Ternary Nanoparticles with a Sheddable Shell Efficiently Deliver

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Ternary Nanoparticles with a Sheddable Shell Efficiently Deliver MicroRNA-34a against CD44-Positive Melanoma Minmin Fan, Ye Zeng, Huitong Ruan, Zhirong Zhang, Tao Gong, and Xun Sun Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00377 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on August 1, 2017

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Molecular Pharmaceutics

Ternary Nanoparticles with a Sheddable Shell Efficiently Deliver MicroRNA-34a against CD44Positive Melanoma Minmin Fan,†,‡ Ye Zeng,† Huitong Ruan,† Zhirong Zhang,† Tao Gong,† and Xun Sun†,* †

Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West

China School of Pharmacy, Sichuan University, Chengdu 610041, China. ‡

State Key Laboratory of Polymer Materials Engineering, Polymer Research Institute of Sichuan

University, Chengdu 610065, China KEYWORDS: PEGylation, ternary nanoparticles, sheddable shell, CD44-positive melanoma, microRNA-34a release

ABSTRACT: PEGylation can stabilize drug delivery systems for cancer therapy by creating repulsive interactions with biological components in vivo. While these interactions reduce nonspecific adsorption of drug-loaded particles onto non-target surfaces, they also inhibit internalization of particles into target cells. To circumvent this so-called “PEG-dilemma”, we have developed nanoparticles with a PEG coating that is shed after arrival in target tissue. Positively charged polycation nanoparticles were assembled with microRNA-34a via

ACS Paragon Plus Environment

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electrostatic interactions, and then coated again via electrostatic interactions with an anionic PEG derivative that separates from the nanoparticle in the acidic tumor microenvironment. The resulting ternary nanoparticles with a sheddable shell have nearly neutral surface charge, which markedly reduces non-specific adsorption. Shedding the PEG coat enhanced nanoparticle uptake into CD44-positive melanoma cells and promoted microRNA-34a release, which down-regulated CD44 expression and thereby inhibited tumor growth. We conclude that nanocarriers with a sheddable shell show promise for cancer therapy.

1. INTRODUCTION Malignant melanoma is among the most aggressive neoplasms; it can be fatal, especially at the metastatic stage. Incidence of malignant melanoma has increased over the last decade,1 highlighting the need for early, more effective treatment. While some treatments against metastatic malignant melanoma have been approved by the US Food and Drug Administration, including interferon-a2b (IFN-a2b),2,

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interleukin-2 (IL-2),4,

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and dacarbazine,6 none clearly

alters the natural history of the disease in the majority of patients. Novel types of targeted therapy and immunotherapy have been developed, but they do not provide durable therapeutic effects because metastases are difficult to target.7 Expression of CD44 on the surface of melanoma cells may contribute to their high metastatic potential,8 suggesting that reducing CD44 expression or activity may inhibit melanoma growth, invasion, and metastasis.9, 10 Recently, the microRNA miR-34a has been shown to repress CD44 expression and thereby inhibit clonogenic expansion of cancer stem cells, tumor regeneration, and metastasis.11 The


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gene encoding miR-34a is a downstream target of p5312-15 and possesses tumor suppressive properties.16 In fact, miRNAs have been implicated in the onset and progression of many types of cancer,17, 18 where they act as tumor suppressors and oncogenes19-22 by regulating a wide array of biological processes such as differentiation, proliferation, and apoptosis.23 The lability and hydrophilicity of miRNAs in vivo means that they cannot be administered directly into the body;24 instead they are typically encapsulated in nanoparticles containing poly(ethylene glycol) (PEG), which reduces protein adsorption and particle aggregation, prolonging lifetime in circulation, thereby improving systemic delivery of drugs and genes.25, 26 While such PEGylation improves delivery of drug to target tissue, it inhibits uptake into tumor cells,27, 28 significantly reducing antitumor efficacy in vivo. One strategy to overcome this socalled “PEG-dilemma” is to coat lipid- or polymer-based carriers with a PEG shell that is sensitive to acidic pH. Such a shell should be shed in the acidic microenvironment of intracellular endosomes or tumors;29-32 average extracellular pH is 6.5 in tumors, compared to physiological pH of 7.4.33,

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Nanocarriers that can be activated at the pH of the tumor

extracellular microenvironment form the basis of a new strategy for tumor-targeted drug delivery.35-37 Various in vitro studies have tested drug delivery systems that, in response to an acidic microenvironment, disassemble to release drug or expose targeting molecules that were shielded during circulation.35-39 Most of these shedding systems, though effective in vitro, have not worked well in vivo. Recently Wang et al.40 reported a negatively charged ternary nanoparticle system with a sheddable shell for tumor acidity-targeted siRNA delivery. This system showed good RNA-interfering and tumor growth suppression in vivo. However, the body normally treats charged particles as foreign and mounts an immune response against them.41 Such a response


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could be avoided by designing drug delivery nanocarriers that are neutral until they reach target tissue, yet such carriers have scarcely been investigated. Here we develop shell-shielded ternary nanoparticles composed of biocompatible cationic βcyclodextrin-PEI600 loaded with miR-34a and coated with a PEGylated anionic polymer (BocPEOn/PAGEm-Cys-DMMA, PPC-DA). The particles have a near-neutral surface and they lose their coating in the acidic tumor microenvironment, facilitating uptake by tumor cells and intracellular release of miR-34a via the enhanced permeation and retention (EPR) effect.42, 43 We show in vitro and in vivo that the sheddable coating enhances particle stability in serum and uptake by cells, and we confirm that the desirable characteristics of the nanoparticles translate to inhibition of CD44-positive melanoma growth in vivo. 2. EXPERIMENTAL SECTION 2.1. Materials and Reagents. Branch PEI600 (Mw 600), branch PEI25K (Mw 25,000), allyl glycidyl ether (AGE), 2,3-dimethylmaleic anhydride (DMMA), succinic anhydride (SA), βcyclodextrin,

