Nucleolin-Targeting AS1411-Aptamer-Modified Graft Polymeric

Dec 18, 2017 - In this article, a novel graft polymeric micelle with targeting function ground on aptamer AS1411 was synthesized. The micelle was base...
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Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Nucleolin-Targeting AS1411-Aptamer-Modified Graft Polymeric Micelle with Dual pH/Redox Sensitivity Designed To Enhance Tumor Therapy through the Codelivery of Doxorubicin/TLR4 siRNA and Suppression of Invasion Shudi Yang,† Zhaoxiang Ren,‡ Mengtian Chen,† Ying Wang,† Bengang You,† Weiliang Chen,† Chenxi Qu,† Yang Liu,† and Xuenong Zhang*,† †

Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, 199 Ren’ai Road, Suzhou 215123, P. R. China ‡ Jiangsu Key Laboratory for Translational Research and Therapy for Neuropsycho-disorders & Department of Pharmacology College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, P. R. China S Supporting Information *

ABSTRACT: In this article, a novel graft polymeric micelle with targeting function ground on aptamer AS1411 was synthesized. The micelle was based on chitosan-sspolyethylenimine-urocanic acid (CPU) with dual pH/redox sensitivity and targeting effects. This micelle was produced for codelivering Toll-like receptor 4 siRNA (TLR4−siRNA) and doxorubicin (Dox). In vitro investigation revealed the sustained gene and drug release from Dox−siRNA-loaded micelles under physiological conditions, and this codelivery nanosystem exhibited high dual pH/ redox sensitivity, rapid intracellular drug release, and improved cytotoxicity against A549 cells in vitro. Furthermore, the micelles loaded with TLR4−siRNA inhibited the migration and invasion of A549. Excellent tumor penetrating efficacy was also noted in the A549 tumor spheroids and solid tumor slices. In vivo, multiple results demonstrated the excellent tumor-targeting ability of AS1411-chitosan-sspolyethylenimine-urocanic acid (ACPU) micelle in tumor tissues. The micelles exhibited excellent antitumor efficacy and low toxicity in the systemic circulation in lung-tumor-bearing BALB/c mice. These results conclusively demonstrated the great potential of the new graft copolymer micelle with targeting function for the targeted and efficient codelivery of chemotherapeutic drugs and genes in cancer treatment. KEYWORDS: polymeric micelles, codelivery, environmentally sensitive, invasion, tumor targeting cells. In addition, compared with that in extracellular fluids, the high concentration of GSH in intracellular compartments also promotes drug and siRNA release.15,16 Thus, the drug delivery systems with reduction-sensitivity have attracted considerable attention for the targeted tumor-specific drug release for cancer therapy. There is now a general consensus that PEI has been proved as an competent gene delivery vehicle in vitro and in vivo.17 As expected, siRNA condenses with PEI via electrostatic interaction and forms compact nanometer polymers. These nanoparticles can adsorb onto a negatively charged cell surface or after some modification, then translocate into cells via endocytic pathways.18 TLR4-mediated signaling pathway makes a critical difference in the process of survival, migration, and invasion in numerous cancers.19 The TLR4 stimulation with lipopolysaccharide (LPS) is well accepted to facilitate the migration and invasion

1. INTRODUCTION Cancer is widely accepted to be characterized by low cure rates, high recurrence rates, and high mortality rates.1−3 Substantial effort has been exerted in cancer therapy. Even so, the design and exploitation of novel, safe, and effective codelivery nanosystems for siRNA and chemotherapeutic drugs to cure cancers continue to face tremendous challenges.4,5 Lung cancer is most frequently histologically detected as nonsmall cell lung cancer (NSCLC) and the material reason for deaths of cancer patients worldwide, and the therapeutic outcome is unsatisfactory.6 The clinical treatments are facing numerous challenges, such as lack of targeting ability, multidrug resistance, migration, and invasion.7,8 Currently, novel delivery nanosystems with controlled drugrelease and targeted drug-delivery properties are highly needed. The pH-sensitive polymers can release drugs or genes to inside tumor tissue or lysosome (pH = 6.8 and 5.3, respectively; weak acid environments).9−14 Polymers that respond to acidic microenvironment and the proton sponge effect of lysosomes accelerate drug and siRNA release, respectively, inside tumor © XXXX American Chemical Society

Received: December 5, 2017 Accepted: December 7, 2017

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DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

