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Oral siRNA Delivery to Treat Colorectal Liver Metastases Sung Hun Kang,† Vishnu Revuri,□ Sang-Joon Lee,‡,§ Sungpil Cho,∥ In-Kyu Park,⊥ Kwang Jae Cho,*,# Woo Kyun Bae,*,¶ and Yong-kyu Lee*,†,∥,□ †
Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea ‡ Department of Biomedical Science, Chonnam National University Medical School, Gwangju 500-757, Republic of Korea § Department of Health Administration, Gwangju Health University, Gwangju, Republic of Korea ∥ KB BioMed Inc., Chungju 380-702, Republic of Korea ⊥ Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju 500-757, Republic of Korea # Department of Otolaryngology, Head & Neck Surgery, College of Medicine, The Catholic University of Korea, Seoul 480-717, Republic of Korea ¶ Department of Hematology-Oncology, Chonnam National University Hwasun Hospital, Hwasun-gun, Jeollanamdo, Republic of Korea □ Department of Green Bio Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea S Supporting Information *
ABSTRACT: Convenient multiple dosing makes oral administration an ideal route for delivery of therapeutic siRNA. However, hostile GI environments and nonspecific biological trafficking prevent achieving appropriate bioavailability of siRNA. Here, an orally administered AuNP−siRNA−glycol chitosan−taurocholic acid nanoparticle (AR-GT NPs) was developed to selectively deliver Akt2 siRNA and treat colorectal liver metastases (CLM). AR-GT NPs are dual padlocked nonviral vectors in which the initially formed AuNP−siRNA (AR) conjugates are further encompassed by bifunctional glycol chitosan-taurocholic acid (GT) conjugates. Covering the surface of AR with GT protected the Akt2 siRNA from GI degradation, facilitated active transport through enterocytes, and enhanced selective accumulation in CLM. Our studies in CLM animal models resulted in the reduction in Akt2 production, followed by initiation of apoptosis in cancer cells after oral administration of Akt2 siRNA-loaded AR-GT. This therapeutic siRNA delivery system may be a promising approach in treating liver-associated diseases. KEYWORDS: oral siRNA delivery, colorectal liver metastases, bile acids, cancer therapy, targeted oral delivery, gold nanoparticles
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nodular regenerative hyperplasia (NRH) with FOLFOX, has a significant impact on patient recovery from treatment.3 RNA interference (RNAi), a molecular tool for silencing genes associated with a disease phenotype, is a promising therapeutic alternative to conventional chemotherapeutics.4 Combining existing chemotherapy with RNAi has shown a synergistic effect in various disease conditions. As a potential strategy to overcome the side effects of FOLFOX or FOLFIRI, application of RNAi has been suggested to treat CLM. Indeed, several studies have demonstrated prevention of micro-
olorectal liver metastases (CLM) form from colorectal cancer (CRC) and are the most common secondary hepatic cancer. Patients with CLM have a median survival of 5 to 20 months when left untreated, and five-year survival is extremely rare.1 While surgical resection of the liver is the only available treatment that allows long-term survival, 60% to 70% of patients with CLM will experience recurrence at intrahepatic and extrahepatic sites.2 When feasible, combination therapy of hepatic resection and systemic chemotherapy is the most effective method for improving the survival of patients that develop CLM. Current systemic chemotherapeutic treatments of CLM rely on 5-fluorouracil (5-FU) and folinic acid associated with either oxaliplatin as “FOLFOX” or irinotecan as “FOLFIRI”. Despite proven effectiveness, hepatic injury, known as sinusoidal obstruction syndrome (SOS) with FOLFIRI and © 2017 American Chemical Society
Received: August 4, 2017 Accepted: September 13, 2017 Published: September 13, 2017 10417
DOI: 10.1021/acsnano.7b05547 ACS Nano 2017, 11, 10417−10429
Article
Cite This: ACS Nano 2017, 11, 10417-10429
Article
ACS Nano
Figure 1. Synthesis and characterization of AR-GT nanoparticles. (A) Schematic illustration of the synthesis of AR-GT NPs. Gold−thiol interactions between citrate-stabilized gold nanoparticles (AuNP) and thiolated Akt siRNA facilitated the synthesis of AuNP−siRNA complex (AR). Fe-TEM image shows the morphology of ARNPs (upper right). Electrostatic interactions between negatively charged AR and GT assisted in the synthesis of AR-GT. AFM images indicate the morphology of AR-GT NPs (bottom right). (B) Effect of feed mole ratios of AuNP to siRNA on the particle size and surface charge of AR. (C) Particle size and surface charge of AR-chitosan and AR-GT NPs (AR-GT50 and AR-GT100). (D) UV−vis absorption spectra of AuNPs, AR, AR-chitosan, AR-GT50, and AR-GT100 NPs. The red shift in the absorption spectra indicates the increase in the particle size of the NPs. (E) Effect of GSH concentration on the release of siRNA from AR and AR-GT100 NPs. (F−H) Changes in the particle size of AR-chitosan and AR-GT100 NPs at various GI pH conditions (pH 2, 5, and 7) for 5 days.
metastases and metastatic growth of CRC in the liver.5−7 Although the results showed promising potential for treatment of CLM, translation of RNAi-based therapies toward clinical settings poses challenges, including intrinsic poor serum stability and nonspecific uptake into biological systems, suggesting a need for the development of efficient delivery systems.8 siRNA delivery systems have been developed using either cationic lipid or polymeric carrier systems for systemic administration through an intravenous (IV) route. The therapeutic potential of these systems has been confirmed by various studies.9−13 However, unsolved issues, such as intrinsic poor serum stability, cytotoxicity of the cationic carriers, and the difficulty in achieving therapeutic siRNA levels, have led to the development of alternative routes of administration.14 As a convenient and possibly multiple-dosing route, the oral (PO) route has been employed to deliver siRNA to target
inflammation in the intestinal region. Some researchers have also introduced chitosan, a cationic polysaccharide, to deliver genes through the PO route.11,15−18 Therefore, the PO route has potential for overcoming problems associated with IV siRNA delivery. However, certain inherent challenges are also associated with the development of a PO siRNA delivery system, such as overcoming the acidic stomach environment and nonspecific targeting to regions of the gastrointestinal (GI) tract. Bile acids are steroidal amphiphilic molecules secreted from hepatocytes in the liver.19 They are stable in the acidic stomach environment and are recycled through enterohepatic circulation with an efficiency of approximately 90%. Apical sodium bile acid transporters (ASBT) on the ileum region of the small intestine are involved in preferential transport of bile acids into enterocytes. