Journal of Controlled Release 192 (2014) 122–130

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Target-specific delivery of siRNA by stabilized calcium phosphate nanoparticles using dopa–hyaluronic acid conjugate Min Sang Lee a, Jung Eun Lee a, Eunkyoung Byun b, Nak Won Kim a, Kyuri Lee a, Haeshin Lee b, Sang Jun Sim c, Doo Sung Lee d, Ji Hoon Jeong a,⁎ a

School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea The Graduate School of Nanoscience & Technology (WCU), Department of Chemistry, KAIST, Daejeon 305-701, Republic of Korea Department of Chemical and Biological Engineering, Korea University, Seoul 136-713, Republic of Korea d School of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 25 December 2013 Accepted 25 June 2014 Available online 1 July 2014 Keywords: Gene delivery Calcium phosphate nanoparticle Targeted delivery 3,4-Dihydroxy-L-phenylalanine Hyaluronic acid

a b s t r a c t Low cytotoxicity and high cellular gene delivery capability are among the most important prerequisites for the selection of a non-viral carrier. Although calcium phosphate (CAP) nanoparticles have been long used for animal cell transfection, its rapid and uncontrollable crystal growth and lack of tissue specificity are among the most challenging problems that limit its use in the clinic. In this study, we report the development of CAP nanoparticles stabilized by a conjugate of the mussel-inspired adhesive molecule, 3,4-dihydroxy-L-phenylalanine (dopa), and a nontoxic hydrophilic natural polymer, hyaluronic acid (HA), for targeted siRNA delivery to tumors. CAP/siRNA/ dopa–HA can form compact nanoparticles that effectively protect siRNA from enzymatic degradation despite the structural drawbacks of siRNA, such as low charge density and short and rigid structure. In addition, stabilized CAP nanoparticles were able to maintain their colloidal stability in a physiological salt condition for over a week. The superior ability of CAP/siRNA/dopa–HA to maintain the integrity of encapsulated siRNA and the stability in solution of the nanoparticles allow this formulation to achieve improved intratumoral accumulation of siRNA and a high level of target gene silencing in solid tumors after systemic administration. Considering its biocompatibility, transfection efficacy, and tumor targeting capability, this stabilized calcium phosphate nanoparticle-based gene delivery platform should be considered a promising candidate carrier for systemic siRNA delivery and targeted cancer therapy. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Despite many studies on the development of efficient siRNA carriers over the last decade, the need for improved in vivo performance of siRNA for use in clinical settings has not yet been met. Although siRNA-based therapeutics share the similar physicochemical characteristics to plasmid DNA, some problems arise from the low charge density and the short and unbendable structure of double-stranded siRNA, the persistence length of which is about 50 nm. The structural characteristics of siRNA make it unable to form tight nano-scale complexes necessary for efficient protection from extracellular nucleases and improved cellular delivery, using conventional polymer- or lipid-based carriers. In addition, issues of potential in vivo toxicity of conventional cationic carriers have not been resolved yet. Calcium phosphate (CAP), a natural inorganic material found in bone and teeth, has long been regarded as a promising vehicle for gene delivery due to its biocompatibility, biodegradability, and ability ⁎ Corresponding author. Tel.: +82 31 290 7783; fax: +82 31 292 8800. E-mail address: [email protected] (J.H. Jeong).

http://dx.doi.org/10.1016/j.jconrel.2014.06.049 0168-3659/© 2014 Elsevier B.V. All rights reserved.

to encapsulate negatively charged genetic material by chelating calcium ions while forming calcium phosphate crystals [1,2]. However, its use as a carrier for siRNA has remained challenging due to lack of tissue specificity and the uncontrollable growth of CAP crystals in a physiological solution, which profoundly limits its utility in vivo. A number of studies have been carried out to control the growth of CAP crystals during the formation of nucleic acid-encapsulating CAP nanoparticles. Most strategies have been based on coating CAP nanoparticles to prevent excessive surface crystal growth through the use of bisphosphonate-derivatized PEG [3], PEGylated anionic polymer [4,5], PEGylated calcium chelators [6], PEGylated siRNA [7], or lipid bilayers [8,9]. In our previous study, we showed that a conjugate of chitosan and 3,4-dihydroxy-Lphenylalanine (dopa), an unusual amino acid found in adherent threads of marine mussels, can effectively stabilize CAP nanoparticles containing plasmid DNA and siRNA [10]. Dopa has been widely investigated as a surface modification agent owing to its excellent adhesive properties to a variety of organic and inorganic materials, especially in wet conditions [11–13]. In addition, a catechol group of dopa provides efficient binding to hydroxyapatite, a crystalline form of calcium phosphate found in bone tissue [12,14].