1,1’-carbonyldiimidazole

(CDI),

3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT), agarose and Trypsin were purchased from Sigma (St Louis, MO, USA). Gold-view was purchased from Solarbio (Beijing, China). The plasmid DNAs coding green fluorescence protein (pEGFP) were amplified in DH5-α Escherichia coli and purified with a Qiagen Giga Endofree plasmid purification kit (USA). FAM-labeled DNA (FAM-DNA), miR34a mimics (5’-UGGCAGUGUCUUAGCUGGUUGU-3’) and Cy5-labeled miRNA (Cy5miRNA) were supplied by RiboBio (Guangzhou, China). Cell culture medium 1640 was bought from Gibco (Grand Island, NY, USA). All the other chemicals and reagents used were of the analytical grade obtained commercially.


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KB cell lines were kindly given by Shanghai Cell Institute, China Academy of Sciences. B16F10 cells (a murine melanoma cell line) were obtained from ATCC and cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin. CD44 positive B16F10 cells (B16F10-CD44+ cells) were sorted by using antimouse CD44 FITC antibody (eBioscience, USA). Then the mean purity of CD44-enriched cells, determined by flow cytometry using the CytomicsTM FC500 flow cytometer (Beckman Coulter, Miami, FL), was 96.4%. Female C57BL/6 mice (6-8 weeks old) were purchased from Vital River Laboratories (Beijing, China). All animal experiments were approved by the Institutional Animal Care and Ethics Committee of Sichuan University. 2.2. Synthesis and Characterization of Polymers 2.2.1 Protection for the Amine group of β-aminoethanol. Sodium hydroxide (10.00 g, 0.25 mol) and β-aminoethanol (7.64 g, 0.125 mol) were dissolved in 50 mL component solvent formed from a mixture of water and methanol (V/V=2/1). While stirring and cooling at an ice bath, 25 mL of methanol solution containing tert-butyl dicarbonate ((Boc)2O, 32.7 g, 0.15 mol) was added dropwise, then the mixture was stirred at room temperature for 24 h. When the reaction was complete, the mixture was filtered through a plug of silica, and the filtrate was extracted three times with ethyl acetate. The ethyl acetate extract was washed with water until neutral, then washed by saturation salt solution, dried over sodium sulfate, and evaporated to dryness in a vacuum, giving the viscous product namely Boc-aminoethanol. The structure of Boc-aminoethanol was verified using 1H NMR. 1H NMR (D2O, ppm): 3.702 (-O-CH2-), 3.286 (−CH2-), 1.445 (-CH3) .


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2.2.2 Synthesis of Boc-PEOn/PAGEm. Boc-aminoethanol (2.64 g, 0.015 mol) was dried in vacuo at 100 °C for 1 h, and dissolved in 15 mL anhydrous 1,4-dioxane. Sodium hydride (60%, w/w) (1.03 g, 0.045 mol) was added while the above solution was heated to 60 °C, and the distilled ethylene oxide (EO, 44 g, 1 mol) was then added dropwise with constant stirring. After stirring the reaction mixture at 60 °C for 36 h, allyl glycidyl ether (AGE, 22.9 g, 0.2 mol) was added to the above solution, and the mixture was further stirred at 60 °C for 24 h. Afterward, the reaction was terminated by the addition of a minimal amount of water, and the mixture was adjusted to pH 7 with hydrochloric acid (HCl, 5 M). After the removal of the solvent under reduced pressure, the residue was dissolved in dichloromethane, and dried over anhydrous sodium sulfate. After removing sodium sulfate by filtration, the clarified filtrate was concentrated in vacuo. The concentrated solution was added to 200 mL cold diethyl ether to precipitate the product and the resulted Boc-PEOn/PAGEm was verified by 1H NMR. 1H NMR (D2O, ppm): 5.886 (-CH=CH2), 5.247 (-CH=CH2), 3.996 (-CH2-CH=CH2), 3.813 (-O-CH-CH2), 3.741-3.473 (-O-CH2-CH2-O-), 2.808 (-NH-CH2-), 1.194 (-CH3). 2.2.3 Synthesis of Boc-PEOn/PAGEm-Cys (PPC). Boc-PEOn/PAGEm-Cys (PPC) was synthesized according to the procedure described by Koyama et al.44 The reaction was performed as follows: Boc-PEOn/PAGEm (0.774 g) dissolved in methanol (4 mL) was added dropwise to a solution of the cysteamine hydrochloride (Cys, 2.27 g, 0.02 mol) in methanol (6 mL). After stirring for 48 h at room temperature, the solvent was removed by rotary evaporation. Impurities were removed by complete dialysis (MWCO 1000 Da) at 4 °C against distilled water for 3 days, and PPC was obtained by lyophilization. 1H NMR (D2O, ppm): 3.813 (-O-CH-CH2-), 3.7413.473 (-O-CH2-CH2-O-), 2.608 (-CH2-CH2-CH2-S-), 2.791 (-S-CH2-CH2-NH3+Cl--), 3.152 (-SCH2-CH2-NH3+Cl--), 1.903 (-CH2-CH2-CH2-), 1.194 (-CH3).


