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phospholipid mixed micelles evaluated in vitro and in vivo in breast cancer
Accepted Manuscript Title: Juglone loaded Poloxamer 188/phospholipid mixed micelles evaluated in vitro and in vivo in breast cancer Author: Xin Jin Youwen Zhang Zhenhai Zhang Danbiao Che Huixia Lv PII: DOI: Reference:
S0378-5173(16)30961-9 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.10.027 IJP 16157
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
16-8-2016 24-9-2016 12-10-2016
Please cite this article as: Jin, Xin, Zhang, Youwen, Zhang, Zhenhai, Che, Danbiao, Lv, Huixia, Juglone loaded Poloxamer 188/phospholipid mixed micelles evaluated in vitro and in vivo in breast cancer.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.10.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Juglone loaded Poloxamer 188/phospholipid mixed micelles evaluated in vitro and in vivo in breast cancer Xin Jin* a,Youwen Zhang a, Zhenhai Zhang c, Danbiao Che a, Huixia Lv *b a
Department of hospital pharmacy, The First Hospital of Suqian, 120 Suzhilu, Suqian 223800,
China b
Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China c
Jiangsu Province Hospital on Integration of Chinese and Western Medicine affiliated with
Nanjing University of Chinese Medicine, 100 Shizijie, Nanjing 210028, China .
Corresponding author: Xin Jin, Tel: (+86)15951896676
E-mail: [email protected]
Huixia Lv , Tel:(+86)02585309785 E-mail: [email protected]
Graphical abstract
Abstract Aim: Investigating the effects of juglone loaded P188/phospholipid mixed micelles
1
(J-MM) in breast cancer. Materials & Methods: In vitro cytotoxicity, apoptotic effects, in vivo therapeutic efficacy and toxicity were used to assess its antitumour effect. Uptake and imaging were used to evaluate the effect on the uptake and passive targeting. Results: Mixed micelle carrier enhanced the targeting and uptake by MB-231 cells. The tumour inhibition rates in tumour xenograft models for paclitaxel, juglone, J-MM (10 mg/kg) and J-MM (40 mg/kg) were 46%, 27%, 39% and 53%, respectively. J-MM (10 mg/kg) exhibited lower toxicity compared with that by free juglone or high dose J-MM. Conclusion: J-MM exhibited low toxicity, improved cellular uptake, passive targeting and anti-cancer effects in breast cancer model. Keywords: juglone, mixed micelles, antitumor, toxicity, uptake
1. Introduction Quinones are widely distributed in nature and have immense scope for clinical application. Many quinones, such as adriamycin, daunorubicin, actinomycin D, mitomycin
C,
and
mitoxantrone
exhibit
anticancer
activity.
Juglone
(5-hydroxy-1,4-naphtha-quinone), a naturally occurring naphthoquinone, is found in the roots, leaves, nut-hulls, bark and wood of J. manshurica Maxim (Matławsk I et al., 2015). Previous studies have demonstrated that juglone could induce apoptosis through different mechanisms including mitochondrial pathway, down regulation of the Bcl-2/Bax ratio and clastogenic action (Xu et al.,2013; Avcı et al., 2016; Meskelevicius et al., 2016; Sajadimajd et al., 2016 ). It could also arrest the cell cycle and inhibit cell proliferation by inactivation of cyclin D1 protein and targeting the 2
transient receptor potential ankyrin subtype 1 channel (Kiran et al., 2009; Fang et al., 2015; Hill et al., 2016). Although juglone should be a promising agent for oncotherapy, its clinical applications were limited owing to its poor solubility in aqueous solutions, low absorption and generalised cytotoxicity to normal tissues (Aithal et al., 2011). Using nano-delivery carriers is an efficient strategy in the field of cancer therapy to overcome the aforementioned limitations by improving the therapeutic efficacy and reducing systemic toxicity (Chaurasiya et al., 2016; Talluri et al., 2016). Mixed micelle system is an example that has attracted our attention because of its specific advantages. Mixed micelles are made from more than one surfactant, often are small in size (Russo et al., 2016; Shi et al., 2015; Singh et al., 2016), and can enhance the drug’s solubility ( Danhier et al., 2015; López-Dávila et al., 2016; Mao et al., 2015). They also have longer circulating half-life because of the micelles’ stable outer hydrophilic shell (Ribeiro et al., 2012; Jindal., 2015; Rezazadeh et al. 2016). These characteristics along with their enhanced permeability and retention (EPR) effect make them attractive drug delivery vehicles for reducing nonspecific uptake by reticuloendothelial system and increasing the uptake by tumour cells (Elvang et al., 2016; Shen et al., 2016; Zhuang et al., 2016). Poloxamer 188 (P188), an amphiphilic copolymer, is composed of two polyethylene oxide (PEO) chains, separated by a polypropylene oxide (PPO) chain (Yan et al., 2010). Food and Drug Administration (FDA) recognized P188 as a safe pharmaceutical excipient. The PEO brushes off P188 like PEGylated surfactant, often 3
used to modify the surface of micelles for minimizing phagocytosis by immune system and increasing the circulating half-life in vivo (Zhang et al., 2015). However, micelles composed of P188 alone have low drug entrapment efficiency and poor stability due to the low lipophilic core. Therefore, these micelles break down easily, which restricts their application. Thus, P188 needs to be combined with other micelle forming polymers to increase the drug loading capacity. The excellent biocompatibility and especially, the amphiphilicity of phospholipids make them important pharmaceutical excipients that are widely used in drug delivery systems such as liposomes and emulsions. Phospholipids with high content of long chain polyunsaturated fatty acids can be added to P188 micelles in order to increase the hydrophobic lumen of the micelle, thus increasing the drug loading capacity of micelles. According to the recently published Global Cancer Statistics, breast cancer alone accounts for 25% of all cancer cases and 15% of all cancer deaths among women. It is the most frequently diagnosed cancer and the leading cause of cancer deaths among women worldwide, with an estimated 1.7 million cases and 521,900 deaths in 2012 (Torre et al., 2015). Therefore, breast cancer was chosen for this study, and MB-231, a breast cancer cell line was used as the experimental model. A mixed micelle formulation comprising poloxamer 188 and phospholipids was developed to incorporate juglone into the lumen of the micelle. The cytotoxicity and apoptotic effects of this formulation were studied in vitro, to assess its antitumor effect. In addition, we investigated the cellular uptake by MB-231 cells and used in vivo 4
imaging to examine for any enhancement in uptake and passive targeting effect. Finally, the therapeutic efficacy and toxicity were evaluated in vivo in a xenograft model.
