Cerasomal Lovastatin Nanohybrids for Efficient Inhibition of Triple

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Cerasomal Lovastatin Nanohybrids for Efficient Inhibition of TripleNegative Breast Cancer Stem Cells to Improve Therapeutic Efficacy Liujiang Song, Xiaojun Tao, Li Lin, Chao Chen, Hui Yao, Guangchun He, Guangyang Zou, Zhong Cao, Shichao Yan, Lu Lu, Huimei Yi, Di Wu, Siyuan Tan, Wanxin Ouyang, Zhifei Dai, and Xiyun Deng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01633 • Publication Date (Web): 06 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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ACS Applied Materials & Interfaces

Cerasomal Lovastatin Nanohybrids for Efficient Inhibition of Triple-Negative Breast Cancer Stem Cells to Improve Therapeutic Efficacy

Liujiang Song1,#, Xiaojun Tao1,#, Li Lin2,#, Chao Chen1, Hui Yao1, Guangchun He1, Guangyang Zou2, Zhong Cao2, Shichao Yan1, Lu Lu1, Huimei Yi1, Di Wu1, Siyuan Tan1, Wanxin Ouyang1, Zhifei Dai2,*, Xiyun Deng1,* 1

Hunan Normal University Medical College, Department of Clinical Medicine (LS),

Department of Pharmacy (XT, DW, ST, WO), Department of Pathology (CC, HY, GH, SY, LL, XD), Changsha, Hunan 410013, China; 2

Department of Biomedical Engineering, College of Engineering, Peking University,

Beijing 100871, China.

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ABSTRACT Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a higher risk in younger women and a poorer prognosis and without targeted therapies available currently. Cancer stem cells (CSCs) are increasingly recognized as the main cause of treatment failure and tumor recurrence. The present paper reported the encapsulation of lovastatin (LV) into cerasomes. Compared with free LV, C-LV nanohybrids showed cytotoxicity to MDA-MB-231 CSCs in a dose- and time-dependent manner. Furthermore, intravenous injection of C-LV nanohybrids resulted in significant tumor size reduction in a dose-dependent manner in xenograft tumors derived from subcutaneous inoculation of MDA-MB-231 cells. Further, histopathological and/or immunohistochemical analysis revealed that C-LV nanohybrids significantly induced mammary gland formation and apoptosis and inhibited angiogenesis, the CSC phenotype, and the epithelial-to-mesenchymal transition (EMT) in xenograft tumors. Most importantly, C-LV nanohybrids were found to be more effective than free LV in inhibiting the growth of breast cancer xenografts and the stemness properties in vivo. To the best of our knowledge, it is the first demonstration of nanohybrids for efficient inhibition of cancer stem cells derived from TNBC, offering a new option for the TNBC treatment.

KEYWORDS: cancer stem cells; cerasome; lovastatin; triple-negative breast cancer; epithelial-to-mesenchymal transition

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ABBREVIATIONS C-LV

Cerasome-encapsulated lovastatin

CSCs

Cancer stem cells

DSPC

Distearoyl phosphatidylcholine

EMT

Epithelial-to-mesenchymal transition

EPR

Enhanced permeability and retention

LV

Lovastatin

PCs

Parental cells

SFCs

Sphere-forming cells

TEM

Transmission electron microscopy

TNBC

Triple-negative breast cancer

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INTRODUCTION Breast cancer is one of the most common malignancies around the world.1-2 In particular, triple-negative breast cancer (TNBC), which accounts for about 15-20% of all breast cancer cases, is a subtype of breast cancer with aggressive behavior and poor prognosis.3 Due to the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), there is currently no targeted therapies available for the treatment of TNBC.4 Nowadays, broad-spectrum anti-cancer drugs remain the mainstay of treatment for TNBC.5 Commonly used chemotherapeutic drugs, such as doxorubicin and taxol, exhibit high dose-limiting toxicity to tumor cells as well as normal cells, which limits the clinical effectiveness of cancer therapy.6-7 Therefore, the development of more favorable targeted drugs for those difficult-to-treat diseases such as TNBC is an urgent task for both clinicians and basic scientists.