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2.2.4 Synthesis of Boc-PEOn/PAGEm-Cys-DMMA (PPC-DA). PPC-DA was synthesized by reaction of PPC with dimethylmaleic anhydride (DMMA), according to the procedure reported by Wang et al.40 PPC (5.3 mg) was dissolved in distilled water (20 mL), and the pH was adjusted to 8.5 by addition of 0.2 M NaOH solution. Afterward, 100 equivalents (to amino groups) of DMMA (148.36 mg) were added in several portions with stirring. The pH of solution was kept between 8 and 9 using 1 M NaOH solution. The reaction mixture was stirred at room temperature for 6 h. At the end, unreacted DMMA was removed by ultrafiltration (Millipore, MWCO 3000 Da), and PPC-DA was obtained by lyophilization. 1H NMR (D2O, ppm): 3.815 (O-CH-CH2-), 3.741-3.473 (-O-CH2-CH2-O-), 3.421 (-S-CH2-CH2-NHC(O)-), 2.753 (-S-CH2CH2-NH-), 2.612 (-CH2-CH2-CH2-S-), 1.912 (-CH2-CH2-CH2-), 1.832 (-C(CH3)=C(CH3)-), 1.194 (-CH3). 2.2.5 Synthesis of Boc-PEOn/PAGEm-Cys-SA (PPC-SA). The synthesis of PPC-SA was very similar to that of PPC-DA, using succinic anhydride (SA) as a substitute for DMMA. 1H NMR (D2O, ppm): 3.815 (-O-CH-CH2-), 3.741-3.473 (-O-CH2-CH2-O-), 3.421 (-S-CH2-CH2NHC(O)-),

2.753

(-S-CH2-CH2-NH-),

2.612

(-CH2-CH2-CH2-S-),

2.463

(-

NHC(O)CH2CH2COOH), 1.912 (-CH2-CH2-CH2-), 1.194 (-CH3). 2.3. Preparation of Ternary Nanoparticles with a Sheddable Shell (S-TNP). S-TNPs were prepared in various mass ratios of PPC-DA/CP600/miR-34a = x : 4 : 1, where x = 0, 40, 80, 120, 160 and 200. Firstly, the aqueous solutions of miR-34a (40 µg/mL) were mixed with CP600 (160 µg/mL) and incubated for 30 min at room temperature. Then the aqueous solutions of CP600/miR-34a nanoparticles were mixed with different volumes of PPC-DA solution (10 mg/mL), giving PPC-DA/CP600/miR-34a nanoparticles (denoted hereafter as S-TNP). Mixtures were incubated for 15 min at room temperature before use in further study.


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2.4. Preparation of Ternary Nanoparticles without a Sheddable Shell (unS-TNP). PPCSA/CP600/miR-34a nanoparticles (unS-TNP) were prepared as described above, except that Boc-PEOn/PAGEm-Cys-SA (PPC-SA) was used instead of PPC-DA. 2.5. Stability Tests of Nanoparticles. The CP600/miR-34a nanoparticles (0.1‰ (w/v), mass ratio = 4 : 1), S-TNP (0.9‰, w/v), or unS-TNP (0.9‰, w/v) were added dropwise to phosphatebuffered saline (PBS) solution (pH 7.4, 0.01 M) with 0.5% bovine serum albumin (BSA). After mixing, the average sizes of nanoparticles were measured at different incubation times by laserlight scattering (Zetasizer 3000, Malvern Instruments, UK). 2.6. Zeta Potential versus pH of S-TNP and unS-TNP. The S-TNP or unS-TNP in PBS either at pH 6.8 or 7.4 was incubated for 0 min, 10 min, 20 min, 30 min, 40 min, 80 min and 120 min at 37 °C. The samples were measured at a final RNA concentration of 1.0 µM. At each time point, 1 mL of nanoparticle suspension was isolated for zeta-potential test using a Malvern Zetasizer 3000. 2.7. Analysis of the Cellular Uptake of S-TNP and unS-TNP under Different pH. For quantitative flow cytometry analysis, B16F10-CD44+ cells (1×105 cells/well) were cultured in 24-well plate in 0.5 mL complete RPMI 1640 medium 24 h prior to transfection. Subsequently, the culture medium was replaced with the conditioned complete RPMI 1640 medium (pH 6.8 or 7.4) supplemented with S-TNP or unS-TNP. After 2 h incubation at 37 °C, cells were washed three times with ice-cold PBS. The cells were then harvested, washed extensively, and analyzed using FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). For confocal analysis, B16F10-CD44+ cells (1×105 cells/well) were seeded directly on poly-D-lysine treated glass coverslips in a 24-well plate and incubated for 24 h. Afterward, the culture medium was replaced


