugated to the liposomal surface. The molecular mass of Tf has been determined to be around 74 kDa and isoelectric point about 5.1 (Dietrich et al., 2010). Therefore, under the neutral condition, Tf was negatively charged. The amount of Tf (assessed by BCA assay) in correlation to the amount of total PL was calculated to be in the range of 35.248-41.724 mg Tf/mmol PL, indicating the success of the post-insertion of the Tf ligand to the liposomal surface. In addition, PC composition seemed to have little effect on the Tf conjugation efficiency of liposomes.
The phase transition temperatures (T m s) of Tf-conjugated liposomes containing FITC-HP-b-CD were determined by DSC. The DSC thermographs are shown in Figure 2. It can be observed that T m s of Tf-C-L-d and Tf-C-L-h showed a phase transition at 42.85 and 54.20 C, respectively. For Tf-C-L-s and P-C-L-s, no phase transition was found in the range 10-80 C.
The in vitro leakage profiles of FITC-HP-b-CD from different liposomes are shown in Figure 3. It was obvious that the leakage of liposomes was markedly affected by both Tf modification and PC composition. It could also be concluded that Tf conjugation to the surface of DCL might significantly enhance the stability of vesicles since more FITC-HP-b-CD were retained in the Tf-C-L-s compared with P-C-L-s. In addition, liposomes obtained from PC with a high T m were more stable than liposomes prepared with a low T m .

Interaction of Tf with HP--CD
Tf is a protein with intrinsic fluorescence resulting from three types of amino acid residues (i.e. Trp, Tyr and phenylalanine) (Zhang et al., 2015). The fluorescent spectrum of Tf can be influenced by the changes in the micro-environment of these amino acid residues. As shown in Figure 4(A), the fluorescent   The results demonstrated that one or more fluorescenceemitting amino acid residues of Tf are involved in the interaction with HP-b-CD. Consequently, Tf modification might result in the decrease of HP-b-CD leakage and the increase of stability of liposomes. More details of which residues are involved in the interaction can be provided by the synchronous fluorescence spectra, in which 4l of 15 and 60 nm indicate the specific microenvironment change of Tyr and Trp residues, respectively. As shown in Figure 4(B) and (C), it is interesting to find that when 4l is set at 15 nm, the addition of HP-b-CD has little effect on the spectra of Tf, while the fluorescence intensity of Tf becomes lower at higher HP-b-CD concentrations when 4 is 60 nm. These results suggest that Tyr residues may not interact with HP-b-CD and the fluorescence spectra changes of Tf may only be attributed to the interaction of Trp residues with HP-b-CD.
It was postulated that the Tf fluorescence quenching was a static quenching process (Zhang et al., 2015). The association constants (Ka) can be calculated from the regression curve based on the following equation (Lehrer, 1971) where F 0 and F are the relative fluorescence intensities of Tf in the absence and presence of the HPb-CD, respectively, Ka is the binding constant, n is the number of binding sites, Q is the concentration of HP-b-CD. The binding constant (Ka ¼ 19.41 M À1 ) of the HP-b-CD was found to be higher than the published values of the drug molecules, such as doxorubicin hydrochloride (Zhang et al., 2011).

Flow cytometry analysis
Flow cytometry was used to verify the effect of modification with Tf. As shown in Figure 5, for two tumor cell lines (A2780 and S180) with over-expressed TfR (Hong et al., 2009), the cellular of FITC-HP-b-CD level for Tf-C-L-s was significantly higher than that of P-C-L-s, indicating the successful modification of Tf. But for the normal cell (L02), the differences between Tf-C-L and P-C-L were slight.
As shown in Figure 5, it was also obvious that the uptake of Tf-modified liposomes varied with PC composition. And the TfR + cells treated with Tf-C-L-s showed higher uptake compared to those with Tf-C-L-h and Tf-C-L-d. The decreasing trend of cell uptake was Tf-C-L-s4Tf-C-L-h4Tf-C-L-d.

Confocal microscopy analysis
The extent of cellular uptake of the different FITC-HP-b-CDloaded liposomes with TfR+ (A2780 and S180) cells was further evaluated by confocal microscopy. As shown in Figure 6, after exposure to TfR + cells for 4 h, Tf-C-L-s was efficiently internalized by the cells and the level of cellular uptake was significantly higher than that of non-targeted liposomes (P-C-L-s). It was suggested that Tf could enhance the cell binding and internalization of Tf-C-L-s by a specific receptor-endocytosis mechanism in TfR overexpressed cells, and also demonstrated the success of conjugation of Tf with the liposomes. Furthermore, PC composition of Tf-modified liposomes influenced the fluorescence intensity of FITC-HPb-CD in TfR + cells. The cellular uptake of Tf-C-L-s was enhanced compared with that of Tf-C-L-h and Tf-C-L-d.

Determination of apparent partition coefficient of PC
The properties of PCs, namely SPC, DPPC and HSPC, are shown in Table 2. The decreasing trend of the log P values was SPC4HSPC4DPPC.

