Selenium-functionalized liposomes for systemic delivery of doxorubicin with enhanced pharmacokinetics and anticancer effect

Accepted Manuscript Selenium-functionalized liposomes for systemic delivery of doxorubicin with enhanced pharmacokinetics and anticancer effect Qian Xie, Wenji Deng, Xue Yuan, Huan Wang, Zhiguo Ma, Baojian Wu, Xingwang Zhang PII: DOI: Reference:

S0939-6411(17)30468-X https://doi.org/10.1016/j.ejpb.2017.10.010 EJPB 12615

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Revised Date: Accepted Date:

13 April 2017 26 July 2017 11 October 2017

Please cite this article as: Q. Xie, W. Deng, X. Yuan, H. Wang, Z. Ma, B. Wu, X. Zhang, Selenium-functionalized liposomes for systemic delivery of doxorubicin with enhanced pharmacokinetics and anticancer effect, European Journal of Pharmaceutics and Biopharmaceutics (2017), doi: https://doi.org/10.1016/j.ejpb.2017.10.010

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Selenium-functionalized liposomes for systemic delivery of doxorubicin with enhanced pharmacokinetics and anticancer effect

Qian Xie, Wenji Deng, Xue Yuan, Huan Wang, Zhiguo Ma, Baojian Wu*, Xingwang Zhang*

Division of Pharmaceutics, College of Pharmacy, Jinan University, 601 West Huangpu Avenue, Guangzhou 510632, China.

*Corresponding authors at: College of Pharmacy, Jinan University, 601 West Huangpu Avenue, Guangzhou 510632, China. Tel.: +86 20 85220084; E-mail: [email protected] (X. Zhang); [email protected] (B. Wu)

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ABSTRACT: Liposomes have shown to be an excellent drug delivery system, but the short in vivo fate discourages their popularity. This work aimed to develop selenium-functionalized liposomes (SeLPs) for doxorubicin (Dox) delivery to prolong the systemic circulation of liposomes by in situ selenium coating and enhance the anticancer effect via the synergy between Dox and selenium. Dox-loaded SeLPs (Dox-SeLPs) were prepared by film hydration/active loading/in situ reduction technique and characterized by particle size, entrapment efficiency and morphology. The resulting Dox-SeLPs were 127 nm around in particle size (uncoated liposomes 107 nm) and were spherical in morphology. It was shown that Dox-SeLPs possessed a sustained release effect for Dox and could increase the cellular uptake of Dox compared with Dox-loaded liposomes (Dox-LPs). The accumulative Dox release from Dox-SeLPs was 46.5% and it was 64.9% for Dox-LPs within 84 h. Moreover, Dox-SeLPs exhibited slower drug release in the fetal bovine serum. Trafficking pathway study revealed that clathrin-mediated endocytosis and macropinocytosis were involved in the cellular uptake process of Dox-SeLPs. The in vitro cytotoxicity and apoptosis test indicated that Dox-SeLPs had higher cytotoxicity than that of free Dox and Dox-LPs. Dox-SeLPs showed a IC50 of 0.92 ± 0.16 μg/mL on A549 cells, far lower than that of free Dox (4.40 ± 0.58 μg/mL) and Dox-LPs (5.68 ± 0.73 μg/mL). Dox-SeLPs significantly improved the pharmacokinetic property and enhanced the antitumor efficacy of Dox in tumor-bearing mice. In conclusion, SeLPs exhibit good sustained release for Dox and have synergic anticancer effect with Dox, which may be promising as drug delivery vehicle.

Keywords: Doxorubicin; liposomes; selenium; anticancer; synergic effect. 2

Introduction Cancer remains one of the biggest threats to human life. Although great efforts have been attempted to overcome cancer over the past decades, it is still challenging to defeat the disease [1]. Chemotherapy is the commonly applied approach for cancer management, but the toxic and side effects make chemotherapy become troublesome [2]. Depending on excellent ability in improving the pharmacokinetics and pharmacodynamics, nanocarriers are attracting enormous attention in drug delivery [3], such as nanoemulsions [4], micelles [5], liposomes [6], and sorts of nanoparticles [7, 8]. Among various vehicles, liposomes that have a similar nature to the biological membrane represent the most acceptable modality of parenteral drug delivery systems by virtue of high biocompatibility and safety. Liposomal drug products have been successfully marketed and applied to the clinical practice. Liposomes can passively accumulate in the cancerous tissue via the enhanced permeability and retention (EPR) effect [9], but they can also be quickly eliminated due to complement binding and phagocytosis by the reticuloendothelial system (RES) [10, 11]. The short circulation half-life of common liposomes depresses the EPR effect. Although stealth liposomes can prolong the body residence time, lack of pharmacological activity as a result of PEGylation has been reported for liposomal Dox [12]. Therefore, great effort is still required to improve the in vivo biofate of liposomes. Selenium is an essential micronutrient for human beings that functions as cofactor for reduction of antioxidant enzymes (e.g., glutathione peroxidases and thioredoxin reductase) [13]. Selenium plays an important role in body growth and life maintenance. The