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with the conditioned complete RPMI 1640 medium (pH 6.8 or 7.4) supplemented with S-TNP or unS-TNP. After 2 h incubation at 37 °C, the cells were washed twice with PBS and fixed on the coverslips with 4% formaldehyde in PBS for 20 minutes at room temperature. After fixing, the cells were stained with 4', 6-diamidino-2-phenylindole (DAPI, 100 ng/mL) according to the standard protocol provided by the suppliers, rinsed three times with PBS and mounted on glass slides with one drop of antifade mounting medium (Sigma, St. Louis, MO). Subsequently, confocal analysis was performed on each sample of cells for no more than 30 minutes. 2.8. In vivo Tumor-Targeted Delivery of Nanoparticles. A B16F10-CD44+ xenograft tumor mouse model was created by injecting B16F10-CD44+ cells (5×106 per mouse, 0.1 mL) subcutaneously into the left oxter of the mouse. When the tumors were palpable and no less than 100 mm3 in volume calculated from caliper measurements, each mouse was given i.v. injection with 200 µL of CP600/Cy5-microRNA nanoparticles, S-TNP, or unS-TNP, containing 20 µg of Cy5-microRNA or PBS. The mice were sacrificed 24 h after injection, and the tumors were carefully collected. Images were acquired with the IVIS Spectrum (Caliper Life Sciences, USA). Excitation and emission wavelengths were 550 nm and 570 nm, respectively, based on the available filter sets on the IVIS Spectrum. In order to visualize Cy5-microRNA distribution in tumor, the tumor tissues were then cryopreserved in tissue freezing medium (Leica) and cut using a microtome (Leica CM1950) into 5 µm slices. Nuclei were stained with DAPI. Microscopical analysis was performed with a confocal laser scanning microscope (CLSM 510 Meta) (Zeiss, Germany). 2.9. Antitumor Efficacy in vivo. As described above, B16F10-CD44+ xenograft tumor mouse model for evaluation of therapeutic efficacy was created. When the tumors in mice reached a diameter of approximately 100 mm3 (around 7 days), mice were randomly assigned to five


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groups (n = 10 per group), which were treated intravenously with phosphate-buffered saline (PBS), naked miR-34a, CP600/miR-34a nanoparticles, or either unS-TNP or S-TNP containing miR-34a. Mice received three injections on days 7, 9 and 11. Each dose contained 20 µg miRNA (1.4 nmol). Tumor size was measured daily, and tumor volume (mm3) was calculated as 0.5 × length × width2. 2.10. Immunohistochemical Study. Twenty four hours after the last intravenous dose, mice were sacrificed and tumors were harvested to prepare paraffin-embedded sections. CD44 expression in the tumors was detected using the rabbit anti-mouse CD44 antibody (Boster, Wuhan, China) at 200-fold dilution in the assay. A streptavidin–biotin complex kit (Wuhan Boster Biological Technology, Wuhan, China) was used for subsequent steps, according to the manufacturer’s protocol. The tumor sections were counterstained with hematoxylin for nuclei coloration and observed by light microscopy (Zeiss Axiovert 40). According to the manufacturer’s protocol, TUNEL method was used to detect the apoptosis of tumor cells after treatments (Roche, Basel, Switzerland). At the end, the slices were observed by confocal laser scanning microscopy. To assess the degree of tumor necrosis, tumors of these mice was taken for pathological specimens by hematoxylin and eosin (H&E) staining and observed by light microscopy (Zeiss Axiovert 40). 2.11. Statistical Analysis. Data were presented as the means ± standard deviation (SD) and experiments were conducted at least in triplicate, unless otherwise noted. Differences between groups were examined for statistical significance with Student's t test. Throughout this manuscript, a P value of p < 0.05 was considered statistically significant. 3. RESULTS AND DISCUSSION


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PEGylation and controlling surface charge can reduce non-specific adsorption of nanoparticles onto serum components,45 increasing their chances of reaching tumor tissue after systemic administration. Unfortunately PEGylation also inhibits uptake of nanoparticles once they have arrived at the tumor, giving rise to the so-called PEG paradox. Here we circumvented this problem by designing a pH-sensitive PEG coating that is shed at the acidic pH characteristic of the tumor microenvironment. The resulting nanoparticles are stable in serum, they are efficiently taken up by melanoma tumor cells, and they efficiently deliver miR-34a that substantially knocks down CD44 expression, which is associated with aggressive, metastatic melanoma. These properties are due mostly to the acid-sensitive linkage within the PEG derivative on the nanoparticle surface, since they were not observed in parallel experiments conducted with unSTNP. 3.1. Synthesis and characterization of pH-sensitive anionic PEG derivatives for modifying the surface of CP600/miR-34a nanoparticles. We used a previously described approach46 to obtain tumor acidity-responsive anionic PPC-DA (Figure S3A). First, we copolymerized ethylene oxide (EO) and allyl glycidyl ether (AGE) monomers using Bocaminoethanol as the initiator. Gel permeation chromatography (GPC) showed that the weightaveraged molecular weight of Boc-PEOn/PAGEm was 4450 Da. The average degree of polymerization was 70 for EO and 10 for AGE, based on the molecular weight and 1H NMR spectrum of Boc-PEOn/PAGEm. Cysteamine was added to the side chains on the double bond in Boc-PEOn/PAGEm, generating the PEG derivative Boc-PEOn/PAGEm-Cys (PPC).44 Complete disappearance of 1H NMR peaks corresponding to the double bond protons suggested the addition of cysteamine. The pH-sensitive PPC-DA was then obtained by reacting PPC with 2,3dimethylmaleic anhydride (DMMA). A pH-insensitive Boc-PEOn/PAGEm-Cys-SA (PPC-SA)


