Journal of Controlled Release 194 (2014) 228–237

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Dual drugs (microRNA-34a and paclitaxel)-loaded functional solid lipid nanoparticles for synergistic cancer cell suppression Sanjun Shi 1, Lu Han 1, Li Deng, Yanling Zhang, Hongxin Shen, Tao Gong, Zhirong Zhang, Xun Sun ⁎ Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, PR China

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Article history: Received 15 April 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: Co-delivery MicroRNA Paclitaxel Synergistic cancer suppression Tumor relapse

a b s t r a c t A co-delivery system that can transport cancer related microRNAs and chemotherapeutics to their distinct targets in the tumors is an attractive strategy to eliminate tumor relapse in lung cancer therapy. In this study, we developed a dual-drug delivery system for an endogenous microRNA (miR-34a) and paclitaxel (PTX) for synergistic cancer therapy. PTX (a meiotic inhibitor) and miR-34a were loaded into cationic solid lipid nanoparticles (miSLNs-34a/PTX) which were used to treat murine B16F10-CD44+ melanoma metastasized to the lungs of mice. This nanoparticle system demonstrated good protection for miR-34a and PTX from degradation in the serum, and had an average size of approximately 220 nm by photon correlation spectroscopy (PCS). In vitro, the parallel activity of PTX and miR-34a show synergistic anticancer efficacy. In vivo, miSLNs-34a/PTX showed passive targetability to the tumor-bearing lung tissues, and was demonstrated to be much more potent in inhibition of B16F10-bearing tumor growth and elimination of cancer cell populations in the lung than single drug-loaded solid lipid nanoparticles. It has been shown that such co-delivery of miR-34a and PTX is promising for enhanced cancer therapy to reduce tumor relapse. © 2014 Elsevier B.V. All rights reserved.

1. Introduction MicroRNAs (miRNAs) are a class of small, endogenous noncoding RNAs that post-transcriptionally control the translation and stability of mRNAs [1–3]. Recently, miRNAs have been acquainted as a novel class of important gene-regulatory molecules involved in many critical biological functions such as cell proliferations, developments, cellular functions and even tumorigeneses [1–7]. Dysregulation of miRNAs has been shown in many tumors and recognized as a hallmark of cancer. MiR-34a is one of the most prominent endogenous miRNAs implicated in the genesis and progression of human cancers and is commonly downregulated in many human cancers [8,9]. Therefore, it functions as a tumor suppressor during tumor initiation and progression and enhanced expression of miR-34a in many tumors can induce cell apoptosis and block the proliferation of cancer cells [7,9]. MiRNA-based anti-cancer therapies are thus raising the hope for the patients. More recently, miR-34a has been found to target CD44 molecule, which plays a key role in determining the tumorigenic and metastatic capacities of many tumor cells (such as prostate and head and neck cancer cells) [10,11]. But the relationship between miR-34a and CD44 in melanoma remains uncertain. Therefore, it is necessary to investigate

⁎ Corresponding author at: West China School of Pharmacy, Sichuan University, Chengdu 610041, Sichuan, PR China. Tel.: +86 28 85502307; fax: +86 28 85501615. E-mail address: [email protected] (X. Sun). 1 Sanjun Shi and Lu Han equally contributed to the work.

http://dx.doi.org/10.1016/j.jconrel.2014.09.005 0168-3659/© 2014 Elsevier B.V. All rights reserved.

the miR-34a function in melanoma. However, its nuclease-labile property and its poor biomembrane permeability limit the delivery efficiency into tumor cells. Many efforts have been made in delivering miR-34a into tumor cells by viral carriers [12], liposomes [13] and nanoparticles [13]. In our previous study, a nanoparticle system based on solid lipid nanoparticles has been exploited for miR-34a to inhibit B16F10 (melanoma) cancer stem cells (CSCs) [14]. The B16F10-CD44+ cells were inhibited by attenuating CD44 expression and the mice survival rate was increased. However, the success of miR-34a in cancer treatment is still limited by the risk of therapeutic relapse, which may be attributable to that of the tumor development and tumorigenicity originating from CSCs is extremely multiple. Even though one single miRNA (miR-34a) can target CD44 followed by induction cell apoptosis, the tumor cell originating from other signaling networks remains alive owing to escaping from the miR-34a treatment. Therefore, delivery of miR-34a alone to target tumors is insufficient to eliminate all the tumor cells. A more efficacious tumor therapy for miR-34a could be achieved by a combinatorial approach with conventional anti-cancer agents such as paclitaxel (PTX) that kills all the tumor cells. Paclitaxel, a chemotherapeutic agent, which showed an exceptional activity against various solid tumors by disrupting the normal tubule dynamics required for cellular division thus provoking cell death [15–17], and could cause serious toxicity in normal tissues that limits the maximum tolerated dose in vivo. In conventional treatment, the co-delivery of PTX and nucleic acids by nanoparticle systems could achieve the synergistic/combined effect of drug and gene therapies based on their respective mechanisms of action on cancers [18,19],

