Ratiometric co-encapsulation and co-delivery of doxorubicin and paclitaxel by tumor-targeted lipodisks for combination therapy of breast cancer

International Journal of Pharmaceutics 560 (2019) 191–204

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Ratiometric co-encapsulation and co-delivery of doxorubicin and paclitaxel by tumor-targeted lipodisks for combination therapy of breast cancer

T

Chunlai Fenga, Haisheng Zhanga, Jiaming Chena, Siqi Wanga, Yuanrong Xina, Yang Qua, ⁎ ⁎⁎ Qi Zhanga, Wei Jia, Fumiyoshi Yamashitab, Mengjie Ruia, , Ximing Xua, a

Department of Pharmaceutics, School of Pharmacy, Jiangsu University, Zhenjiang 212013, PR China Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-shimoadachi cho, Sakyo-ku, Kyoto 606-8501, Japan

b

ARTICLE INFO

ABSTRACT

Keywords: Co-loaded lipodisk Tumor-targeted ratiometric drug delivery Combination therapy Doxorubicin Paclitaxel Synergistic anticancer effect Drug resistance

Combination therapy is a promising treatment for certain advanced drug-resistant cancers. Although effective inhibition of various tumor cells was reported in vitro, combination treatment requires improvement in vivo due to uncontrolled ratiometric delivery. In this study, a tumor-targeting lipodisk nanoparticle formulation was developed for ratiometric loading and the transportation of two hydrophobic model drugs, doxorubicin (DOX) and paclitaxel (PTX), in one single platform. Furthermore, a slightly acidic pH-sensitive peptide (SAPSP) incorporated into lipodisks effectively enhanced the tumor-targeting and cell internalization. The obtained coloaded lipodisks were approximately 30 nm with a pH-sensitive property. The ratiometric co-delivery of two drugs via lipodisks was confirmed in both the drug-resistant MCF-7/ADR cell line and its parental MCF-7 cell line in vitro, as well as in a tumor-bearing mouse model in vivo compared with a cocktail solution of free drugs. Coloaded lipodisks exerted improved cytotoxicity to tumor cells in culture, particularly to drug-resistant tumor cells at synergistic drug ratios. In an in vivo xenograft mouse model, the anti-tumor ability of co-loaded lipodisks was evidenced by the remarkable inhibitory effect on tumor growth of either MCF-7 or MCF-7/ADR tumors, which may be attributed to the increased and ratiometric accumulation of both drugs in the tumor tissues. Therefore, tumor-specific lipodisks were crucial for the combination treatment of DOX and PTX to completely exert a synergistic anti-cancer effect. It is concluded that for co-loaded lipodisks, cytotoxicity data in vitro could be used to predict their inhibitory activity in vivo, potentially enhancing the clinical outcome of synergistic therapy.

1. Introduction Over many decades, numerous compounds that are either synthesized chemically or naturally occurring have been documented as possessing antitumor activities. Although the number of potential antitumor agents is notably increasing, considerable attention is focused on specific problem areas, such as poor aqueous solubility (O'Driscoll and Griffin, 2008; Williams et al., 2013), adverse effects and multidrug resistance issues (Choi and Yu, 2014; Kathawala et al., 2015; Pan et al., 2016), which have limited the development of drug candidates from the bench to the bedside. Initially, these complex issues can be attributed to a bewildering range of factors, such as physicochemical properties, drug metabolism, in vivo pharmacokinetics and biodistribution. However, with a better understanding of therapeutic failures, we proposed

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that the application of rational-designed drug carriers is a possible solution to these issues in light of previous reports (Blanco et al., 2015; Danhier et al., 2010; Parveen et al., 2012). In addition to increased aqueous solubility and sustainable release behavior, nanocarriers easily enable the alteration of drug distribution in favor of improved bioavailability and reduced toxicity (Liu et al., 2014; van der Meel et al., 2017; Wang et al., 2017d; Youn and Bae, 2018). These benefits of nanoparticles are partially the result of a narrow size range, from approximately 10 nm to 200 nm, which capitalize on the well-known enhanced permeability and retention (EPR) effect (Fang et al., 2011; Maeda et al., 2013; Shi et al., 2017). Because of the leaking property of underdeveloped tumor vasculature, suitable entities can escape the circulation and accumulate in the tumor bed. Based on passive targeting, active recognition of nanocarriers by tumor cells can be further

Correspondence author at: School of Pharmacy, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu Province 212013, PR China. Correspondence author at: School of Pharmacy, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu Province 212013, PR China. E-mail addresses: [email protected] (M. Rui), [email protected] (X. Xu).

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https://doi.org/10.1016/j.ijpharm.2019.02.009 Received 31 July 2018; Received in revised form 31 January 2019; Accepted 4 February 2019 Available online 12 February 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.

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achieved through specific interaction between receptors expressed on cell surfaces and the modified targeting moieties of entities, including antibodies, peptides or some protein ligands (Bar-Zeev et al., 2017; Dissanayake et al., 2017; Huo et al., 2017; Tang et al., 2014; Yao et al., 2016). More importantly, nanocarriers targeting tumor cells may help address some of the major challenges faced by cancer chemotherapy, which include inherent or acquired multidrug resistance (MDR). Tumor MDR in clinical treatment has been discovered to be explained by complicated mechanisms (Han et al., 2016; Li et al., 2016b), showing that blocking a single pathway may not be sufficient to combat MDR. One hypothesis is that synergistic combination of two chemotherapeutic agents may more effectively break the defense system of tumor cells that depend upon a variety of MDR mechanisms (Chen et al., 2009; Guo et al., 2017; Hu et al., 2016; Khan et al., 2017; Li et al., 2016b). Of note, previous reports showed that combined drugs at optimized drug ratios and dosages demonstrate significant synergistic, rather than additive or antagonistic, effects (Hu et al., 2016; Li et al., 2016c; Rui et al., 2017a). This finding indicates that distinct pharmacokinetics and distributions of individual drugs in combination therapy or conventional cocktail administration explicitly result in deviation from the prediction, posing a real challenge for bridging the gap between the optimized synergistic effects in vitro and unsatisfactory therapeutic outcomes in vivo. For instance, a typical combination of doxorubicin (DOX) and paclitaxel (PTX), widely applied in the treatment of many tumors (Baabur-Cohen et al., 2017; Chen et al., 2015b; Lv et al., 2014), has shown diverse plasma elimination half-lives and biodistributions after systemic administration. Therefore, to address these problems, it is necessary to precisely load drugs with different physicochemical properties into one designed nanoscaled system and co-deliver these drugs to cancer cells at a prefixed ratio, in other words, ratiometric coencapsulation and co-transportation. Also, this is the reason why only a small number of combination therapies are successful. Previously, in our laboratory, we developed a variety of lipid-based nanocarriers that can deliver multiple drugs, such as DOX, genetic materials, and peptide-drug conjugates (Feng et al., 2017b; Rui et al., 2017b; Shi et al., 2016). Recently, reports revealed that the morphology of drug delivery systems, including spheres, disks, or rods, can significantly influence the pharmacokinetics and adsorption properties (Decuzzi et al., 2010; Sharma et al., 2010; Truong et al., 2015; Venkataraman et al., 2011; Zhang et al., 2014). Therefore, in the present study, discoidal lipid-based lipodisks provided us a strategy to codeliver a wide range of drugs with diverting physicochemical property. Lipodisks are discoidal bilayers within which phospholipids establish the flat lipid surface and another important component, PEGylated lipids, are located particularly at the highly curved rim. According to reported results, the physiochemical properties of lipodisks render them potential carriers for a wide range of drugs or biomolecules. For example, the amphiphilic peptide melittin was favorably delivered by lipodisks (Reijmar et al., 2016; Zetterberg et al., 2011), showing improved stability against enzymatic degradation in blood circulation, as well as rehabilitated antibacterial activity compared with the behavior of the free peptide in vivo. Additionally, various studies verified that the lipodisk formulation is applicable for the loading and delivery of anticancer drugs, such as doxorubicin and curcumin (Ahlgren et al., 2017a; Zhang et al., 2014). With the conjugation of targeting or trafficking moieties, functionalized PEGylated lipids enable lipodisks to preferentially localize to the sites of action. Several studies suggested that targeting lipodisks to tumor cells is enhanced by inserting epidermal growth factor, which also minimizes off-target toxicity during systemic administration (Ahlgren et al., 2017a, 2017b; Zetterberg et al., 2016a). Based on this rationale, in this report, we describe the design of tumor-targeted lipodisks in which not only precise ratiometric loading and co-delivery of DOX and PTX were achieved, but a slightly acidic pH-sensitive peptide (SAPSP) as a targeting moiety was also conjugated

to the terminal end of PEG molecules (Fig. 1A). With regard to systemic toxicity, certain targeting peptides, such as cell-penetrating peptides (Copolovici et al., 2014; Dissanayake et al., 2017; Madani et al., 2011; Yuan et al., 2013), were reported to bind both tumor and normal tissues cells without significant discrimination. In the sequence of peptide SAPSP, glutamic acid residues were elaborately inserted to neighbor histidine residues and were applied to stabilize the protonated form of histidine. As a result, the pKa value of histidine residues in SAPSP increased from 6.0 to approximately 6.5, facilitating the protonation of histidine in response to slightly acidic pH (> 6.5). In other words, SAPSP could switch its negative charge under physiological conditions to a positive charge under some weakly acidic conditions. Since the tumor microenvironment is reported to be slightly acidic (pH 6.5–6.8), this pH-triggered charge-reversal peptide seems an ideal targeting moiety, which had the ability to direct lipodisks towards tumor tissues more specifically and improve the anti-tumor efficacy (Feng et al., 2017b; Hama et al., 2015; Itakura et al., 2016; Suzuki et al., 2017). After the preparation process, co-loaded lipodisks were characterized by their hydrodynamic diameter, size distribution, morphology and release behavior in vitro. Moreover, as a prerequisite for the simultaneous delivery of two drugs, ratiometric encapsulation of DOX and PTX was subsequently verified. We further proposed that these tumor-specific lipodisks could be ratiometrically directed to the site of tumor at an optimized ratio. This hypothesis was examined by cellular uptake studies in vitro, pharmacokinetics behavior, and biodistribution in tumor-bearing mice in vivo. Breast cancer cells and its drug-resistant strain were initially used to systemically evaluate the synergistic antitumor efficacy of two drugs encapsulated in lipodisks. Moreover, the ability of lipodisks to maintain the synergistic ratios of the combined drugs in vitro was thoroughly investigated in tumor xenograft models compared with a free drug cocktail administration. This study provides additional insights showing that tumor-targeted lipodisks have potential for combinatorial drug delivery, resulting in synergistic therapeutic effects against drug-resistant tumors, both in vitro and in vivo. 2. Materials and methods 2.1. Materials Doxorubicin hydrochloride (Dox·HCl, 98.0–102.0% purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and paclitaxel (PTX, 99% purity) was from Meilun Biotechnology Co., Ltd. (Dalian, China). 1,2-Distearoyl-sn-grycero-3-phosphocholine (DSPC), 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-maleimide) were obtained from Avanti Polar Lipids (Alabaster, AL). The fluorescent lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)-fluorescein (DSPE-PEG-FITC), was purchased from Xi'an Ruixi Biological technology Co., Ltd. (Xi'an, China). Cholesterol, chlorpromazine, indomethacin, colchicine, and quercetin were purchased from Aladdin Bio-Chem Co., LTD (Shanghai, China). The SAPSP peptide (Ac-CHGAHEHAGHEHAAGEHHAHE-NH2) was synthesized by GL Biochem (Shanghai, China). Other reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used as received. 2.2. Conjugation of SAPSP peptide to DSPE-PEG2000-maleimide We synthesized this lipid derivative based on a previous report (Chen et al., 2011; Saw et al., 2013). In brief, the SAPSP peptide and DSPE-PEG2000-maleimide were separately dissolved in phosphate buffered saline (20 mM sodium phosphate, 150 mM NaCl, pH 7.4). Next, the peptides were reacted with DSPE-PEG2000-maleimide at the molar ratio of 1.5:1 at ambient temperature for 24 h with continuous stirring. To remove unreacted SAPSP peptide and other impurities, the 192

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Fig. 1. Fabrication and characterization of co-loaded lipodisks. (A) Preparation of co-loaded lipodisks containing DOX and PTX via thin-film hydration method. Two drugs could be ratiometrically loaded in lipodisk, simultaneously transported into tumor tissues, and eventually exert strong synergistic anti-cancer effect. (B) Hydrodynamic diameters of blank lipodisks and co-loaded lipodisks. The values are mean ± SD, n = 3. (C) Typical TEM image of co-loaded lipodisk.

mixture was subjected to dialysis for two days against distilled water using SnakeSkin Dialysis bags with a 3500 molecular weight cut-off (SnakeSkin, Thermo Fisher). After dialysis, the final samples were freeze-dried and identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (4800, AB Sciex), followed by the storage at −20 °C until used.

1 h in the dark to remove the hydrochloric acid. Next, the desalted DOX and PTX were mixed in chloroform at the appropriate molar ratio followed by the addition of lipid chloroform solution. The remaining preparation process for the lipodisks composed of DSPC/DSPEPEG2000/DSPE-PEG-SAPSP/ drugs (DOX + PTX) (75:21:4:5, mol/mol) was the same as that of the blank lipodisks. To purify the loaded drugs, the obtained lipodisks were subjected to centrifugal ultrafiltration using an Amicon Ultra-50K device (MWCO = 50 kDa, Millipore) to remove drug precipitates. For subcellular distribution study, 0.5% of total lipids in co-loaded lipodisks were replaced with fluorescent lipids, DSPE-PEGFITC. To demonstrate the tumor-specific property of SAPSP, the nontargeting co-loaded lipodisk was composed of DSPC/DSPE-PEG2000/ drugs (DOX + PTX) (75:25:5, mol/mol), and prepared using the similar procedure as described above.

2.3. Preparation of co-loaded lipodisks The blank targeting lipodisks were composed of DSPC/DSPEPEG2000/DSPE-PEG-SAPSP (75:21:4, mol/mol). Initially, lipids, including DSPC, DSPE-PEG2000 and DSPE-PEG-SAPSP, were weighed and dissolved in chloroform. The total lipid amount in the starting materials equaled to 4 μmol per mL of buffer; however, in some cases the amount was slightly changed to investigate the optimal encapsulation efficiency. Next, the lipid film was prepared by mixing the lipid solutions and evaporating the solvent under a vacuum for at least 1 h. The obtained lipid film was hydrated in 2 mL of HBS (HEPES buffered saline, 10 mM, pH 7.4) at 60 °C for half an hour. The hydrated samples were subjected to sonication on ice for 30 min using an ultrasonic cell disruptor (JY-92-II, Ningbo Xinzhi Instruments, China). After sonication, metal debris from the probe tip was removed by centrifugation at 480g for 2 min. Finally, the targeting lipodisk samples containing SAPSP moiety were stored at 4 °C until further use. Before preparing the co-loaded lipodisks, DOX·HCl in dichloromethane was stirred with a three-fold excess of triethylamine for

2.4. Characterization The hydrodynamic diameter, size distribution and surface zeta potential of co-loaded lipodisks were determined by dynamic light scattering (DLS) technique using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). Samples were initially diluted 1:5–1:10 in HBS (10 mM, pH 7.4) buffer, containing approximately 0.4–1 mM lipids. All the measurements were carried out in triplicate. The morphology of co-loaded lipodisks was observed using transmission electron microscopy (TEM). A drop of sample was placed on one carbon formvar-coated copper grid, negatively stained with 193

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were seeded in glass bottom dishes at a density of 1 × 105 cells/dish and cultured for 24 h. After the removal of original medium, cells were incubated with FITC-labeled co-loaded lipodisks for 2 h. The concentration of DOX was kept at 5 μM. Then cells were stained with Hoechst 33342 (1 μg/mL) for half an hour, and LysoTracker Deep Red (50 nM) (Thermo Fisher Scientific, USA) for 5 min, followed by three washes with PBS. Finally, the subcellular distributions of DOX and lipodisks were visualized by CLSM. To further investigate the endocytotic mechanism of co-loaded lipodisks incorporating SAPSP peptides, a series of uptake inhibition experiments were performed in MCF-7 cells, which were seeded into 6well plates as described above. After cultivation for 24 h, cells were separately pre-treated with different endocytosis inhibitors, including chlorpromazine (50 μM), indomethacin (50 μM), colchicine (50 μM), or quercetin (50 μM), for 1 h. Conversely, cells were incubated at 4 °C for 1 h. Subsequently, cells were treated with co-loaded lipodisks decorated with or without SAPSP peptides for another 1 h. Concentrations of DOX inside the cells were detected using the same method described above.

phosphotungstic acid (2%, w/v) for 30 s, and then visualized using a JEOL JEM-2100 instrument (JEOL, Tokyo, Japan). To study the encapsulation efficiency of DOX and PTX, the encapsulated drugs were released from the lipodisks by incubation with 1% Triton X-100 solution that was applied to disrupt the integrity of lipodisk. DOX was determined with a RF-5301PC spectrofluorometer (Shimadzu, Kyoto, Japan) with an excitation wavelength of 470 nm and an emission wavelength of 590 nm. PTX inside the lipodisks was determined using C18 high-performance liquid chromatography (HPLC). The mobile phase consisted of a mixture of acetonitrile and water (3:1, v/v), and the flow rate was 1 mL/min. Twenty microliters of sample was injected into a C18 column (5 μm, 4.6 mm × 250 mm), and PTX was detected at 227 nm. The drug loading efficiency was calculated according to the following formula:

Encapsulation efficiency% =

amount of loaded drug × 100% amount of feeding drug

2.5. In vitro release behavior

2.8. In vitro cytotoxicity assay

The release profiles of DOX and PTX from co-loaded lipodisks were estimated using the conventional dialysis method at 37 °C. In brief, samples were transferred into dialysis bags (SnakeSkin, 10 kDa MWCO), and these bags were immersed in 50 mL of HBS containing 0.1% (w/v) Tween 80. In this release medium, the surfactant Tween 80 was used to maintain sink condition so that two drugs could be released freely from co-loaded lipodisk. Under continuous agitation at 100 rpm, 1 mL of medium was withdrawn at predetermined time points and replaced with fresh medium to maintain a sink condition. The removed medium was quantified for the concentration of DOX (by spectrofluorometer) and PTX (by HPLC). The release experiments were performed in triplicate.

The cytotoxicity of co-loaded lipodisks with or without the SAPSP targeting moiety, single drug loaded lipodisks and free drugs towards MCF-7 and MCF-7/ADR cells was evaluated by CCK-8 assay (Dojindo, Japan). Cells were seeded in 96-well plates at approximately 6000 cells per well and later incubated for 48 h under normal cell culture conditions. After removal of the cell medium, cells were incubated with various formulations containing a series of concentrations of DOX or equivalent DOX for another 48 h, as indicated. Next, cells were subjected to a CCK8 assay according to the manufacturer's instructions, and the absorbency of the solution was detected at 450 nm on a microplate reader. The relative cell viability was calculated by (Asample/Acontrol), where Asample and Acontrol represent the absorbances of samples and cell controls, respectively. Data were presented as the average ± SD (n = 5). The values of the inhibitory concentration at 50% growth (IC50) were calculated using GraphPad Prism 6 (GraphPad Software Inc., San Diego, US). To investigate dual-drug interactions in combinatorial studies, a combination index (CI) analysis was used to classify the interactions into types of synergy, additivity or antagonism. Based on the ChouTalalay method (Chou, 2006, 2010), CI values were calculated using the following equation:

2.6. Cell culture Two types of human cancer cell lines, including DOX-sensitive breast carcinoma (MCF-7) and its DOX-resistant counterpart (MCF-7/ ADR), were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were maintained at 37 °C in 5% CO2 in a humidified incubator. 2.7. Cellular uptake study To investigate the cellular uptake behavior of co-loaded lipodisks, cells were seeded in 6-well plates at a density of 5 × 105 cells per well and incubated overnight. Co-loaded lipodisks with three DOX/PTX ratios were diluted with DMEM (10% FBS) and adjusted to 10 μg/mL Dox concentration. The free drug combination was dissolved in DMSO to make a stock solution, and it was appropriately diluted with DMEM thereafter and used as a control. Cells were treated with either coloaded lipodisks or free drug solutions at 37 °C for 0.5, 2, and 4 h. After incubation, cells were washed twice with PBS (pH 7.4) and later lysed with lysis buffer (1% Triton X-100, 20 mM Tris·HCl, 100 mM NaCl, 1 mM EDTA, pH 7.5) at 37 °C for 30 min. All samples were subsequently diluted with acetonitrile at the appropriate ratios. The concentration of PTX was quantified by HPLC as described above. For the determination of DOX, the chromatographic separation was also performed on the same C18 column using acetonitrile-methanol-water (32:50:18, v/v/v) as a mobile phase at a flow rate of 1.0 mL/min, and DOX was detected at a wavelength of 482 nm. Finally, the concentrations of the two drugs were normalized to the whole protein concentration in the cell lysates, which was measured using a BCA Protein Assay Kit (Beyotime, China). In light of results of cellular uptake study, the observation of the internalization process was carried out using a confocal laser scanning microscope (CLSM, Laser TCS SP5 II, Germany). Briefly, MCF-7 cells

CI =

(D )1 (D )2 + (D x )1 (DX )2

where (D)1 and (D)2 are the respective dose of each drug in a combination that yield an effect of 50% cell growth inhibition, and (Dx)1 and (Dx)2 are the corresponding concentration (IC50) of the single drug that leads to the same cytotoxic effect. A CI value of less than, equal to, or more than 1 represent drug interactions that are synergistic, additive or antagonistic, respectively. 2.9. Animal study Nude mice (female, 5–7 weeks) were purchased from the Shanghai Laboratory Animal Center (SLAC, China) and housed in a pathogenreduced room. All animal procedures were performed in accordance with guidelines and approved by the Institutional Animal Care and Use committee of Jiangsu University. 2.10. Pharmacokinetics study First, nude mice were inoculated subcutaneously with MCF-7 breast cancer cells. Subsequently, tumor-bearing mice were randomly assigned to two groups with 6 mice each. These groups were the co194