completely dissolved in distilled water (20 mL, 25 °C). Moderate EDC·HCl (1.437 g) and NHS (0.861 g) were added to dithiodipropionic acid (40 °C, 0.5 h). Then, the aqueous solution of CSO (0.6 g, 20 mL) was added dropwise (40 °C, 12 h). The product was dialyzed via distilled water (MWCO: 1000 Da, 48 h) and then lyophilized for 2 days to obtain CSO-ss. To conjugate CSO-ss to PEI (CP), 1 g of CSO-ss was dissolved in distilled water (100 mL, 25 °C). Approximately 2 g of PEI, 2.395 g of EDC·HCl, and 1.435 g of NHS were solubilized to the aforementioned solution (pH 6, 12 h, 25 °C). The dialysis method was performed as previously mentioned. CSO-g-PEI-g-UA (CPU) was synthesized by CP (0.6 g) and UA (0.43 g) under the catalysis of EDC·HCl (1.19 g) and NHS (0.71 g). Briefly, the carboxyl groups of UA were activated as previously mentioned. The reaction solution was added dropwise to aqueous solution of CP (24 h, 25 °C). The reaction product was dialyzed as before to obtain CPU after lyophilizing.32 For AS1411 conjugation, the carboxyl units were activated in the same methods in distilled water for 6 h. Then, AS1411 (5 OD) was added to the nanocluster solution (24 h, lucifuge). ACPU was purified against PBS (MWCO: 500 Da) for dialysis. The chemical structures of CPU and ACPU (dissolved in D2O) were characterized by 1H NMR (400 MHz, Varian, Palo Alto, CA, USA). The observation of ACPU morphology was by TEM (TecnaiG220, FEI Company, Hillsboro, Oregon, USA). The surface zeta potential of the ACPU micelles was observed by DLS at 25 °C using ZEH 3600 (Malvern Instruments, Malvern, UK). The AS1411 Apt (0.5 mg/mL) and the ACPU micelle were measured by CD spectropolarimeter (JASCO Pty Ltd., Japan).33 2.3. pH/Redox Response Test for the Polymeric Micelle. CPU micelles (2 mg/mL) were obtained by the method of probe sonication. To investigate the pH/redox response of conjugates, we performed DLS to track the size and electric-potential changes of the micelles. The micelles tested were dissolved in different solutions34,35 2.4. Evaluation of Dox−siRNA-Loaded Micelles. The particle distribution and morphology of Dox−siRNA−CPU micelles and Dox−siRNA−ACPU micelles were investigated by DLS. 2.4.1. Dox and TLR4 siRNA Loading. Dox-loaded ACPU micelles (Dox−ACPU micelles) were prepared after obtaining hydrophobic Dox (500 mL of DMSO with 1 mg of Dox·HCl and 0.5 mL of triethylamine), then added to blank-micelles (5 mL, ultrasonic agitation) under stirring for 30 min with dialysis. siRNA-loaded ACPU micelles (siRNA−ACPU micelle) were prepared via mixing siRNA and micelleplex for 30 min at room temperature. siRNAFAM−ACPU micelles were then prepared similarly. The amounts of Dox micelle were measured using a fullwavelength microplate reader (Infinite M1000 PRO, TECAN, Switzerland) with wavelengths of 484 nm (excitation) and 598 nm (emission), respectively. The drug encapsulation efficiency (EE%) and the drug loading capacity (LC%) of the micelles were calculated as

of multiple malignant tumor cells like lung cancer cells, hepatocellular carcinoma cells, and so on. However, gene delivery, especially siRNA, faces massive barriers before accumulating in the targeted cytoplasm. These barriers include negative charges and large molecular weight (which render the siRNA difficult to cross cellular membranes), short half-life in blood (due to the rapid regression by nucleases), and poor uptake rate (causing decreased accumulation in cytoplasm).20 To overcome these obstacles, scholars have extensively explored various delivery vectors to enhance the delivery of genes. In this study, we attempted to synthesize chitosan-sspolyethylenimine-urocanic acid (CSO-g-PEI-g-UA; CPU) graft polymer as carriers loaded with gene and chemotherapy drug. CSO is covalently linked to the PEI by disulfide bonds (-ss-) in the disulfide dipropionic acid to weaken the powerful positive charge of PEI. Urocanic acid (UA) was used as a hydrophobic core for doxorubicin (Dox) loading. Additionally, in order to alleviate the toxic side effects of CPU micelles as safe and efficient anticancer carriers, researchers must use exquisite methods that would increase the specificity of these micelles in targeting cells. This step would improve the therapeutic efficacy and diagnostic capability of CPU micelles. AS1411 Apt (aptamers) is one targeting legend with a high specially binding to nucleolin, which is highly-expressed in a plasma membrane of cancer cells (not in normal tissues) such as A549 cells.21−29 Thus, the combination of polymeric micelles with Apt AS1411 for nucleus-targeted cancer therapy is favorable for guaranteeing high tumor specificity.25,30,31 According to a general consensus, the codelivery of chemotherapy and siRNA with targeting function in an individual delivery system is a considerable strategy for cancer treatment. AS1411-chitosan-ss-polyethylenimine-urocanic acid (ACPU) codelivering TLR4 siRNA and Dox with dual-pH/ redox sensitivity and targeting effect was designed to suppress TLR4 expression, overcome migration and invasion, and enhance the antitumor effect of Dox.