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which achieves the possibility of co-delivering PTX and genes to the same cells. However, the safety concerns are still considered due to the high administrated dosage of PTX. The combination of two or more drugs within a single nanocarrier can overcome toxicity and other side effects. Thus, we hypothesized that miR-34a could target CD44-positive tumor cells and achieve a synergistic effect with PTX. With this in mind, there is an urgent need for investigating an enhanced cancer therapy strategy that utilizes simultaneous delivery of miR-34a and a relatively low PTX dose. Herein, we present an interesting strategy for fighting against tumors by using a combination therapy with the synergistic effect, which could enhance cellular uptake and increase the drug concentration in the cancerous lung. The physical–chemical characteristics of this dual drug co-delivery system was characterized and the cellular uptake was studied in the stem-like cancer cell line B16F10-CD44+. The antitumor efficacy and the CD44 phenotype were also tested in a B16F10-CD44+-bearing mouse model. 2. Materials and methods 2.1. Materials Glyceryl monostearate (GMS) was purchased from Taiwei Pharmaceutical Co., Ltd (Shanghai, China), and cholesterol (Chol) was obtained from Boao Biotech Co., Ltd (Shanghai, China). Soy phosphatidylcholine (SPC, S100) was obtained from Germany Lipoid Company. Dimethyldioctadecylammonium bromide (DDAB) and coumarin-6 were purchased from Sigma-Aldrich (St. Louis, MO). Paclitaxel (PTX) was obtained from Haoxuanbio. Co., Ltd (Xian, China). Mmu-miR-34a mimics and control miRNA were synthesized by GenePharm (Shanghai, China) and Cy3/Cy5-labeled miRNA was supplied by RiboBio (Guangzhou, China). 2.2. Cell culture and animals B16F10 cells (a murine melanoma cell line) were obtained from ATCC and cultured in RPMI-1640 with 10% fetal bovine serum, 1% penicillin/streptomycin. Female C57BL/6 mice (6–8 weeks) were supplied by the Laboratory Animal Center of Sichuan University (Chengdu, China). All animal experiments were approved by the Institutional Animal Care and Ethic Committee of Sichuan University. 2.3. Fabrication and physicochemical characteristics of dual drug loadedcationic solid lipid nanoparticles First, PTX-loaded SLNs containing DDAB were prepared using a filmultrasonic method [14]. SLNs/PTX were derived from a thin film consisting of GMS (15 mg), SPC (15 mg), Chol (10 mg), DDAB (10 mg) and 1 mg of PTX, followed by sonication (80 W, 60 s) with 5 mL of RNase-free water. Second, 50 μL of miRNA solution was added to an isovolumetric SLNs/PTX colloidal solution and incubated for 30-min at room temperature to prepare the dual drug delivery system (miSLNs34a/PTX). The physicochemical properties of miSLNs-34a/PTX were characterized in terms of size distribution, zeta potential, morphology, entrapment efficiency, loading capacity and release profiles. Particle size distribution and zeta potential were measured in triplicate by photon correlation spectroscopy (PCS) (Malvern zetasizer Nano ZS90, UK). Transmission electron microscopy (TEM) specimens were prepared by depositing the SLN suspensions onto a copper grid, stained with 1% phosphotungstic acid (pH 6.5) for 10 s. Then the morphology of SLNs was observed by using an H-600 TEM instrument (Hitachi, Japan). The entrapment efficiency was obtained by measuring the amount of drug that was encapsulated in SLNs. Briefly, 3 mL of SLNs was dissolved in methanol and analysed by high-performance liquid chromatography (HPLC) (Kromasil ODS-1 C18 column (150 × 4.6 mm, 5 μm);

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the mobile phase: CH3CN: H2O = 55:45(v/v); flow rate: 1 mL/min; wavelength: 227 nm). The non-entrapped RNA in the aqueous phase was separated by the ultrafiltration method (300 kD, 3000 g, 30 min). Cy3-labeled RNA was used to enable fluorometric measurements of RNA concentration. Drug loading yield (DL) and encapsulation efficiency (EE) of miSLNs were calculated by the following equations. weight of drug in SLNs  100. Encapsulation efficiency % ¼ weight of the feeding drug nmols of RNA in miSLNs RNA loading yield ðnmol=mg lipidsÞ ¼ weight . of total lipids in miSLNs encapsulated PTX inSLNs  100. PTX loading yield % ¼ weight of total weight of SLNs

To investigate the release kinetics of FAM-labeled miR-34a from miSLNs-34a/PTX, the nanoparticles were suspended in 1 mL of deionized water and sealed in an Eppendorf tube. At designated time intervals, miSLNs-34a/PTX were subjected to ultrafiltration (100 KDa). The concentration of released miR-34a was measured by using a Microplate Reader (Thermo, Varioskan Flash). PTX release profiles from SLNs and miSLNs-34a/PTX were monitored by dialysis in the presence of 0.2% Tween 80 [19]. Briefly, 1 mL of SLNs/PTX (0.2 mg of PTX) was dialysed using a molecular weight cut-off of 8–12 kDa (Millipore, USA), against 50 mL PBS (pH 7.4) with 0.2% Tween 80. At designated time intervals, aliquots were removed from the dialysate and replaced by an equal volume of PBS/Tween buffer. The amount of PTX in the dialysate was determined by HPLC (Agilent Technologies, Santa Clara, CA). 2.4. In vitro sera stability of miSLNs-34a/PTX In order to investigate the stabilities of miR-34a and PTX in the presence of sera, real-time PCR [14] and HPLC method were carried out to measure miR-34a copy number and PTX content, respectively, before and after treatment with sera. Free miR-34a, PTX and miSLNs-34a/PTX (DDAB/RNA, 8:1) containing 0.5 μg of RNA were incubated with 100 μL of serum from C57BL/6 mice and 5% FBS at 37 °C for 6 h, respectively. The samples were then lyophilized in a Modulyo freezedryer (Thermo Savant, USA) to obtain the miR-34a/PTX powder. For PTX stability measurements, the powder was redissolved in 1 mL 70% methanol and the concentration of PTX was measured by HPLC as mentioned above. For miR-34a stability measurements, each sample was redissolved in 600 μL of cell lysis buffer containing a demulsifier (TianGen, Bejing, China) which can break down the lipid matrix of SLNs to release miRNA. The miR-34a was extracted from the lysates using an RNA isolation kit (TianGen). The stability of miR-34a in sera was further confirmed by real-time PCR (iCycler iQ™ 5, Bio-Rad, USA) in terms of copy number. 2.5. Differential scanning calorimetry (DSC) Differential scanning calorimetry (DSC) was performed in a DSC analyzer (Netzsch DSC 200 PC, Germany). The dried samples including blank SLNs, PTX and SLNs/PTX were sealed in the aluminum crimp cell, and heated from 0–300 °C at a rate of 10 °C/min under nitrogen atmosphere. 2.6. Uptake of miSLNs-34a/PTX by B16F10-CD44+ cells The cellular uptake and distribution of the drug formulations were examined by flow cytometry and confocal microscopy. The CD44enriched B16F10 (B16F10-CD44+) cells were seeded in a 12-well culture plate and grown to about 70% confluence prior to transfection. For detection by flow cytometry and confocal microscopy, coumarin-6 was incorporated in the place of PTX (SLNs/PTX(coumarin-6)), followed by incubation with Cy5-labeled RNA as described above, to obtain the final dual fluorescence-labeled nanoparticle system (miSLN-34a(Cy5)/ PTX(coumarin-6)). The fluorescence-labeled formulations including free miR-34a(Cy5) and miSLN-34a(Cy5)/PTX(coumarin-6) were added directly to the cells without serum for a 4-h incubation at 37 °C. Then cells

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were harvested, washed with PBS, and analysed by flow cytometry (Cytomics™ FC500, Beckman Coulter, Miami, FL) and confocal laser scanning microscopy (TCS SP5, Leica, Germany). For quantitative analysis, B16F10-CD44+ cells were seeded in a 12well culture plate and grown to about 70% confluence prior to transfection. After 4 h incubation of 350 μL miSLNs-34a/PTX, the cells were washed three times with PBS. Subsequently, 400 μL of cell lysis buffer was added to all the wells to lyse the cells and miR-34a was extracted from the lysates using an RNA isolation kit (TianGen). The miR-34a and PTX contents in the lysates were determined by real-time PCR (iCycler iQ™ 5, Bio-Rad, USA) and LC–MS/MS (Agilent 1200), respectively. The protein concentration was measured with the Braford protein assay (KeyGEN BioTech, China). the drugs in cells Cellular uptake ¼ total weight of protein. uptake content  100. cellular uptake effeciency ¼ theCellular total drugs administrated

2.9. miSLNs-34a/PTX suppression of tumorigenicity of B16F10-CD44+ cells To evaluate the effect of miSLNs-34a/PTX on B16F10-CD44+ cells, tumorigenicity was studied in mice subcutaneously injected with B16F10-CD44+ cells [21]. Cell treated with different formulations were injected into different parts of the mouse [22]. In brief, the cells were first seeded in a 12-well culture plate and grown to about 70% confluence prior to transfection. Cells were treated with different formulations: miSLNs-34a (150 nM), SLNs/PTX (10 μg · mL−1) and miSLNs-34a/PTX (150 nM of miR-34a/10 μg · mL− 1 of PTX). At 4 hpost transfection, the B16F10-CD44+ cells were re-fed with fresh medium for a further 48-h culture. The treated cells were harvested, washed with PBS, diluted in serum-free medium and subcutaneously injected into C57BL/6 mice at a density of 5 × 104 cells/100 μL, and the tumor size at 23 days was measured and recorded. 2.10. In vivo therapeutic experiments

2.7. Assessment of cell proliferation and apoptosis Proliferation of drug-treated B16F10-CD44+ cells was determined by an MTT assay. The cells were plated in 96-well culture plates and allowed to attach for 24 h. The following day, cells were transfected in triplicate with miSLNs-34a/PTX (equal to 150 nM of miR-34a) for 4 h with three concentrations of PTX (2.5, 10, 20 μg/mL). The miSLNs-34a samples served as the positive control in these experiments. After miSLNs-34a/PTX transfection, the B16F10-CD44+ cells were re-fed with fresh medium for a further 72-h culture, followed by the addition of 20 μL of MTT solution (5 mg/mL) to each well. The cells were incubated in total darkness for 4 h, and precipitates were resuspended with 150 μL of DMSO in order to ensure solubilization of the formazan crystals. Cell proliferation was assessed by measuring the absorbance at 570 nm with a microplate reader (Thermo, Varioskan Flash). Cell apoptosis was assessed after staining with the highly specific and sensitive fluorescent DNA-binding dye 4′6-diamidino-2phenylindole (DAPI) [14,20]. B16F10-CD44 + cells were treated with the miR-34a/PTX formulations (at a concentration of 150 nM RNA and of 10 μg/mL PTX) and replaced with fresh medium at 4 h post-transfection. After 72 h of culture, cells were rinsed with PBS and the cell nuclei were then incubated with DAPI for 10 min at a final concentration of 1 μg/mL. The nuclear morphology of cells was analysed using a Zeiss fluorescence microscope (ex 358 nm, em 461 nm, Axiovert 40, German). Cells were judged to be apoptotic or not based on the nuclear morphology changes including chromatin condensation, fragmentation and apoptotic body formation.

2.8. Nanoparticle biodistribution miSLNs-34a (Cy3) /PTX (coumarin-6) were prepared as the dual fluorescence-labeled delivery system as described above. B16F10CD44 + -bearing mice were injected via the lateral tail vein with free drugs (miR-34a(Cy3) and coumarin-6), miSLNs-34a(Cy3), SLNs/ PTX (coumarin-6) and miSLNs-34a(Cy3) /PTX (coumarin-6) at the equivalent of 62.5 μg coumarin-6/kg body weight for in vivo imaging. During necropsy, organs of interest (heart, liver, spleen, tumor-bearing lung, kidney and brain) were dissected followed by fluorescence tissue distribution measurements using an IVIS® Spectrum system (Caliper, Hopkington, MA). The fluorescence signals of coumarin-6 (Ex = 498 nm; Em = 520 nm) and Cy3 (Ex = 550 nm; Em = 570 nm) were recorded respectively based on the available filter sets on the IVIS spectrum. The tumor-bearing lung tissues were then cryopreserved in tissue freezing medium (Leica) and cut using a microtome (Leica CM1950) into cross-sections, which were stained with DAPI for 5 min and mounted on glass slides for fluorescence microscopy analysis (TCS SP5, Leica, Germany).

An in situ murine lung metastasis model was established by i.v. injection of 2 × 105 B16F10-CD44+ cells via the lateral tail vein into C57BL/6 mice [13]. Starting from day 9, B16F10-bearing mice were given i.v. injections with miR-34a/PTX formulations via tail vein every day at a rate of 0.5 mg of miR-34a per kg of body weight and 1 mg of PTX per kg of body weight until day 15. On day 24, animals were sacrificed after seven injected administrations, and the B16F10-bearing lungs were collected and analyzed. For the survival analysis study, B16F10-bearing mice were also treated with miSLNs-34a/PTX on days from 9 to 15, and the death date of mice was recorded and statistically analyzed with SPSS 19.0 software. 2.11. Immunohistochemistry In order to verify the mechanism of antitumor activity in mice, CD44 expression was detected by immunohistochemistry. After treatment with the different formulations, normal lungs and B16F10CD44+-loaded lungs were harvested and prepared for paraffinembedded sectioning. CD44 expression in the tissues was evaluated using a biotinylated rabbit anti-mouse CD44 antibody (Boster, Wuhan, China) at a 200-fold dilution, and a kit (Streptavidin-biotin complex detection system, Boster). Tissue sections were counterstained with hematoxylin for nuclei coloration and observed by light microscopy (Zeiss Axiovert 40). 2.12. Statistics All quantitative data were expressed as mean ± standard deviation from at least 3 separate experiments performed in triplicate, unless otherwise noted. Differences between two groups were evaluated by oneway ANOVA followed by Student's t-test and the level of significance was set as P b 0.05. 3. Results 3.1. Nanoparticle design and characterization In this study, paclitaxel (PTX) was encapsulated into solid lipid nanoparticles (SLNs) first to obtain SLNs/PTX, and then miSLNs-34a/ PTX were self-assembled by charge–charge interaction, which not only exhibited the ability of simultaneous loading of miR-34a and PTX but also protected the stabilities of drugs from degradation in the presence of sera (Fig. 1). The cationic lipid DDAB was used to condense miR34a via charge interaction; miRNA-loaded SLNs/PTX (miSLNs-34a/PTX) were then prepared by co-incubation of SLNs/PTX with miRNA at a ratio of 8:1 (DDAB/RNA, w/w). By photon correlation spectroscopy (PCS) the average size of SLNs/PTX was 141.6 ± 2.5 nm, while miSLNs-34a/PTX containing both drugs had an average size of 218.2 nm and zeta

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Fig. 1. In vitro characterization of miSLNs-34a/PTX. (A) Schematic of miSLNs-34a/PTX assembly. (B) Blank SLNs, SLNs/PTX and miSLNs-34a/PTX visualized by transmission electron microscopy (TEM), Scale bar: 100 nm. (C) The encapsulation efficiency and drug loading yield of miSLNs-34a/PTX. (D) Release profiles of PTX and miR-34a from miSLNs-34a/PTX in PBS with 0.2% Tween 80. (E) The remaining PTX content of various miR-34a/PTX formulations after incubation in mouse serum or FBS. (F) The copy numbers of various miR-34a formulations after incubation in mouse serum or FBS. (G) Differential scanning calorimetry (DSC) thermograms of PTX and SLNs/PTX. Data in C–F are expressed as the mean ± SD (n = 3).