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loaded lipodisk group and free drug combination group. The free drug solution was prepared by diluting the DMSO stock solution with HBS buffer. Then, mice were intravenously injected with doses of 5.0 mg/kg DOX and 7.9 mg/kg PTX. At the scheduled time points (0.25, 0.5, 0.75, 1, 2, 4, 8, 12, and 24 h) after administration, blood samples were withdrawn through orbital venous plexus. These samples were immediately centrifuged at 1000 rpm for 10 min to harvest plasma, which was stored at −70 °C until further use. Before drug extraction, plasma samples were spiked with docetaxel and daunorubicin, which were used as internal standards for PTX and DOX, respectively. Next, both drugs in the plasma were separately measured using the HPLC method after liquid-liquid extraction, as reported previously (Chen et al., 2015a; Rui et al., 2017a). Pharmacokinetic parameters were calculated with the program PKSolver (Zhang et al., 2010) using a noncompartmental analysis. All results were presented as means ± SD (n = 6) unless otherwise stated.

between two groups, whereas one-way ANOVA with a Turkey's post hoc test was used for differences between more than two groups. A value of * p < 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Preparation and characterization of co-loaded lipodisks The combination of PTX and DOX has been applied as a first-line therapy against metastatic breast cancer. However, cardiac risk along with drug resistance seriously limit clinical use of the PTX-DOX regimen (Perez et al., 2008; Tan-Chiu et al., 2005). In addition, the poor solubility of PTX and DOX in water hampers their ability to reach sites of action in vivo. In this study, to overcome the associated toxicities and drug resistance, two drugs were loaded into lipodisks. Based on previous studies (Reijmar et al., 2016; Zetterberg et al., 2016b), our lipodisks consisting of DSPC, DSPE-PEG2000 and a targeting moiety were prepared at an optimized molar ratio using the thin-film hydration method (Fig. 1A). Initially, a targeting peptide, SAPSP, the charge of which is responsive to the tumor microenvironment, was coupled to a PEGylated lipid based on a fast reaction between the thiol groups of peptides and the maleimide moiety in the terminal of DSPE-PEG2000Mal. DSPE-PEG-SAPSP conjugate was successfully synthesized and further confirmed by the MALDI-TOF mass, showing a molecular weight of approximately 5225 Da. Next, both drugs, DOX and PTX, were efficiently encapsulated into lipodisks, mainly by hydrophobic interaction, achieving the simultaneous encapsulation of two drugs with a predesignated ratio in one lipodisk. As presented in Table 1 and Fig. 1B, the average hydrodynamic diameter of the co-loaded lipodisks was 38.39 ± 6.53 nm, which was similar to that of the blank lipodisk at 33.12 ± 4.11 nm. Furthermore, the polydispersity index (PDI) of coloaded lipodisks or blank lipodisks was < 0.25, indicating narrow size distributions. Because of the pH-sensitive peptide SAPSP, lipodisks with or without drugs were negatively charged under physiological conditions, and these nanoparticles demonstrated a positive surface charge at pH 6.8, which represented a slightly acidic extracellular pH of the tumor microenvironment (Table 1). The surface charge reversal of lipodisks guaranteed their high affinity for tumor cell membranes that possess a net negative charge but not to normal tissues, resulting in high tumor targeting efficiency and low systemic toxicity. From the TEM images (Fig. 1C), we observed that the sizes of the lipodisks were monodispersed and between 30 and 40 nm, which was consistent with the DLS measurement results. The rounded shape represented the lipodisks observed en face; however, unlike disc-shaped high-density lipoproteins, these lipodisks could not be viewed from the rim clearly. One possible reason was that lipodisks with PEG chains were not able to stand up on the copper grid using the TEM technique. Compared to previous reports (Johansson et al., 2005; Reijmar et al., 2016; Zetterberg et al., 2016b), the similar diameters of nanoparticles suggested that we successfully prepared co-loaded lipodisks.

2.11. In vivo biodistribution analysis To investigate the biodistribution profiles of co-loaded lipodisks, MCF-7 tumor-bearing mice were randomly divided into two groups (3 mice per group) and later intravenously injected with either co-loaded lipodisks or free drug combination at doses of 5.0 mg/kg DOX and 7.9 mg/kg PTX. At time intervals of 1 h, 6 h, and 24 h post-injection, several organs and tissues, including the lung, liver, spleen, heart, kidney and tumor, were collected after sacrifice. Tissues were washed twice with saline and then weighed. Tissues of appropriate weight were resuspended with 2 mL methanol, with docetaxel and daunorubicin being added as internal standards, and were vortexed for 1 min. After homogenization, the mixtures were centrifuged (4000 rpm, 20 min) at 4 °C to collect the organic layer. After repeating the extraction again, the total organic solvents were evaporated to dryness followed by resuspending in 100 μL of acetonitrile. Subsequently, concentrations of DOX and PTX were determined as described in the above section. 2.12. In vivo anticancer efficacy To evaluate the anti-tumor effects, female nude mice were inoculated subcutaneously with MCF-7 or MCF-7/ADR cells. When the tumor volumes reached approximately 100–150 mm3, the mice were divided into six groups (n = 6), including normal saline, free drug solution (5 mg/kg DOX, 7.9 mg/kg PTX, DOX/PTX = 1:1, m/m), free drug solution (6.7 mg/kg DOX, 5.3 mg/kg PTX, DOX/PTX = 2:1, m/m), co-loaded lipodisk without SAPSP moiety (6.7 mg/kg DOX, 5.3 mg/kg PTX, DOX/ PTX = 2:1, m/m), and co-loaded lipodisk containing SAPSP (5 mg/kg DOX, 7.9 mg/kg PTX, DOX/PTX = 1:1, m/m), and co-loaded lipodisk containing SAPSP (6.7 mg/kg DOX, 5.3 mg/kg PTX, DOX/PTX = 2:1, m/ m). Either formulation was intravenously administered every three days for twelve consecutive days. Tumor width and length were recorded every three days for three weeks, and the tumor volume was calculated as (length × width2)/2. Additionally, the body weights of mice were also measured every three days. At the end of the experiment, all mice were sacrificed, and the tumors were collected and weighed.

3.2. Drug loading efficiency To investigate the synergistic effects of dual-drug lipodisks, the high encapsulation efficiency of both drugs is an essential prerequisite for the precise control of dual-drug ratios. The results identified that the molar ratio of the total lipids to the two drugs was important to regulate the encapsulation efficiency of the two drugs. As shown in Fig. 2 for

2.13. Statistical analysis Quantitative data were presented as the mean ± standard deviation. Student's t test was used to analyze the significance of differences Table 1 Characterization of various lipodisks.

Blank lipodisk Co-loaded lipodisk

Size (nm)

Polydispersity Index (PDI)

Zeta potential at pH 7.4 (mV)

Zeta potential at pH 6.8 (mV)

33.12 ± 4.11 38.39 ± 6.53

0.229 ± 0.047 0.164 ± 0.017

−10.6 ± 2.52 −8.78 ± 0.49

6.52 ± 1.02 7.47 ± 1.25

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Fig. 2. Effect of drug/lipid molar ratio on the encapsulation efficiency of DOX and PTX in co-loaded lipodisks. Drug-to-lipid ratios varied from 0.05 to 0.6. Each point represented an average of three measurements. Fig. 3. In vitro release kinetics of DOX and PTX from co-loaded lipodisks under different conditions. (A) Concentrations of DOX and PTX that were released from co-loaded lipodisks to HBS buffer at a series of pH values (7.4, 6.5, and 5.0) at 37 °C. (B) Release profiles of DOX and PTX from either co-loaded lipodisks or single drug lipodisks in HBS buffer at pH 7.4. Each point represented an average of three measurements.