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physiological functions of selenium in cancer chemoprevention and chemotherapy have been confirmed [14, 15]. Selenium nanoparticles as anticancer agent and drug delivery carrier have been tentatively developed and evaluated [16, 17]. They can be internalized into cancer cells through endocytosis because of higher density, thus inducing cell death by triggering mitochondria-mediated apoptosis [18]. However, the combination of nano selenium and liposomal doxorubicin (Dox) for cancer intervention has not been investigated. In this study, we developed selenium-plated liposomes (SeLPs) for systemic delivery of Dox to improve the pharmacokinetics and potentiate the anticancer effect of Dox through a synergy with selenium. Dox-loaded SeLPs (Dox-SeLPs) were prepared by electrostatic complexation/in situ reduction technique based on liposomes. We characterized Dox-SeLPs with particle size, drug entrapment and morphology and performed the in vitro release and cellular uptake experiments. The cellular trafficking mechanism, cytotoxicity and apoptotic effect on Dox-SeLPs were investigated in A549 cells. Finally, the pharmacokinetics and antitumor efficacy of Dox-SeLPs were evaluated in rats and A549 tumor-bearing mice, respectively.

Materials and methods Materials Doxorubicin hydrochloride was purchased from Nanjing Jingzhu Bio-technology Co., Ltd. (Nanjing, China). Lecithin S100 was the product of Lipoid (Ludwigshafen, Germany), and 1,2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP) was from A.V.T. Corp., (Shanghai, China). Cholesterol was obtained from Sinopharm Chemical Reagent (Beijing, 4

China). Reduced glutathione (GSH) and sodium selenite (Na2SeO3) were provided by Aladdin Reagent (Shanghai, China). Chlorpromazine, simvastatin, Filipin, and ethylisopropyl amiloride (EIPA) were purchased from Sigma–Aldrich (Shanghai, China). Sucrose was kindly gifted by Guangzhou Standard Pharma Ltd. (Guangzhou, China). MTT and Annexin V-FITC apoptosis detection kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). All other chemicals were of analytical grade and used as received. Preparation of Dox-SeLPs Dox-loaded liposomes (Dox-LPs) were prepared by the ammonium sulfate gradient method [19]. Briefly, lecithin S100, DOTAP and cholesterol were dissolved in 3 mL of chloroform and evaporated to form a thin lipid film under vacuum at 30°C. Then, the lipid film was hydrated in 10 mL of ammonium sulfate solution (250 mM) at 40°C for 30 min. The resulting vesicles were further homogenized to form fine liposomes using microfluidizer (Nano DeBee, MA, USA) for 5 circles at 12000 psi. Afterwards, liposomes were dialyzed against 0.9% NaCl solution at 4°C to make a pH gradient between inside and outside liposomes. The dialysis medium was exchanged every 2 h for 6 times. After that, Dox-LPs were prepared through mixing the blank liposomes with Dox solution (10 mg/mL) at a drug/lipid ratio of 1 : 6, which was performed in a water bath of 40°C for 30 min. To prepare Dox-SeLPs, an appropriate amount of Na2SeO3 was introduced into Dox-LPs followed by addition of reduced GSH. The stoichiometric ratio of Na2SeO3 to GSH was 1 : 4 [20]. The reaction was maintained for 12 h at 4°C in the system buffered at pH 7.4 and then dialyzed against 0.9% NaCl to remove the excess reactants and unloaded Dox. The amount of

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Na2SeO3 used in the system was optimized for obtaining a preferred formulation. Characterization of Dox-SeLPs Dox-SeLPs were characterized through particle size, ζ potential, morphology, entrapment efficiency (EE) and drug loading (DL). Particle size and ζ potential of liposomes were determined by Zetasizer Nano ZS (Malvern, UK). Morphology of liposomes was inspected by transmission electron microscopy (TEM) (H-7650, Hitachi, Japan). The above samples were prepared by diluting them with appropriate amount of deionized water. TEM micrographs were acquired at the acceleration voltage of 100 kV. The centrifugal filtration technique was employed to determine DL and EE. Free Dox was separated from liposomes at 8,000 g for 10 min by a centrifugal filter device (Amicon®, MWCO 50K, Millipore, USA) [21]. Dox in the filtrate was quantified by HPLC established below. DL and EE were calculated according to the following equations: EE (%)  (1 

Mtot  Mfre Mfre  100 )  100 ; DL(%)  ( Mtot  Mexc) Mtot

where Mfre, Mtot and Mexc denote the amount of free Dox, total Dox and excipients used in the formulation, respectively. Assay of Dox and selenium Doxorubicin in the samples was analyzed by a Dionex HPLC system (Ultimate 3000, Thermo Scientific, USA). The sample was eluted against a C18 column (Syncronis C18, 5μm, 4.6 × 250 mm) guarded with a precolumn at 40ºC and monitored at 233 nm. The injection volume was 10 μL. The mobile phase consisted of 60% methanol and 40% phosphoric acid/water

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solution (0.05%, v/v) pumped at a flow rate of 1.0 mL/min [22]. Selenium in Dox-SeLPs was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) [23]. Briefly, the sample was digested with 5 mL of concentrated nitric acid and 1 mL of H2O2 at 180°C for 3 h. The digested products were reconstituted into 25 mL of deionized water and then subjected to ICP-AES analysis. In vitro release study In vitro release of Dox from Dox-LPs and Dox-SeLPs was studied using the dialysis method [21]. Briefly, 1 mL of Dox solution, Dox-LPs or Dox-SeLPs suspensions was placed in a dialysis bag (MWCO 100 kDa). Then, the dialysis bag was immersed into 100 mL of pH 7.4 phosphate buffer solution (PBS) and dialyzed at 37°C under agitation. At predetermined intervals, 0.2 mL of release medium was withdrawn and replenished with 0.2 mL of fresh medium. The concentration of Dox in the medium was measured using HPLC. To simulate the in vivo release of liposomal Dox, the experiments were simultaneously performed in the fetal bovine serum. Cellular uptake and internalization Human lung carcinoma A549 cells from ATCC were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS, 100 IU/mL of penicillin and streptomycin at 37°C under 5% CO2 atmosphere. Cells were passaged upon reaching an 80% confluence in the culture flask. For cellular uptake study, A549 cells were seeded in 12-well plate at a density of 2 × 105 cells/well and cultured for 24 h. Subsequently, the culture medium was removed and replaced with DMEM-diluted Dox, Dox-LPs or Dox-SeLPs equivalent to 5 μg/mL of Dox. The cells 7

were separately treated for 1 h, 2 h and 4 h at 37 °C, then washed twice with cold PBS (pH 7.4), and followed by tyrisinization to detach. Afterwards, the cells were suspended and centrifuged at 2000 rpm for 3 min under 4°C. The supernatants were removed, and the cells were washed twice again with cold PBS. After centrifugation, the cells were resuspended in 0.5 mL of PBS. Then, the cellular fluorescent intensity was measured with a flow cytometer (Gallios, Beckman, USA). Each assay was performed in triplicate. Another batch of A549 cells were cultured in 12-well plate with round microslides for 24 h. Then, the cells were treated with free Dox, Dox-CLPs and Dox-SeLPs (5 μg/mL) at 37°C for 4 h. After that, the cells were washed three times with cold PBS and fixed with 4% paraformaldehyde for 20 min. Cell nuclei were dyed with DAPI for 15 min. Cellular internalization of Dox-SeLPs was characterized using confocal laser scanning microscopy (CLSM) (Zeiss LSM510 Meta, Oberkochen, Germany). Cellular trafficking pathway study Cellular trafficking pathway of Dox-SeLPs was analyzed based on the cellular uptake in the presence of transport inhibitors [24]. A549 cells were cultured in 12-well plates at a density of 2 × 105 cells/well and grew for 24 h. Inhibitors (hypertonic sucrose, chlorpromazine, simvastatin, Filipin and EIPA) were prepared with serum-free medium and added into the cells to make final concentrations of 0.4 M, 30 μM, 50 μM, 1.5 μM and 20 μM, respectively. After pre-incubation for 0.5 h, Dox-SeLPs were added and incubated for another 4 h at a Dox level of 5 μg/mL. To investigate the effect of temperature on cellular uptake, cells were treated with Dox-SeLPs at 4°C for 4 h. Then, the treated cells were washed with cold PBS,