Cancer stem cells (CSCs), which have the potential for self-renewal and multidirectional differentiation, are considered the primary cause of tumor formation, metastasis, and drug resistance.8-9 Since the first isolation of CD44+ CD24-/low breast cancer stem cells from patients via pleural effusion by Al-Hajj et al.,10 a large number of studies on breast cancer stem cells have demonstrated their importance in tumor resistance and recurrence.11 Recently, the antibiotic salinomycin and its derivatives have shown selective targeting effect of CSCs in different types of human cancers.12-13 In breast cancer, however, they don’t show preference to TNBC over non-TNBC. Furthermore, although these antibiotics show great promise in targeting CSCs, they have shown significant toxicity in vivo.14-15

Lovastatin (LV) is the first statin approved by the FDA to treat hyperlipidemia with little toxic profile.16-17 Over the past decades, evidence has emerged that LV can inhibit proliferation and induce differentiation or apoptosis of tumor cells. In breast cancer, in vitro studies have shown that LV can induce apoptosis10, 18 and inhibit cell migration and invasion.19-20 In mice, LV can inhibit the primary growth and pulmonary metastasis of breast cancer cells. Recently, we have shown that LV could decrease the expression level of CD44 and inhibit the stemness properties of nasopharyngeal carcinoma CSCs, and sensitize these cells to chemotherapy and photodynamic therapy in vitro.21 Further, we have demonstrated that LV could 4


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reverse the epithelial-to-mesenchymal transition (EMT) phenotype of breast cancer stem cells by altering the levels of EMT-related proteins and also confer enhanced sensitivity to certain chemotherapeutic drugs such as doxorubicin in TNBC [manuscript in preparation]. Therefore, LV is expected to be a candidate drug for the treatment of TNBC through targeting breast cancer stem cells.

Currently available formulation of LV used in the clinic is the orally administered preparation, which often fails to achieve the desired effect due to its poor water solubility and bioavailability.22 One possible solution to this problem is to use liposomes as a drug carrier to increase the solubility of LV. Liposomes can fuse with the cell membrane to promote the delivery and release of a variety of hydrophilic or lipophilic drugs.23-24 However, it is difficult to prevent premature drug release from the nanocomposite due to the poor stability of traditional liposomes.25 A cerasome (partially ceramic-coated liposome) nanoparticle, which is a lipid-like vesicle with significantly improved stability, has been developed in recent years.26-27 While retaining the excellent biocompatibility related with liposomes, the cerasomes offer the advantages of enhanced morphological stability compared with traditional liposomes and reduced overall rigidity and density compared with the silica nanoparticles (liposils).28-29 The cerasomal nanohybrids have been shown to be more stable in our previous studies in terms of better controlled release of the loaded drug,30 prolonged storage stability, and resistance against Triton X-100 solubilization.31 We have used this biomaterial to generate a cerasomal doxorubicin nanocomposite and found that this cerasomal hybrid system retained the encapsulated drug over a period of 90 days in solution and could effectively kill human ovarian cancer cells in vitro.32 With a diameter of about 100 nm, the cerasome nanoparticles can pass through the vascular endothelium via the enhanced permeability and retention (EPR) effect, passively targeting the tumor tissue.33 Compared with non-encapsulated free drug, the cerasomal nanohybrids were distributed to the tumor site at a much higher efficiency.34 In the present study, we prepared a cerasome-LV nanocomposite and investigated its effectiveness in targeting TNBC stem cells using in vitro and in vivo models to enhance therapeutic efficacy.

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MATERIALS AND METHODS Reagents Distearoyl Phosphatidylcholine (DSPC) was purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N -polyethylene glycol 2000 (DSPE-PEG2000) was obtained from A.V.T. Pharmaceutical Co., Ltd.(Shanghai, China). Chlorpromazine, amiloride hydrochloride, sucrose, methylated-β-cyclodextrin were purchased from Nacalai (Nijo Karasuma, Kyoto, Japan). Lovastatin (LV, ab120614), obtained from Abcam (Cambridge, UK), was dissolved in DMSO as a 30 mM stock solution and stored at -20°C before use. Routine chemicals including chloroform, ethanol, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, potassium chloride, sodium acetate, glacial acetic acid, sodium hydroxide, hydrochloric acid, and Tween-80 were obtained from Tianjin Guangfu Fine Chemical Research Institute (Nankai, Tianjin, China).