Table 2 Effect of drug-to-lipid ratios on encapsulation efficiency of drugs. Drug-to-lipidmolar ratio

Encapsulation efficiency (%) DOX

0.05 0.1 0.2 0.4 0.6

95.67 87.03 73.27 64.73 52.73

PTX ± ± ± ± ±

2.38 2.64 1.70 2.15 4.00

97.17 83.73 75.57 61.43 47.40

± ± ± ± ±

1.59 2.12 2.34 5.77 4.10

suggesting that both DOX and PTX were preferentially buried in the hydrophobic core of the lipodisk and not absorbed on surfaces. Approximately 16% or 21% of both drugs were gradually released from carriers within the first 6 h at pH 7.4 or pH 6.5. Furthermore, the accumulated amount of each drug reached approximately 30% or 42% at 48 h at pH 7.4 or pH 6.5. However, the leakage of DOX and PTX from lipodisks was accelerated under more acidic conditions (pH 5.0), showing a relatively strong pH-dependent release profile. Compared with the release profile for the other two conditions, the cumulative release amounts in pH 5.0 reached 82.4 ± 5.2% for DOX and 79.3 ± 3.8% for PTX. Therefore, lipodisks can reduce the undesired release during blooding circulation but specifically improve the drug release in the endo/lysosomes of tumor cells. Additionally, we studied whether one drug affected the release behavior of the other drug from the same vehicle, such as decelerating or accelerating its release. As shown in Fig. 3B, DOX leaked slightly faster from DOX-only lipodisks than that from co-loaded carriers; however, no significant difference was observed between these two groups. Similar release kinetics were observed with lipodisks loaded with either PTX alone or both drugs, suggesting that the two drugs retained their own release behavior after ratiometric encapsulation into the same lipodisks.

DOX, the encapsulation efficiency was gradually reduced from nearly 100% at a low drug-to-lipid ratio of 0.05 to < 60% at high ratio of 0.6 when DOX and PTX were loaded at 1:1 ratio (Table 2). Additionally, a similar trend for the encapsulation efficiency was observed for PTX as the drug-to-lipid ratio was increased. Therefore, the optimal drug-tolipid ratio was selected as 0.05, and the loading efficiencies of DOX and PTX were determined to be as high as 95.6% and 97.2%, respectively, indicating the high-affinity capacity of hydrophobic discoidal cores for DOX and PTX (Table 2). To confirm that the two drugs are ratiometrically co-loaded into lipodisks, further studies were conducted when the feed molar ratio between DOX and PTX was altered from 1:2 to 2:1. It was found that the three measured molar ratios between DOX and PTX were almost identical to the feed molar ratios. For example, 0.56 vs 0.5, 1.12 vs 1, and 2.07 vs 2. Therefore, these results showed that because the encapsulation efficiency of a single drug was highly comparable to another, the ratiometric co-encapsulation of distinct drug models in lipodisk cores was achieved over a relatively wide range of dual-drug ratios.

3.4. Cellular uptake behavior Next, we investigated the ratiometric delivery property of co-loaded lipodisks to breast MCF-7 cancer cells as well as its drug-resistant counterpart MCF-7/ADR. In vitro cellular uptake results of free drugs in combination, as shown in Fig. 4A, indicated that MCF-7 cells were more likely to take up DOX than PTX, suggesting that the initial ratio and actual intracellular ratio of the free drug cocktail solution was not the same. Compared with the free cocktail solution, the results revealed that both lipodisks, with or without SAPSP peptides, ratiometrically transported drugs into cells and maintained the predetermined ratio of both drugs inside the tumor cells. In particular, targeting lipodisks with SAPSP peptide exhibited a significantly enhanced delivery efficiency of both drugs. For example, the uptake of DOX was increased by

3.3. In vitro release behavior of co-loaded lipodisks The release kinetics of DOX and PTX from co-loaded lipodisks were investigated in vitro via dialysis in solutions of various pH values at 37 °C for 48 h. To simulate different conditions, the pH values of the solutions were 7.4, 6.5 and 5.0, which represented physiological conditions, the tumor microenvironment (Danhier et al., 2010; Li et al., 2016a; Wang et al., 2018), and endo/lysosomes (Wang et al., 2017a, 2017c), respectively. The release profiles of co-loaded lipodisks are reflected in Fig. 3A. Notably, the lipodisk demonstrated promising sustained drug release behavior with a negligible initial burst release, 196

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Fig. 4. Ratiometric cellular delivery of dual drugs from co-loaded lipodisks and the related uptake mechanism of lipodisk modified with SAPSP peptides. Cellular accumulation of lipodisks encapsulating DOX and PTX in drug-sensitive breast cancer MCF-7 cells (A) and drug-resistant MCF-7/ADR cells (B). Subcellular distributions of DOX and lipodisk in MCF-7 cells (C). Cells were treated with FITC-labeled co-loaded lipodisks at a DOX concentration of 5 μM for 2 h. Nuclei were stained with Hoechst 33342 (blue), endo/lysosomes were stained by LysoTracker Deep Red (red), lipodisks were labeled with FITC (green) and DOX was visualized in pseudocolored orange. Scale bar: 10 μm. Effects of a variety of endocytosis inhibitors on cellular uptake in drug-sensitive MCF-7 cells treated with either normal lipodisks (D) or lipodisks decorated with SAPSP peptide (E). The values are mean ± SD, n = 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

approximately 5.8-fold at 0.5 h, 4.3-fold at 1 h, and 2.6-fold at 3 h compared with DOX delivered by normal lipodisks (Fig. 4A). Based on these observations, the SAPSP peptide was beneficial for the increased accumulation of drugs in tumor cells. Compared with some targeting moieties that were previously applied (Chen et al., 2012; Copolovici et al., 2014; Huo et al., 2017; Zhang et al., 2016), the SAPSP peptide not only improved cellular uptake in vitro but also potentially altered the drug distribution in vivo and reduced the systemic toxicity that often restricts the clinical application of targeting nanocarriers (Feng et al., 2017a; Hama et al., 2015). It also showed favorable advantages for the development of more specific and safer delivery systems.

To address the drug resistance issue, the ratiometric uptake of both drugs by drug-resistant cells is a prerequisite for combination therapy (Aryal et al., 2011; Chen et al., 2017; Hu et al., 2010; Hu and Zhang, 2012; Luo et al., 2015). The cellular uptake of drugs in SAPSP-lipodisks was subsequently compared with that of the cocktail solution (Fig. 4B). As expected, the uptake of DOX was significantly decreased in MCF-7/ ADR cells compared with the parent MCF-7 cells. In addition, not only the absorption of PTX but also the molar ratio between DOX and PTX was significantly affected by drug-resistant mechanisms in MCF-7/ADR cells. In contrast, SAPSP-lipodisks enabled ratiometric control over the cellular uptake of DOX and PTX in drug-resistant cell lines. Notably, the 197

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Fig. 5. In vitro cytotoxicity of co-loaded lipodisks and single-loaded lipodisks against drug-sensitive MCF-7 cells (A) and drug-resistant MCF-7/ADR cells (B) after 48 h incubation. Viability of cells were determined using CCK-8 assay at three DOX-to-PTX ratios, including 2:1, 1:1, and 1:2. Also, in vitro cytotoxicity of free drug solutions against MCF-7 cells (C) and MCF-7/ADR cells (D) were detected. Data were presented as mean ± SD, n = 5.

uptake of both drugs from SAPSP-lipodisks was slightly reduced by approximately 21.4%, confirming the benefit of the tumor-specific property of the SAPSP peptide sequence.

internalized by MCF-7 cells and came up with the release of loaded DOX from lysosome to cytoplasm. These results clearly indicated that SAPSP could facilitate the uptake of lipodisks via endocytosis to some extent. To clarify the uptake mechanism of lipodisks underlying the ratiometric transportation of two drugs, the endocytotic pathway was investigated with a variety of endocytosis inhibitors, such as chlorpromazine for clathrin-dependent endocytosis (Cheng et al., 2018; Richard et al., 2005; Wang et al., 1993), indomethacin for caveolae-dependent endocytosis (Gumbleton et al., 2000; Liu et al., 2017), colchicine for macropinocytosis (Huang et al., 2017; Tiwari et al., 2017), and quercetin for non-clathrin and non-caveolae mediated endocytosis, such as phosphoinositide-3 kinase (PI3K) mediated endocytosis (Kong and Yamori, 2008; Nagahama et al., 2009; Walker et al., 2000). Cells were pre-incubated with various endocytotic inhibitors for 1 h, followed by another 1 h incubation with co-loaded lipodisks decorated with or without SAPSP peptides. To examine the energy-dependent endocytosis pathway, cells were first incubated at 4 °C and then treated with two types of lipodisks. Compared with the control group, the cellular

3.5. Analysis of cell uptake mechanism The cellular uptake behavior of SAPSP-decorated co-loaded lipodisks was initially investigated in MCF-7 cells using confocal laser scanning microscopy (CLSM). For subcellular observation, cellular nuclei were stained with Hoechst 33342 (blue), and lipodisks were labeled with FITC (green), and then endosome and lysosome were stained with LysoTracker Deep Red, which is selective for acidic organelles, for example, endosome and lysosome (red) (Fig. 4C). After incubation for 2 h, co-loaded lipodisks labeled with green fluorescent dye were observed as yellow dots inside cancer cells, indicating that these nanoparticles colocalized with endo/lysosomes that were stained with red fluorescent probes. Also, the fluorescence of DOX was distributed widely in the cytoplasm, suggesting that co-loaded lipodisks were rapidly 198

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Table 3 In vitro cytotoxicity and combination index (CI) of various formulations against MCF-7 and MCF-7/ADR cells for 48 h incubation. Drug formulation

Co-loaded lipodisk DOX-lipodisk PTX-lipodisk Free dual-drug solution Free DOX solution Free PTX solution

DOX-to-PTX molar ratio

MCF-7

MCF-7/ADR

IC50 DOX

IC50 PTX

CI (50%)

IC50 DOX

IC50 PTX

CI (50%)

2:1 1:1 1:2

1.004 1.935 2.879 4.431

0.502 1.935 5.757

0.309 0.756 1.599

2.246 4.388 3.343 6.620

1.123 4.388 6.687

0.435 1.039 1.078

2:1 1:1 1:2

4.119 4.942 7.460 4.020

2.059 2.471 3.730

1.222 1.466 2.213

20.233 24.827 27.393 35.660

10.117 12.413 13.697

6.063

10.440

internalization of normal lipodisks was significantly inhibited by low temperature, chlorpromazine, colchicine, and quercetin, whereas indomethacin did not affect the uptake of normal lipodisks (Fig. 4D). These results indicated that the cellular internalization of disk-shaped nanocarriers was mainly regulated by several endocytosis processes, including energy-dependent, clathrin-dependent, micropinocytosis-dependent, and other pathways. Similarly, the effects of these inhibitors were examined on the intracellular trafficking of the SAPSP peptide modified lipodisks (Fig. 4E). Compared with the normal lipodisks, chlorpromazine significantly interfered with the uptake of SAPSP lipodisks, indicating that a major internalization pathway is clathrinmeditated endocytosis. Additionally, the inhibition effects of cold temperature and quercetin suggested that energy-mediated and PI3Kdependent endocytosis were also involved, to various extents, in the uptake of tumor-specific lipodisks. Therefore, the conjugation of SAPSP peptide could alter the uptake mechanism of lipodisks to a certain degree and subsequently improve the tumor-binding efficiency of nanocarriers. In addition, the endocytosis process of lipodisks is a key to escape the efflux and barrier functions of the P-gp transporter, resulting in the ratiometric delivery of two drugs to tumor cells.

11.680 1.150 1.415 1.562

17.260

MCF-7 cells via the endocytosis pathway and showed its inhibitory effect after DOX molecules were released from the nanoparticles. However, as a result of the overexpression of the drug efflux pump P-glycoprotein (P-gp) in the membranes of MCF-7/ADR cells, the resistance of MCF-7/ADR to DOX was confirmed by a higher IC50 value for the free DOX solution (Table 3). The increased IC50 value of free PTX on MCF-7/ ADR may be partially explained by the same reason. Notably, any drug combination at three ratios loaded into lipodisk, could reverse drugresistance, resulting in stronger cell inhibition effects compared with the free dual-drug solution and single drug groups. The reversal of drug resistance indicated that tumor-targeted lipodisks could effectively transport drug molecules into MCF-7/ADR cells and circumvent the drug efflux effect caused by the P-gp efflux pump. Furthermore, according to the Chou-Talalay method (Chou, 2007, 2010), synergy studies revealed that all CI values for the free drug solutions against MCF-7 and MCF-7/ADR cells at three ratios were larger than 1, indicating that the combination of two free drugs achieves unfavorable antagonistic effects. The impaired chemotherapeutic results may be due to the uncoordinated cellular uptake profiles of the two drugs by tumor cells. By contrast, when both drugs were ratiometrically loaded into lipodisks, the CI50 values of co-loaded lipodisks against MCF-7 cells were calculated as 0.309 at a DOX/PTX ratio of 2:1, and 0.756 at a DOX/PTX ratio of 1:1 with significant synergistic antitumor efficacy (Table 3). Notably, co-loaded lipodisks at a ratio of 2:1 presented pronounced synergism against MCF-7/ADR cells. The clear synergistic outcomes of lipodisks in vitro may be explained by the coordination and cooperation of the transfer and release behaviors of the two drugs, such as ratiometric cellular uptake, controlled and effective

3.6. In vitro cytotoxicity studies To determine whether improved cellular uptake of co-loaded lipodisks could translate into better therapeutic outcomes, the anti-proliferative effects of various preparations against drug-resistant MCF-7 cells and its parent MCF-7/ADR cells were determined. As shown in both Fig. 5A and B, lipodisks showed negligible toxicity towards MCF-7 and MCF-7/ADR cells across a large range of concentrations of total lipids. In particular, SAPSP peptides incorporated into lipodisks did not induce any further toxic effects on tumor cells, indicating that tumorspecific lipodisks were safe and biocompatible. Next, the in vitro cytotoxicity of drug-loaded lipodisks and free drugs against two cell lines were detected. After incubation for 48 h, all formulations demonstrated dose-dependent inhibitory activities against drug-sensitive MCF-7 cells (Fig. 5C) and drug-resistant MCF-7/ADR cells (Fig. 5D). Compared with the single drug solution, the combination of DOX and PTX enhanced the inhibitory effects on the growth of tumor cells to various extents. Among all drug formulations, co-loaded lipodisks exhibited the highest cytotoxicity at all tested concentrations. Furthermore, the IC50 values of different formulations were summarized in Table 3, revealing that the IC50 value of the DOX-lipodisk against DOX-sensitive MCF-7 cells was slightly higher than free DOX. This may be partly attributed to different cellular uptake pathways of encapsulated DOX and free DOX, as well as the sustained release behavior of DOX from lipodisks. More specifically, free DOX could rapidly cross the cell membranes of MCF-7 cells by passive diffusion to exert its antitumor effects inside cells (Du et al., 2017; Feng et al., 2017b; Wang et al., 2017b). By contrast, DOX-lipodisks were mainly taken up by