2. Materials and methods 2.1 Materials Juglone, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide (DiR), coumarin-6 (C-6) and 4',6-diamidino-2-phenylindole (DAPI) were all purchased from Aladdin Scientific Inc. (Shanghai, China). Phospholipids were purchased from Shanghai Advanced Vehicle Technology Co., Ltd. (Shanghai, China). Poloxamer 188 was obtained from BASF China Co., Ltd. 5-diphenyl-2H-terazolium bromide (MTT), propidium iodide (PI), fluorescein isothiocyanate (FITC)-AV and Triton X-100 were all obtained from Nanjing KeyGen Biotech Inc. (Nanjing, China). All other solvents used in this investigation were of HPLC grade. 2.2 Cell lines and animals The human breast cancer cells (MB-231) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), which contained 10% FBS, streptomycin (100 μg/mL) and penicillin (100 U/mL). The cells were maintained under an atmosphere of 5% CO2 at 37 °C. Nude mice (20 ± 2 g body weight) were purchased from SLEK Lab Animal Center of Shanghai (Shanghai, China). All animal experiments were performed in accordance with the principles of care and use of laboratory animals and were approved by the 5
experimental animal administrative committee of China Pharmaceutical University. 2.3 Preparation and characterisation of juglone mixed micelles (J-MM) J-MM were prepared by thin film dispersed method. Juglone, P188 and phospholipids were all dissolved in anhydrous ethanol with the help of ultrasound to form the settled solution. Uniform thin films were obtained by removing the solvent by rotary vacuum evaporation at 37 °C. Subsequently, 10 ml deionized water was added and dispersed to hydrate the films overnight at 25°C. Finally, free juglone was removed by filtration with a 0.45 μm filter membrane to obtain J-MM suspension. C-6 mixed micelles (C-6-MM) or DiR mixed micelles (DiR-MM) were prepared by the same method by substituting Juglone with C-6 and DiR respectively. Hydrodynamic diameter and zeta potential of J-MM were measured using Malvern zetasizer nano series instrument (Zetasizer Nano ZSP, Melvin, England). The morphology of J-MM was observed using a transmission electronic microscope (JEM-2000EX, JEOL, Japan). The samples were diluted 10 times with deionized water, placed on a copper grid coated with carbon film, and air-dried. The samples were then stained with 2% phosphotungstic acid for 3 min and air dried prior to TEM analysis. 2.4 In vitro antitumor effect 2.4.1 In vitro cytotoxicity studies The cytotoxicity of juglone and J-MM against MB-231 breast tumour cells was determined using the MTT dye assay. The cells were incubated at 37 °C in a 5% CO2 incubator for 24 h with 5×103 cells/well plated in 96-well plates. Samples were 6
diluted with culture media to make various concentrations of juglone or J-MM in quintuple (0.25-64 μM). Control wells were treated with equivalent volumes of free media. The supernatant was removed after 24 h. MTT (0.5 mg/ml) in culture medium (100 μl) was added to each well and incubated for 4 h. The unreduced MTT and medium were then discarded. Each well was washed with DMSO to dissolve the MTT formazan crystals. Plates were shaken for 10 min. The absorbance at 570 nm was used to assess the relative cell viability. 2.4.2 Cell cycle analysis MB-231 cells seeded on six-well plates were treated with juglone or J-MM (juglone concentration of 10 μM) at 37 °C for 24 h. The cells treated with serum free culture medium served as control. At the end of incubation, cells were harvested by treatment with 0.05% trypsin, washed twice with cold PBS, fixed with 70% cold ethanol and immobilized at 4 °C for 12 h. Cells were centrifuged again, washed with cold PBS twice, incubated and stained with PI staining solution in the dark for 0.5 h at 37 °C. The DNA content was measured by flow cytometry, and the percentage of cells in each phase of the cell cycle was evaluated using the ModFit software (Becton Dickinson, CA, USA). 2.4.3 In vitro wound healing assay The MB-231 tumour cells in medium containing 10% FBS were seeded in 24-well plates. When the cells reached confluence, a 1-mm wide scratch was created in the cell monolayer of each well by using a 200-μl pipette tip. The medium and floating cells were removed. The attached cells were washed with PBS. Then, new medium 7
containing juglone or J-MM (10 μM) was added. The pictures of the scratches were taken again after 24 h of incubation. The distance of cell migration was measured under microscope and compared with that of the cells in control wells. 2.4.4 TUNEL analysis DNA fragmentation was recorded by TUNEL to detect apoptosis in situ. Briefly, MB-231 cells were harvested by treatment with 0.05% trypsin,after treated with juglone or J-MM (10 μM) for 24 h. Collectted cells by centrifugation, washed with PBS, and resuspended cells at a concentration of 2×105 cells/ML in PBS. Transferred resuspended cells on microscopic slides, then immobilized in 4% paraformaldehyde for 20 min after air seasoning. After permeated in Triton-X100 at 4 °C for 5 min, the cells were treated with reaction buffer at 37 °C for 60 min and washed twice with PBS. After that, the cells were treated with stain buffer at 37 °C for 30 min in dark and washed twice with PBS. Fragmented DNA that was dyed brown was photographed using a microscope (OLYMPUS IX51). 2.4.5 Cell apoptosis assay Cell apoptosis was detected by the Annexin V/PI staining assay. MB-231 cells were treated with juglone or J-MM at juglone concentration of 10 μM for 24 h. Untreated cells acted as control for this experiment. Both non-adherent and adherent MB-231 cells were collected, washed with cold PBS, and re-suspended in 200 μl binding buffer that contained 5 μl Annexin V-FITC (Sigma–Aldrich St. Louis, MO, USA). Cells were gently mixed, incubated at 25°C in dark for 10 min, and stained with 10-μl PI solution. Cells were analysed immediately by FACS FCM (Beckman Coulter). 8
Annexin-V-FITC+/PI- cells were considered as early apoptotic cells, and Annexin-V-FITC+/PI+ cells were considered as late apoptotic cells. 2.4.6 Western blot analysis Expression of apoptosis related proteins in MB-231 cells was detected by western blot analysis. MB-231 cells were treated with free juglone solution or J-MM at juglone concentration of 10 μM for 24 h. Untreated cells acted as control for this experiment. Cells were collected, washed, and lysed. Proteins separated by Polyacrylamide gel electrophoresis were transferred onto membrane and probed with Bax, Bcl-2, Cleaved-Caspase-3, -8, -9 or Cleaved-PARP. Antibodies were all obtained from Cell Signaling Technologies (Beverly, MA). The protein bands were visualized by enhanced chemiluminescence detection kit (Beyotime, Shanghai, China). Densitometric measurements of the scanned bands were performed using Image pro plus (IPP) software program. Data were normalized to GAPDH. 2.5 Uptake and targeted studies 2.5.1 Cellular uptake and intracellular localization in vitro MB-231 cells were seeded in 24-well plates at a density of 1×105 cells/well and incubated for 24 h. The medium was then changed to medium without serine. Free C-6 was used as a model drug. To study the effect of mixed micelles on uptake, MB-231 cells were incubated with free C-6 or C-6-MM at the same concentrations (C-6 concentration: 500 ng/mL) for 4 h at 37 °C. The culture medium was then removed and cells were washed thrice with PBS. The cells were fixed with 4% formaldehyde for 20 min at 25°C, and the cell nuclei were stained with DAPI for 20 9
min. The cellular uptake was observed under fluorescent microscope (IX71; Olympus Corp, Tokyo, Japan). The intracellular localization was observed by laser scanning confocal microscope (TCS SP5; Leica, Heidelberg, Germany). 2.5.2 Targeted delivery to subcutaneous tumour To assess the targeting efficiency of P188/Phospholipids mixed micelles, free DiR and DiR-MM (20 μg/ml of DiR) in normal saline were intravenously injected into mice (n = 3) bearing subcutaneous tumours of 200 mm3 size via tail vein. The mice were anesthetized with sevoflurane and monitored by fluorescence imaging using IVIS Spectrum at 1 h, 3 h, 6 h, 12 h, and 24 h. At the end of the experiment, the mice were sacrificed, and major organs were collected and imaged. 2.6 Assessment of in vivo therapeutic efficacy and toxicity Day 0 was defined as the day on which the tumour volume reached about 50 mm3. The animals were randomly assigned to six groups of six each. Each group was injected through the tail vein with one of the following: normal saline, 10 mg/kg PTX, 10 mg/kg juglone, 40 mg/kg juglone, 10 mg/kg J-MM and 40 mg/kg J-MM at days 0, 3, 6, 9, and 12. The tumour volume and body weight of each mouse was monitored at 3-day intervals. On day 15th, mice were sacrificed, and the tumour, liver, kidney tissues and blood were removed for subsequent assays. The tissues were stored in 4% paraformaldehyde for a week, embedded in paraffin, and cut into sections. The paraffin embedded sections were used for hematoxylin and eosin (H&E assay). The blood samples were drawn from the ophthalmic vein and examined by a routine blood examination. White blood cells (WBC), red blood cells 10
(RBC) and platelets (PLT) were counted using blood cell counting chamber. Aspartate transaminase (AST), alanine aminotransferase (ALT) and blood urea nitrogen (BUN) levels were assayed by Elisa. Briefly, plasma was collected and followed by centrifugation at 3000 g for 15 min. The concentration of AST, ALT and BUN in the supernatants was quantitated by their ELISA kit according to the manufacturer's instructions on a microplate reader. 2.7 Statistical analysis Data were expressed as the mean ± SD. Student’s two-sample t-test was utilized for statistical analysis (spss 12.0, American). In all cases, P <0.05 was considered as significant.