Synthesis of the cerasome-lovastatin nanocomposite The cerasome-forming lipid, N-[N-(3-triethoxysilyl)propylsuccinamoyl]-dihexadecylamine, was synthesized according to the literature.35 LV was encapsulated into cerasome using the thin film hydration method in combination with the sol-gel reaction and self-assembly process according to our published procedure.28, 36 Briefly, the cerasome-forming lipid, DSPC and DSPE-PEG2000 were dissolved in chloroform solution. The molar proportion of DSPE-PEG2000 was fixed at 5% while the molar proportions of DSPC were varied at 30%, 40% and 50%, respectively. LV was dissolved in ethanol solution and added to the above mixed solution at a drug/lipid ratio of 1:5 or 1:10. The mixture was allowed to slowly evaporate at 90 rpm for 30 min to remove the solvent and a uniform film formed on the flask wall. Then, ultrapure water was added to the flask, and the film was hydrated in a water bath at 55°C for 30 min. The flask was vortexed for 5 min so that the lipid film was completely peeled off to form a milky white suspension. After treatment in ultrasonic water bath for 10 min, the mixture was subjected to probe ultrasound for another 5 min (1/2’ probe, amplitude 30%, interval 3 s). The unloaded LV was removed by centrifugation at 6,000 rpm for 15 min. The resulting supernatant was cerasome-encapsulated LV solution, which was designated C-LV.

Transmission electron microscopy 6


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The transmission electron microscope (TEM) examination of C-LV nanoparticles was performed as described.37 Briefly, 0.1 mL of C-LV solution was dropped on the carbon film-sprayed copper mesh and allowed to adsorb for 10 min. After incubation with 2% uranyl acetate negative dye for 10 min followed by absorbance of most of the dye solution with filter paper, the copper mesh was removed. After drying for about 20 min, the morphology of C-LV vesicles was observed under the transmission electron microscope.

Measurement of the particle size and zeta potential The particle size and zeta potential of C-LV were analyzed by the Zeta PALS Analyzer (Brookhaven, Holtsville, NY, USA) at room temperature. Measurement of each sample was repeated three times and the average obtained.

Evaluation of drug encapsulation efficiency and drug-loading efficiency The content of unloaded LV was analyzed by HPLC according to the standard curve of the LV preparations (0.003, 0.03, 0.3, 3, 30, and 300 µM). Drug entrapment efficiency was calculated using the formula:

The immediately prepared C-LV solution was added to 1 M hydrochloric acid solution (v/v, 1:9) and this mixture was stirred vigorously overnight to allow complete release of the wrapped LV. Then, the sample was subjected to HPLC and the LV content in C-LV was calculated according to the standard curve of LV. The drug-loading efficiency was calculated using the formula:

In vitro drug release 2 mL of the C-LV solution was dialyzed against 50 mL of PBS (pH 7.4). At predetermined time intervals, 2 mL of the release medium was collected and replaced with an equal volume of the fresh buffer. The amount of LV released from C-LV was determined by HPLC and the cumulative release percentage (CRP%) of the drug was measured as follows:

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Where Ve is the displacement volume of release medium; V0 is the total volume of release medium; Cn is the concentration of LV in the release solution at each sampling point (µg/mL); n is the number of times of replacing release medium; MLV is the total amount of cerasome in C-LV (µg).

Cell culture and treatment TNBC cell lines (MDA-MB-231, MDA-MB-468, MX-1, BT-549) and non-TNBC cell lines (MDA-MB-453, ZR-75-1, BT-474, MCF-7) were obtained from the Cell Resources Center of the CAS Shanghai Institute of Life Sciences (Shanghai, China). These cell lines were routinely cultured in DMEM or RPMI 1640 supplemented with 10% FBS at 37°C with 5% CO2 in a humidified incubator. None of the cell lines used in this study was listed in the database of commonly cross-contaminated or misidentified cell lines (http://iclac.org/databases/cross-contaminations/). They were routinely monitored to ensure absence of mycoplasma contamination. For drug treatment, the cells were treated with LV under normoxic (21% O2) or hypoxic (1% O2) culture conditions depending on the experimental purposes. Hypoxia was achieved in a hypoxic chamber (Thermo Fisher Scientific, Waltham, MA, USA) by inflation of N2 into the air-filled chamber until 1% O2 was reached.

Derivation of breast cancer stem cells Sphere-forming cells (SFCs) were derived from MDA-MB-231 parental cells (PCs) according to our published technique for tumorsphere culture with some modifications of the recipe.21 The culture medium consisted of DMEM/F-12 (Life Technologies, Carlsbad, CA, USA) supplemented with 1 × B27 (w/o Vit A, Life Technologies), 20 ng/mL EGF (Prospec, East Brunswick, NJ, USA), 20 ng/mL bFGF (Prospec), 0.4% BSA (Sigma, St. Louis, MO, USA), 4 µg/mL insulin (Genview, Beijing, China). CD44+ CD24-/low CSCs were further enriched by magnetic separation using the human CD24-CD44+ breast cancer stem cells isolation kit (MAGH111, RND Systems, Minneapolis, MN, USA). Cell spheres were collected and passaged for more than 3 times to use as CSCs. The stemness properties of these CSCs were verified by subcutaneous inoculation into nude mice, which gave rise to the tumor-forming ability of SFCs two orders of magnitude stronger compared with PCs. 8


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Cell proliferation assay To examine the effects of LV and C-LV on cell proliferation, MDA-MB-231-PCs or MDA-MB-231-SFCs were seeded at a density of 1 × 104 cells/well (for PCs) or 4 × 103 cells/well (for SFCs) in 96-well plates. Next day, LV or C-LV was added to the cells at different concentrations and the culture continued for up to 72 h under normoxia or hypoxia. Cell proliferation was detected using an AlamarBlue cell viability assay kit (G8081, Promega, Madison, WI, USA). The values of half maximal inhibitory concentration (IC50) were calculated using the GraphPad Prism software (La Jolla, CA, USA).

Xenograft model of breast cancer tumor growth Female athymic null mice (BALB/c) aged 5 – 6 weeks obtained from SJA Experimental Animals Co. Ltd (Changsha, Hunan, China) were housed in sterile cages and maintained in SPF aseptic rooms with the 12h/12h light/dark cycle. MDA-MB-231 cells were trypsinized into single-cell suspension and resuspended in PBS and 100 µl of cell suspension (containing 1 × 106 cells) was injected subcutaneously into each nude mouse. The animals were randomly divided into five groups (5 – 6 mice/group) about two weeks after cancer cell injection. LV or C-LV diluted in NS was administered twice weekly via oral gavage (for LV) or tail vein injection (for C-LV). Control mouse received a similar volume of NS via oral gavage or tail vein injection. The tumor growth was monitored twice weekly by measuring the major (a) and minor (b) axes of the tumor using a caliper. The tumor volume (V) was calculated by the equation V = (a × b2)/2 as described.38 Three weeks after drug treatment, the mice were sacrificed and the tumors resected, weighed, and photographed. Part of the tumor tissue was fixed in 4% buffered formaldehyde and subjected to histopathological and immunohistochemical analyses. All animal studies were approved by the Hunan Normal University Animal Care Committee and performed according to the NIH Guide for the Care and Use of Laboratory Animals.

Immunohistochemistry The tumor tissues were formaldehyde-fixed paraffin-embedded and cut into 4-µm-thick tissue sections. Immunohistochemical staining was carried out using the PV-9000 plus Poly-HRP Anti-Mouse/Rabbit IgG Detection System as described in 9



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our previous study.39 The details of antibodies used for immunohistochemistry were summarized in Table 1. After immunohistochemical staining, the tissue section was scanned using Automated Quantitative Pathology Imaging System (Vectra, PerkinElmer, Hopkinton, MA, USA) and the staining intensity (H-score) was calculated each from six randomly chosen images at 20× magnification. SMA was scored by counting the positive cells in the entire field of view.

TUNEL staining Apoptosis of tumor tissues was detected using the in situ cell death detection kit-POD (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, the paraffin-embedded tissue sections were dewaxed and rehydrated after baking in a drying oven at 60°C for 2 h. The sections were incubated with proteinase K (40 µg/mL) for 30 min at 37°C and then with TUNEL reaction mixture in a dark humidity chamber for 1 h at 37°C. Next, the sections were incubated with Converter-POD for 30 min at 37°C, followed by coloration with the DAB/H2O2 color development system and counterstaining with hematoxylin. For the negative control, TUNEL reaction mixture was replaced with PBS. The positive control section was pre-treated with DNase I for 10 min at 15-25°C followed by TUNEL staining. Cells with tan granules in the nucleus were regarded as positive for TUNEL.

Statistical analysis All quantitative data were presented as mean ± SEM and obtained from at least two independent experiments. One-way ANOVA followed by the Holm Sidak post hoc test was used for multiple group comparisons of means. P < 0.05 was considered statistically significant.