Fig. 6. Pharmacokinetic profiles of DOX and PTX in tumor-bearing mice after intravenous injection of co-loaded lipodisks and free cocktail solution, respectively. Dosage was 5 mg/kg DOX and 7.9 mg/kg PTX. Data were presented as mean ± SD, n = 6. 199

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Table 4 The main pharmacokinetic parameters of co-loaded lipodisks or free drug solutions (n = 6). Pharmacokinetic parameters

t1/2 (h) Tmax (h) Cmax (μmol/L) AUC0-t (μmol/L*h) AUC0-inf_obs (μmol/L*h) Vz_obs ((mg/kg)/(μmol/L)) Cl_obs ((mg/kg)/(μmol/L)/h)

Free drug solution

Co-loaded lipodisk

DOX

PTX

DOX

PTX

1.811 ± 0.373 0.250 ± 0.000 65.100 ± 3.847 76.897 ± 4.971 76.544 ± 4.411 0.168 ± 0.043 0.064 ± 0.004

0.656 ± 0.118 0.300 ± 0.100 53.400 ± 8.089 61.102 ± 5.958 61.138 ± 5.948 0.124 ± 0.030 0.130 ± 0.013

10.990 ± 2.601 1.150 ± 0.436 27.820 ± 4.510 299.131 ± 7.255 381.020 ± 43.785 0.205 ± 0.027 0.013 ± 0.001

13.413 ± 1.901 1.150 ± 0.436 33.344 ± 1.208 339.555 ± 6.777 479.297 ± 23.077 0.316 ± 0.032 0.016 ± 0.001

t1/2: half-life of drug. Cmax: maximum plasma concentration. Tmax: time of maximum plasma concentration. AUC0-t: area under the drug concentration-time curve values from time 0 till time t. AUC0-inf_obs: area under the drug concentration-time curve values from time 0 till infinity. Vz_obs: apparent volume of distribution. Cl_obs: clearance.

Fig. 7. Biodistribution of DOX (A) and PTX (B) in tumor-bearing mice at 1, 6, and 24 h after intravenous injection of co-loaded lipodisks or free drug solution. Dosage was 5 mg/kg DOX and 7.86 mg/kg PTX. Data were expressed as mean ± SD, n = 3.

intracellular drug release, and multiple inhibitory effects at their individual sites of action. Taken together, co-loaded lipodisks at a DOX/ PTX ratio of 2:1 have potential to treat drug-resistant tumor cells.

As shown in Fig. 6, a significant difference in drug concentrations versus time profiles was evident for co-loaded lipodisks. For free solutions, DOX and PTX were rapidly eliminated from the systemic blood circulation after administration, whereas drugs encapsulated into lipodisks achieved higher blood levels of the two drugs and longer circulation times. Thus, a higher plasma AUC was achieved in lipodisk samples compared with free drug solution. As expected, the AUC0-t values of DOX and PTX in lipodisks were 4.0-fold and 5.8-fold greater

3.7. Pharmacokinetics and biodistribution studies The pharmacokinetics of co-loaded lipodisks and free drug solutions were investigated using tumor-bearing nude mice via tail vein injection. 200

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Fig. 8. In vivo anti-tumor efficiency of various formulations in nude mice bearing xenograft tumors. Various formulations, including normal saline, free drug solution (5 mg/kg of DOX and 7.9 mg/kg of PTX, DOX/PTX = 1:1, m/m), free drug solution (6.7 mg/kg of DOX and 5.3 mg/kg of PTX, DOX/PTX = 2:1, m/m), co-loaded lipodisk without SAPSP moiety (6.7 mg/kg of DOX and 5.3 mg/kg of PTX, DOX/PTX = 2:1, m/ m), and co-loaded lipodisk containing SAPSP (5 mg/kg of DOX and 7.9 mg/kg of PTX, DOX/PTX = 1:1, m/m), and co-loaded lipodisk containing SAPSP (6.7 of mg/kg DOX and 5.3 mg/kg of PTX, DOX/ PTX = 2:1, m/m), were administered on days 0, 3, 6, 9, and 12. Tumor volume changes were measured in MCF-7 tumor bearing mice (A) and MCF-7/ADR tumor bearing mice (B) after administration of ** different formulations. p < 0.01, *** p < 0.001. (C) Weight of excised tumors at sacrifice. (D) Changes in body weight were recorded during treatment. Data were expressed as mean ± SD, n = 5.

than that of free DOX and DOX, respectively (Table 4). These results demonstrated that co-loaded lipodisks with a sustained release property could significantly improve the bioavailability of both chemotherapeutic agents. To further evaluate the ratiometric delivery efficacy of lipodisks in vivo, the biodistribution profiles of DOX and PTX were determined in tumor-bearing mice after the administration of either free dual-drug solution or co-loaded lipodisks. Free DOX and PTX showed little accumulation in the tumor after 24 h but relatively high deposition in other organs, such as the lung, liver, spleen and kidney, indicating that the administration of free cocktail solution may not exert its full synergistic anti-tumor effect (Fig. 7A and B). The low amount in the tumor and high concentration in the normal tissues, particularly in heart, would contribute to severe systemic toxicity. By contrast, the concentration of DOX and PTX from lipodisks in tumors was 4.16-fold and 5.70-fold higher than the free drug solution, respectively (Fig. 7A and B). This increased uptake of drugs by tumor tissues was most likely due to the enhanced permeability and retention (EPR) effect, as well as the

selective binding ability of the SAPSP peptide. Notably, the concentration of DOX and PTX in tumors was further analyzed using a grouped t-test, showing that there was no significant difference between the two drugs. This result indicated that the two drugs loaded in lipodisks were delivered into tumors at predefined synergistic ratios after 24 h post-injection. Conversely, compared with PTX, the higher uptake of DOX in the tumor after injection with the free solution was observed. According to previous reports, variations in pharmacokinetics and in vivo biodistribution would partly result in the failure of combination therapy. Taken together, these results indicated that the benefits of lipodisks are to coordinate the distinct pharmacokinetics of two drugs and maintain the ratios of the drugs for systemic delivery to tumor tissues. 3.8. In vivo anti-tumor effect of co-loaded lipodisks in a xenograft tumor model As discussed above, one of the benefits of lipodisks was to 201

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ratiometrically deliver two drugs into tumor tissues with an in vitro predetermined ratio to obtain an improved anti-cancer efficacy in vivo. Thus, various formulations were further investigated in MCF-7 and MCF-7/ADR breast tumor-bearing mouse model. Tumors were allowed to develop until the tumor volumes reached approximately 100 mm3, followed by treatment with a total of 5 injections of either co-loaded lipodisk or free solutions at different DOX/PTX molar ratios. As shown in Fig. 8A, compared with the uncontrolled tumor growth in the saline treatment, free cocktail solution with a DOX/PTX molar ratios of either 1:1 or 2:1 demonstrated little inhibitory effect at the same injection schedule, likely a result of the low accumulation in tumor tissues. The normal co-loaded lipodisk without SAPSP moiety, labeled as non-targeting lipodisk, showed a modest anti-cancer activity at the dual-drug ratio of 2:1, while much stronger therapeutic responses were observed in mice that were treated with SAPSP-modified co-loaded lipodisks at DOX/PTX molar ratios of 2:1 and 1:1, indicating the improved inhibitory efficacy of DOX and PTX in combination and the benefit of tumor-specific SAPSP group. More specifically, the tumor weight of the co-loaded lipodisk treated groups at DOX/PTX molar ratios of 1:1 and 2:1 were 19.3% and 17.4% of the control group at the end of in vivo experiment, respectively, which were also approximately 2.5-fold smaller than mice treated with the free dual-drug solution at two DOX/ PTX ratios (Fig. 8C). However, when free cocktail solution was used to treat the drugresistant MCF-7/ADR tumor at a DOX/PTX ratio of either 1:1 or 2:1, tumor inhibition was significantly compromised (Fig. 8B), and the tumor weight on the last day of measurement was not significantly different from the control group (Fig. 8C). For the same dosage, all the loaded drugs in lipodisks with or without SAPSP showed better antitumor effect compared with free drug solutions. Among all groups, tumor-targeted co-loaded lipodisk at the DOX/PTX molar ratio of 2:1 exhibited the best anti-tumor efficacy, achieving 65.1% inhibition efficacy for tumor volume (Fig. 8B) and 35.7% for tumor weight (Fig. 8C) compared with the saline treated group. These results suggested that the combination of many factors, including tumor-selective binding peptide, co-loaded and co-delivery property of lipodisk, and synergistic drug ratios, was responsible for the apparent antitumor effects observed in animal study. In addition, changes in body weight are considered a crucial indicator of systemic safety (Chen et al., 2018). As shown in Fig. 8D and E, slight weight loss was observed in groups treated with free drug solutions at two DOX/PTX ratios. By contrast, all co-loaded lipodisk groups, whether they contained tumor-specific SAPSP or not, did not cause a reduction in body weight, and there was no significant difference compared with the saline group. Collectively, these results emphasize the importance of the EPR effect, the selective binding property of SAPSP moiety, and the synergistic effect of two drugs based on the lipodisk formulations.

Conflict of interest The authors declare no competing financial interests. Acknowledgments Financial support was from the National Natural Science Foundation of China (Nos. 81402880, 81602656, and 51703086), the Natural Science Foundation of Jiangsu Province for Youth (Nos. BK20160496, BK20160546, BK20170533) and Natural Science Foundation of Jiangsu Province (General Program: BK20181445), and the Scientific Research Foundation of Jiangsu University (Nos. 14JDG163 and 16JDG030). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2019.02.009. References Ahlgren, S., Fondell, A., Gedda, L., Edwards, K., 2017a. EGF-targeting lipodisks for specific delivery of poorly water-soluble anticancer agents to tumour cells. RSC Adv. 7, 22178–22186. Ahlgren, S., Reijmar, K., Edwards, K., 2017b. Targeting lipodisks enable selective delivery of anticancer peptides to tumor cells. Nanomedicine 13, 2325–2328. Aryal, S., Hu, C.M., Zhang, L., 2011. Polymeric nanoparticles with precise ratiometric control over drug loading for combination therapy. Mol. Pharm. 8, 1401–1407. Baabur-Cohen, H., Vossen, L.I., Kruger, H.R., Eldar-Boock, A., Yeini, E., Landa-Rouben, N., Tiram, G., Wedepohl, S., Markovsky, E., Leor, J., Calderon, M., Satchi-Fainaro, R., 2017. In vivo comparative study of distinct polymeric architectures bearing a combination of paclitaxel and doxorubicin at a synergistic ratio. J. Control. Release 257, 118–131. Bar-Zeev, M., Livney, Y.D., Assaraf, Y.G., 2017. Targeted nanomedicine for cancer therapeutics: towards precision medicine overcoming drug resistance. Drug Resist. Updat. 31, 15–30. Blanco, E., Shen, H., Ferrari, M., 2015. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941–951. Chen, B., Cheng, J., Wu, Y., Gao, F., Xu, W., Shen, H., Ding, J., Gao, C., Sun, Q., Sun, X., Cheng, H., Li, G., Chen, W., Chen, N., Liu, L., Li, X., Wang, X., 2009. Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and 5-bromotetrandrine in xenograft nude-mice. Int. J. Nanomed. 4, 73–78. Chen, C.-W., Lu, D.-W., Yeh, M.-K., Shiau, C.-Y., Chiang, C.-H., 2011. Novel RGD-lipid conjugate-modified liposomes for enhancing siRNA delivery in human retinal pigment epithelial cells. Int. J. Nanomed. 6, 2567–2580. Chen, C., Tao, R., Ding, D., Kong, D.L., Fan, A.P., Wang, Z., Zhao, Y.J., 2017. Ratiometric co-delivery of multiple chemodrugs in a single nanocarrier. Eur. J. Pharm. Sci. 107, 16–23. Chen, Y., Cheng, Y., Zhao, P., Zhang, S., Li, M., He, C., Zhang, X., Yang, T., Yan, R., Ye, P., Ma, X., Xiang, G., 2018. Co-delivery of doxorubicin and imatinib by pH sensitive cleavable PEGylated nanoliposomes with folate-mediated targeting to overcome multidrug resistance. Int. J. Pharm. 542, 266–279. Chen, Y., Yuan, L., Zhou, L., Zhang, Z.H., Cao, W., Wu, Q., 2012. Effect of cell-penetrating peptide-coated nanostructured lipid carriers on the oral absorption of tripterine. Int. J. Nanomed. 7, 4581–4591. Chen, Y., Zhang, W., Huang, Y., Gao, F., Sha, X., Fang, X., 2015. Pluronic-based functional polymeric mixed micelles for co-delivery of doxorubicin and paclitaxel to multidrug resistant tumor. Int. J. Pharm. 488, 44–58. Cheng, B., Pan, H., Liu, D., Li, D., Li, J., Yu, S., Tan, G., Pan, W., 2018. Functionalization of nanodiamond with vitamin E TPGS to facilitate oral absorption of curcumin. Int. J. Pharm. 540, 162–170. Choi, Y.H., Yu, A.M., 2014. ABC transporters in multidrug resistance and pharmacokinetics, and strategies for drug development. Curr. Pharm. Des. 20, 793–807. Chou, T.C., 2006. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 58, 621–681. Chou, T.C., 2007. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug-combination studies (vol 58, pg 621, 2006). Pharmacol. Rev. 59, 124. Chou, T.C., 2010. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 70, 440–446. Copolovici, D.M., Langel, K., Eriste, E., Langel, U., 2014. Cell-penetrating peptides: design, synthesis, and applications. ACS Nano 8, 1972–1994. Danhier, F., Feron, O., Preat, V., 2010. To exploit the tumor microenvironment: passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J. Control. Release 148, 135–146. Decuzzi, P., Godin, B., Tanaka, T., Lee, S.Y., Chiappini, C., Liu, X., Ferrari, M., 2010. Size and shape effects in the biodistribution of intravascularly injected particles. J. Control. Release 141, 320–327.

4. Conclusions We developed a tumor-specific lipodisk delivery system for coloading and co-delivery of DOX and PTX. Discoidal lipodisks also encapsulate DOX and PTX together. A tumor-selective binding profile of lipodisks was shown in response to tumor extracellular acidic stimuli (pH 6.5–6.8), but this formulation showed favorable stability under physiological conditions (pH 7.4). At the cellular level, co-loaded lipodisks displayed improved cell internalization, ratiometric tumor penetration, and significant cytotoxicity. At the in vivo tumor level, the coloaded lipodisks were significantly accumulated in the tumor due to the EPR effect and SAPAP peptides. Ultimately, co-loaded lipodisks efficiently inhibited the growth of both MCF-7 and MCF-7/ADR tumors. Therefore, our co-loaded lipodisks with SAPSP peptides may represent a synergistic tumor therapy. 202

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