2. MATERIALS AND METHODS 2.1. Materials. 3′-Carboxyl-AS1411 Apt with oligonucleotide sequence 5′-GGTGGTGGTGGTTGTGGTGGTGGTGG-COOH-3′ (Shanghai Sangon Biotech Co., Ltd.), siRNAs (Santa cruze), CSO with 3−5 kDa molecular weight (Nantong Xingcheng Biochemical Co., Ltd.), dithiodipropionic acid and UA (Beijing Acros Technology Co., Ltd.), PEI (Aladdin-Reagent Co., Ltd.), GSH, EDC·HCl, and NHS (Aladdin-Reagent Co., Ltd.), doxorubicin hydrochloride (Dox·HCl, Dalian Meilun Biotech Co., Ltd.), MTT (Sigma− Aldrich), FBS and RPMI-1640 medium (Hyclone), an d Did (Beijing Solarbio Science and Technology Co., Ltd.). All other chemicals were analytically pure. The human lung adenocarcinoma cell line luc-A549 was obtained from the Department of Pharmacology of Soochow University, which was maintained in RPMI-1640 media with 10% (v/v) FBS (5% CO2, 37 °C). Female and male nude mice (BALB/C, nu/nu) (18 ± 2 g) were from the Experimental Animal Center of Soochow University (Suzhou, China). All animals were raised according to the standards of Suzhou University Laboratory. All animal dispositions were fulfilled following the agreements approved by the Institutional Animal Care. 2.2. Synthesis and Characterization of Graft Polymeric Micelle. First, dithiodipropionic acid (1.6 g) was B

EE% =

W0 × 100 W1

(1)

LC% =

W0 × 100 W

(2) DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics where W0 is loaded Dox; W1 is total weight of Dox; and W is total weight of Dox-loaded micelles. Agarose gels (1.5% (w/v)) were used to determine the binding ability of CPU and siRNA. Heparin sodium with DEPC was dissolved and mixed with a series of equal-volume solutions of different concentration gradients. At the same amount of siRNA−CPU micelle, 15 μL of each sample and 3 μL of buffer (6 × ) mixture were mixed. A gel imaging system was used for observation. Nuclease (with 1 U of RNase I/mg siRNA) and serum stability (20% serum) of siRNA were demonstrated by incubating siRNA−CPU micelle. Afterward, the disassembling of the siRNA micelle with heparin (36 U/mg siRNA, 15 min) was detected by a gel retardation assay. 2.4.2. Dox and TLR4 siRNA Release. Briefly, Dox−ACPU micelle (5 mL) was added into a dialysis bag (MWCO = 1000). This dialysis bag was then placed into a jar with PBS with various pH and GSH levels (pH 7.4, pH 7.4 + GSH [10 mM], pH 6.8, pH 6.8 + GSH [10 mM], pH 5.3, and pH 5.3 + GSH [10 mM]) as corresponding medium and stirred at 37 °C. The group of the free Dox was set as control.35 The siRNAFAM−ACPU micelle was incubated in PBS at different values (pH 7.4 and 5.3) with moderate shaking at 37 °C. The supernatant was removed after centrifugation (30,000 rpm, 45 min, 4 °C) for the fluorescence measurement of the released siRNAFAM at different time (excitation, 480 nm; emission, 500−600 nm). 2.5. Cytotoxicity Assay. The cytotoxicity of Dox and different formulations on A549 cells were performed by MTT assay. Briefly, A549 cells (5000 cells/well) were incubated with free Dox, Dox−CPU micelle, Dox−ACPU micelle, Dox− siRNA−CPU micelle, and Dox−siRNA−ACPU micelle at various concentrations for 48 h. Cells were incubated with 5 mg/mL MTT solution for 4 h and then dissolved by DMSO. After shaking for three times, the plates explored at 570 nm with a microplate reader. The cell viability (%) of the treated cells was computed. The cytotoxicity was determined as described above. Meanwhile, we examined the cytotoxicity of blank micelles on the tumor cell A549 and mouse fibroblast L929 to examine safety of vector. 2.6. Cellular Uptake. 2.6.1. Dynamic Uptake. Briefly, the cell nuclei of A549 cells were stained with Hoechst 33258 for 90 min, followed by injection of Dox−siRNA−CPU and Dox− siRNA−ACPU (Dox, 5 μg/mL; siRNA, 100 nM). The images were captured every 20 s for 3 h by a live cell station (Cell’ R, Olympus).36 2.6.2. Uptake Kinetics. We assumed that the micelles came into the cells and were eliminated from the cells at the speed of K0 and K, respectively, as follows:37