3. Results 3.1 Characterisation of the juglone loaded mixed micelles Optimized formulation of drug loaded mixed micelles was achieved at the concentration of 1.48 mg/ml juglone, and 1 to 4 ratio of P188 to phospholipids. Its physicochemical characterization was examined and showed in table 1. This formulation yielded 89.67% entrapment efficiency. The hydrodynamic diameter of the micelles thus formed was 108.5 ± 9.7 nm. J-MM displayed homogeneous particle sizes of 108.5 ± 9.7 nm with 0.231 polydispersity index (PDI) (Fig. 1A) and a negative surface (Fig. 1B) charge of about -9.78 mV. The solubility of juglone in the form of J-MM was almost 48 times higher than that of the free form. The TEM images (Fig. 1C) further illustrated that the J-MM had homogeneous spherical shapes 11
and smooth surfaces without cracks. 3.2 In vitro antitumor effect 3.2.1 Cytotoxicity of free juglone and J-MM against MB-231 cells In vitro cytotoxicity of free juglone and J-MM against MB-231 cells was investigated by MTT assay after 24 h of incubation (Fig. 2A). J-MM exhibited stronger inhibition rates compared with that by free juglone, at every concentration. The IC50 value of J-MM was almost 45% lower than that of free juglone (10.35 μM vs. 5.67 μM). The cytotoxicity of J-MM was significantly higher than that of free juglone, suggesting that the drug’s uptake by MB-231 cells was enhanced in the form of mixed micelles. 3.2.2 Cell cycle analysis Cells were treated with juglone or J-MM for 24 h and cell cycle was studied using flow cytometry following PI staining. The results showed that juglone increased the proportion of cells in G2/M phase and decreased that of those in G0/G1 and S phase. The increased rate of arrest at G2/M phase is an indication of the inhibition of cell division and slowing of cell growth. The various cell cycle phases of MB-231 cells after treatment with juglone or J-MM, are shown in Fig. 2B, which shows that J-MM caused significantly greater accumulation of cells in the G2/M phase after 24 h of incubation, compared to that by juglone. 3.2.3 Effects of J-MM on migration The cells were incubated with medium, juglone or J-MM, and the migration of cells was monitored over 24 h (Fig. 2C). The migration of MB-231 cells treated with free 12
juglone was lower compared to that of control cells. Treatment with J-MM resulted in greater inhibition of migration, probably due to increased cellular uptake of juglone. 3.2.4 Apoptotic effects induced by J-MM Induction of apoptosis is an important strategy used in cancer chemotherapy (Wong, 2011). In order to investigate the underlying mechanisms involved in the antitumor effect of J-MM, we performed a series of studies including cell morphology observation, Annexin V/PI dual staining, and examination of apoptosis related proteins. DNA was stained with haematoxylin to observe nuclear condensation. The indicators of apoptosis, including reduction in nuclear size, chromatin condensation, and nuclear crack, are stained brown (Emanuele et al., 2007). Observation under a microscope revealed that the percentage of apoptotic cells in the J-MM treated group was significantly higher than that in the free juglone group, as showed in Fig. 3A. Early and late apoptosis were quantified by the Annexin V/PI dual staining method. During apoptosis, apoptotic cells present transmembrane phosphatidylserine, which can be labelled by Annexin V-FITC. Increased membrane permeability in the late apoptotic cells allows PI to cross the cell membranes. Therefore, Annexin-V+ and PIstaining indicates early apoptotic cells, whereas Annexin-V+ and PI+ staining indicates late apoptotic cells. As shown in Fig. 3B, few apoptotic cells were observed after treatment with blank medium. However, treatment with free juglone or J-MM for 24 h significantly induced cell apoptosis, with more severe effects observed in the J-MM treated group than in the free juglone group. 13
Cleavage of effector caspases, bcl-2/bax ratio, and nuclear substrates including Poly (ADP-ribose) polymerase (PARP) are involved in the induction of apoptosis. In this study, the effects of J-MM on cleaved caspase-3, 8, 9 activation, bcl-2/bax ratio and PARP in MB-231 cells were assayed by western blot analysis. As shown in Fig. 3C, untreated control had very low levels of cleaved caspase and cleaved PARP. In contrast, cleaved caspase and cleaved PARP levels were elevated in cells treated with J-MM. J-MM treatment also decreased the bcl-2/bax ratio significantly. Collectively, these data demonstrated that J-MM enhanced the apoptotic effect of juglone against MB-231 cells. 3.3 Uptake and targeting studies 3.3.1 Cellular uptake and intracellular localization in vitro Due to the easier internalization and longer half-life of nano-sized micelles, increased cellular uptake has been regarded as an important reason for the superior anti-cancerous effect of drug-loaded micelles. To determine the uptake and intracellular localization of mixed micelles in MB-231 cells, we performed double labelling experiments using fluorescence microscopy and laser scanning confocal microscopy. C-6 exhibits green fluorescence. The nuclei were visualized with blue fluorescence after DAPI staining. As shown in Fig. 4A, the cellular uptake of C-6 mixed micelles after 4 h incubation was remarkably higher than that of free C-6. Fig 4B shows the localization of the C-6 in the cytoplasm rather than in the nucleus. The fluorescence intensity of free C-6 in cytoplasm was lower than that of C-6 MM. Therefore, it can be inferred that mixed micelles of P188 and phospholipid could 14
promote the uptake of drugs. This can explain the enhanced antitumor effect of J-MM compared to that of free juglone in vitro. 3.3.2 In vivo tumour targeting effect of mixed micelles The tumour targeting effects of mixed micelles were evaluated by DiR labelling and the subsequent observation using near infrared (NIR) fluorescence imaging system (Fig. 5). No signals were observed in the tumours in free DiR treated group. However, DiR-MM displayed better targeting efficiency compared to that of free DiR and attained maximum absorbance at 6 h. This result indicated that mixed micelle carriers could improve the targeting of DiR to the tumour site. Concurrent with the increased uptake by the tumour cells, the fluorescence intensity of liver and spleen also increased, probably because of the nonspecific uptake, partly by the reticuloendothelial system. Ex vivo images of excised organs and tumours also showed that the uptake of DiR by the tumour cells increased up to 24 h due to the passive targeting effect. The group treated with DiR-MM also exhibited strong fluorescence. Thus, both the in vivo and in vitro results were consistent and suggested that P188/ phospholipid mixed micelle was a promising drug carrier for promoting absorption in antitumour therapy. 3.4 Assessment of in vivo therapeutic efficacy and toxicity Finally, the in vivo antitumour efficacy was tested in an MB-231 xenograft nude mouse model. Strong suppression of tumour volume was observed in the group receiving J-MM (40 mg/kg) compared to that in the saline group (Fig. 6A). At the end of the study, the mean tumour size was 297 mm3, which was higher than the average 15