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trypsinized with trypsin-EDTA (0.25%) solution, and centrifuged at 2,000 rpm to prepare cell pellets. Cellular fluorescence intensity was quantified by flow cytometer, and the cellular trafficking pathway was interpreted according to the relative uptake rate under the action of functional inhibitors or low temperature. Cell cytotoxicity assay The cytotoxicity of free Dox, Dox-LPs and Dox-SeLPs against A549 cells was investigated by MTT assay [25]. Cells were seeded in the 96-well plate containing 100 μL of DMEM and 10% FBS at a density of 1 × 104 cells/well and cultured for 24 h. Then, the medium was replaced with 100 μL of free Dox, liposomes (LPs), SeLPs, Dox-LPs or Dox-SeLPs (diluted with fresh DMEM in advance). After incubation for 24 h at 37 °C, 10 μL of MTT solution (5 mg/mL) was added into each well and continued to incubate for 4 h. Then, the medium was discarded, and 150 μL of dimethyl sulfoxide was added to dissolve the resultant formazan. The absorbance was measured using a microplate reader (BioTek , USA) at 490 nm. Cell viability (%) was calculated as Atri/Acon × 100%, where Atri and Acon denote the absorbance of trial group and control group, respectively. Apoptosis detection A549 cells were cultured in 12-well plates at 2 × 105 cells/well for 24 h. The cells were then treated with free Dox, LPs, SeLPs, Dox-CLPs or Dox-SeLPs for 24 h at 1 and 2 μg/mL of Dox or selenium. Afterwards, the cells were trypsinized and washed twice with cold PBS. To mark the cells in different apoptosis phase, 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) were added in turn and successively incubated for 10 min and 5 min at 4 °C in the 9

dark. Finally, apoptosis analysis was performed on the flow cytometry (Gallios, Beckman, USA). Pharmacokinetics study Male SD rats (200 ± 20 g) were randomly divided into three groups (n = 5). The rats were fasted overnight prior to administration but freely accessible to water. Free Dox, Dox-LPs and Dox-SeLPs were dosed via the jugular vein at a dose of 3 mg/kg equivalent to Dox. At the time of 5, 15, 30, 60, 120, 240, 360, 480 and 720 min, 200 μL of blood were collected from the jugular vein and centrifuged at 5000 rpm for 8 min to collect the plasma. The experimental protocol was reviewed and approved by the Experimental Animal Ethical Committee of Jinan University (Guangzhou, China). The use of animals was conducted as per the Guidelines on the Care and Use of Animals for Scientific Purposes (2004, Singapore). Doxorubicin in the plasma was extracted through a deproteinization procedure. Briefly, 500 μL of acetonitrile mingled with 100 μL of plasma. The mixture was then eddied and centrifuged for 10 min at 12,000 g. The supernatants were transferred to new tubes and subjected to evaporation under vacuum using concentrator (Eppendorf, NY, USA). The residuals were reconstituted in 100 μL of 30% acetonitrile, in which daunorubicin was added as an

internal standard

(0.05 μg/mL).

Dox concentration was determined by

UPLC–QTOF/MS (Xevo G2 QTOF, Waters, Milford, USA) according to the reported method [26]. Antitumor efficacy of Dox-SeLPs in xenograft tumor mice Female BALB/c nude mice were used for the in vivo antitumor efficacy study. Two hundred 10

microliter of A549 cells, suspended in the serum-free medium (about 2 × 106 cells), were subcutaneously injected into the right flank of mice. When the tumor grew to 200 mm3, the mice were randomly divided into four groups (n = 5). At the day of 8, 10, 12, 14, 16 and 18 after inoculation, physiological saline, free Dox, Dox-LPs or Dox-SeLPs was injected into mice via the tail vein at a dose of 5 mg/kg of Dox. The mice were observed and recorded for weight loss and tumor change every two days. Tumor volume was measured with the formula: V = (L×W2)/2 (mm3), where L and W represent the longest and shortest diameter of the tumor. Treatment lasted for 24 days. Afterwards, the tumor, heart, liver, spleen, lung and kidney of mice were excised. Tumors were photographed and weighed to evaluate the therapeutic efficacy. To appraise the physiological implication of different formulations, the tumor and vital organs were prepared into tissue slides and histologically investigated by ematoxylin and eosin (H.E.) staining.

Results and discussion Preparation and characterization In the preliminary experiment, we ascertained the mole ratio of phospholipid/cholesterol and drug/lipid at 2 : 1 and 1 : 6 that could produce stable liposomes and achieve high drug loading. Another important factor affecting the formulation property and drug release is the amount of Na2SeO3 involved in the system. When Se4+ is reduced to Se, a surface coating takes place on liposome. The participation of cationic DOTAP imparts liposomes a positive charge that causes SeO32- to gather on the surface of liposomes due to electrostatic interaction. GSH functions as a reductant and can well react with Na2SeO3 at the mole of 4 : 1 [20]. Based on 11