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Hyaluronic acid (HA) is a natural polysaccharide composed of alternating N-acetyl-D-glucoamine and D-glucuronic acid units and is known to be abundant in extracellular matrix (ECM) and synovial fluid [15]. HA plays important roles in organization of ECM, cell motility and adhesion, and differentiation [16,17]. HA is also involved with the regulation of tumor angiogenesis and its receptors such as CD44 and receptor for hyaluronate-mediated motility (RHAMM) are abundantly presented in various cancer cells [18,19]. Owing to its biocompatibility, biodegradability, cancer cell targeting property, HA has been popularly used in designing drug and gene delivery systems for specific tumor targeting [20–22]. In this study we took advantage of the unique functions of dopa and hyaluronic acid (HA) to control CAP growth and target siRNA to diseased cells. A dopa-modified hyaluronic acid (dopa–HA) was synthesized and used as a particle stabilizer that can be adsorbed onto the surface of CAP/siRNA nanoparticles. The dopa unit serves to interact with the surface of CAP particles, and the hydrophilic HA backbone acts as a protective shell that prevents excessive growth of CAP nanoparticles as well as serving as a targeting moiety for CD44 + cancer cells. The cancer-targeting properties and cellular siRNA delivery capabilities of the cancer-targeting CAP nanoparticle were assessed. 2. Materials and methods 2.1. Materials Hyaluronic acid (HA, MW = 780 kDa) was purchased from Lifecore Co (Chaska, MN). Calcium chloride (CaCl2), sodium phosphate dibasic (Na2HPO4), acetic acid, branched polyethylenimine (PEI, Mw 25,000), dopamine hydrochloride, N-(3-Dimethylaminopropyl)N′-ethylcarbodiimide hydrochloride (EDC), 4′,6-diamidino-2phenylindole (DAPI) and (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide) (MTT) were obtained from Sigma-Aldrich (St. Louis, MO). Cell culture materials, including Roswell Park Memorial Institute 1640 medium (RPMI 1640) and fetal bovine serum (FBS), were from Invitrogen (Carlsbad, CA). Luciferase-silencing siRNA (sense: GGAC GAGGACGAGCACUUCUU; antisense:AAGAAGUGCUCGUCCUCGUCC), non-targeting double stranded RNA with a scrambled sequence (scRNA, sense: CCUACGCCACCAAUUUCGU; antisense: ACGAAAUUGGUGGCGU AGG), and Cy5.5-labeled scRNA were synthesized and purified by Bioneer (Daejeon, Korea). All other chemicals and solvents were of analytical grade and were used without further purification. 2.2. Synthesis of dopa-conjugated hyaluronic acid (dopa–HA) To prepare the dopa–HA conjugate, HA (1 g, 2.5 mmol) was dissolved in 200 ml of degassed deionized water, and the solution was kept under nitrogen. EDC (959 mg, 5 mmol) and NHS (575 mg, 5 mmol) were slowly added to the solution. After 20 min stirring, dopamine hydrochloride (766 mg, 5 mmol) was added to the mixture. The pH value of the solution was monitored and adjusted to keep the value between 4 and 5 for 3 h. Subsequently, the solution was allowed to react overnight at room temperature. After the reaction, the solution was purified by dialysis (MWCO = 12,000 Da) four times under acidic conditions and lyophilized. The resulting white powder was stored at −20 °C before use. The catechol content was confirmed by UV–vis spectrometer (Agilent 8453, Agilent Technologies, Santa Clara, CA), measuring absorbance at 280 nm, and quantitative measurement was performed with a dopamine standard. In addition, nuclear magnetic resonance (NMR) analysis was performed (Bruker 400 MHz, Bruker, Billerica, MA). 2.3. Cytotoxicity of dopa–HA The cytotoxicity of dopa–HA was investigated by MTT assay to measure mitochondrial enzyme activity. Cells were seeded in a 96-well

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plate at a density of 1 × 104 cells/well in medium containing 10% FBS and incubated at 37 °C in a humidified 5% CO2 atmosphere for 24 h. The culture medium was removed and replaced with medium containing 10% FBS and the desired amount of dopa–HA. After 48 h incubation, 100 μl of fresh growth medium containing 50 mg MTT was added to each well and cells were further incubated for 4 h. Insoluble formazan crystals were dissolved by the addition of 50 μl dimethylsulfoxide (DMSO), followed by incubation at 37 °C for 2 h. Absorbance was measured at 590 nm using a microplate reader (EL808, Bio-Tek Instrument, Winooski, VT). Cell viability percentage was calculated vs. mock-treated cells (100% survival). 2.4. Preparation and characterization of CAP/siRNA/dopa–HA nanoparticles Thirty microliters of CaCl2 solution (36 mM, pH 9.0) and 35 μl of siRNA dissolved in deionized water were mixed with 30 μl of HEPESbuffered saline solution (HBS, 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 7.1) and incubated for 1 min. The predetermined amount of dopa–HA dissolved in HBS was subsequently added to the resulting solution and allowed to react for 1 h. The final pH of the solution containing CAP/siRNA/dopa–HA nanoparticles was approximately 7.0. To determine the amount of incorporated siRNA, the nanoparticle solution was centrifuged at 15,000 rpm for 1 h to precipitate the nanoparticles and the supernatant was collected. The amount of unincorporated siRNA was measured at 260 nm using a spectrophotometer. The amount of incorporated siRNA was determined by subtracting the unincorporated siRNA from the total siRNA concentration. The hydrodynamic diameter and surface zeta-potential values of the nanoparticles were determined at room temperature using a dynamic light scattering device equipped with a He–Ne laser at a wavelength of 633 nm (Zetasizer Nano, Malvern, Worcestershire, UK). To visualize the morphology of CAP/siRNA/dopa–HA (dopa–HA to siRNA ratio = 60, w/w) nanoparticles, the nanoparticle solution was dropped onto a 300-mesh carboncoated copper grid and dried on filter paper at room temperature. The grid was then stained with 2% uranyl acetate and observed under a transmission electron microscope (TME, JEM-3010, ZEOL, Tokyo, Japan).

2.5. Long-term stability of CAP/siRNA/dopa–HA nanoparticles The stability of CAP/siRNA/dopa–HA (dopa–HA to siRNA ratio = 60, w/w) was assessed in phosphate-buffered saline (PBS, pH 7.4) by monitoring changes in size at predetermined intervals (0, 1, 2, 3, 4, and 7 days). The samples were prepared by adding the same volume of 2 × PBS, and the size was determined by a light scattering method as described above.

2.6. In vitro transfection and gene silencing Human colon carcinoma cells stably expressing firefly luciferase (HT29-luc) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified 5% CO2 atmosphere. Before transfection, the cells (1.5 × 105/well) were seeded in a 12-well plate and incubated for 24 h. The cell culture medium was replaced with fresh serum-free medium or medium with 10% FBS before adding the desired formulation of CAP/siRNA/dopa–HA. The final concentration of siRNA was fixed at 100 nM. After 4 h of incubation, the transfection medium was removed and supplemented with fresh medium containing 10% FBS. Twenty-four hours after transfection, luciferase gene silencing efficiency was determined by measuring cellular luciferase activity using a commercial luciferase assay kit (Promega, Madison, WI) and GloMax 20/20 luminometer (Promega). The siRNA-mediated target gene silencing efficiencies were normalized to the total cell protein concentration as determined by BCA assay and expressed as percent luciferase activity of mock-treated cells.