bottom Petri dish and treated with different formulations (Dox 5 μg/mL), the fluorescent intensity was monitored by CLSM 2 h later. The frozen slices of tumor tissues were obtained 24 h after the administration with Dox−CPU, Dox−ACPU, Dox− siRNA−ACPU, and Dox to investigate Dox distribution in solid tumor tissues.38 The solid tumor tissue slices were prepared for observation with a CLSM. 2.7. In Vitro Tumor Cell Motility Study. 2.7.1. Migration Assay. A549 cells were seeded in six-well plates to achieve a confluence of 80%, and subsequently, 10 μL pipet tip was used to scratch the cells to get vertical streaks.39 The medium was replaced with fresh low serum medium (2% fetal bovine serum) after washing twice with PBS. The pictures were observed by fluorescence microscopy (IX51, Olympus), and the scratch width was measured at predetermined times (0 and 48 h). 2.7.2. Invasion Assay. The invasion of A549 cells was measured via a Transwell insert composed of 8 μm pores. Both sides were coated with 10 μg/mL fibronectin. Then, A549 cells were suspended with serum-free medium and then inoculated into the upper chamber and cultured for 48 h (1 × 105/well). By contrast, the invasion medium in the lower wells was the same medium containing 10% FBS.13 2.8. Quantitative Real-Time PCR (Q-PCR) and Western Blot. 2.8.1. Q-PCR. RNA was extracted from the A549 cells after 48 h incubated with CPU micelle and siRNA−CPU micelle, and PBS using TRIzol reagent (TaKaRa Biotechnology, Dalian, China) was used to extract total RNA from A549. Then, 1000 ng of total RNA was reversed in accordance with the manufacturer’s instructions.40,41 mRNA expression quantitative tests were performed by SYBR Premix II (Bio-Rad, Hercules, CA, USA). GAPDH was used as the endogenous control. TLR4 specific primers: forward primer 5′-AAGCCGAAAGGTGATTGTTG-3′; reverse primer 5′-CTGAGCAGGGTCTTCTCCAC-3′. 2.8.2. Western Blot. The cells were incubated with RIPA cell lysis buffer (Cell Signaling Technology, USA) for 30 min on ice. Then, the protein concentration was determined by BCA Protein Assay Kit (Beyotime, China).40,42 The blots were probed by anti-TLR4 antibody (1:500, Santa Cruz, USA) and anti-α-tubulin antibody (1:10000, Sigma-Aldrich, Germany). Densitometry was quantified by ImageJ software. 2.9. In Vivo Study NIR Fluorescence Imaging. 2.9.1. Construction of Orthotopic Lung Tumor. Orthotopic lung tumor was constructed in accordance with Yuan et al. with slight modifications.43 Orthotopic lung tumor was established and all the above-mentioned operations were carried out in SPF level lab. The bioluminescence in situ was observed using an IVIS Spectrum. 2.9.2. NIRF Imaging. The targeting properties were investigated by a near-infrared fluorescent (NIRF) imaging system. First, Did-labeled micelles Did−CPU and Did−ACPU (50 μg/mL Did) were intravenously injected through the tail vein. The fluorescence distribution in vivo was tested by the NIRF imaging system at 6 and 12 h. The tumors and major organs were also imaged (Did: λex = 644 nm; λem = 663 nm). 2.9.3. Antitumor Effect. The antitumor activity of Dox− ACPU micelles was evaluated via bioluminescence imaging. Meanwhile, the body weight of mice in each group was recorded. More specifically, the mice (at least 10 mice in each group) bearing orthotopic lung tumors were divided to six groups: saline, blank micelle, Dox, Dox micelle, siRNA micelle, and Dox−siRNA micelle. Ten mg/kg Dox was injected through the tail vein every other day.33

k0 → X → k

Then, k was changed in a proportional manner according to the increasing micelles uptake (X) by A549 cells with incubation time and k0 as well k were calculated as follows:

X = k 0 − kX

(3)

X = k 0(1 − e−kt )/k

(4)

2.6.3. Dox Distribution in Tumor Spheroids and Solid Tumors. A549 cells were seeded on 96-well plates coated with 80 μL of agarose (2%) at a density of 600 cells/well to obtain the 3D tumor spheroids. After incubation for approximately 4 days, the 3D tumor spheroids were transferred to a glass C

DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 1. (A) Synthesis procedure of CPU. (B) 1H NMR spectra of CSO (a), CSO-ss (b), PEI (c), CP (d), and CPU (e). (C) TEM micrograph of ACPU micelle (60,000× original magnification). (D) CD spectra of AS1411 and ACPU-micelle. (E) Particle size changes in micelles after incubation with PBS (pH 7.4) + GSH, PBS (pH 5.3), and PBS (pH 5.3) + GSH for 1 h.

2.10. Statistical Analysis. All quantitative data are expressed as means ± standard deviation (SD). Data were analyzed by ANOVA. Values of p < 0.05 were considered statistically significant.