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potential of 45.05 ± 2.85 mV. The latter size is similar to that which we previously reported for miSLNs-34a (~200 nm) [14]. Transmission electron microscopy (TEM) revealed that miSLNs-34a/PTX were spherical in morphology (Fig. 1B). Detailed characterization studies revealed that PTX and miRNA were incorporated on the SLNs with extraordinarily high encapsulation efficiencies of 93.05 and 95.13%, respectively (Fig. 1C). The PTX and miRNA loading yields were 1.82% and 1.79 nmols per mg lipid, respectively (Fig. 1C). Paclitaxel showed a sustained drug release from SLNs/PTX up to 96 h (Fig. 1D). In the initial 12 h, the release amount of free PTX reached 90%, while it was less than 50% for SLNs/PTX. 3.2. Stability of miR-34a and PTX in the presence of sera Paclitaxel is a hydrophobic chemotherapeutic molecule and miRNAs are nuclease-labile in serum; accordingly, it is important to maintain their stability during administration. For an ideal drug delivery system, it is essential to protect the drug from degradation in the in vivo biological environment. In this study, the sera stabilities of miR-34a and PTX were evaluated in terms of copy number and HPLC, respectively. As can be seen from Fig. 1E, PTX in miSLNs-34a/PTX was stable when treated with fetal bovine serum (FBS) (102.12%) and mouse serum (91.22%), as compared to the untreated PTX group (100%). Simultaneously, the miR-34a copy numbers of miSLNs-34a/PTX treated with FBS and serum were 452.9 and 560.5 ng respectively, while those of the free miR-34a group were 190.8 and 27.7 ng, respectively (Fig. 1F). Even though the miR-34a copy numbers in both free miR-34a and miSLNs-

34a/PTX group were decreased compared with untreated miR-34a (951.4 ng), SLNs can protect the miRNA from degradation more efficient than free miR-34a in the sera. These data support that the resistance of the dual drugs to degradation was improved after incorporating into SLNs. 3.3. Differential scanning calorimetry (DSC) analysis DSC is a very useful technique in the investigation of thermal properties of nanoparticles, which can provide relevant physicochemical data concerning the drug within the nanoparticles. Unfortunately in our experiments, the amount of miRNA in the nanoparticles was below the threshold for accurate DSC analysis. Therefore, in the present study, we limited our DSC analysis to investigating the thermal property of PTX in the nanoparticles. The thermogram of PTX showed a decomposition peak at 244 °C (Fig. 1G) which is the melting point of PTX [23]. However, this peak was absent in the PTX-loaded SLNs, indicating that the drug was likely to be dispersed in the lipid matrix molecularly after nanoparticle preparation. 3.4. Cellular uptake of dual fluorescence-labeled SLNs The cellular uptake of miSLN-34a(Cy5)/PTX(coumarin-6) was then evaluated by flow cytometry and confocal microscopy. We first assessed the influence of DDAB on the cellular uptake of the miSLNs-34a/PTX (Fig. 2A). Increasing the DDAB/RNA ratio up to 8:1 (DDAB/RNA, w/w)

Fig. 2. Cellular uptake of miSLNs and miSLNs-34a/PTX. (A) The mean fluorescence intensity of different miR-34a concentrations associated with cells measured by flow cytometry. (n = 3). (B) B16F10-CD44+ cellular uptake of miSLNs-RNA(Cy5)/PTX(coumarin-6) measured by flow cytometry. (C) Confocal laser scanning microscopy images of cellular uptake of miSLNs-RNA(Cy5)/ PTX(coumarin-6) 4 h after incubation with B16F10-CD44+ cells at 37 °C. (D&E) Quantitative analysis of cellular uptake of miSLN-34a/PTX on B16F10 cells.

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resulted in enhanced cellular uptake of nanoparticles, but there was no further increase beyond 8:1, consistent with the results of our previous studies [14]. When the DDAB/RNA ratio reached 8:1, its cellular uptake was significantly increased compared to that of free RNA and DDAB/ RNA at a ratio of 4:1 (P b 0.001). Considering these data along with the increased risk of cyotoxicity with increasing concentration of cationic lipid, the DDAB/RNA ratio was set at 8:1 (w/w). To facilitate detection of SLNs in cellular uptake studies, coumarin-6 and Cy5 labeled RNA were incorporated into the SLNs (miSLNs-RNA(Cy5)/PTX(coumarin-6)). At 4 h-post administration, cells were collected and analysed by flow cytometry. Fig. 2B shows that virtually 100% (91.6% + 8.4%) of cells were coumarin-6-positive and 91.6% of cells were Cy5positive, suggesting that miSLNs-34a/PTX were efficiently taken up by B16F10-CD44 + cells. Confocal microscopy demonstrated that the coumarin-6 and Cy5 fluorescent signals were mainly localized in the cytoplasm (Fig. 2C), indicating that miSLNs-34a/PTX could achieve cytosolic delivery of these two drugs. The positive charge and pertinent particle size (~ 200 nm) contributed to the endocytosis of miSLNs. Fig. 2D&E show the quantitative analysis of cellular uptake of miR34a and PTX by B16F10-CD44+ cells. Higher cellular uptake efficiency was evidently observed at nanoparticle groups compared with free miR-34a group and free PTX group (Fig. 2D&E). For miR-34a analysis, the cellular uptake content of miSLNs-34a/PTX was 11.38 pg/μg protein versus 11.87 pg/μg protein in miSLNs-34a group (P N 0.05). For PTX analysis, a similar trend in the cellular content was observed between miSLNs-34a/PTX group (2.1 ng/μg protein) and SLNs/PTX group (2.4 ng/μg protein) (P N 0.05). These results suggested that the adsorption of negative miR-34a to positive SLNs/PTX did not affect the uptake of these two drugs by B16F10 cells (P N 0.05).