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was prepared by conjugating PPC with SA in order to have a structure similar to PPC-DA. Analysis of proportionality of hydrogen atoms at respective sites indicated that about 75% of DMMA or SA was conjugated to PPC, forming the pH-sensitive PPC-DA or pH-insensitive PPC-SA. Wang et al.40, 47 showed that amide bonds formed between an amino group and DMMA are easily cleaved at pHs present in the acidic tumor microenvironment. They also showed that this cleavage exposes the cationic amino groups. To test this idea with our novel nanoparticle carriers, we incubated PPC-DA at pH 6.8 and tracked acid-induced cleavage of the amide bond. As incubation proceeded, the area under the peak for proton Ha gradually fell, while the area under the Hb peak increased (Figure S3B); the Ha peak disappeared entirely by 2 h. We attribute this change in Ha and Hb areas to transformation of the carboxyl groups to amino groups as a result of rapid, acid-induced degradation of the amide bonds. We investigated the cytotoxicity of the anionic PEG derivatives on B16F10-CD44+ cell lines. Relative viabilities of cells treated with increasing concentrations of the polymers were measured using the MTT assay after 24 h; cells were treated with commercial PEG4K as a control. The anionic PEG derivatives PPC-DA and PPC-SA showed no appreciable toxicity at any of the concentrations tested, identifying them as good candidates for coating cationic CP600/miR-34a nanoparticles via electrostatic interactions. 3.2. Preparation and characterization of S-TNP. We used the anionic PEG derivative PPCDA to coat cationic CP600/miR-34a nanoparticles, generating the final ternary nanoparticles with a sheddable shell (Figure 1). In order to select the optimal ratio of CP600 to miRNA, CP600/miRNA nanoparticles were prepared by mixing constant amounts of miRNA with


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different amounts of CP600 polymer. The resulting particle sizes and zeta potentials are presented in Figure 2A. The size of the nanoparticles gradually decreased as mass ratio increased, eventually stabilizing within a narrow range. A mass ratio of 4 gave the smallest CP600/miRNA nanoparticles (84.3±15.1 nm). All nanoparticles were cationic and showed zeta potentials ranging from 15 to 39 mV.

Figure 1. Schematic of the structure and mechanism of acid-responsive ternary nanoparticles with a sheddable shell.


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To investigate the ability of anionic PEG derivatives to shield the positive charges on the surface of CP600/miRNA nanoparticles, we coated CP600/miRNA nanoparticles (mass ratio = 4) with various amounts of PPC-DA, then measured particle size and zeta potential (Figure 2B). Adding PPC-DA increased the size of nanoparticles and gradually reduced their zeta potential. This suggests that PPC-DA effectively shielded the positive charges of CP600/miRNA. A mass ratio of PPC-DA/CP600/miRNA = 40 : 4 : 1 gave the smallest nanoparticles. The surface of the ternary nanoparticles was nearly neutral. Similar trends in size and zeta potential were observed for PPC-SA/CP600/miRNA nanoparticles without a sheddable shell (data not shown). We used a gel retardation assay to investigate whether electrostatic interaction between the negatively charged PEG derivatives and PEI might cause release of RNA cargo (Figure 2C). CP600 efficiently condensed miRNA at a mass ratio of CP600/miRNA of 4 : 1. Introducing different amounts of anionic PPC-DA did not cause obvious release of RNA from the resulting ternary nanoparticles, suggesting that PPC-DA does not affect the ability of CP600 to bind miRNA. The satisfactory miRNA-condensing ability of PPC-DA/CP600/miRNA ternary nanoparticles prepared at a mass ratio of 40 : 4 : 1 led us to adopt this ratio in subsequent experiments. PPC-DA/CP600/miRNA ternary nanoparticles prepared with this ratio are hereafter denoted as S-TNP, while the corresponding PPC-SA/CP600/miRNA nanoparticles are hereafter denoted as unS-TNP. Transmission electron microscopy (TEM) showed that CP600/miRNA nanoparticles, S-TNP and unS-TNP are spherical particles with a smooth surface (Figure 2D). The presence of anionic PPC-DA or PPC-SA polymers did not affect the particle shape. Particle size increased only slightly due to PEGylated anionic polymer on the surface of CP600/miRNA nanoparticles.


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Figure 2. (A) Size and zeta potential of CP600/RNA nanoparticles. (B) Size and zeta potential of PPC-DA/CP600/RNA ternary nanoparticles with a sheddable shell. (C) Agarose gel electrophoresis assay of CP600/RNA and PPC-DA/CP600/RNA nanoparticles. (D) Transmission electron micrographs of CP600/RNA, unS-TNP and S-TNP (magnification, × 60,000). 3.3. PEG shielding of positively charged nanoparticles enhances serum stability. We expected that the ability of anionic PEG to shield the cationic charge of CP600/miRNA in STNP would reduce undesirable interactions. We tested this hypothesis by examining the stability of S-TNPs in serum and measuring their half-life in circulation (Figure 3A, 3B). CP600/miRNA nanoparticles, S-TNP or unS-TNP were added to PBS (pH 7.4) containing 0.25 mg/mL of bovine serum albumin (BSA), the mixtures were shaken gently for different periods, and particle sizes were measured. Size of S-TNP and unS-TNP did not change observably during incubation. In contrast, CP600/miRNA nanoparticles gradually became larger and aggregated during incubation. This indicates that coating CP600/miRNA nanoparticles with a PEG layer enhances their stability in serum.