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RESULTS Preferential effect of lovastatin on TNBC vs. non-TNBC cells We first evaluated the effect of LV on the proliferation of different TNBC and non-TNBC cell lines using the standard AlamarBlue assay. Human TNBC cells and non-TNBC cells were treated with different concentrations of LV for 72 h under normoxia or hypoxia. We found that LV, when used within a clinically relevant dose rang,40 exhibited cytotoxicity on TNBC cells as shown by reduced proliferation (Figure 1, top) and morphological changes such as cell rounding (data not shown). These inhibitory effects of LV were not observed in all the non-TNBC cell lines examined, except for some marginal inhibition of MCF-7 cells by high concentration of LV (10 µM) under hypoxia (Figure 1, bottom). These data indicate that LV preferentially exerted its inhibitory effect on TNBC cells rather than non-TNBC cells.

Fabrication and characterization of C-LV nanohybrids LV was encapsulated into cerasomes with 5% molar proportion of DSPE-PEG2000 and different molar proportions of DSPC (30%, 40%, and 50%, respectively) by using the thin film hydration method in combination with sol-gel and self-assembly process according to our published procedure.28, 36 The size and morphology of the C-LV nanoparticles were characterized by TEM and zeta potential analysis. A representative TEM image of C-LV was shown in Figure 2A, which showed the formation of spherical vesicles with a diameter ranging from 100 to 150 nm. The size distribution of C-LV was further confirmed by Zeta PALS measurement (Figure 2B), which showed the average size of 128 ± 0.3 nm. The zeta potential of C-LV as an indication of nanoparticle surface charge was evaluated to be -23.1 ± 0.8 mV (Figure 2C). Their high surface charges were similar to that of the original “empty” cerasomes due to the negatively charged hydroxyl groups of the polysiloxane networks formed on their surface.41

Release behavior of C-LV As shown in Table 2, cerasomal composition can have proud impact on the encapsulation efficiency (EE) and drug loading content (DLC). When the C-LV nanohybrids were prepared by including 30%, 40%, and 50% molar proportions of DSPC, the EE was evaluated to be 65.22%, 69.87%, 78.38%, 63.51%, respectively. The DLC of the C-LV nanohybrids was 5.43%, 5.82%, 6.53%, 5.24%, respectively. A 11



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DSPC ratio of 40% in C-LV yielded the highest encapsulation efficiency and drug-loading content.

The in vitro release behavior of LV from C-LV incorporating different molar proportions of DSPC was examined in PBS at 37°C and pH 7.4, and the release curves were presented in Figure 2D. There was a rapid release at the beginning (up to 12 h) and the sustained release maintained until 120 h for all four C-LV preparations. Generally, the higher the ratio of DSPC in C-LV, the faster the drug releases. At the end of the whole examination period, a DSPC ratio of 40% and 50% released about 80% and 95% of LV, respectively. The increased release rates and LV amounts from the C-LV nanohybrids could be assigned to the increased membrane permeability because of the incorporation of DSPC. Thus, the LV release profiles from the C-LV nanohybrids could be modulated by varying the incorporated DSPC molar proportions. Considering the release behavior and the high level of encapsulation and drug-loading efficiency, the C-LV nanohybrid with 40% DSPC was selected as the best nanoformulation for all the in vitro and in vivo experiments in this study.

Cytotoxicity of LV and C-LV in breast cancer stem cells in vitro The AlamarBlue cell viability assay was used to detect the toxicity of free LV and C-LV to breast cancer stem cells, and the results were shown in Figure 3. In general, the survival rate of MDA-MB-231-SFCs decreased gradually with the increase of the treatment time and the concentration of free LV or C-LV used. Compared with the control group, at all the three time points, the proliferation of MDA-MB-231-SFCs was significantly inhibited when the concentration of free LV or C-LV was 0.1 µM or above (P < 0.05). Furthermore, the inhibitory effects on SFCs were similar between LV and C-LV at all the time points examined, suggesting a similar uptake and cytotoxicity level between free LV and encapsulated LV in vitro.

Effects of LV and C-LV on breast cancer xenograft growth and stem cell properties Effect of LV and C-LV on breast cancer xenograft growth Next, a nude mouse model of breast cancer xenograft tumor was used to evaluate the effect of LV and C-LV on MDA-MB-231 breast cancer in vivo. The schedule of the animal experiment was illustrated in Figure 4A. Two weeks after cancer cell 12


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inoculation, LV or C-LV was administered twice weekly for three consecutive weeks. At the end of the experiment, the mice were euthanized and the tumors resected. Tumor volumes and weights were obtained and tumor tissues were subjected to formaldehyde-fixation and paraffin-embedding and sectioning for histopathological examination and immunohistochemistry. As shown in Figures 4B & 4C, compared with the control mice, the tumor growth rate and tumor volume were significantly inhibited by treatment with LV (10 mg/kg) or C-LV (2 and 10 mg/kg). Tumor weight measurement also revealed that the inhibition of tumor growth was more prominent in mice treated with C-LV than those treated with the same dose of LV (Figure 4D). These results indicate that LV and C-LV could inhibit breast cancer xenograft tumor growth and that C-LV was more effective than LV in inhibiting breast cancer xenograft growth in nude mice.