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2.7. Free HA competition assays To study HA receptor (CD44)-mediated cellular uptake of CAP/ siRNA/dopa–HA in HT29-luc cells, a transfection experiment was carried out in the presence of free HA. For competition assays, unmodified HA (10 mg/ml) was added to transfection medium 1 h before transfection. Transfection was performed in serum-free medium as described above except for the addition of free HA. The gene silencing efficiency was determined by monitoring the remaining luciferase activity as described above. 2.8. Flow cytometry and confocal microscopy The cellular uptake of CAP/siRNA/dopa–HA was quantitatively and qualitatively assessed using flow cytometry analysis and confocal laser

scanning microscopy. Cy5.5-labeled siRNA was used for the preparation of CAP/siRNA/dopa–HA nanoparticles. Prior to transfection, the culture medium was removed and replaced with fresh serum-free medium, into which the nanoparticles were added. Cell transfection was carried out with or without the addition of unmodified HA (10 mg/ml). After 4 h incubation, the transfection medium was removed and replaced with fresh medium containing 10% FBS. The cells were then cultured for an additional 24 h. For flow cytometry, the transfected cells were washed three times with PBS and trypsinized. The cellular uptake of Cy5.5-labeled siRNA was monitored using a flow cytometer (Guava easyCyte™ Flow Cytometer, Merck Millipore, Billerica, MA) and InCyte software for data acquisition and analysis. For confocal microscopy, transfected cells were washed three times with PBS and treated with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) for nuclear staining. After 5 min incubation at room temperature, the cells were washed

Fig. 1. (A) Synthesis of the dopa–HA conjugate. (B) UV–vis spectra of the dopa–HA conjugate. The single absorbance peak appears at 280 nm. (C) 1H-NMR analysis of the dopa–HA conjugate. The peaks of the catechol protons appear around 6.7 ppm, and the proton peak of the \CH2-close to the catechol ring appears between 2.5 and 3.0 ppm.

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three times with PBS and fixed by submerging the cells in 10% buffered formaldehyde solution (Hedwin Corp., Baltimore, MD, USA) at 4 °C for 30 min. The fixed cells were washed three times with PBS and visualized using a Zeiss LSM 510 laser scanning confocal fluorescence microscope (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany). 2.9. Serum stability of siRNA encapsulated in CAP/siRNA/dopa–HA The stability of siRNA encapsulated in CAP/siRNA/dopa–HA nanoparticles was tested by incubating the nanoparticles (dopa–HA to siRNA ratio = 60, w/w) in RPMI medium containing 50% FBS at 37 °C for predetermined periods of time (0, 1, 2, 4, 6, 8, 12, and 24 h). The stability of the siRNA was analyzed by electrophoresis in a 1.5% agarose gel containing ethidium bromide.

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removing insoluble tissue debris by centrifugation (4 °C, 15,000 rpm, 10 min), luciferase activity was measured using a luciferase assay kit (Promega) and GloMax 20/20 luminometer (Promega). To observe the biodistribution profile of CAP/siRNA/dopa–HA after systemic injection, tumor-bearing mice were injected with the desired nanoparticle formulation containing Cy5.5-labeled siRNA via the tail vein. The dose of Cy5.5-siRNA was 0.6 mg/kg. Four hours after administration, the animals were sacrificed by cervical dislocation. Tumors, as well as major organs including the liver, lungs, and spleen, were collected and visualized under an in situ bio-imaging system equipped with Analysis Workstation software (Optix III, Advanced Research Technologies, Montreal, Canada) for quantitative analysis. 3. Results and discussion

2.10. Animal experiments

3.1. Synthesis of dopa–HA

To generate animal tumor models, 1 × 106 HT29-luc cells were subcutaneously injected into one flank of female Balb/c nude mice (nu/nu, 7 weeks old, Japan SLC, Hamamatsu, Japan). To evaluate the in vivo target gene silencing ability of CAP/siRNA/dopa–HA, each formulation of CAP/siRNA/dopa–HA containing luciferase-silencing siRNA (luc-siRNA) was injected into the tumor-bearing mice via the tail vein once the volume of the tumor xenograft reached ca. 400 mm3. The dose of luc-siRNA was 0.6 mg/kg. Twenty-four hours after injection, the tumor was excised and homogenized in lysis buffer (Promega, Madison, WI). After