such ratio, Dox-SeLPs was optimized in preparation. Table 1 shows the particle size and polydispersity index (PDI) changes of Dox-SeNPs with different levels of Na2SeO3 in the system upon preparation. Below 0.5 mg/mL, the particle size of Dox-SeNPs increased with the increase of Na2SeO3 concentration. Above 0.5 mg/mL, the particle size and PDI of Dox-SeLPs sharply raised, indicating that selenium overload easily induced liposomes unstable. Therefore, 0.5 mg/mL of Na2SeO3 was finally determined for the preparation of Dox-SeLPs via the in situ reduction technique [27]. The particle size distribution and morphology of resulting Dox-SeLPs are presented in Figure 1. Untreated liposomes (Dox-LPs) possessed an average particle size of 107.1 nm with a PDI of 0.137. Upon coating with selenium, the particle size increased compared with Dox-LPs, up to 127 nm around. TEM micrograph revealed Dox-SeLPs to be spherical in morphology. Dox-SeLPs exhibited a decreased ζ potential (28.9 mV) relative to Dox-LPs (42.0 mv), indicating a physiochemical change occurring on the surface of liposomes. Of note, the surface engineering of liposomes did not cause sharp decline of interfacial potential, and Dox-LPs was still stable as colloidal dispersions (absolute potential > 25 mV) [28]. It has been shown that nanoparticles with slightly positive charge and smaller particle size easily accumulate toward the tumor tissues [29]. Our developed Dox-SeNPs exhibited the formulation advantages. In addition, the EE of Dox-LPs and Dox-SeLPs was all greater than 95% with DL of ~9%, showing high drug entrapment capacity. Concentrations of Dox and selenium in Dox-SeLPs suspensions were determined to be 3.2 mg/mL and 0.167 mg/mL, which satisfied for the requirements of injection volume and dose.

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In vitro release of Dox The release profiles of Dox from Dox-LPs and Dox-SeLPs are shown in Figure 2. As a control, Dox solution almost released all free drug within 2 h, indicating less release resistance from the dialysis bag. As entrapped in liposomes, the release of Dox apparently became slow. However, the release profiles between Dox-LPs and Dox-SeLPs displayed a clear distinction. The accumulative release of Dox-LPs was up to 64.9% within 84 h, while it was merely 46.5% for Dox-SeLPs. Dox release from Dox-SeLPs was evidently slower than from Dox-LPs in the later phase. This may be attributed to selenium coating that retards diffusion outward of Dox from the phospholipid bilayer. The T50 values of Dox-LPs and Dox-SeLPs, representing the time for 50% drug release, were calculated to be 35.2 h and 54.8 h according to the first-order kinetic equation. In addition, Dox-SeLPs exhibited a slower release process than Dox-LPs in the fetal bovine serum. Otherwise, the release profiles of Dox-LPs and Dox-SeLPs were different from those in pH 7.4 PBS, probably due to high viscosity pertaining to the serum. Taking together, there existed release differences among free Dox, Dox-LPs and Dox-SeLPs. The in vitro release study indicates that Dox-SeLPs are provided with good sustained release for Dox owing to selenium coating. Cellular uptake and internalization Figure 3 shows the cellular uptake of free Dox, Dox-LPs and Dox-SeLPs in A549 cells. Free Dox exhibited the highest cellular uptake due to free access to cells in the form of molecule with no release lag. Dox uptake in A549 cells increased with the time in the cases of free Dox, Dox-CLPs and Dox-SeLPs. Nevertheless, the cellular uptake of free Dox slowed down

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latterly compared with Dox-LPs and Dox-SeLPs, which may be associated with drug efflux. For liposomal Dox, there was an obvious time-dependent uptrend in uptake, indicating that Dox could be incessantly assimilated into the cells under the mediation of liposomes. Liposomes shield the unfavorable characteristics of drug that increases the cellular uptake by reducing the drug efflux. By contrast, the cellular uptake happening to Dox-SeLPs was significantly higher than that of Dox-LPs at 2 h and 4 h. These results demonstrate that SeLPs can improve the cellular delivery of Dox. Figure 4 exhibits the fluorescence intensity and distribution of Dox in A549 cells in different formulation forms. As seen, Dox (red) was co-localized with the cell nucleus (blue) and displayed a pink in the merged image. The cellular fluorescence intensity treated with Dox-SeLPs was significantly stronger than that of free Dox and Dox-LPs. It is shown that Dox-SeLPs have better cellular internalization and cytoplasmic transport ability. Cellular trafficking pathway Cellular trafficking of nanoparticles greatly depends on the membrane mobile transport, such as pinocytosis and endocytosis [30]. To clarify the cellular trafficking mechanism of Dox-SeLPs, we investigated the cellular uptake of Dox-SeLPs in the presence of different physiological inhibitors or under lower temperature in A549 cells. Figure 5 presents the cellular uptake of Dox-SeLPs in limited conditions, expressed as relative fluorescence intensity (%). The cellular uptake of Dox-SeLPs was significantly inhibited by hypertonic sucrose, chlorpromazine and EIPA, whereas simvastatin and Filipin almost exerted no effect on the cellular uptake. Hypertonic sucrose and chlorpromazine, functionally as non-specific