initial volume of 50 mm3. Notably, when free juglone (40 mg/kg) was administered, there was significant mortality due to toxicity. After being encapsulated in the mixed micelles, the toxicity of juglone decreased significantly. At a drug dose equivalent to 10 mg/kg free juglone, J-MM, endowed with high permeability and targeted effect, could significantly increase the antitumour effect. The treatment in any of the groups did not affect the normal activity or body weight of mice. At the end of the study, the tumours, livers and kidneys were excised. The weights of the tumour were consistent with the tumour growth curve (Fig. 6B). The tumour inhibition rates of free PTX, juglone, J-MM (10 mg/kg), and J-MM (40 mg/kg) were 46%, 27%, 39%, and 53%, respectively, which indicated that the antitumor effect better than the PTX group was observed only in J-MM (40 mg/kg) group. The tissues were then further analysed by haematoxylin and eosin staining (Fig. 6C). As expected, J-MM demonstrated strong apoptotic effect on the tumours. It also exhibited lower toxicity toward liver and kidney. Serious side effects including haematotoxicity, nephrotoxicity, and hepatotoxicity are factors that limit the clinical application of juglone. In order to systematically evaluate these side effects in the experimental animals in this study, certain clinical parameters that indicate the physiological status of the blood, liver, and kidneys were evaluated (Fig. 6D). The results were consistent with our microscopy findings, and indicated that J-MM exhibited lower toxicity compared with that of free juglone. However, J-MM in high dose caused side effects similar to those in the PTX group. These findings reinforce the conclusion that J-MM had greater therapeutic efficacy and lower toxicity than that 16
by juglone alone.
4. Discussion The aim of the current study was to prepare novel and simple mixed micelles from poloxamer 188 and phospholipids for passive targeted delivery of juglone. Micelles ≤ 200 nm in size and high drug solubility were obtained. These properties were reported to increase the retention and bioavailability of the drug by preventing nonspecific uptake, increasing its penetration through surfactant, and promoting its uptake (Kaklotar et al., 2016). The in vitro studies of the cellular uptake and targeting of J-MM demonstrated that using mixed micelle carriers could promote the uptake of drugs. In the in vivo tumour targeting study, intense whole body fluorescence was observed continuously up to 24 h after injection, indicating the prolonged circulation time of the DiR-MM. The results also demonstrated the effective accumulation of the mixed micelles in tumour tissue. This appreciable tumour targeting ability may have resulted from the prolonged circulation time achieved by the PEO brushes surface, the small size, the stability of the mixed micelles and the EPR effect in tumour tissue (Mayol et al., 2015; Zhang et al., 2016). Apoptosis and cell proliferation are common clinical indicators for the assessment of tumour prognosis and tumour response to therapy. Both in vitro and in vivo results in the current study have showed that targeted J-MM inhibited cell proliferation and increased apoptosis in MB-231 cells. Enhanced apoptosis combined with reduced proliferation rate was correlated with extensive tumour necrosis in breast cancer. 17
Therefore, the results of the present study can be attributed to the enhanced antitumour activity. Biocompatibility is also a major consideration for developing drug carriers. Poloxamer 188, as a triblock copolymer, has been approved by the FDA for use in injections. Intravenous injection formulation containing poloxamer 188 as excipient was well tolerated and proven safe in clinical studies (Adams-Graves et al., 1997). Phospholipids have often been used to exert a membrane “sealing effect”. Insertion of phospholipids into poloxamer 188 micelles may increase the lipid packing density, decrease the leakage and side effects (Wu et al., 2009; Tagami et al., 2015). The discovery of juglone could not be exploited for therapeutic purposes early on, because of its high levels of cytotoxicity, until the poloxamer 188/phospholipid mixed micelles were introduced to lessen its adverse effects. Although J-MM in high dose group showed certain side effects, intravenous administration of J-MM at low dose (10 mg/kg) did not cause any significant adverse effects in the vital haematological parameters by the end of the monitoring period, indicating absence of high toxicity. The results of the histopathological evaluation of livers and kidneys, consistent with those of haematological evaluation, suggest that J-MM could be safely injected systemically into mice without adverse effects. The results of our studies demonstrate that the mixed micelles with better biocompatibility could decrease the cytotoxicity of juglone.
Conclusion 18
This study suggested that passively targeted J-MM has the potential to serve as an effective and tolerable chemotherapy for breast cancer treatment. It could reduce tumour volume and exert enhanced antitumour activity without apparent toxicity, compared with that by non-targeted formulations.
Financial & competing interests disclosure There are no disclosures or any conflict of interests.
Acknowledgments This work was supported by National Natural Science Foundation of China (Grant No. 81403119), 333 project of Jiangsu Province and the Science and Technology Support Project of Suqian (S210520).
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24
Figure caption Fig 1 Characterizations of J-MM for average sizes (A), zeta potential (B) and TEM (C). Fig 2 In vitro cytotoxicity assays. MTT assay (A), cell cycle (B) and wound healing assay (C). Fig 3 Promote apoptotic effects induced by J-MM. TUNNEL (A), apoptosis analysis (B), and apoptosis-related protein expressions (C). Fig 4 Determination of cellular uptake (A) and intracellular localization (B) for mixed micelles on MB-231 cells. Fig 5 In vivo and ex vivo NIRF optical images of MB-231 nude mice injected intravenously with different formations. Fig 6 Tumor volumes as functions of time (A), tumor weight at the end of experiments(B), images of H&E-stained sections of tumor, liver and kidney excised from subcutaneous tumor-bearing mice (C) and
the toxicity studies for evaluating
the WBC, RBC, PLT, AST, ALT and BUN at the end of the experiments(D).
25
Fig 1
26
Fig 2
27
Fig 3
28
Fig4
29
Fig 5
30
Fig 6
31
Table 1 Physicochemical characterization of juglone loaded mixed micelles Note: Values are expressed as mean ± SD (n=3).
Formulation
DL(% )
EE(%)
0.231±0.0 3.13±0 /phospholipids 108.5 ± 9.7 -9.78±1.32 2 .28 (n:n=1:4) Abbreviations: Juglone loaded mixed micelles, micelles composed of Poloxamer 188/ phospholipids (n:n =1:4) loaded with juglone; DL, drug loading efficiency; EE, encapsulation efficiency; SD, standard deviation.