To synthesize dopa–HA, dopamine was conjugated to the carboxyl group of HA by the EDC/NHS coupling reaction (Fig. 1A). The reaction was performed in degassed water in a nitrogen-purged environment overnight and the pH of the reaction was maintained at 5. The characteristic absorption peak of dopa–HA appeared at 280 nm. The degree of substitution was approximately 6%, as determined by comparison with dopamine standard solutions (Fig. 1B). 1H-NMR analysis demonstrated that the protons in the catechol ring newly appeared at 6.7 ppm, and that the proton peak between 2.5 and 3.0 ppm was due

Scheme 1. Schematic illustration of the formation and cellular uptake of CAP/siRNA/dopa–HA nanoparticles.

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Fig. 3. (A) TEM images of CAP/siRNA and (B) CAP/siRNA/dopa–HA (dopa–HA to siRNA ratio = 60, w/w). (C) EDS analysis of CAP/siRNA/dopa–HA (Scale bars: A = 100 nm; B = 500 nm; and C (inset) = 50 nm).

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Fig. 2. (A) Hydrodynamic diameter and (B) surface zeta-potential of CAP/siRNA/dopa–HA nanoparticles at various dopa–HA/siRNA weight ratios. The concentration of siRNA was fixed at 100 nM. Values are given as the mean ± SD of triplicates.

to a \CH2-group close to the catechol ring. The results confirmed that the conjugation of dopa to HA was successful (Fig. 1C). 3.2. Formation of CAP/siRNA/dopa–HA nanoparticles Stabilized CAP/siRNA/dopa–HA nanoparticles were generated by mixing a CaCl2 solution containing siRNA with HEPES-buffered saline containing Na2HPO4 in mildly alkaline conditions (pH 9.0), followed by subsequent addition of dopa–HA (Scheme 1). The incorporation efficiency of siRNA was about 84% at the dopa–HA to siRNA weight ratio of 60. The siRNA loading efficiency depending on the feed ratio is shown in Fig. 1S (see Supplementary data). The catechol moiety of dopa–HA may play a critical role in binding to the surface of CAP/siRNA nanoparticles [14]. The catechol moiety may also interact with calcium ion and enrich the organic–inorganic interface with calcium ion, facilitating the formation of the stable interface via further mineralization. The catechol-assisted bio-mineralization phenomenon was suggested in a previous report [12]. The negatively charged and hydrophilic HA located at the outer most layer of the nanoparticle can effectively prevent inter-particular aggregation and excessive crystal growth during the mineralization process. CAP/siRNA nanoparticles prepared without the addition of dopa–HA became large and neutral aggregates and started to form visible precipitates within 30 min (Fig. 2). In contrast, CAP/siRNA/dopa–HA nanoparticles varied in size, ranging from 63 to 278 nm, and in negatively charged surface potential,

depending on the amount of dopa–HA. The polydispersity of CAP/ siRNA/dopa–HA nanoparticles also become smaller as the dopa–HA/ siRNA weight ratio increased (Table 1S, see Supplementary data). The addition of unmodified HA instead of dopa–HA in the formulation (CAP/siRNA/HA) failed to prevent excess growth of CAP crystals (Fig. 2S, see Supplementary data), suggesting that the dopa moiety may play a critical role in binding of HA to CAP for the surface stabilization of the nanoparticles. The CAP/siRNA/dopa–HA nanoparticles were well-dispersed spherical particles, as observed by TEM (Fig. 3). EDS scanning analysis demonstrated successful formation of CAP particles. The incorporation of siRNA

Fig. 4. Colloidal stability of CAP/siRNA/dopa–HA nanoparticles (dopa–HA to siRNA ratio = 60, w/w) in PBS as a function of time. Values are given as the mean ± SD of triplicates.