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Systemic circulation time of S-TNP was measured using rhodamine-B (RB) as a fluorescent probe, which shows an inclusion constant of 4580 M-1 for β-cyclodextrins.48 Naked CP600/miRNA nanoparticles loaded with RB served as a negative control. The RB fluorescence signal dropped below the limit of detection at 24 h in the case of S-TNP, whereas it fell to that limit within 6 h in the case of naked CP600/miRNA nanoparticles, reflecting their rapid aggregation in serum. This indicates that S-TNP is retained in circulation better than unshielded CP600/miRNA nanoparticles. 3.4. Removal of the PEG shell at tumor pH. The amide bond between an amino group and DMMA is relatively weak and easily cleaved under acidic conditions similar to those in tumor microenvironments.49 To verify that our S-TNP can shed the PEG layer at tumor pH, we tracked the surface charge conversion from nearly neutral to cationic, reflecting the positive charge shielding loss of the anionic PPC-DA on the nanoparticle surface. Prolonged incubation of STNP at pH 6.8 caused their zeta potential to increase much more than incubation at pH 7.4 (Figure 3C). In contrast, the zeta potential of unS-TNP showed no obvious change during incubation at either pH 7.4 or 6.8. These results confirm the pH sensitivity of amide bonds in PPC-DA and suggest that the ternary nanoparticles become net cationic in acidic microenvironments.


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Figure 3. (A) Stability of CP600/RNA nanoparticles, unS-TNP and S-TNP in serum at pH 7.4. (B) Plasma concentration-time profiles of RB-loaded S-TNPs in mice after intravenous injection (n = 5). (C) Changes in zeta potential of unS-TNP and S-TNP during incubation in PBS at pH 6.8 and 7.4. 3.5. Removal of the PEG shell improves cellular uptake. The exposure of cationic charges upon shedding of the PEG coat is expected to facilitate internalization of the nanoparticles by target cells as well as promote their escape from endosomes once inside cells.51 To verify this


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hypothesis, B16F10-CD44+ cells were cultured with S-TNP or unS-TNP containing Cy5-labeled miRNA at pH 6.8 or 7.4. Intracellular fluorescence due to S-TNP internalization was markedly greater after 2 h at pH 6.8 than at pH 7.4 (Figure 4A). In contrast, intracellular fluorescence due to unS-TNP internalization was similar after 2 h at either pH (Figure 4B). Internalization of S-TNP and unS-TNP was investigated in further detail using confocal laser scanning microscopy. Culturing cells with S-TNP for 2 h at pH 6.8 led to abundant intracellular red fluorescent nanoparticles in the cytoplasm (Figure 4C); in contrast, culturing at pH 7.4 led the nanoparticles to accumulate on the intracellular side of the cell membrane. These results support data from the zeta potential experiments that the pH-sensitive ternary nanoparticles become positively charged at pH 6.8, which strengthens nanoparticle interaction with cells and enhances their internalization.


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Figure 4. Uptake of S-TNP and unS-TNP by B16F10-CD44+ cells. (A-B) Flow cytometric evaluation of uptake efficiency of Cy5-miRNA-loaded S-TNP (A) or unS-TNP (B) at different pH values, based on mean fluorescence intensity (MFI). (C) Confocal laser scanning micrographs of B16F10-CD44+ cells incubated for 2 h at different pH values in the presence of nanoparticles carrying Cy5-miRNA (red). Scale bar, 25 µm.


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3.6. Removal of the PEG shell enhances the level of mature miR-34a and reduces CD44 expression. B16F10-CD44+ cells were incubated with S-TNP or unS-TNP containing miR-34a for 4 h at pH 6.8 or 7.4, after which the medium was changed and the cultures were incubated another 24 h. Then the level of miR-34a was measured using quantitative real time-PCR (qRTPCR). The level of mature miR-34a was dramatically higher after incubation with S-TNP at pH 6.8 than at pH 7.4 (Figure 5A), whereas the level of mature miR-34a after incubation with unSTNP was similar at the two pH values. This reflects the fact that unS-TNP does not shed its PEG coat at either pH. Since miR-34a represses expression of adhesion molecule CD44 and thereby acts as a tumor suppressor,11,

50

we tested whether the existence of miR-34a in B16F10-CD44+ cells down-

regulates CD44 expression. CD44 expression was substantially down-regulated after incubation with S-TNP at pH 6.8 (Figure 5B). Such down-regulation was not appreciable relative to the negative control after incubation with unS-TNP containing miR-34a at pH 6.8 or 7.4, or following incubation with S-TNP at pH 7.4.

Figure 5. (A) The level of mature miR-34a in B16F10-CD44+ cells after treatment with S-TNP or unS-TNP loaded with miR-34a; cells were transfected at different pH values for 4 h, then


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cultured in fresh complete RPMI 1640 medium for 24 h. (B) Flow cytometric detection of CD44 expression in B16F10-CD44+ cells after various treatments. Data are presented as mean ± SD (n = 3). 3.7. Removal of the PEG shell inhibits tumor cell progression. The ability of S-TNPs to reduce CD44 expression should translate to reduce the migration and proliferation of B16F10CD44+ cells, which can easily be detected in wound healing assays.51 Incubating cultures at pH 6.8 with S-TNP loaded with miR-34a inhibited cell migration and did so to a greater extent than incubation at pH 7.4 (Figure 6A). Incubating cultures with unS-TNPs inhibited cell migration to a much smaller extent, and this weak inhibition was similar at the two pH values. These results suggest that shedding of the PEG layer from ternary nanoparticles may increase antitumor efficacy. To understand the mechanisms of the reduced cell migration and proliferation in the wound healing assay, we investigated the influence of miR-34a-loaded S-TNP on tumor cell apoptosis. Nuclear morphology was evaluated using DAPI staining, and the proportion of apoptotic cells was determined by flow cytometric analysis of cells double-stained with annexin V-FITC and PI. Incubating tumor cells with miR-34a-loaded S-TNP at pH 6.8 led to extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation in (Figure 6B); these processes were observed to a much smaller extent after incubation with S-TNP at pH 7.4, and they were rarely observed in control cultures. Consistent with these results, the apoptotic cell fraction was 83.7% after incubation with S-TNP at pH 6.8 and 43.6% after incubation at pH 7.4 (Figure 6C). The corresponding fractions were 51.2% and 44.1% after incubation with unS-TNP. Statistical analysis showed that the proportion observed after incubation with S-TNP at pH 6.8 was significantly greater than under all other conditions (P < 0.05; Figure 6D). Toxic effects