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3.5. In vitro synergistic cytotoxicity and apoptosis Cell proliferation was assessed by an MTT assay. We chose 8:1 as the DDAB/RNA ratio, and then tested the effect of PTX concentration on the B16F10-CD44+ cell viability. Untreated B16F10-CD44+ cells were used as the negative control. Fig. 3A shows that increasing the PTX content in the nanoparticles resulted in an enhanced antitumor effect, implying that cell inhibition was concentration-dependent. For free PTX and SLNs/PTX group, the antitumor effect (cell viability b 60%) was not evident until the cells were treated with a relatively high concentration (20 μg/mL) of PTX. However, treatment with miSLNs-34a/PTX (10 μg/mL of PTX) resulted in a cell viability of 52.75%, which was significantly lower than that of SLNs/PTX (10 μg/mL of PTX, 70.32%) (P b 0.05). More importantly, even when the high concentration of PTX (20 μg/mL) in SLNs/PTX was used, its antitumor effect was similar to that of miSLNs-34a/PTX (10 μg/mL of PTX) (P N 0.05), the results of which were much better than that of the miSLNs-34a not containing PTX (74.25%, P b 0.05). These findings suggested that co-delivery of miR-34a and PTX by SLNs improved the antitumor effect, compared with miR-34a or PTX separately delivered by SLNs/PTX or miSLNs-34a, respectively. To investigate the influence of the miR-34a/PTX formulations on cell apoptosis, we stained the nuclei of B16F10-CD44+ cells with DAPI and evaluated cell morphology changes by fluorescence microscopy. Viable cells display nuclei with bright blue fluorescence and homogeneous chromatin, whereas apoptotic cells show characteristic morphologic modifications such as nuclear condensation and pyknosis, chromatin fragmentation and formation of apoptotic bodies [20]. As shown in Fig. 3B, the miSLNs-34a/PTX sample produced the strongest cell apoptosis induction against B16F10-CD44+ cells among all formulations. The

Fig. 3. Antitumor effects of miSLNs-34a/PTX on B16F10-CD44+ cells in vitro. (A) MTT viability assay for B16F10-CD44+ cells after treatment with different formulations of PTX and miSLNs-34a (n = 3). n.s., not significant. (B) Cell apoptosis induction and morphological changes in B16F10-CD44+ cells produced by different formulations of PTX and miSLNs-34a (arrows: cell apoptosis). (C) Schematic illustration of B16F10-CD44+ tumor killing by miSLNs-34a/PTX based on the synergistic effect of paclitaxel and miR-34a.

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nuclei of the negative control groups remained spherical and integrated with homogeneous chromatin after DAPI staining. By contrast, in the PTX, SLN/PTX, miSLNs-34a and miSLNs-34a/PTX groups, it was readily evident that substantially greater numbers of cells exhibited extreme chromatin condensation and nuclear fragmentation, indicating that the nuclei were further distributed into apoptotic bodies.

3.6. Nanoparticle biodistribution via i.v. injection It can be seen from Fig. 4A that SLNs (miSLNs-34a, SLNs/PTX and miSLNs-34a/PTX) could increase their targeted accumulation in the tumor-bearing lungs compared with free drugs. To investigate the miR34a influence on the biodistribution and lung targeting characteristics

Fig. 4. In vivo fluorescence images of miSLNs-34a(Cy3)/PTX(coumarin-6) in mice. (A) Ex vivo biodistribution of free drugs, miSLNs-34a(Cy3), SLNs/PTX(coumarin-6) and miSLNs-34a(Cy3)/ PTX(coumarin-6) at 2 h after treatment. (B) Time-dependent intensity images of major organs of miSLNs-34a(Cy3)/PTX(coumarin-6). (C) Fluorescence microscopy images of tumor-bearing lung tissue sections at 4 h after i.v. injection of dual-fluorescence-labeled SLNs (blue fluorescence shows nuclear staining with DAPI, and green fluorescence shows the location of PTX (coumarin-6), and red fluorescence shows the location of RNA (Cy3)). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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of the dual drug formulation, the negative control oligonucleotide of miR-34a (GenePharm, China) was used to prepare miSLNs-NC/ PTX(coumarin-6) and its ex vivo imaging was also carried out. Fig. 4A shows that there was no evident difference between miSLNs-34a/ PTX (coumarin-6) and miSLNs-NC/PTX (coumarin-6). A gradual increase in fluorescence signal was observed in the tumor-bearing lungs from 1 to 4 h after i.v. injection (Fig. 4B). The ex vivo imaging of the lungs at 4 h (Fig. 4B) showed a relatively higher intensity than that in the lungs at any time point post-administration. Furthermore, these results demonstrated that miSLNs-34a(Cy3)/PTX(coumarin-6) had a higher selectivity for lung tissues than the other organs, resulting in a locally elevated apparent drug concentration in the lung. Coronal sections observed by confocal microscopy showed that both coumarin-6 and Cy3 fluorescence could distribute within the tumors, and that a robust intracellular delivery of both molecules occurred in the tumor cells (Fig. 4C).

function of time. Specifically, the survival time of 56 days for miSLNs34a/PTX, was much longer than that of SLNs/PTX (39 days) and miSLNs-34a (40 days) (P b 0.05), while there was no statistically significant difference between SLNs/PTX and miSLNs-34a (P N 0.05). Moreover, the survival time of the PTX group was 34 days, with very weak tumor inhibitory effect. The B16F10-based tumor nodules that were detectable in the lungs of PTX-, SLNs/PTX- and miSLNs-34a-treated groups were absent in those treated with co-delivery of miR-34a and PTX (Fig. 6B and D), suggesting an enhanced therapeutic efficacy. Notably, the average lung weight of mice treated with miSLNs-34a/PTX was significantly lower than those in the PBS and PTX groups (P b 0.05) (Fig. 6C).