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CAP may overcome the inherent problems stemming from the short and rigid structure of siRNA, which hinders the proper formation of compact polyplexes or lipoplexes with conventional cationic polymers or lipids. In addition, the stabilized nanoparticles maintained their size and narrow distribution in a physiological saline solution even after a week (Fig. 4). This suggests that the interaction between CAP and the catechol group of dopa–HA is strong enough to tether HA to the surface of CAP particles for extended periods of time. The hydrophilic HA can then provide effective surface shielding via its hydration layer, which prevents excessive particle growth and inter-particular aggregation, and improves the solubility of the particles in an aqueous medium via hydrogen bonding [23]. The increase in size of the nanoparticles after 48 h may be due to the formation of secondary aggregates following the partial detachment of HA from the nanoparticles. A similar phenomenon was observed in a previous report on CAP nanoparticles stabilized by a block copolymer of PEG and a pH-dependent charge-conversional polymer [24].

3.3. Cell cytotoxicity and in vitro transfection Fig. 5. Effect of the amount of dopa–HA on the efficiency of luciferase gene silencing efficiency in vitro by CAP/siRNA/dopa–HA in HT29-luc cells. PEI 25 kDa (PEI/siRNA weight ratio = 1) and mock treated cells were used as positive and negative controls, respectively. CAP/siRNA/HA, siRNA/dopa–HA, siRNA/HA, and CAP/scRNA/dopa–HA were formulated at a HA-to-siRNA weight ratio of 60 and used as controls. The final concentration of siRNA was 100 nM. Gene silencing efficiency was expressed as percent luciferase activity relative to that of mock-treated cells (100%). Values are given as the mean ± SD of triplicates. *p b 0.05.

within the nanoparticles was confirmed by measuring the amount of encapsulated siRNA (Fig. 1S, see Supplementary data). These results support our initial hypothesis that physical encapsulation of siRNA in

CAP and HA have long been used as biomaterials for clinical purposes due to their excellent biocompatibility and biodegradability. The cytotoxicity of dopa–HA was further assessed using MTT assays. As shown in Fig. S3 (see Supplementary data), the cytotoxicity profile of dopa–HA was as low as that of unmodified HA. In addition, CAP/ siRNA/dopa–HA was able to achieve a much higher level of siRNAmediated target gene silencing in CD44 + human colon carcinoma cells stably expressing luciferase (HT29-luc) than were controls such as siRNA alone or CAP/siRNA (Fig. 5). Higher transfection and target gene silencing were observed in the nanoparticle formulations with higher contents of dopa–HA, suggesting that smaller particles with higher ligand density, i.e., of HA, may be more efficiently taken up by

Fig. 6. (A) Gene silencing efficiency of CAP/siRNA and CAP/siRNA/dopa–HA (dopa–HA to siRNA ratio = 60, w/w) in HT29-luc cells in the absence and presence of free HA. Gene silencing efficiency was expressed as percent luciferase activity relative to that of mock-treated cells (100%). Values are given as the mean ± SD of triplicates. Flow cytometry (B) and confocal microscopy (C) analyses of the cellular uptake of CAP/siRNA and CAP/siRNA/dopa–HA (dopa–HA to siRNA ratio = 60, w/w) in the absence and presence of free HA.