Discussion
Drug-in-CD-in-liposome approaches have been proved to improve the stability of liposomal vesicles in most cases. However, CDs, particularly the methylated derivatives, are known to remove lipid components from liposomal membranes by forming inclusion complexes with them, especially cholesterol (Beseničar et al., 2008). This could destabilize the bilayers to some extent, enabling partial or complete leakage of drug content from liposomes. Therefore, in the present paper, cholesterol was not included in the lipid composition. Moreover, it had been demonstrated that the lipophilicity of CDs rather than the size of CD cavity, seems to be the most important influencing factor on the destabilization of liposomes (Hatzi et al., 2007). So HP-b-CD, a b-CD derivative with high hydrophilicity, is usually applied for the preparation of DCL (Chen et al., 2015;Mendonça et al., 2012;Zhou et al., 2012). Additionally, in contrast to the disaccharides, HPb-CD had been demonstrated to stabilize the liposomal membranes during the drying process of both spray drying and freeze-drying of the PEGylated liposomes (van den Hoven et al., 2012). The large number of hydrogen donors and acceptors in the structure of HP-b-CD likely attributes to the efficiency of replacement of the water molecules at the liposomal surface during drying of the formulation, thereby protecting the liposomal membranes from damage and keeping its structure intact.
The TfR is up-regulated on the surface of many cancer types and is internalized efficiently, thus providing a useful means of targeting antitumor drugs to these cells (Deilarduya & Düzgünes, 2013). Consequently, the Tf-TfR serves as a potential target for anticancer therapy. By modifying the surface of PEGylated DCL with Tf, the resulting liposomes targeted at these receptors may be considered to be an effective carrier for tumor-specific targeting (Chen et al., 2015). Furthermore, since the coated albumin corona has been found to inhibit the release of the nanoparticles and enhance the biostability in organs (Peng et al., 2015), the Tf modification might also help to enhance the vesicle stability of DCL. It should be noted that vesicle stability is the key factor affecting the therapeutic efficacy of DCL (Chen et al., 2014). In the present paper, interaction between Tf and HP-b-CD was investigated by fluorescence analysis. Such interaction renders the Tf corona as a chemical barrier controlling the release of HP-b-CD. This may be the reason for the slower leakage of FITC-HP-b-CD after Tf modification of liposomes.
It is well known that the phosphatidylcholine (PC) used to prepare targeted liposomes has significant effect on the properties of resulting liposomes. PC is an important consideration with liposome stability, and the state of liposomes at physiological temperature can be altered by inclusion of PC with different T m s. The T m s of HSPC and DPPC were determined to be 42 and 53 C, respectively (Chen et al., 2013). Consequently, the T m s of Tf-C-L-d and Tf-C-L-h were measured to be 42.85 and 54.20 C, respectively. It was obvious that the T m of PC was the key factor affecting the T m of liposomes. In addition, the T m of SPC was much lower than 0 C, so no phase transition was found in the range of 10-80 C.
It had been revealed that drug release rate increased with the decrease of T m of PCs. Liposomes with higher T m appear to be more stable in PBS, indicating the increase of T m is directly proportional to vesicle stability (Anderson & Omri, 2004). PC composition is also shown to affect cytotoxicity of DCL. Cytotoxicity of gefitinib DCLs composed of egg phosphatidylcholine (EPC, T m 50 C) or HSPC was compared in A549 tumor cells. The IC 50 values for gefitinib EPC and HSPC DCL were determined to be 149 ± 28 and 205 ± 12 mM, respectively (Zhou et al., 2012). In the present paper, cellular uptake of Tf-modified liposomes with different PC composition was compared. The T m s of liposomes were verified by DSC analysis. It was found that the cellular uptake of FITC-HP-b-CD could be significantly enhanced by liposomal encapsulation and Tf modification. And SPC liposomes modified with Tf seemed to be more easily internalized into tumor cells than DPPC and HSPC in spite that Tf density on the liposomal surface was similar.
Two mechanisms, endocytosis or fusion, were included in the process of cellular uptake of liposomes and lipid composition was proved to affect the rate of liposomes uptake and internalization by cells (Dini et al., 1998). In the present paper, the log P values of SPC, DPPC and HSPC were measured and compared, and the lipophilicity of SPC was found to be much higher than DPPC and HSPC. It is thus possible that, after Tf-TfR conjugation, PC composition plays a key role on the cellular endocytosis or fusion process of the Tf-modified liposomes, especially the latter. The PC with higher lipophilicity might facilitate the liposomes to pass through the lipid cell membrane.
The lipophilicity of antitumor drugs had been proved to greatly affect cellular uptake and optimal lipophilicity may be a critical factor in the design of analogs with high antitumor activity (McKeage et al., 2000). Similarly, the lipophilicity of active targeted liposomes should also be optimized by changing the PC composition.

Conclusion
FITC, a fluorescence dye, was successfully conjugated with HP-b-CD molecules to facilitate analysis. Then the FITC-HP-b-CD was encapsulated into liposomes and Tf modification was carried out on the liposomal surface. Compared to PEGylated liposomes, FITC-HP-b-CD-loaded liposomes modified with Tf had been proved to significantly increase vesicle stability and specific cellular uptake. Furthermore, the effect of PC composition on the properties of Tf-modified liposomes containing FITC-HP-b-CD was investigated. And SPC which is more lipophilic than DPPC and HSPC was found to be more easily internalized into tumor cells in spite that Tf density on the liposomal surface was similar. In summary, by the optimization of PC composition, the development of DCL modified with Tf might represent a potential strategy for the antitumor application of lipophilic drugs.

Declaration of interest
The authors report no declarations of interest. The authors alone are responsible for the content and writing of this article.