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and specific inhibitors of clathrin-mediated endocytosis, reduced the cellular uptake to 76.97% and 72.18% relative to the control. This indicates that clathrin-mediated endocytosis gets involved in the transport process of Dox-SeLPs. Simvastatin and Filipin as non-specific and specific inhibitors of caveolae-mediated endocytosis did not reduce the cellular uptake, indicating no caveolae-mediated endocytosis involved in the cellular trafficking of Dox-SeLPs. EIPA is an inhibitor of macropinocytosis. In the presence of EIPA, the cellular uptake of Dox-SeLPs was reduced by 13.95% compared with the control. Likewise, under 4ºC, the cellular uptake of Dox-SeLPs was significantly inhibited, resulting in a reduction of 77.54%. Macropinocytosis and endocytosis are generally believed as active transport processes of substances, in which energy is required [31]. These results suggest that membrane mobile transport plays an important role in the cellular trafficking of Dox-SeLPs, especially the clathrin-mediated endocytosis and macropinocytosis. In vitro cytotoxicity The in vitro cytotoxicity of free Dox, Dox-LPs and Dox-SeLPs against A549 cells is shown in Figure 6A. Three Dox formulations exhibited a concentration-dependent cytotoxic effect. However, the cytotoxicity of Dox-SeLPs was more notable in comparison with free Dox and Dox-LPs. The 50% inhibitory concentration (IC50) value of Dox-SeLPs on A549 cells was 0.92 ± 0.16 μg/mL, significantly lower than that of free Dox (4.40 ± 0.58 μg/mL) and Dox-LPs

(5.68 ± 0.73 μg/mL). Dox-SeLPs demonstrated a higher cytotoxicity against A549 cells. Figure 6B shows the cytotoxicity of blank liposomes and SeLPs. Blank liposomes displayed almost no cytotoxicity relative to the culture medium. Surprisingly, SeLPs exhibited a

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definite cytotoxicity against A549 cells. The IC50 value of SeLPs was 17.37 ± 1.28 μg/mL in terms of selenium concentration. It has been verified that selenium nanoparticles themself

possess anticancer activity [32]. As far as our study concerned, this is the initial discovery on the antitumor activity of selenium-coated liposomes. SeLPs not only can control the release rate of Dox, but also enhance the cytotoxicity of cargo through a synergic effect with selenium. Apoptosis analysis To evaluate the effect of liposomal Dox and carrier on apoptosis, we quantified the proportion of apoptotic and necrotic cells by annexin V-FITC and PI double staining. Figure 7 shows the cell distribution in different phases after treatment. Free Dox induced considerable apoptosis of cells, containing later apoptotic cells in Q2 and early apoptotic cells in Q4. The apoptosis rate was positively related with the drug concentration. Dox-LPs also resulted in conspicuous apoptosis of A549 cells, but the apoptotic rate induced by Dox-LPs was a little inferior to that of free Dox. More significant apoptosis of A549 cells was caused by Dox-SeLPs. The apoptotic rates were separately up to 68.5% and 88.24 % at 1 and 2 μg/mL of Dox level, which were approximately 1.5~3.5 times as much as that of free Dox and Dox-LPs. In addition, the apoptotic cells treated with SeLPs (blank carrier) aggravated to some extent compared with the control, which was consistent with the outcomes of cytotoxicity test. Apoptosis assay indicates that Dox-SeLPs should have more potent anticancer effect than Dox alone.