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cells than are larger particles [5]. When the ratio of dopa–HA to siRNA reached 60, the gene silencing efficiency of CAP/siRNA/dopa–HA was comparable to that achieved with PEI 25 kDa. The addition of unmodified HA (CAP/siRNA/HA) failed to improve siRNA-mediated gene silencing. The incorporation of double stranded RNA with a scrambled sequence (scRNA) did not show inhibition of the target gene expression, suggesting target sequence specific gene silencing. The receptor mediated cellular uptake of CAP/siRNA/dopa–HA was further investigated by observing the influence of a competitor ligand, free HA, in transfection medium. The gene silencing efficiency of CAP/ siRNA/dopa–HA was significantly reduced by the presence of free HA, while that of CAP/siRNA and PEI 25 kDa/siRNA were not affected by free HA (Fig. 6A). Flow cytometry analysis and confocal microscopy using fluorescence-labeled siRNA also demonstrated the critical role of receptor-mediated endocytosis in the cellular uptake of CAP/siRNA/ dopa–HA (Fig. 6B and C). Receptor-mediated endocytosis is reported as a major mechanism for cellular uptake of HA and HA-based nanoparticles [25–27]. The enhanced gene silencing of CAP/siRNA/dopa–HA can be explained by the increased cellular uptake via receptor mediated endocytosis. This result agrees well with previous observations about enhanced cancer cell-specific cellular uptake of nanoparticles of which surface was decorated with HA derivatives [28–30]. After the cellular uptake via endocytosis, CAP/siRNA/dopa–HA should escape from the endosomal compartment and release siRNA in the cytoplasm. Endosomal escape and release of siRNA were previously hypothesized that the dissolution of CAP core in the acidic environment of the endosome may cause the increase in the endosomal concentration of calcium and phosphate ions, leading to swelling and disruption of the endosomal membrane due to increased internal osmotic pressure [8]. The dissolution of CAP core and the release of siRNA in the endosome-like acidic environment were observed at physiological (pH 7.4), early endosomal (pH 6.5), and late endosomal pH (pH 5.0) [31]. While only 10% of entrapped siRNA was released from CAP/ siRNA/dopa–HA after 8 h incubation at pH 7.4, 56 and 82% of siRNA was released within 2 h incubation at pH 6.5 and 5.0, respectively (Fig. 4S, see Supplementary data), suggesting the release of siRNA from CAP/siRNA/dopa–HA in the endosome by the dissolution of CAP core. The acid-facilitated dissolution of CAP can also be found in previous observations [8,32]. 3.4. Serum stability of CAP/siRNA/dopa–HA

Fig. 7. (A) Effect of serum protein on gene silencing efficiency and (B) encapsulated siRNA stability of CAP/siRNA and CAP/siRNA/dopa–HA (dopa–HA to siRNA ratio = 60, w/w). Gene silencing efficiency was expressed as percent luciferase activity relative to that of mock-treated cells (100%). Values are given as the mean ± SD of triplicates. Transfection experiments and siRNA stability assays were performed in the presence of 10% and 50% FBS, respectively.

in the tumor tissue. This also showed the distinct roles of dopa moiety and HA for the binding to CAP particles and the targeted siRNA delivery to tumor, respectively. The in vivo distribution of CAP/siRNA/dopa–HA after systemic administration is presented in Fig. 9. CAP/siRNA/dopa–HA containing

Before carrying out in vivo experiments, the stability of CAP/siRNA/ dopa–HA nanoparticles in the presence of serum proteins was assessed. As shown in Fig. 7A, the siRNA delivery capability of CAP/siRNA/ dopa–HA was not significantly affected by serum proteins. In contrast, a dramatic reduction in gene silencing efficiency was observed with PEI 25 kDa/siRNA in the presence of 50% serum. The reduced gene silencing efficiency may be due to inter-particular aggregation and destabilization of complexes by non-specific adsorption of serum proteins to the positively charged polyplexes [33]. In addition, CAP/ siRNA/dopa–HA also provides effective protection to siRNA from degradation by serum-derived nucleases (Fig. 7B). In conjunction with the transfection data, these results suggest the possibility of utilizing CAP/ siRNA/dopa–HA for systemic targeted delivery of siRNA to CD44positive cancers. 3.5. In vivo siRNA delivery and gene silencing In vivo siRNA delivery and target gene silencing by CAP/siRNA/ dopa–HA was evaluated in animals bearing HT29-luc tumor xenografts. As shown in Fig. 8, CAP/siRNA/dopa–HA containing luciferase-silencing siRNA significantly suppressed the expression of the target gene in the tumor xenograft after a single dose (0.6 mg/kg) administered through the tail vein. In contrast, CAP/siRNA, CAP/siRNA/HA, and CAP/scRNA/ dopa–HA could not induce the silencing of luciferase gene expression

Fig. 8. In vivo gene silencing effects of CAP/siRNA/dopa–HA (dopa–HA to siRNA ratio = 60, w/w) in an HT29-luc tumor xenograft model. CAP/siRNA, CAP/siRNA/HA, and CAP/scRNA/ dopa–HA (dopa–HA (or HA) to siRNA (or scRNA) ratio = 60, w/w) were used as controls. The gene silencing efficiency was expressed as percent luciferase activity in solid tumor relative to that of mock-treated animals (100%). Values are given as the mean ± SD (n = 3 to 4).