89.67± 9.15
juglone loaded mixed micelles
Composition
Zeta-average size (nm)
Poloxamer 188
32
Zeta potential (mv)
polydispe rsity
S0378-5173(16)30961-9 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.10.027 IJP 16157
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
16-8-2016 24-9-2016 12-10-2016
Please cite this article as: Jin, Xin, Zhang, Youwen, Zhang, Zhenhai, Che, Danbiao, Lv, Huixia, Juglone loaded Poloxamer 188/phospholipid mixed micelles evaluated in vitro and in vivo in breast cancer.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.10.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Juglone loaded Poloxamer 188/phospholipid mixed micelles evaluated in vitro and in vivo in breast cancer Xin Jin* a,Youwen Zhang a, Zhenhai Zhang c, Danbiao Che a, Huixia Lv *b a
Department of hospital pharmacy, The First Hospital of Suqian, 120 Suzhilu, Suqian 223800,
China b
Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China c
Jiangsu Province Hospital on Integration of Chinese and Western Medicine affiliated with
Nanjing University of Chinese Medicine, 100 Shizijie, Nanjing 210028, China .
Corresponding author: Xin Jin, Tel: (+86)15951896676
E-mail: [email protected]
Huixia Lv , Tel:(+86)02585309785 E-mail: [email protected]
Graphical abstract
Abstract Aim: Investigating the effects of juglone loaded P188/phospholipid mixed micelles
1
(J-MM) in breast cancer. Materials & Methods: In vitro cytotoxicity, apoptotic effects, in vivo therapeutic efficacy and toxicity were used to assess its antitumour effect. Uptake and imaging were used to evaluate the effect on the uptake and passive targeting. Results: Mixed micelle carrier enhanced the targeting and uptake by MB-231 cells. The tumour inhibition rates in tumour xenograft models for paclitaxel, juglone, J-MM (10 mg/kg) and J-MM (40 mg/kg) were 46%, 27%, 39% and 53%, respectively. J-MM (10 mg/kg) exhibited lower toxicity compared with that by free juglone or high dose J-MM. Conclusion: J-MM exhibited low toxicity, improved cellular uptake, passive targeting and anti-cancer effects in breast cancer model. Keywords: juglone, mixed micelles, antitumor, toxicity, uptake
1. Introduction Quinones are widely distributed in nature and have immense scope for clinical application. Many quinones, such as adriamycin, daunorubicin, actinomycin D, mitomycin
C,
and
mitoxantrone
exhibit
anticancer
activity.
Juglone
(5-hydroxy-1,4-naphtha-quinone), a naturally occurring naphthoquinone, is found in the roots, leaves, nut-hulls, bark and wood of J. manshurica Maxim (Matławsk I et al., 2015). Previous studies have demonstrated that juglone could induce apoptosis through different mechanisms including mitochondrial pathway, down regulation of the Bcl-2/Bax ratio and clastogenic action (Xu et al.,2013; Avcı et al., 2016; Meskelevicius et al., 2016; Sajadimajd et al., 2016 ). It could also arrest the cell cycle and inhibit cell proliferation by inactivation of cyclin D1 protein and targeting the 2
transient receptor potential ankyrin subtype 1 channel (Kiran et al., 2009; Fang et al., 2015; Hill et al., 2016). Although juglone should be a promising agent for oncotherapy, its clinical applications were limited owing to its poor solubility in aqueous solutions, low absorption and generalised cytotoxicity to normal tissues (Aithal et al., 2011). Using nano-delivery carriers is an efficient strategy in the field of cancer therapy to overcome the aforementioned limitations by improving the therapeutic efficacy and reducing systemic toxicity (Chaurasiya et al., 2016; Talluri et al., 2016). Mixed micelle system is an example that has attracted our attention because of its specific advantages. Mixed micelles are made from more than one surfactant, often are small in size (Russo et al., 2016; Shi et al., 2015; Singh et al., 2016), and can enhance the drug’s solubility ( Danhier et al., 2015; López-Dávila et al., 2016; Mao et al., 2015). They also have longer circulating half-life because of the micelles’ stable outer hydrophilic shell (Ribeiro et al., 2012; Jindal., 2015; Rezazadeh et al. 2016). These characteristics along with their enhanced permeability and retention (EPR) effect make them attractive drug delivery vehicles for reducing nonspecific uptake by reticuloendothelial system and increasing the uptake by tumour cells (Elvang et al., 2016; Shen et al., 2016; Zhuang et al., 2016). Poloxamer 188 (P188), an amphiphilic copolymer, is composed of two polyethylene oxide (PEO) chains, separated by a polypropylene oxide (PPO) chain (Yan et al., 2010). Food and Drug Administration (FDA) recognized P188 as a safe pharmaceutical excipient. The PEO brushes off P188 like PEGylated surfactant, often 3
used to modify the surface of micelles for minimizing phagocytosis by immune system and increasing the circulating half-life in vivo (Zhang et al., 2015). However, micelles composed of P188 alone have low drug entrapment efficiency and poor stability due to the low lipophilic core. Therefore, these micelles break down easily, which restricts their application. Thus, P188 needs to be combined with other micelle forming polymers to increase the drug loading capacity. The excellent biocompatibility and especially, the amphiphilicity of phospholipids make them important pharmaceutical excipients that are widely used in drug delivery systems such as liposomes and emulsions. Phospholipids with high content of long chain polyunsaturated fatty acids can be added to P188 micelles in order to increase the hydrophobic lumen of the micelle, thus increasing the drug loading capacity of micelles. According to the recently published Global Cancer Statistics, breast cancer alone accounts for 25% of all cancer cases and 15% of all cancer deaths among women. It is the most frequently diagnosed cancer and the leading cause of cancer deaths among women worldwide, with an estimated 1.7 million cases and 521,900 deaths in 2012 (Torre et al., 2015). Therefore, breast cancer was chosen for this study, and MB-231, a breast cancer cell line was used as the experimental model. A mixed micelle formulation comprising poloxamer 188 and phospholipids was developed to incorporate juglone into the lumen of the micelle. The cytotoxicity and apoptotic effects of this formulation were studied in vitro, to assess its antitumor effect. In addition, we investigated the cellular uptake by MB-231 cells and used in vivo 4
imaging to examine for any enhancement in uptake and passive targeting effect. Finally, the therapeutic efficacy and toxicity were evaluated in vivo in a xenograft model.