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Pharmacokinetics The pharmacokinetic curves of free Dox, Dox-LPs and Dox-SeLPs in SD rats are shown in Figure 8. Free Dox and Dox-LPs exhibited a rapid drug elimination after injection. There was no significant difference in pharmacokinetics between free Dox and Dox-LPs, though they possessed different in vitro Dox release. It may be ascribed to intense phagocytosis of Dox-LPs by the macrophages in RES. Additionally, common liposomes possess a fragile structure and are easily affected by the plasma components [33]. These factors result in rapid clearance of Dox-LPs from the blood together, thus showing a similar pharmacokinetic curve to free Dox. Overtly, Dox-SeLPs exhibited a strikingly different pharmacokinetics compared with Dox-SeLPs and free Dox. Table 2 presents the main pharmacokinetic parameters calculated by the two-compartmental model. The half-life time (t1/2α , t1/2β) and mean residence time (MRT) of Dox-SeLPs were significantly longer than that of free Dox and Dox-CLPs. Reversely, the clearance (CL) of Dox-SeLPs was significantly smaller than that of free Dox and Dox-CLPs, demonstrating a long-circulation effect of Dox-SeLPs in the blood. The plasma peak concentration (Cmax) of Dox-SeLPs was also higher than that of free Dox and Dox-CLPs. The in vivo pharmacokinetics results correlated well with the in vitro release. After injection, liposomes are quickly diluted and distribute into different tissues and organs (e.g., liver and lung). There is only a small percentage of liposomes retaining in the blood [10, 34]. Thus, it is critical to reduce the body clearance and prolong the plasma circulation of liposomes for anticancer agent delivery. Selenium-coated liposomes depending on excellent plasma stability greatly improved the pharmacokinetic property of Dox. It can be

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expected that Dox-SeLPs can increase Dox accumulation in the tumor via the EPR effect on the base of elongated circulation. Antitumor efficacy of Dox-SeLPs Figure 9 shows the antitumor efficacy of free and liposomal Dox in A549 cells xenograft model after treatment for 14 days with tumor volume, body weight and tumor weight as indicators. Free Dox and two kinds of liposomal Dox exhibited significant tumor inhibitory effect compared with the control (saline). From the tumor growth curves, it could be seen that the tumor of saline group grew fairly fast; free Dox and Dox-LPs inhibited the tumor growth to some extent; and Dox-SeLPs showed the strongest tumor inhibitory effect. The tumor volume of free Dox, Dox-LPs and Dox-SeLPs group was 63.2%, 69.7% and 41.7% of the saline group on the 24th day of inoculation, respectively. Dox-LPs yielded an antitumor effect similar to free Dox, indicating that common liposomes have inadequate drug delivery efficiency. The tumor weight from the sacrificed mice at the end of experiment also indicates that Dox-SeLPs possess more potent antitumor activity. As for free Dox and Dox-CLPs, Dox is quickly and widely distributed into a variety of tissues and organs after injection and lack of tumor specificity, thus resulting in systemic toxicity and low antitumor effect. Dox-SeLPs have higher plasma stability and can deliver more Dox into the tumor tissue via the EPR effect. The outcomes of in vivo antitumor are in agreement with the implication of in vitro cytotoxicity test. There are possibly two reasons responsible for enhanced antitumor efficacy of Dox-SeLPs: (1) selenium has direct tumor inhibitory activity and allows synergy with Dox [35]; (2) Dox-SeLPs possess improved pharmacokinetics that result in high distribution of

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Dox into the tumor [36]. To evaluate the systemic toxicity of free Dox, Dox-LP and Dox-SeLPs, the body weight changes of trial animals were recorded (Figure 9C). Dox-SeLPs posed less body weight loss compared with free Dox and Dox-LPs. This indicates that Dox-SeLPs are provided with low systemic toxicity, and selenium may have the potential to alleviate the body burden caused by Dox. In addition, we checked the histomorphological changes of tumor and vital organs of tumor-bearing mice after treatment at the end of the experiment by H.E. staining. As observed in Figure 10, free Dox and Dox-LPs caused slight damage to the cardiac muscle, whereas Dox-SeLPs did not exhibit obvious cardiotoxicity. Apart from the heart, other organs were less affected. However, Dox-SeLPs induced severe necrosis of tumor cells where appeared considerable cell swelling and plasmorrhexis compared with other formulations. Anyway, Dox-SeLPs display better antitumor efficacy and lower systemic toxicity.

Conclusions In this study, we successfully develop Dox-SeLPs by in situ precipitation of selenium on the surface of liposomes. Selenium coating reinforces the plasma stability of liposomes and reduces the premature release of Dox from liposomes. Dox-SeLPs demonstrate preferable sustained release effect for Dox in vitro and higher AUC value, longer t1/2 and MRT in vivo compared to free Dox and Dox-LPs. Moreover, Dox-SeLPs substantially increase the cellular uptake of Dox through clathrin-mediated endocytosis and exhibit stronger cytotoxicity and apoptosis induction against A549 cells. More importantly, Dox-SeLPs show higher antitumor efficacy and lower systemic toxicity in vivo. Our research provided a new strategy for 19

liposome engineering to efficiently deliver anticancer agents, which may be constructive for the design of chemotherapeutic medications.

Acknowledgment This work was supported by the National Natural Science Foundation of China (81673604) and the Fundamental Research Funds for the Central Universities (21617473).