Effect of LV and C-LV on mammary gland formation and angiogenesis Mammary gland formation is one of the characteristic features of normal breast tissues and can be used as an indication of normalization of breast tumor tissues.42 We found that both LV and C-LV increased the formation of mammary glands in the mice bearing MDA-MB-231 breast cancer xenograft compared with the control mice which had almost no mammary glands at all (Figure 5, top panel). We further carried out TUNEL and immunohistochemical staining to evaluate the effect of LV and C-LV on apoptosis and angiogenesis in the xenograft tumors, respectively. We found that both LV and C-LV significantly induced apoptosis as demonstrated by increased TUNEL staining (Figure 5, middle panel) and suppressed tumor angiogenesis as demonstrated by decreased immunostaining of VWF (a marker of vascular endothelium) (Figure 5, bottom panel). Quantitation of immunohistochemical staining revealed that C-LV (not LV) almost completely eradicated angiogenesis in the xenograft tumors. Without exception, C-LV had a better effect than LV in restoring mammary gland formation and inhibiting tumor angiogenesis in MDA-MB-231 xenograft tumors.

Effect of LV and C-LV on breast cancer stem cell properties Finally, we evaluated the effect of LV and C-LV on breast cancer stemness-related markers including the general stem cell markers (CD44 and Nanog) and the EMT markers (Snail, Twist, and Vimentin), which are closely associated with the CSC properties,43-44 by immunohistochemistry. As shown in Figure 6, immunostaining for 13



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all these markers was significantly decreased in the mice treated with C-LV compared with the control mice. Consistent with the above results, C-LV had a similar or better effect in terms of inhibiting breast cancer stem cell properties than LV.

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DISCUSSION Currently, chemotherapy is the mainstay of treatment of various cancer types. However, routine chemotherapeutic drugs lack tissue selectivity, leading to toxic side effects and greatly reduced treatment efficacy. Therefore, through passive targeting owing to the EPR effect of nanoparticles at the tumor site, the efficacy of drugs on cancer treatment can be improved. Recently, we have demonstrated that LV confers enhanced sensitivity to conventional chemotherapeutic drugs such as doxorubicin [manuscript in preparation]. However, the problem of poor solubility and bioavailability severely restricts the potential clinical application of LV. Zhang et al.22 used a mesoporous nanomatrix-supported lipid layer to encapsulate LV into the liposome, which solved the issue of the water solubility of LV to some extent. However, the issue of instability of unmodified liposome is still a problem for the potential application in the body. Allen et al.45 used PEG to modify the liposomes, which can reduce the destruction of liposomes by the reticuloendothelial system. However, the introduction of PEG will lead to the so-called “hand-foot syndrome” and other toxic reactions.46-47 Meanwhile, PEGylation can also reduce the phagocytosis of liposomes by tumor cells and thus limits the use of the liposome-LV nanocomposite. Thus, formulations that enhance the water solubility, maintain the stability, and, at the same time, enhance tumor cell phagocytosis of LV can promote the clinical use of LV.

Compared with the traditional liposome, the cerasome with the silane on the surface of the liposome has greatly increased mechanical and thermal stability, which can ensure the survival of the nanocomposite under the harsh pH and salt ion conditions in the tumor microenvironment. Jin et al.32 used cerasomes to load doxorubicin and found that the drug-loaded cerasomes retained 92.1% of the drug load even after 90 days of storage and exhibited sustained release of doxorubicin over 150 h. These data, together with ours, suggest that the cerasome performs much better than liposomes and liposils and is indeed an ideal drug carrier to increase the stability and the controlled release of the loaded drug.