2. Materials and methods 2.1 Materials Juglone, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide (DiR), coumarin-6 (C-6) and 4',6-diamidino-2-phenylindole (DAPI) were all purchased from Aladdin Scientific Inc. (Shanghai, China). Phospholipids were purchased from Shanghai Advanced Vehicle Technology Co., Ltd. (Shanghai, China). Poloxamer 188 was obtained from BASF China Co., Ltd. 5-diphenyl-2H-terazolium bromide (MTT), propidium iodide (PI), fluorescein isothiocyanate (FITC)-AV and Triton X-100 were all obtained from Nanjing KeyGen Biotech Inc. (Nanjing, China). All other solvents used in this investigation were of HPLC grade. 2.2 Cell lines and animals The human breast cancer cells (MB-231) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), which contained 10% FBS, streptomycin (100 μg/mL) and penicillin (100 U/mL). The cells were maintained under an atmosphere of 5% CO2 at 37 °C. Nude mice (20 ± 2 g body weight) were purchased from SLEK Lab Animal Center of Shanghai (Shanghai, China). All animal experiments were performed in accordance with the principles of care and use of laboratory animals and were approved by the 5
experimental animal administrative committee of China Pharmaceutical University. 2.3 Preparation and characterisation of juglone mixed micelles (J-MM) J-MM were prepared by thin film dispersed method. Juglone, P188 and phospholipids were all dissolved in anhydrous ethanol with the help of ultrasound to form the settled solution. Uniform thin films were obtained by removing the solvent by rotary vacuum evaporation at 37 °C. Subsequently, 10 ml deionized water was added and dispersed to hydrate the films overnight at 25°C. Finally, free juglone was removed by filtration with a 0.45 μm filter membrane to obtain J-MM suspension. C-6 mixed micelles (C-6-MM) or DiR mixed micelles (DiR-MM) were prepared by the same method by substituting Juglone with C-6 and DiR respectively. Hydrodynamic diameter and zeta potential of J-MM were measured using Malvern zetasizer nano series instrument (Zetasizer Nano ZSP, Melvin, England). The morphology of J-MM was observed using a transmission electronic microscope (JEM-2000EX, JEOL, Japan). The samples were diluted 10 times with deionized water, placed on a copper grid coated with carbon film, and air-dried. The samples were then stained with 2% phosphotungstic acid for 3 min and air dried prior to TEM analysis. 2.4 In vitro antitumor effect 2.4.1 In vitro cytotoxicity studies The cytotoxicity of juglone and J-MM against MB-231 breast tumour cells was determined using the MTT dye assay. The cells were incubated at 37 °C in a 5% CO2 incubator for 24 h with 5×103 cells/well plated in 96-well plates. Samples were 6
diluted with culture media to make various concentrations of juglone or J-MM in quintuple (0.25-64 μM). Control wells were treated with equivalent volumes of free media. The supernatant was removed after 24 h. MTT (0.5 mg/ml) in culture medium (100 μl) was added to each well and incubated for 4 h. The unreduced MTT and medium were then discarded. Each well was washed with DMSO to dissolve the MTT formazan crystals. Plates were shaken for 10 min. The absorbance at 570 nm was used to assess the relative cell viability. 2.4.2 Cell cycle analysis MB-231 cells seeded on six-well plates were treated with juglone or J-MM (juglone concentration of 10 μM) at 37 °C for 24 h. The cells treated with serum free culture medium served as control. At the end of incubation, cells were harvested by treatment with 0.05% trypsin, washed twice with cold PBS, fixed with 70% cold ethanol and immobilized at 4 °C for 12 h. Cells were centrifuged again, washed with cold PBS twice, incubated and stained with PI staining solution in the dark for 0.5 h at 37 °C. The DNA content was measured by flow cytometry, and the percentage of cells in each phase of the cell cycle was evaluated using the ModFit software (Becton Dickinson, CA, USA). 2.4.3 In vitro wound healing assay The MB-231 tumour cells in medium containing 10% FBS were seeded in 24-well plates. When the cells reached confluence, a 1-mm wide scratch was created in the cell monolayer of each well by using a 200-μl pipette tip. The medium and floating cells were removed. The attached cells were washed with PBS. Then, new medium 7
containing juglone or J-MM (10 μM) was added. The pictures of the scratches were taken again after 24 h of incubation. The distance of cell migration was measured under microscope and compared with that of the cells in control wells. 2.4.4 TUNEL analysis DNA fragmentation was recorded by TUNEL to detect apoptosis in situ. Briefly, MB-231 cells were harvested by treatment with 0.05% trypsin,after treated with juglone or J-MM (10 μM) for 24 h. Collectted cells by centrifugation, washed with PBS, and resuspended cells at a concentration of 2×105 cells/ML in PBS. Transferred resuspended cells on microscopic slides, then immobilized in 4% paraformaldehyde for 20 min after air seasoning. After permeated in Triton-X100 at 4 °C for 5 min, the cells were treated with reaction buffer at 37 °C for 60 min and washed twice with PBS. After that, the cells were treated with stain buffer at 37 °C for 30 min in dark and washed twice with PBS. Fragmented DNA that was dyed brown was photographed using a microscope (OLYMPUS IX51). 2.4.5 Cell apoptosis assay Cell apoptosis was detected by the Annexin V/PI staining assay. MB-231 cells were treated with juglone or J-MM at juglone concentration of 10 μM for 24 h. Untreated cells acted as control for this experiment. Both non-adherent and adherent MB-231 cells were collected, washed with cold PBS, and re-suspended in 200 μl binding buffer that contained 5 μl Annexin V-FITC (Sigma–Aldrich St. Louis, MO, USA). Cells were gently mixed, incubated at 25°C in dark for 10 min, and stained with 10-μl PI solution. Cells were analysed immediately by FACS FCM (Beckman Coulter). 8
Annexin-V-FITC+/PI- cells were considered as early apoptotic cells, and Annexin-V-FITC+/PI+ cells were considered as late apoptotic cells. 2.4.6 Western blot analysis Expression of apoptosis related proteins in MB-231 cells was detected by western blot analysis. MB-231 cells were treated with free juglone solution or J-MM at juglone concentration of 10 μM for 24 h. Untreated cells acted as control for this experiment. Cells were collected, washed, and lysed. Proteins separated by Polyacrylamide gel electrophoresis were transferred onto membrane and probed with Bax, Bcl-2, Cleaved-Caspase-3, -8, -9 or Cleaved-PARP. Antibodies were all obtained from Cell Signaling Technologies (Beverly, MA). The protein bands were visualized by enhanced chemiluminescence detection kit (Beyotime, Shanghai, China). Densitometric measurements of the scanned bands were performed using Image pro plus (IPP) software program. Data were normalized to GAPDH. 2.5 Uptake and targeted studies 2.5.1 Cellular uptake and intracellular localization in vitro MB-231 cells were seeded in 24-well plates at a density of 1×105 cells/well and incubated for 24 h. The medium was then changed to medium without serine. Free C-6 was used as a model drug. To study the effect of mixed micelles on uptake, MB-231 cells were incubated with free C-6 or C-6-MM at the same concentrations (C-6 concentration: 500 ng/mL) for 4 h at 37 °C. The culture medium was then removed and cells were washed thrice with PBS. The cells were fixed with 4% formaldehyde for 20 min at 25°C, and the cell nuclei were stained with DAPI for 20 9
min. The cellular uptake was observed under fluorescent microscope (IX71; Olympus Corp, Tokyo, Japan). The intracellular localization was observed by laser scanning confocal microscope (TCS SP5; Leica, Heidelberg, Germany). 2.5.2 Targeted delivery to subcutaneous tumour To assess the targeting efficiency of P188/Phospholipids mixed micelles, free DiR and DiR-MM (20 μg/ml of DiR) in normal saline were intravenously injected into mice (n = 3) bearing subcutaneous tumours of 200 mm3 size via tail vein. The mice were anesthetized with sevoflurane and monitored by fluorescence imaging using IVIS Spectrum at 1 h, 3 h, 6 h, 12 h, and 24 h. At the end of the experiment, the mice were sacrificed, and major organs were collected and imaged. 2.6 Assessment of in vivo therapeutic efficacy and toxicity Day 0 was defined as the day on which the tumour volume reached about 50 mm3. The animals were randomly assigned to six groups of six each. Each group was injected through the tail vein with one of the following: normal saline, 10 mg/kg PTX, 10 mg/kg juglone, 40 mg/kg juglone, 10 mg/kg J-MM and 40 mg/kg J-MM at days 0, 3, 6, 9, and 12. The tumour volume and body weight of each mouse was monitored at 3-day intervals. On day 15th, mice were sacrificed, and the tumour, liver, kidney tissues and blood were removed for subsequent assays. The tissues were stored in 4% paraformaldehyde for a week, embedded in paraffin, and cut into sections. The paraffin embedded sections were used for hematoxylin and eosin (H&E assay). The blood samples were drawn from the ophthalmic vein and examined by a routine blood examination. White blood cells (WBC), red blood cells 10
(RBC) and platelets (PLT) were counted using blood cell counting chamber. Aspartate transaminase (AST), alanine aminotransferase (ALT) and blood urea nitrogen (BUN) levels were assayed by Elisa. Briefly, plasma was collected and followed by centrifugation at 3000 g for 15 min. The concentration of AST, ALT and BUN in the supernatants was quantitated by their ELISA kit according to the manufacturer's instructions on a microplate reader. 2.7 Statistical analysis Data were expressed as the mean ± SD. Student’s two-sample t-test was utilized for statistical analysis (spss 12.0, American). In all cases, P <0.05 was considered as significant.