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Cy5.5-labeled siRNA significantly accumulated in HT29-luc tumors, relative to major organs such as the spleen and lungs. The accumulation of the nanoparticles in the liver may be attributed to the presence of the hyaluronan receptor for endocytosis (HARE receptor) in hepatocytes [34]. However, no sign of hepatic toxicity after a systemic administration of CAP/siRNA/dopa–HA was detected in histological evaluation and liver function tests (Fig. 7S, see Supplementary data). In addition, CAP/siRNA/dopa–HA accumulated in tumors to a much higher degree than did the CAP/siRNA formulation (Fig. 9B). The tumor-to-liver ratio of the CAP/siRNA/dopa–HA was 1.13 ± 0.13, while that of the CAP/ siRNA was 0.26 ± 0.04 (Fig. 9C). The increased tumor-to-liver ratio reflects the enhanced penetration of CAP/siRNA/dopa–HA nanoparticles through the looser tumor vasculature [35], followed by prolonged retention in tumor cells, possibly owing to CD44-mediated cellular uptake [36]. These results suggest that CAP/siRNA/dopa–HA has the potential to be used as a safe and effective siRNA carrier for systemic cancer therapy. Our previous study showed that CAP nanoparticles can be effectively stabilized by simply adding dopa–chitosan conjugate to CAP formulation [10]. In the present study, the use of dopa–HA makes it possible to give systemic cancer targeting property to CAP nanoparticles by simply changing the hydrophilic polymer. This result suggests that a variety of hydrophilic natural or synthetic functional polymers can also be employed for the preparation of surface-stabilized CAP nanoparticles without compromising their own properties.

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4. Conclusion In this study, we introduced a new siRNA delivery system based on CAP nanoparticles stabilized with the biological adhesion moleculenatural polymer conjugate, dopa–HA. The surface-stabilized CAP/ siRNA/dopa–HA can readily form solid nanoparticles containing siRNA by a simple mixing procedure, successfully resolving the inherent problems of siRNA, such as its short and rigid structure and low charge density, which often hinder the formation of compact nanoparticles using conventional cationic polymer- or lipid-based siRNA delivery systems. In addition, owing to the unique adhesive properties of dopa and the non-toxic hydrophilic layer conferred by HA, dopa–HA can efficiently inhibit excessive growth of CAP to stabilize nanoparticles in physiological salt conditions for extended periods of time, and provide effective protection to siRNA from enzyme-mediated digestion, which may allow effective accumulation and siRNA-mediated target gene silencing in solid tumors. Hence, the CAP/siRNA/dopa–HA formulation holds promise as a platform for systemic siRNA delivery and targeted cancer therapy.

Acknowledgments This research was supported by the National Research Foundation of Korea (2011-0019775, 2010-0027955, NRF-2013R1A2A2A04016796)

Fig. 9. (A) In vivo bio-distribution of Cy5.5-siRNA delivered by CAP/siRNA and CAP/siRNA/dopa–HA. (B) Normalized organ distribution of Cy5.5-siRNA delivered by CAP/siRNA and CAP/ siRNA/dopa–HA. Normalized organ fluorescence intensity was obtained by dividing the fluorescence intensity of the organ by the area of the organ using image analysis software. (C) Tumor-to-liver ratio of Cy5.5-siRNA fluorescence intensity. Animals were sacrificed and organs were collected 4 h after systemic administration of Cy5.5-siRNA formulated in CAP/ siRNA or CAP/siRNA/dopa–HA (dopa–HA to siRNA ratio = 60, w/w). The quantitative values are given as the mean ± SD (n = 3 to 4).

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Target-specific delivery of siRNA by stabilized calcium phosphate nanoparticles using dopa-hyaluronic acid conjugate.

Low cytotoxicity and high cellular gene delivery capability are among the most important prerequisites for the selection of a non-viral carrier. Altho...
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