3.7. miSLNs-34a/PTX inhibition of growth and metastasis of tumors

The CD44 molecule plays a key role in determining the tumorigenic capacity of tumor cells [10,11], which could be considered as a reliable maker of tumorigenicity. Immunohistochemistry confirmed that CD44, a cell adhesion protein, was overexpressed in PBS-, PTX- and SLNs/PTX-treated groups (Fig. 6E), suggesting that the tumors in these groups contained numerous cancer stem-like cells. However, CD44 expression was attenuated in the miSLNs-34a group and especially in the miSLNs-34a/PTX group. Accordingly, it can be found that the reduction of CD44 expression depended on miR-34a activity by comparing the results between the groups containing miR-34a and the groups excluding miR-34a (Fig. 6E). Even if PTX and SLNs/PTX showed an antitumor effect on B16F10-CD44+-bearing lungs, the spontaneous appearance of tumors would happen when the treatment is stopped due to the inefficiency in eliminating the CD44-positive cell population in the tumor mass. By contrast, miSLNs-34a and miSLNs-34a/PTX treatments resulted in smaller tumor nodules than those in control groups, leading to a relatively low possibility of tumor relapse. Simultaneously, it can be seen from Fig. 6E that CD44 expression in the miSLNs-34a/PTX-treated tumor-bearing lungs was down-regulated more efficiently than that in miSLNs-34a-treated tumor-bearing lungs. Thus, these findings suggest that miSLNs-34a and miSLNs-34a/PTX suppress tumor growth by regulating CD44 expression, and the enhanced antitumor efficiency of the co-delivery system depends on the lethality of PTX in all of the cancer cells by accompanying miR-34a bioactivity against CD44-positive cells.

Before in vivo antitumor assessment, the tumorigenicity of B16F10CD44+ cells in vivo was first investigated. SLNs/PTX-, miSLNs-34a-, and miSLNs-34a/PTX-treated B16F10-CD44+ cells were reduced in their tumorigenic capacity in vivo (Fig. 5). Tumors arising from miSLNs-34a-treated and SLNs/PTX-treated B16F10-CD44+ cells were 63.48 mm 3 and 60.61 mm3 in volume, respectively, much smaller than those in the untreated group (903.9 mm3, P = 0.0223, P = 0.0211). However, when cells were treated with miSLNs-34a/ PTX, no tumors were detected in the injected sites of C57BL/6 mice. Both PTX and miR-34a can induce cancer cell death; accordingly, the cell viability would be affected after administration of miSLNs34a, SLNs/PTX and especially miSLNs-34a/PTX, resulting suppression of the tumorigenicity of cancer stem-like B16F10 cells (miSLNs-34a/ PTX N miSLNs-34a ≈ SLNs/PTX N untreated). Aiming to verify whether miSLNs-34a/PTX has enhanced antitumor efficacy in the B16F10-CD44+-bearing mice, their survival rates along with lung weight and histological analysis were analyzed. Mice treated with seven consecutive injections of miSLNs-34a/PTX exhibited pronounced life-span extension (Fig. 6A). Treatment of mice with free PTX (commercial Taxol), SLNs/PTX, miSLNs-34a and miSLNs-34a/PTX produced significantly prolonged survival times, as compared with the saline control group, which exhibited relatively rapid death as a

3.8. CD44 downregulation by miSLNs-34a/PTX suppresses tumor growth in vivo

Fig. 5. Co-delivery of miR-34a and PTX (miSLNs-34a/PTX) enhances suppression of tumorigenicity of B16F10-CD44+ cells in mice. In both (A) and (B), 5 × 104 treated cells were injected subcutaneously into C57BL/6 mice, and tumor growth was monitored for 23 days after injection. (A) Tumor volume on day 23. Data are expressed as the mean ± SD. (B) Representative tumors in a mouse injected with B16F10-CD44+ cells that received the indicated treatments.

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Fig. 6. Co-delivery of miR-34a and PTX (miSLNs-34a/PTX) enhances inhibition of growth and metastasis of tumors. (A) Survival analysis of B16F10-CD44+-bearing mice. Survival times shown in right panel are the means ± SDs (n = 7). (B) Images of the B16F10-CD44+-bearing lungs on day 21 after seven consecutive i.v. injections of miSLNs-34a/PTX (n = 5–6). (C) Antitumor effects of different treatments evaluated by B16F10-CD44+-bearing lung weight (n = 5–6). (D) Histological staining of the B16F10-CD44+-bearing lungs after various treatments (arrows: tumor nodules). (E) Representative CD44 immunohistochemistry images of B16F10-CD44+-bearing lungs after treatment with the different formulations.