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were negligible for S-TNP and unS-TNP at the concentrations used (data not shown), indicating that the enhanced apoptosis caused by S-TNP was unlikely to reflect cytotoxicity.

Figure 6. (A) Representative images of wound healing assays to assess motility and migration of B16F10-CD44+ cells after treatment at different pH values with S-TNP or unS-TNP loaded with miR-34a. Red lines indicate the scratches made at the start of the assay. Scale bar, 50 µm. (B) DAPI staining of treated cells showing fragmented chromatin or apoptotic bodies. Scale bar, 50 µm. (C) Flow cytometric analysis of necrosis and apoptosis using the combination of Annexin V and propidium iodide. (D) Percentages of apoptotic cells after various treatments (n = 3). As an additional in vitro test of antitumor efficacy before moving to in vivo experiments, we examined whether ternary nanoparticles can inhibit sphere formation by B16F10-CD44+ cells. Sphere-forming assays depend on the ability of cancer stem cells to self-renew and differentiate.


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Treating cells with naked miR-34a did not affect their ability to form spheres relative to the ability of untreated control cells (Figure S4). Treating cells with S-TNP at pH 6.8 led to significantly fewer spheres than treating them at pH 7.4. Treating cells with unS-TNP at either pH inhibited sphere formation to a similar extent as treating them with S-TNP at pH 7.4. These results further suggest that the sheddability of the PEG coat in S-TNP enhances antitumor activity. 3.8. Removal of the PEG shell enhances tumor cell uptake of miRNA in vivo. To examine effects of S-TNP in vivo, nanocarriers containing Cy5-labeled miRNA (Cy5-miRNA) were injected intravenously into mice bearing B16F10-CD44+ melanoma xenografts and miRNA accumulation in the tumor was monitored after 24 h. Treating mice with S-TNP or unS-TNP led to strong fluorescence in the tumor, much stronger than the fluorescence observed after treatment with CP600/Cy5-miRNA nanoparticles (Figure 7A). These results correlate well with pharmacokinetics data (Figure 3B), confirming that PEGylation substantially improves the stability of S-TNP and unS-TNP in systemic circulation, allowing more tumor accumulation. Fluorescence due to S-TNP was stronger than that due to unS-TNP, consistent with the idea that shedding of the protective PEG layer results in more efficient internalization of S-TNP. Quantitation of tumor fluorescence using the IVIS spectrum system (Figure 7B) indicated stronger miRNA signal in tumors after treatment with S-TNP than after treatment with unS-TNP (Figure 7C), despite the fact that both nanoparticles have similar size and surface charge.


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Figure 7. (A) Representative images of tumors after intravenous injection with different Cy5labeled formulations. Each mouse was given i.v. injection with 200 µL of CP600/Cy5microRNA nanoparticles, S-TNP, or unS-TNP, containing 20 µg of Cy5-microRNA or PBS. (B) Quantification of tumor fluorescence in panel A. (C) Confocal scanning micrographs of tumor sections. Scale bar, 10 µm. The acid-sensitive PEG shell enhanced the accumulation of miRNA in tumors, probably reflecting the increased interaction between tumor and cationic S-TNP after PEG shedding. Similar tumor accumulation was reported by Poon et al.52 using different acid-sensitive PEG linkages based on nanoparticles assembled layer-by-layer. The importance of acid-induced PEG shedding is highlighted by the fact that even though both S-TNP and unS-TNP should be able to


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accumulate in tumors via the EPR effect, such accumulation was much greater in the case of STNP. 3.9. Removal of the PEG shell enhances antitumor activity. After these promising in vivo results on tumor accumulation, we next examined the ability of S-TNP to reduce CD44 expression, induce histopathology in tumor tissue and slow tumor growth. Mice treated with STNP containing miR-34a showed reduced CD44 levels, much lower than the slightly reduced levels observed with unS-TNP (Figure 8A). Mice treated with miR-34a or CP600/miR-34a nanoparticles showed similar CD44 levels as control animals treated with PBS. These flow cytometric results were consistent with the results of immunohistochemisty against CD44. Treating tumors with CP600/miR-34a or unS-TNP/miR-34a down-regulated CD44 expression to a similar extent, and treating them with S-TNP/miR-34a down-regulated CD44 even more (Figure 8B). Tumor cell apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The proportion of TUNEL-positive (green) tumor cells was higher after treatment with unS-TNP/miR-34a than after treatment with free miR-34a. The proportion was higher with S-TNP/miR-34a than with unS-TNP/miR-34a. Hematoxylin and eosin (H&E) staining revealed extensive pathology and necrosis in most of the tumor area following treatment with unS-TNP/miR-34a and CP600/miR-34a, which was even greater with S-TNP/miR-34a. S-TNP carrying miR-34a inhibited tumor cell proliferation and tumor growth to the greatest extent of all treatments (Figure 8C, P < 0.05).