In summary, in this study, we introduced the formulation of C-LV, a cerasome-based nanocomposite to encapsulate LV, a hydrophobic lipid-lowering drug. C-LV had very 15



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high encapsulation and drug-loading efficiency and exhibited excellent drug release performance. Although the inhibitory effect on breast cancer stem cells was similar between free LV and C-LV in vitro, C-LV was more effective than free LV in inhibiting the growth of breast cancer xenografts and the stemness properties in vivo. To our knowledge, this represents the first demonstration of a nanocomposite that can inhibit cancer stem cells derived from TNBC. These findings provide a novel possibility for cancer stem cell-based therapy and warrant further clinical studies for the treatment of TNBC.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; *E-mail: [email protected]. Author Contributions #

: These authors contributed equally to this work

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (81472496 to XD), the National Key Research and Development Program of China (No. 2016YFA0201400 to ZD), the Young Scholar Program of Department of Education of Hunan Province (14B112 to LS), and the Key Project of Department of Education of Hunan Province (14A089 to XD).

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(26) Matsui, K.; Sando, S.; Sera, T.; Aoyama, Y.; Sasaki, Y.; Komatsu, T.; Terashima, T.; Kikuchi, J. Cerasome as an infusible, cell-friendly, and serum-compatible transfection agent in a viral size. J Am Chem Soc 2006, 128 (10), 3114-5. (27) Yue, X.; Dai, Z. Recent advances in liposomal nanohybrid cerasomes as promising drug nanocarriers. Adv Colloid Interface Sci 2014, 207, 32-42. (28) Katagiri, K.; Hamasaki, R.; Ariga, K.; Kikuchi, J. Layered paving of vesicular nanoparticles formed with cerasome as a bioinspired organic-inorganic hybrid. J Am Chem Soc 2002, 124 (27), 7892-3. (29) Ma, Y.; Dai, Z.; Gao, Y.; Cao, Z.; Zha, Z.; Yue, X.; Kikuchi, J. Liposomal architecture boosts biocompatibility of nanohybrid cerasomes. Nanotoxicology 2011, 5 (4), 622-35. (30) Cao, Z.; Yue, X.; Jin, Y.; Wu, X.; Dai, Z. Modulation of release of paclitaxel from composite cerasomes. Colloids Surf B Biointerfaces 2012, 98, 97-104. (31) Zhang, C. Y.; Cao, Z.; Zhu, W. J.; Liu, J.; Jiang, Q.; Shuai, X. T. Highly uniform and stable cerasomal microcapsule with good biocompatibility for drug delivery. Colloids Surf B Biointerfaces 2014, 116, 327-33. (32) Jin, Y.; Yue, X.; Zhang, Q.; Wu, X.; Cao, Z.; Dai, Z. Cerasomal doxorubicin with long-term storage stability and controllable sustained release. Acta Biomater 2012, 8 (9), 3372-80. (33) Katagiri, K.; Hashizume, M.; Ariga, K.; Terashima, T.; Kikuchi, J. Preparation and characterization of a novel organic-inorganic nanohybrid "cerasome" formed with a liposomal membrane and silicate surface. Chemistry 2007, 13 (18), 5272-81. (34) Jing, L.; Shi, J.; Fan, D.; Li, Y.; Liu, R.; Dai, Z.; Wang, F.; Tian, J. (177)Lu-Labeled Cerasomes Encapsulating Indocyanine Green for Cancer Theranostics. ACS Appl Mater Interfaces 2015, 7 (39), 22095-105. (35) Maeda, H.; Nakamura, H.; Fang, J. The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv Drug Deliv Rev 2013, 65 (1), 71-9. (36) Cao, Z.; Ma, Y.; Yue, X.; Li, S.; Dai, Z.; Kikuchi, J. Stabilized liposomal nanohybrid cerasomes for drug delivery applications. Chem Commun (Camb) 2010, 46 (29), 5265-7. (37) Kumar, D.; Shankar, S.; Srivastava, R. K. Rottlerin-induced autophagy leads to the apoptosis in breast cancer stem cells: molecular mechanisms. Mol Cancer 2013, 12 (1), 171, DOI: 10.1186/1476-4598-12-171. (38) Ho, B. Y.; Pan, T. M. The Monascus metabolite monacolin K reduces tumor progression and metastasis of Lewis lung carcinoma cells. J Agric Food Chem 2009, 57 (18), 8258-65. (39) Li, H.; He, G.; Yao, H.; Song, L.; Zeng, L.; Peng, X.; Rosol, T. J.; Deng, X. TGF-beta Induces Degradation of PTHrP Through Ubiquitin-Proteasome System in Hepatocellular Carcinoma. J Cancer 2015, 6 (6), 511-8. (40) Thibault, A.; Samid, D.; Tompkins, A. C.; Figg, W. D.; Cooper, M. R.; Hohl, R. J.; Trepel, J.; Liang, B.; Patronas, N.; Venzon, D. J.; Reed, E.; Myers, C. E. Phase I study of lovastatin, an inhibitor of the mevalonate pathway, in patients with cancer. Clin Cancer Res 1996, 2 (3), 483-91. 19