mV. To identify the secondary structure of AS1411, we performed a CD experiment (Figure 1D). The CD spectrum of single AS1411 showed the characteristic peaks (265 and 241 nm) of G-quartet structures in AS1411.33 The two peaks of ACPU had no obvious differences. Hence, the secondary structure of AS1411 was potentially maintained after conjugating with CPU micelle. The ionization environment change may due to the thin fluctuation in the ACPU micelle group.33 Besides, ACPU copolymer showed a low critical micelle concentration (CMC) value of 7.94 × 10−3 mg/mL. This value suggested that the copolymer could self-assemble into micelles with excellent stability in vitro (Figure S2). The pH/redox-responsive property of ACPU was tested via the changes in the micelle size during incubation separately in weak acidic PBS and PBS with GSH (10 mM). The ACPU micelles swelled from 124.6 nm to 365.3 and 622.6 nm after incubation with PBS (pH 7.4) + GSH and PBS (pH 5.3) for 1 h, respectively (Figure 1E). A double peak was attained for PBS (pH 5.3) + GSH, which implied that micelles lose stability. The changes in particle size were due to the proton sponge of imidazole perssad in acidic solution. Then, GSH destroyed the structure of disulfide bonds and promoted a rapid drugs and genes release. 3.2. Evaluation of Dox−siRNA-Loaded Micelles. The EE% and LC% of Dox-loaded micelles were 85.81 ± 1.22% and 14.26 ± 1.09%, respectively. Meanwhile, siRNA was compressed into the PEI skeleton via electrostatic interaction. siRNA was efficiently tied to the micelles at an N/P ratio of 6/1 identified (Figure 2A).44 Heparin with a strong electronegativity can be used as a clearing agent, and the siRNA can be resolved from the micelles. When the amount of heparin increased from 50 to 70 μg/μL, the band brightness no longer increased. Hence, 50 μg/μL is the heparin amount at which siRNA can be completely dissociated from the micelle. In blood, various nucleases degraded siRNA in the rapid way.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of ACPU. As shown in Figure 1A, CPU copolymer was synthesized via the acylation reaction between the primary amines of CSO or PEI and the carboxyl groups of dithiodipropionic acid or UA activated by EDC·HCl and NHS. Byproducts could be easily removed by dialysis and filtration. Branched PEI, containing abundant primary amines in the structure, was convenient for reaction with CSO-ss and UA. As shown in Figure 1B, the 1H NMR spectrum signals of CSO appeared at δ = 4.67 (H1), 3.20−4.00 ppm (sugar ring in CSO), and 2.01 (−CH2). The signals at δ = 2.74, 2.93 ppm (−CH2CH2−) were attributed to the specific proton peaks of the pentaheterocyclic group in dithiodipropionic acid (Figure 1B-b). This notion suggested the successful grafting of dithiodipropionic acid to CSO. The proton peaks at δ = 2.52−3.47 ppm (−NHCH2CH2− of PEI) in the spectrum of CP, indicating that PEI was grafted to the CSO-ss (Figure 1B-d). The spectrum signals of UA are located at δ = 8.23 (Ha), δ = 7.61 (Hb), δ = 7.5 (Hc), and δ = 6.51 (H1),35 which suggested that UA was indeed introduced into CP (Figure 1Be). Given the spectra of CPU, the peaks assigned to all the results described above imply the successful synthesis of the CPU copolymer. As shown in Figure S1, AS1411 and CPU had been covalently combined carboxyl groups (175−185 ppm), rather than by physical mixing. The graft ratios of CSO and UA calculated from the 1H NMR spectrum were 3.33% and 44.4%, respectively. The TEM (60,000×) of the ACPU micelle in Figure 1C revealed a particle size of 124.6 ± 1.068 nm as a united spherical structure. The mean zeta potential was 24.2 ± 0.529 D

DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 2. (A) Binding ability of blank ACPU micelles and siRNA at different ratios (channel 1−6, N/P ratios were 0, 1, 2, 4, 5, and 6, respectively). (B) Decomplexation results of siRNA−ACPU micelle by agarose gel retardation assay. (C) Serum stability results of the siRNA−ACPU micelle incubated with 20% serum (channel 1, naked siRNA; channel 2, naked siRNA incubated with 20% serum for 30 min; channels 3−6, siRNA−ACPU micelle incubation with 20% serum for 30, 60, 90, and 120 min). (D) Serum stability results of siRNA−ACPU micelle incubated with 1 U of RNase I/μg siRNA. (channel 1, naked siRNA; channel 2, naked siRNA incubated 1 U of RNase I/μg for 15 min; channels 3−6, siRNA−ACPU micelle incubated with 1 U of RNase I/μg for 30, 60, 90, and 120 min). Values are presented as mean ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001 vs control).

Therefore, the poor stability of siRNA was the key problem needing to be overcome.36 In comparison with the consequences of the naked siRNA, siRNA was fully protected by the micelle from nuclease and serum (Figure 2C,D).45 Dox−siRNA−CPU micelle possessed a mean particle size of 133.40 ± 1.88 nm (PDI = 0.226) (Figure 3A) and a zeta potential of 31.91 ± 1.05 mV, while the mean particle size of Dox−siRNA−ACPU micelle was 125.90 ± 1.42 nm (PDI = 0.122) and the zeta potential was 24.66 ± 1.25 mV, which was suitable for the EPR effect. Moreover, the results in Figure S3 showed that AS1411 was taken up into A549 cells through a receptor pathway. The release of Dox was shown in Figure 3B. The release of Dox from Dox−ACPU micelle at pH 7.4 PBS was observably inhibited by the self-assembly of micelles with an accumulated release of 26.83 ± 1.48% within 48 h. However, in pH 5.3 PBS or with 10 mM GSH, drugs were rapidly released from micelles. Furthermore, the cumulative release ratio of Dox from Dox micelle was as 75.47 ± 2.88% in pH 5.3 PBS with GSH (10 mM). This release ratio almost doubled that in pH 7.4 PBS. The protonation of UA in acidic environment has been thought to contribute to the rapid release that leads to the instability of

micellar structure. Fracture of disulfide bonds under reductive conditions is also considered a factor and results in the imbalance of hydrophilicity/hydrophobicity. Results indicate a stable extracellular and intracellular tumor release that benefits targeted cancer therapy. The siRNA release from micelleplex was measured at various pH and reducing conditions (Figure 3C). siRNA adsorbed onto the PEI skeleton or compressed into the core at pH 7.4 also with 10 mM GSH. This phenomenon prevented siRNA release from the siRNA micelle in the systemic circulation. Nevertheless, the significant increase of fluorescence intensity showed the micelle became unstable and released siRNA at pH 5.3 and pH 5.3 + GSH medium.45 These results demonstrated that the polymeric micelle could avoid siRNA from enzymatic degradation in a physiological environment but accelerate intracellular siRNA release, which could inhibit the transcription of TLR4 and suppressed TLR4 expression. The difference of Dox and siRNA release between ACPU and CPU micelles were negligible (Figure S4). 3.3. Cytotoxicity Assay. Figure 4A showed that the different formulations of Dox inhibited cell viability dosedependently. Furthermore, the micelles loaded with TLR4 E

DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 3. (A) Size of Dox−siRNA−CPU micelle and Dox−siRNA−ACPU micelle. (B) Release of Dox from ACPU micelles after incubation with PBS (pH 7.4), PBS (pH 7.4) + GSH, PBS (pH 6.8), PBS (pH 6.8) + GSH, PBS (pH 5.3), and PBS (pH 5.3) + GSH for 48 h. (C) Release of siRNA from ACPU micelles after incubation with (a) PBS (pH 5.3), (b) PBS (pH 5.3) + GSH, (c) PBS (pH 7.4), (d) PBS (pH 7.4) + GSH. Values are presented as mean ± SD.

Figure 4. (A) Cytotoxic effect of different formulations of Dox against A549 cells. (B) Cytotoxicity of two blank micelles against A549 and L929 cells. Values are presented as mean ± SD.

siRNA no longer enhanced the cell viability suppression. Dox− ACPU micelles displayed the strongest cell viability inhibition of A549 cells, followed by Dox−CPU micelles. This inhibition may have resulted from the higher uptake and faster intracellular release of drug from Dox−ACPU micelles. Dox with low cellular uptake exhibited obvious cytotoxicity against A549 cells, which might be due to the rapid colocation with the cell nuclei and the induced cell apoptosis. The safety of the two

kinds of polymers, namely, CPU and ACPU, was assessed by MTT assay (Figure 4B). Of the cells, 90.54% and 91.35%, respectively, survived after treatment with these two kinds of micelles for 48 h against L929 and A549 cells. This finding demonstrated the desirable safety of the vehicles. In Figure S5, hemolysis in the CPU and ACPU micelle groups is far below that in the Tween 80 group at the same concentration. Thus, the polymer micelles possess blood compatibility and safety. F

DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 5. (A) Dynamic uptake images of micelles, where red, green, and blue areas correspond to Dox, siRNAFAM, and cell nucleus, respectively, in (a) CPU and (b) ACPU. (B) Kinetics of fluorescence intensities of (a) CPU and (b) ACPU, and (c) micelle uptake kinetic parameters obtained by fluorescence change over time. (C) Penetrating evaluation: fluorescence distribution of A549 tumor spheroids after incubation with Dox in different formulations at pH 7.4 for 2 h. (a) Dox, (b) Dox−CPU, (c) Dox−ACPU, and (d) Dox−siRNA−ACPU. Values are presented as mean ± SD.

variations were measured and fitted to nonlinear cellular uptake kinetics (Figure 5B). The Dox−siRNA−ACPU micelle revealed the highest uptake rate but the lowest elimination speed compared with the Dox−siRNA−CPU micelle. The divergence was attributed to the interaction of AS1411 and nucleolin, which could increase intracellular drug concentrations. Furthermore, the cellular uptake of the CPU and ACPU micelles was quantitatively examined by flow cytometry (Figure S7). 3.5. Tumor Penetrability Evaluation. The targeting evaluation reflected the accumulation of ACPU micelle in the tumor site but did not ensure the further distribution in solid tumor. Therefore, the distribution of Dox−ACPU micelle and other formulations in A549 tumor spheroids and solid tumor slices was further investigated via CLSM. After a 2 h

3.4. Cellular Uptake. The cellular uptake and subcellular distribution of micelles in vitro were confirmed by CLSM (Figure S6). A greater amount of Dox colocated with the nuclei treated with Dox−ACPU than with the nuclei treated with Dox−CPU after 3 h of incubation. The preponderant cellular uptake was further dynamically confirmed by live cell station (Figure 5A). That is, Dox and siRNA were delivered by micelles in the cells and then released into the nucleus and cytoplasm. Concomitantly, the fluorescence of Dox and siRNA in ACPU was stronger than that in CPU. The fluorescence distribution table indicated that the Dox−siRNA-loaded micelle could simultaneously deliver drugs and genes into cells. These results were consistent with the aforementioned observations. After treatment with different micelles, the intracellular fluorescence from Dox and siRNA dynamically changed. These G

DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 6. (A) In vitro tumor cell motility study including migration assay (a) and relative cell migration rate (b). (B) Tumor invasion assay (a) and total number of invading cells under the microscope (×400) (b). (C) Analysis of TLR4 mRNA level and (D) protein expression. A549 cells were transfected with different siRNA−TLR4 doses in micelleplex. The concentration of siRNA−TLR4 with lipofectamine 2000 and NC-siRNA with micelleplex (NC-micelle) was 100 nM. Transfection tests were performed at least three times. Values are presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). (#p < 0.05, ##p < 0.01, ###p < 0.001 vs PBS).