3. Results 3.1 Characterisation of the juglone loaded mixed micelles Optimized formulation of drug loaded mixed micelles was achieved at the concentration of 1.48 mg/ml juglone, and 1 to 4 ratio of P188 to phospholipids. Its physicochemical characterization was examined and showed in table 1. This formulation yielded 89.67% entrapment efficiency. The hydrodynamic diameter of the micelles thus formed was 108.5 ± 9.7 nm. J-MM displayed homogeneous particle sizes of 108.5 ± 9.7 nm with 0.231 polydispersity index (PDI) (Fig. 1A) and a negative surface (Fig. 1B) charge of about -9.78 mV. The solubility of juglone in the form of J-MM was almost 48 times higher than that of the free form. The TEM images (Fig. 1C) further illustrated that the J-MM had homogeneous spherical shapes 11
and smooth surfaces without cracks. 3.2 In vitro antitumor effect 3.2.1 Cytotoxicity of free juglone and J-MM against MB-231 cells In vitro cytotoxicity of free juglone and J-MM against MB-231 cells was investigated by MTT assay after 24 h of incubation (Fig. 2A). J-MM exhibited stronger inhibition rates compared with that by free juglone, at every concentration. The IC50 value of J-MM was almost 45% lower than that of free juglone (10.35 μM vs. 5.67 μM). The cytotoxicity of J-MM was significantly higher than that of free juglone, suggesting that the drug’s uptake by MB-231 cells was enhanced in the form of mixed micelles. 3.2.2 Cell cycle analysis Cells were treated with juglone or J-MM for 24 h and cell cycle was studied using flow cytometry following PI staining. The results showed that juglone increased the proportion of cells in G2/M phase and decreased that of those in G0/G1 and S phase. The increased rate of arrest at G2/M phase is an indication of the inhibition of cell division and slowing of cell growth. The various cell cycle phases of MB-231 cells after treatment with juglone or J-MM, are shown in Fig. 2B, which shows that J-MM caused significantly greater accumulation of cells in the G2/M phase after 24 h of incubation, compared to that by juglone. 3.2.3 Effects of J-MM on migration The cells were incubated with medium, juglone or J-MM, and the migration of cells was monitored over 24 h (Fig. 2C). The migration of MB-231 cells treated with free 12
juglone was lower compared to that of control cells. Treatment with J-MM resulted in greater inhibition of migration, probably due to increased cellular uptake of juglone. 3.2.4 Apoptotic effects induced by J-MM Induction of apoptosis is an important strategy used in cancer chemotherapy (Wong, 2011). In order to investigate the underlying mechanisms involved in the antitumor effect of J-MM, we performed a series of studies including cell morphology observation, Annexin V/PI dual staining, and examination of apoptosis related proteins. DNA was stained with haematoxylin to observe nuclear condensation. The indicators of apoptosis, including reduction in nuclear size, chromatin condensation, and nuclear crack, are stained brown (Emanuele et al., 2007). Observation under a microscope revealed that the percentage of apoptotic cells in the J-MM treated group was significantly higher than that in the free juglone group, as showed in Fig. 3A. Early and late apoptosis were quantified by the Annexin V/PI dual staining method. During apoptosis, apoptotic cells present transmembrane phosphatidylserine, which can be labelled by Annexin V-FITC. Increased membrane permeability in the late apoptotic cells allows PI to cross the cell membranes. Therefore, Annexin-V+ and PIstaining indicates early apoptotic cells, whereas Annexin-V+ and PI+ staining indicates late apoptotic cells. As shown in Fig. 3B, few apoptotic cells were observed after treatment with blank medium. However, treatment with free juglone or J-MM for 24 h significantly induced cell apoptosis, with more severe effects observed in the J-MM treated group than in the free juglone group. 13
Cleavage of effector caspases, bcl-2/bax ratio, and nuclear substrates including Poly (ADP-ribose) polymerase (PARP) are involved in the induction of apoptosis. In this study, the effects of J-MM on cleaved caspase-3, 8, 9 activation, bcl-2/bax ratio and PARP in MB-231 cells were assayed by western blot analysis. As shown in Fig. 3C, untreated control had very low levels of cleaved caspase and cleaved PARP. In contrast, cleaved caspase and cleaved PARP levels were elevated in cells treated with J-MM. J-MM treatment also decreased the bcl-2/bax ratio significantly. Collectively, these data demonstrated that J-MM enhanced the apoptotic effect of juglone against MB-231 cells. 3.3 Uptake and targeting studies 3.3.1 Cellular uptake and intracellular localization in vitro Due to the easier internalization and longer half-life of nano-sized micelles, increased cellular uptake has been regarded as an important reason for the superior anti-cancerous effect of drug-loaded micelles. To determine the uptake and intracellular localization of mixed micelles in MB-231 cells, we performed double labelling experiments using fluorescence microscopy and laser scanning confocal microscopy. C-6 exhibits green fluorescence. The nuclei were visualized with blue fluorescence after DAPI staining. As shown in Fig. 4A, the cellular uptake of C-6 mixed micelles after 4 h incubation was remarkably higher than that of free C-6. Fig 4B shows the localization of the C-6 in the cytoplasm rather than in the nucleus. The fluorescence intensity of free C-6 in cytoplasm was lower than that of C-6 MM. Therefore, it can be inferred that mixed micelles of P188 and phospholipid could 14
promote the uptake of drugs. This can explain the enhanced antitumor effect of J-MM compared to that of free juglone in vitro. 3.3.2 In vivo tumour targeting effect of mixed micelles The tumour targeting effects of mixed micelles were evaluated by DiR labelling and the subsequent observation using near infrared (NIR) fluorescence imaging system (Fig. 5). No signals were observed in the tumours in free DiR treated group. However, DiR-MM displayed better targeting efficiency compared to that of free DiR and attained maximum absorbance at 6 h. This result indicated that mixed micelle carriers could improve the targeting of DiR to the tumour site. Concurrent with the increased uptake by the tumour cells, the fluorescence intensity of liver and spleen also increased, probably because of the nonspecific uptake, partly by the reticuloendothelial system. Ex vivo images of excised organs and tumours also showed that the uptake of DiR by the tumour cells increased up to 24 h due to the passive targeting effect. The group treated with DiR-MM also exhibited strong fluorescence. Thus, both the in vivo and in vitro results were consistent and suggested that P188/ phospholipid mixed micelle was a promising drug carrier for promoting absorption in antitumour therapy. 3.4 Assessment of in vivo therapeutic efficacy and toxicity Finally, the in vivo antitumour efficacy was tested in an MB-231 xenograft nude mouse model. Strong suppression of tumour volume was observed in the group receiving J-MM (40 mg/kg) compared to that in the saline group (Fig. 6A). At the end of the study, the mean tumour size was 297 mm3, which was higher than the average 15
initial volume of 50 mm3. Notably, when free juglone (40 mg/kg) was administered, there was significant mortality due to toxicity. After being encapsulated in the mixed micelles, the toxicity of juglone decreased significantly. At a drug dose equivalent to 10 mg/kg free juglone, J-MM, endowed with high permeability and targeted effect, could significantly increase the antitumour effect. The treatment in any of the groups did not affect the normal activity or body weight of mice. At the end of the study, the tumours, livers and kidneys were excised. The weights of the tumour were consistent with the tumour growth curve (Fig. 6B). The tumour inhibition rates of free PTX, juglone, J-MM (10 mg/kg), and J-MM (40 mg/kg) were 46%, 27%, 39%, and 53%, respectively, which indicated that the antitumor effect better than the PTX group was observed only in J-MM (40 mg/kg) group. The tissues were then further analysed by haematoxylin and eosin staining (Fig. 6C). As expected, J-MM demonstrated strong apoptotic effect on the tumours. It also exhibited lower toxicity toward liver and kidney. Serious side effects including haematotoxicity, nephrotoxicity, and hepatotoxicity are factors that limit the clinical application of juglone. In order to systematically evaluate these side effects in the experimental animals in this study, certain clinical parameters that indicate the physiological status of the blood, liver, and kidneys were evaluated (Fig. 6D). The results were consistent with our microscopy findings, and indicated that J-MM exhibited lower toxicity compared with that of free juglone. However, J-MM in high dose caused side effects similar to those in the PTX group. These findings reinforce the conclusion that J-MM had greater therapeutic efficacy and lower toxicity than that 16
by juglone alone.
4. Discussion The aim of the current study was to prepare novel and simple mixed micelles from poloxamer 188 and phospholipids for passive targeted delivery of juglone. Micelles ≤ 200 nm in size and high drug solubility were obtained. These properties were reported to increase the retention and bioavailability of the drug by preventing nonspecific uptake, increasing its penetration through surfactant, and promoting its uptake (Kaklotar et al., 2016). The in vitro studies of the cellular uptake and targeting of J-MM demonstrated that using mixed micelle carriers could promote the uptake of drugs. In the in vivo tumour targeting study, intense whole body fluorescence was observed continuously up to 24 h after injection, indicating the prolonged circulation time of the DiR-MM. The results also demonstrated the effective accumulation of the mixed micelles in tumour tissue. This appreciable tumour targeting ability may have resulted from the prolonged circulation time achieved by the PEO brushes surface, the small size, the stability of the mixed micelles and the EPR effect in tumour tissue (Mayol et al., 2015; Zhang et al., 2016). Apoptosis and cell proliferation are common clinical indicators for the assessment of tumour prognosis and tumour response to therapy. Both in vitro and in vivo results in the current study have showed that targeted J-MM inhibited cell proliferation and increased apoptosis in MB-231 cells. Enhanced apoptosis combined with reduced proliferation rate was correlated with extensive tumour necrosis in breast cancer. 17
Therefore, the results of the present study can be attributed to the enhanced antitumour activity. Biocompatibility is also a major consideration for developing drug carriers. Poloxamer 188, as a triblock copolymer, has been approved by the FDA for use in injections. Intravenous injection formulation containing poloxamer 188 as excipient was well tolerated and proven safe in clinical studies (Adams-Graves et al., 1997). Phospholipids have often been used to exert a membrane “sealing effect”. Insertion of phospholipids into poloxamer 188 micelles may increase the lipid packing density, decrease the leakage and side effects (Wu et al., 2009; Tagami et al., 2015). The discovery of juglone could not be exploited for therapeutic purposes early on, because of its high levels of cytotoxicity, until the poloxamer 188/phospholipid mixed micelles were introduced to lessen its adverse effects. Although J-MM in high dose group showed certain side effects, intravenous administration of J-MM at low dose (10 mg/kg) did not cause any significant adverse effects in the vital haematological parameters by the end of the monitoring period, indicating absence of high toxicity. The results of the histopathological evaluation of livers and kidneys, consistent with those of haematological evaluation, suggest that J-MM could be safely injected systemically into mice without adverse effects. The results of our studies demonstrate that the mixed micelles with better biocompatibility could decrease the cytotoxicity of juglone.
Conclusion 18
This study suggested that passively targeted J-MM has the potential to serve as an effective and tolerable chemotherapy for breast cancer treatment. It could reduce tumour volume and exert enhanced antitumour activity without apparent toxicity, compared with that by non-targeted formulations.
Financial & competing interests disclosure There are no disclosures or any conflict of interests.
Acknowledgments This work was supported by National Natural Science Foundation of China (Grant No. 81403119), 333 project of Jiangsu Province and the Science and Technology Support Project of Suqian (S210520).
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24
Figure caption Fig 1 Characterizations of J-MM for average sizes (A), zeta potential (B) and TEM (C). Fig 2 In vitro cytotoxicity assays. MTT assay (A), cell cycle (B) and wound healing assay (C). Fig 3 Promote apoptotic effects induced by J-MM. TUNNEL (A), apoptosis analysis (B), and apoptosis-related protein expressions (C). Fig 4 Determination of cellular uptake (A) and intracellular localization (B) for mixed micelles on MB-231 cells. Fig 5 In vivo and ex vivo NIRF optical images of MB-231 nude mice injected intravenously with different formations. Fig 6 Tumor volumes as functions of time (A), tumor weight at the end of experiments(B), images of H&E-stained sections of tumor, liver and kidney excised from subcutaneous tumor-bearing mice (C) and
the toxicity studies for evaluating
the WBC, RBC, PLT, AST, ALT and BUN at the end of the experiments(D).
25
Fig 1
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Fig 2
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Fig 3
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Fig4
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Fig 6
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Table 1 Physicochemical characterization of juglone loaded mixed micelles Note: Values are expressed as mean ± SD (n=3).
Formulation
DL(% )
EE(%)
0.231±0.0 3.13±0 /phospholipids 108.5 ± 9.7 -9.78±1.32 2 .28 (n:n=1:4) Abbreviations: Juglone loaded mixed micelles, micelles composed of Poloxamer 188/ phospholipids (n:n =1:4) loaded with juglone; DL, drug loading efficiency; EE, encapsulation efficiency; SD, standard deviation.
89.67± 9.15
juglone loaded mixed micelles
Composition
Zeta-average size (nm)
Poloxamer 188
32
Zeta potential (mv)
polydispe rsity