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FIGURE LEGENDS Figure 1. LV preferentially inhibits the proliferation of TNBC cells over non-TNBC cells. Human TNBC (MDA-MB-231, MDA-MB-468, MX-1, BT-549) and non-TNBC (MDA-MB-453, ZR-75-1, BT-474, MCF-7) cells were treated with LV or vehicle for 72 h and cell proliferation was analyzed by the AlamarBlue assay. Lower-case letters denote statistically significant difference among the different groups, with up-right and italic letters representing normoxia and hypoxia, respectively.

Figure 2. Characterization of the C-LV nanovesicles. (A) Assessment of in vitro release of LV from C-LV. C-LV with different concentrations of DSPC was dialyzed against PBS (pH 7.4) and the kinetics of LV release from C-LV was analyzed by measuring the LV concentration in the dialysate at different time points by HPLC. (B, C) Analysis of the morphology and size of C-LV. The morphology and size of C-LV were examined by transmission electron microscopy (TEM) (B) and ZetaPALS (C), respectively. (D) The apparent zeta potential of C-LV was determined by ZetaPALS.

Figure 3. Inhibition of proliferation by C-LV in MDA-MB-231 cancer stem cells. MDA-MB-231-SFCs were treated with different concentrations of LV or C-LV for 24 h (A), 48 h (B), or 72 h (C) and cell proliferation was analyzed by the MTT assay. Lower-case letters denote statistically significant difference among the different groups, with up-right and italic letters representing LV and C-LV, respectively.

Figure 4. C-LV suppresses tumor growth of MDA-MB-231 breast cancer xenografts in nude mice. (A) Schematics of animal experimental design. (B) Change of tumor volume over time from different groups. (C) Photographs of tumors isolated from the nude mice. (D) The tumor weights of the mice from different groups. Data were shown as mean ± SEM (n = 5 – 6/group). Lower-case letters or symbol (*) denote statistically significant difference among the different groups. n.s.: not significant.

Figure 5. C-LV treatment results in induction of mammary gland formation and apoptosis and suppression of angiogenesis. Tissue sections were examined for the formation of mammary glands (top panel) and stained for apoptosis (TUNEL) and 21



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angiogenesis (VWF). Shown on the right are quantifications of the glands and the TUNEL or VWF staining. Lower-case letters denote statistically significant difference among the different groups.

Figure 6. C-LV treatment results in the downregulation of cancer stem cell properties and reversal of EMT. Tissue sections were stained for the markers of cancer stem cell phenotype (CD44 and Nanog) and EMT (Snail, Twist, and Vimentin) by immunohistochemistry. Shown on the right are quantifications of each respective marker. Lower-case letters denote statistically significant difference among the different groups.

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Table of Contents Graphic:

23



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Figure 1

3

10

LV (M)

LV (μM)

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1 2 3 4 TNBC 5 6 MDA-MB-231 7 8 15000 9 a a 10 10000 b 11 a c 12 b b c 13 5000 14 15 0 16 0 0.3 1 3 17 LV (μM) 18 19 20Non-TNBC 21 22MDA-MB-453 Normoxia 23 Hypoxia 24 25000 b b b 25 20000 a 26 27 15000 28 10000 b b b 29 a 30 5000 31 0 32 0 0.3 1 3 33 LV (M) LV (μM) 34 35 36 37 38 39 40 41

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Figure 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

A

C

B

D



Figure 3

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Figure 4

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Table 1. Primary antibodies used for immunohistochemistry

Antibody Name VWF CD44 Nanog

Company BOSTER Abcam Abcam

Cat# PB0273 ab51037 ab109250

Dilution 1:100 1:100 1:100

Snail

ProteinTech

13099-1-AP

1:100

Twist Vimentin

Abcam CST

ab50581 5741

1:100 1:100


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Table 2. Encapsulation efficiency and drug-loading content of C-LV at different molar proportions of DSPC.

DSPC Molar proportions 20%

Encapsulation efficiency 65.22%

Drug loading content 5.43%

30%

69.87%

5.82%

40%

78.38%

6.53%

50%

63.51%

5.24%


Cerasomal Lovastatin Nanohybrids for Efficient Inhibition of Triple

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