contrast, the number was 60 ± 5 in the control group. Therefore, TLR4 siRNA alone significantly inhibited the migration and invasion of the A549 cells (Figure 6B). 3.6.2. Analysis of TLR4 Expression. The intracellular TLR4 mRNA level and protein expression were measured by Q-PCR and Western blot, respectively, to detect siRNA−ACPU micelle transfection efficiency. Both mRNA level (Figure 6C) and protein expression of TLR4 (Figure 6D) decreased dosedependently when incubated with siRNA−ACPU micelle. A more significant knockdown efficacy was observed in 150 nM TLR4 siRNA treatment group compared to another two lower concentration groups. The transfection efficiency of the siRNA−ACPU micelle was similar to lipofectamine 2000.

pretreatment, the Dox−ACPU and Dox−siRNA−ACPU micelle group exhibited the strongest Dox fluorescence and showed no dramatic decrease from the edge to the deep section of tumor spheroids (Figure 5C). These results demonstrated that the drug and siRNA codelivery micelle exerted excellent penetrating effects. 3.6. Assessment of the Gene Silencing Effect in Vitro. 3.6.1. Motility Study of Tumor Cells. Migration assay and invasion assay were utilized to evaluate whether siRNA micelles could effectively reduce the ability of tumor cell motility. As shown in Figure 6A, siRNA-CPU and siRNA-ACPU could prevent the migration of tumor cells on the scratch area of control in 48 h. Transwell invasion assay showed that the invasive cells was 31 ± 2 after silencing with TLR4 siRNA. By H

DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 7. Targeting property and antitumor effect of micelles toward A549 tumors in vivo. (A) NIRF images of subcutaneously transplanted A549 tumors treated with (a) Did−CPU micelle and (b) Did−ACPU micelle. (B) Tissue ratios of Did−CPU micelle and Did−ACPU micelle in tumorbearing mice at different postinjection times. (C) Fluorescence intensities of isolated organs (heart, liver, spleen, lung, kidney, and intestine) from the tumor-bearing mice at 12 h postinjection. At least six mice for each group were included (*p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 8. (A) Variation in the bioluminescence of orthotopic tumor under various Dox formulations in vivo. (B) Body weights of tumor-bearing mice during the 22 days’ treatment. (C) Fluorescence intensities of the lung organs of the tumor-bearing mice during the 22 days’ treatment at sample postinjection. (D) Survival rates of the mice after treatment with saline, blank−ACPU micelle, free Dox, Dox−ACPU micelle, siRNA−ACPU micelle, and Dox−siRNA−ACPU micelle. Data are presented as mean ± SD. At least 10 mice for each group were included (*p < 0.05, **p < 0.01, and ***p < 0.001).

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DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 9. Histological images of the main organs of the mice treated with saline, blank−ACPU micelle, free Dox, Dox−ACPU micelle, siRNA− ACPU micelle, and Dox−siRNA−ACPU micelle. Data are presented as mean ± SD. At least 10 mice for each group were included.

This finding suggested the highly effective gene vector of the ACPU micelle. 3.7. Study of Targeting Properties. The targeting efficacy of CPU and ACPU were evaluated by a NIR fluorescence imaging system using Did as probe. After CPU and ACPU treatment, the fluorescent signals significantly accumulated in the liver within 6 h (Figure 7A). This accumulation may be due to the rapid swallowing by rich mononuclear phagocytes. After 12 h postinjection, the fluorescence in the tumor was stronger than that in the live cells for both of the two micelles. This result implied that the codelivery micelles could circulate in the blood and accumulate in the tumor through the increased EPR effect. In addition, compared with the Did−CPU group, the tumor-bearing nude mice intravenously treated with Did−ACPU exhibited a more remarkable fluorescence accumulation in tumor within 12 h after injection. Thus, the micelles with AS1411 exerted a superior targeting effect toward the tumor. The fluorescence of the tumor and other organs (heart, liver, spleen, lung, and kidney) also confirmed the effectiveness of the micelles in tumor-targeted delivery (Figure 7B,C). 3.8. Antitumor Effect. The in vivo antitumor efficacies of Dox, Dox micelle, siRNA micelle, and Dox−siRNA micelle were evaluated by the luc-A549 cell orthotopic tumor model. Figure 8A showed the differentiations in bioluminescence of orthotopic tumors treated weekly with multiple Dox formulations. The intensity of orthotopic tumors’ bioluminescence presented an evolutionary tendency over the therapeutic period. After 12 days, the tumor gradually transferred from the inoculated site to another part of lungs. The excised organs from mice after the treatments were revealed in Figure 8A. Quantitative analysis of tumor piece in the lungs revealed that the bioluminescence intensity in the lungs treated with Dox− siRNA micelle was twice lower than Dox (p < 0.01).43 The volume of tumor tissues was greatly suppressed in the Dox group, but the weight of the mice in such group significantly