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Figure 8. (A) Flow cytometric analysis of CD44 expression in B16F10-CD44+ xenograft tumors after miR-34a treatment. (B) Representative micrographs of tumor tissue from mice treated as shown. Tissue sections were subjected to immunohistochemistry against CD44, stained with TUNEL reagents or stained with H&E. Scale bar, 100 µm. (C) Antitumor effects of different formulations evaluated based on measured volumes of B16F10-CD44+ xenograft tumors. *p < 0.05, **p < 0.005 (n = 10). Nevertheless, S-TNP did not show the dramatically better antitumor efficacy than unS-TNP that we expected. This likely reflects the low CD44 expression when B16F10-CD44+ cells differentiate after subcutaneous transplantation, as well as the high metastatic ability of B16F10-


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CD44+ cells, which show a cancer stem cell-like phenotype.53-55 In principle, expression of ectopic miR-34a should efficiently inhibit clonogenic expansion, tumor regeneration and metastasis based on experiments with CD44+ prostate cancer cells.11 Our results suggest that miR-34a-based therapy against melanoma should take into account the already low expression of CD44 on tumors. This may mean that miR-34a may not be sufficient on its own and should therefore be combined with chemotherapy. Our ternary nanoparticles with a sheddable shell may be suitable for such combination therapy. Furthermore, the nanoparticles described here may be adaptable to treat other cancers. The βCD in the nanoparticle core can engage in host-guest interactions with a variety of hydrophobic drugs56 and prior to encapsulation it can be conjugated with various drugs to construct a new class of colon-targeting prodrug.57 4. CONCLUSION In summary, we have successfully developed ternary nanoparticles with a sheddable shell for tumor-specific delivery of microRNAs. The nanoparticles have a nearly neutral surface charge, allowing them to persist in systemic circulation without detection by the immune system; in the acidic tumor microenvironment, the shell is lost, exposing the particle’s positive charges, which facilitates uptake by target cells and intracellular delivery of the therapeutic cargo. We validate this system in vitro and in vivo by loading the particles with miR-34a to inhibit expression of CD44 in B16F10-CD44+ melanoma cells. This system showed minimal toxicity and promising antitumor efficacy in a mouse xenograft model, and it should be adaptable for carrying other therapeutic cargoes against other cancers. ASSOCIATED CONTENT


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Supporting Information. Synthesis of β-cyclodextrin-PEI600 (CP600), preparation of CP600/DNA complexes, cellular uptake and gene transfection assays of CP600/DNA, synthesis and characterization of polymers, Cytotoxicity of CP600 and the Anionic PEG Derivatives (MTT assay), preparation of CP600/miR-34a nanoparticles, agarose retardation assay, Real-Time PCR quantification of miR-34a expression, scratch wound healing assay, cell apoptosis assay, expression of CD44 by flow cytometry, sphere formation assay, and pharmacokinetic analysis. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Phone: +86-28-85502307. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 81422044). ABBREVIATIONS PEG, poly(ethylene glycol); PPC-DA, Boc-PEOn/PAGEm-Cys-DMMA; AGE, allyl glycidyl ether; DMMA, 2,3-dimethylmaleic anhydride; SA, succinic anhydride;

CDI, 1,1’-


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carbonyldiimidazole; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide; FAM-DNA, FAM-labeled DNA; Cy5-miRNA, Cy5-labeled miRNA; FBS, fetal bovine serum; S-TNP, ternary nanoparticles with a sheddable shell; unS-TNP, ternary nanoparticles without a sheddable shell; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DAPI, 4', 6diamidino-2-phenylindole; CDI-CD, β-cyclodextrin-carbonate-benzotriazole; EO, oxide;

PPC,

Boc-PEOn/PAGEm-Cys;

PPC-SA,

Boc-PEOn/PAGEm-Cys-SA;

ethylene TEM,

Transmission electron microscopy; RB, rhodamine-B; qRT-PCR, quantitative real time-PCR; TUNEL, transferase-mediated dUTP nick end-labeling. . REFERENCES (1) Gray-Schopfer, V.; Wellbrock, C.; Marais, R. Melanoma biology and new targeted therapy. Nature 2007, 445, 851-857. (2) Kirkwood, J. M.; Manola, J.; Ibrahim, J.; Sondak, V.; Ernstoff, M. S.; Rao, U. A pooled analysis of eastern cooperative oncology group and intergroup trials of adjuvant high-dose interferon for melanoma. Clin. Cancer Res. 2004, 10, 1670-1677. (3) Tarhini, A. A.; Agarwala, S. S. Cutaneous melanoma: available therapy for metastatic disease. Dermatol. Ther. 2006, 19, 19-25. (4) Atkins, M. B.; Lotze, M. T.; Dutcher, J. P.; Fisher, R. I.; Weiss, G.; Margolin, K.; Abrams, J.; Sznol, M.; Parkinson, D.; Hawkins, M. High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J. Clin. Oncol. 1999, 17, 2105-2105.


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In this study, a ternary miR-34a delivery system with a shell-sheddable PEG corona was designed. The PEG layer protects the CP600/miR-34a nanoparticles in the systemic circulation, and reveals the cationic entities at the tumor sites which facilitate the delivery of miR-34a. Accordingly, the melanoma growth suppression efficiencies are enhanced due to the promoted carrier uptake by the tumor cells. ToC figure

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Ternary Nanoparticles with a Sheddable Shell Efficiently Deliver

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