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Table 1. Effect of Na2SeO3 level in the system upon preparation on the particle size and PDI of Dox-SeLPs Na2SeO3 (mg/mL)

Particle size (nm)

PDI

0.125

110.2 ± 3.4

0.139 ± 0.02

0.250

114.0 ± 4.5

0.143 ± 0.03

0.375

121.0 ± 3.6

0.164 ± 0.03

0.500

127.0 ± 8.3

0.190 ± 0.05

0.625

225.2 ± 12.8

0.309 ± 0.16

0.750

384.7 ± 121.4

0.543 ± 0.13

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Table 2. Pharmacokinetic parameters of free Dox, Dox-LPs and Dox-SeLPs after intravenous injection to rats. Parameter

Free Dox

Dox-CLPs

Dox-SeLPs

Cmax (ng/mL)

0.878 ± 0.060

1.285 ± 0.635

2.505 ± 0.540*

AUC0→t (μg•min/mL)

26.09 ± 6.12

26.92 ± 6.09

87.03 ± 20.18**

t1/2α (min)

3.78 ± 0.26

3.91 ± 0.78

5.40 ± 1.06*

t1/2β (min)

166.41 ± 8.48

177.64 ± 23.46

227.72 ± 19.64**

CL (μL/min/kg)

115.48 ± 46.35

110.15 ± 25.83

25.14 ± 1.50**

MRT (min)

57.17 ± 13.11

59.20 ± 16.67

217.56 ± 25.21**

Data expressed as mean ± SD (n = 5). ANVOA, *p < 0.05; **p < 0.01 compared with Dox-LPs.

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Figure legends Figure 1. Size distribution of Dox-LPs (A) and Dox-SeLPs (B) measured by dynamic light scattering; morphology of Dox-LPs (C) and Dox-SeLPs (D) observed by TEM. The scale bar is 200 nm. Figure 2. Release profiles of free Dox, Dox-LPs and Dox-SeLPs in pH 7.4 PBS (A) and in fetal bovine serum (B). Data expressed as mean ± SD (n = 3), *P < 0.05, **P < 0.01, paired t-test in A (point to point); ANOVA in B (line to line), compared with free Dox and Dox-LPs. Figure 3. Cellular uptake of free Dox, Dox-LPs and Dox-SeLPs in A549 cells at 5 μg/mL of Dox for 2 h and 4 h by flow cytometric analysis. Data expressed as mean ± SD (n = 3). *p < 0.05; **p < 0.01, Paired t-test. Figure 4. Confocal microscopy images of A549 cells after treated with free Dox, Dox-CLPs, and Dox-SeLPs at the concentration of 5 μg/mL for 4 h. Red represents the intrinsic fluorescence of Dox, and blue denotes the fluorescence of DAPI for nuclei staining. Scale bar is 50 μm. Figure 5. Cellular trafficking pathway of SN-PMCNs characterized by the relative cellular uptake in the presence of various inhibitors or under 4ºC. Data expressed as mean ± SD (n = 3); *p < 0.05, **p < 0.01, compared with the control, Paired t-test. Figure 6. In vitro cytotoxicity of preparations (A) and carriers (B) against A549 cells by MTT assay after 24 h incubation. Data expressed as mean ± SD (n = 5), *P < 0.05, **P < 0.01, paired t-test, compared with free Dox and Dox-LPs. Figure 7. Distribution of A549 cells in different apopotic phases after treatment with free

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Dox, Dox-LPs, Dox-SeLPs and SeLPs for 24 h analyzed by flow cytometry based on V-FITC and PI double staining. Figure 8. Pharmacokinetic profiles of free Dox, Dox-LPs and Dox-SeLPs in rats following intravenous injection at the dose of 3mg/kg Dox. Data expressed as mean ± SD (n = 5), P < 0.05, ANOVA (data from 30 min to 720 min). Figure 9. Changes in tumor volume (A) and body weight (B) of A549 xenograft mice with the time of treatment upon injection of saline, free Dox, Dox-LPs and Dox-SeLPs. Average tumor weight (C) and typical tumor masses (D) at the end of experiment. Data expressed as mean ± SD (n = 5). ** p < 0.01; ***p < 0.001, ANOVA. Figure 10. Histopathological analysis by H.E. staining for the heart, liver, spleen, lung, kidney and tumor isolated from A549 xenograft mice after treatment with saline, free Dox, Dox-LPs and Dox-SeLPs for 24 day. Lesions are indicated by the yellow arrow. The heart shows a slight cardiac muscle damage for free Dox and Dox-LPs groups; the tumor takes on obvious cell swelling and plasmorrhexis for Dox-SeLPs group. The scale bar is 100 nm for all graphes.

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Graphical abstract

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