decreased with respect to those of the other micelle groups (Figure 8B). This result suggested the higher safety of the micelle. However, the fluorescence intensity of the mice in the Dox−siRNA micelle-treated group was obviously weaker than Dox and the other treatment groups. All the results showed that the tumor inhibition effect of the Dox−siRNA micelle was much superior to those of Dox and the other groups (Figure 8C). Significant variation in survival rates was observed among the groups treated with saline solution, blank micelle, Dox, Dox micelle, siRNA micelle, and Dox−siRNA micelle (Figure 8D). All the mice in the Dox group were sacrificed after 30 days of treatment and revealed the intrinsic toxicity of free Dox.33 Histological analysis used to evaluate the biotoxicities and tumor-inhibition effects of blank micelle, Dox, Dox micelle, siRNA micelle, and Dox−siRNA micelle. The necrosis of tumor tissue increased and the proliferation of tumor cells slowed down after treatment with Dox and its nanoconjugations, especially for the Dox micelle and Dox−siRNA micelle groups (Figure 9). Obvious pathological changes were apparent in different organs (heart, liver, spleen, lung, and kidney) in the saline and blank micelle groups.33 Tumor mass and broken alveoli were obviously found in the lung tissues. By contrast, no significant lesion was observed in the spleens of the Dox− siRNA micelle group. Hence, the ligand-conjugated nanomedicine achieved reduced biotoxicity. Particularly, the in situ tumors of the lungs were significantly improved.33

4. CONCLUSIONS In summary, novel polymeric micelles with dual pH and reduction sensitivity were successfully prepared for tumor therapy. These micelles enable the rapid and steady intracellular release of drugs and genes. Currently, the invasion and metastasis of tumor cells are major reasons that lead to treatment failure. Thus, novel anticancer therapeutic schedules and nanosystems with powerful tumor cytotoxicity and invasion inhibition activity were highly needed. The ability of tumor J

DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

substitution; DLS, dynamic light scattering; CD, circular dichroism; TEM, transmission electron microscopy; Dox− CPU micelle, Dox-loaded CPU micelle; Dox−ACPU micelle, Dox-loaded ACPU micelle; siRNA−CPU micelle, siRNAloaded CPU micelle; siRNA−ACPU micelle, siRNA-loaded ACPU micelle; siRNAFAM−CPU micelle, FAM-labeled siRNA− CPU micelle; Dox−siRNA−CPU micelle, Dox−siRNA-loaded CPU micelle; Dox−siRNA−ACPU micelle, Dox−siRNAloaded ACPU micelle; NC-micelle, negative-control siRNA micelle; EE%, encapsulation efficiency; LC%, drug loading content; MTT, 5-diphenyl-2H-tetrazolium bromide; CLSM, confocal laser scanning microscopy; Q-PCR, quantitative realtime PCR; NIRF, near-infrared fluorescent; EPR, enhanced permeability and retention; CMC, critical micelle concentration

specificity and deep tissue penetration were verified in different steps. Using biological dye, we directly visualized the biodistribution of the Dox−siRNA−ACPU micelle in a tumor-bearing mice model and localized the tumor sites accurately. Additionally, the Dox−siRNA−ACPU micelle was revealed to actively accumulate in tumor cells. Dox entered the nucleus, and the siRNA entered the cytoplasm after short incubation times. The deep tumor penetration of micelles was confirmed in the 3D tumor spheroid. Moreover, the Dox− siRNA−ACPU micelle improved the tumor therapeutic efficacy by down-regulating the TLR4 protein level to inhibit tumor invasion meanwhile reflecting excellent antitumor activity. The tumor therapy efficacy of the Dox−siRNA−ACPU micelle was also enhanced in orthotopic tumor model. The present study provided new strategies to design drug−siRNA-loaded micelles with ameliorated efficacy against tumor survival and invasion. This novel codelivery nanosystem is expected to prompt the further exploration of these polymeric micelles for combination therapy in other related diseases.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b01093. 13 C nuclear magnetic resonance (13C NMR) spectroscopy; critical micelle concentration (CMC) of CPU; experiment of competitive receptors inhibition; release of Dox and siRNA from CPU micelles; hemolysis activity of micelles in vitro; subcellular distribution of micelles; flow cytometry to quantitatively examine the cellular uptake (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86 (0512) 65882087. ORCID

Xuenong Zhang: 0000-0003-1150-5314 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to National Natural Science Foundation projects of China (NSFC 81773183, 81571788, and 81273463), Jiangsu Science and Technology Support Plan (BE2011670), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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ABBREVIATIONS GSH, glutathione; TLR4, Toll-like receptor 4; Dox, doxorubicin; NSCLC, nonsmall cell lung cancer; PEI, polyethylenimine; CSO, chitosan; LPS, lipopolysaccharide; Apt, aptamer; CPU, chitosan-ss-polyethylenimine-urocanic acid; ACPU, AS1411-chitosan-ss-polyethylenimine-urocanic acid; EDC· HCl, 1-(3-(dimethylamino) propyl)-3-ethyl carbon carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; 1H NMR, 1 H nuclear magnetic resonance spectroscopy; DS, degree of K

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DOI: 10.1021/acs.molpharmaceut.7b01093 Mol. Pharmaceutics XXXX, XXX, XXX−XXX