4. Discussion Numerous efforts have been made to treat the various types of cancer; however, the long-term spontaneous recurrence of tumors represents a major challenge in the field. Recently, microRNA-34a was found to inhibit cancer stem cell (CSC) growth, differentiation and metastasis by directly repressing CD44 and can therefore be developed as a therapeutic agent [11], raising hopes for a long-term lung cancer treatment. Subsequently, miR-34a delivery by systemic injection of nanoparticles was developed in order to inhibit B16F10-CD44+ growth, but this strategy still suffers from the risk of tumor relapse because such stand-alone therapy only target CSCs [14]. The successful elimination of cancer requires anticancer therapy that inhibits all the cell population. There is indeed a long way ahead of us to accomplish efficient cancer therapy from the laboratory to the clinic, which is with a lot of scope for investigation. In this work, we present a compelling strategy for fighting tumors by employing a combination therapy against B16F10-CD44+ cells that has a potentially synergistic effect. In this delivery system, miR-34a and PTX

were co-incorporated into SLNs (miSLNs-34a/PTX) to enhance anticancer therapy. miR-34a, a cancer-specific anti-oncogene, is known to negatively affect the expression of the CSC-specific protein CD44 [11]; PTX is a broad-spectrum chemotherapeutic agent that could affect all the tumor cells. The in vitro characterization of miSLNs-34a/PTX is shown in Fig. 1, in which we established not only the feasibility of simultaneous loading of miR-34a and PTX into SLNs, but also that the SLN formulation enhanced the stabilities of the drugs by protecting them from degradation in the presence of sera. A predominant feature of a desirable drug delivery system is that it possesses high cellular uptake efficiency. We have demonstrated that co-incorporation of miR-34a and PTX in SLNs (miSLNs-34a/PTX) increases the uptake of these nanoparticles by B16F10-CD44+ cells and that they induced more cell death than their stand-alone preparations (miSLNs-34a or SLNs/PTX) (Figs. 2 and 3). PTX, a mitotic inhibitor, promotes tubulin polymerization and formation of dysfunctional microtubules, disrupting the normal tubule dynamics required for cellular division thus provoking cell death [15,16]. miR-34a inhibits tumor growth by regulating multiple tumor-related genes such as SIRT1,

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Myc, Notch, p53, c-Met, survivin, CyclinD1 and CD44 [11,13,24–28]. Although both PTX and miR-34a alone are able to induce death in CD44+ cancer cells, the combination of these two drugs with different mechanisms can cooperatively and more efficiently inhibit B16F10-CD44+ development (Fig. 3C). That the antitumor effect of PTX or miR-34a alone in B16F10 cells was restricted is likely due to the fact that tumorigenesis and tumor inhibition are a complex process with multiple biochemical pathways. Because each of these anticancer drugs used alone was insufficient for managing the complex processes of tumor development and tumorigenicity, a dual-drug-based cancer therapy was developed to improve the antitumor efficacy by exploiting the synergistic effect of the two drugs (Fig. 3). Our studies demonstrate that SLNs can mediate delivery of the two drugs into the lung. We hypothesize that, due to physical entrapment of the SLNs in the capillary bed of the lung, the lung may act as a drug reservoir for miR-34a and PTX, leading to the formation of a drug concentration gradient between the lung and tumor, and thus providing the driving force for drug migration. That is, miSLNs-34a(Cy3)/ PTX(coumarin-6) was apt to penetrate into tumor tissues because of large gaps in the tumor and, equally importantly, it could migrate from the high concentration (lung tissue) side to the low concentration (tumor tissue) side. The well-documented enhanced permeation and retention (EPR) effect provides a satisfactory explanation for the uptake of nanoparticles by tumor tissues [29,30]. Our studies have further shown that the dual drug delivery system could improve the efficacy of anti-cancer therapy based on the synergistic or combined effect of miR-34a and PTX, as compared with their separate delivery by SLNs/PTX and miSLNs-34a. In the dual-drug case, a relatively high concentration in the tumor site (Fig. 4) would promote a more efficient uptake of both miR-34a and PTX by B16F10-CD44+ cells and a synergistic induction of tumor cell death. In addition, previous in vivo data showed an evident antitumor efficacy by administration of PTX formulations, but at relatively high drug doses (5–10 mg per kg of body weight) [18,19,31], which readily resulted in limited clinical outcomes with adverse effects. Conversely, the dual drug delivery system employed in our study, administrated at a comparatively low dose of PTX (1 mg PTX per kg of body weight), may reduce the potential for deleterious side-effects in normal cells, which we would attribute to relatively high drug levels in the tumor site due to the EPR effect, and the relatively low PTX dose used in vivo. MiSLNs-34a/PTX showed enhanced anti-cancer efficiency by regulating CD44 expression (miR-34a activity) (Fig. 6E), accompanied by the toxicity of PTX to all of the cancer cells (PTX activity). Therefore, miSLNs-34a/PTX cannot only result in a major impact on the treatment of cancer and side effects of PTX, but also enhance the therapeutic outcome of PTX. Altogether, the dual-drug delivery system offers a compelling strategy for the enhanced cancer therapy. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81130060 and 81173011) and the Science Foundation for Youths of Sichuan Province (No.2012JQ0024). We also thank Dr. Chester Provoda from the University of Michigan for his help in preparing this manuscript. References [1] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116 (2004) 281–297. [2] J. Lu, G. Getz, E.A. Miska, E. Alvarez-Saavedra, J. Lamb, D. Peck, A. Sweet-Cordero, B.L. Ebert, R.H. Mak, A.A. Ferrando, J.R. Downing, T. Jacks, H.R. Horvitz, T.R. Golub, MicroRNA expression profiles classify human cancers, Nature 435 (2005) 834–838. [3] V. Ambros, The functions of animal microRNAs, Nature 431 (2004) 350–355. [4] R.I. Gregory, R. Shiekhattar, MicroRNA biogenesis and cancer, Cancer Res. 65 (2005) 3509–3512.

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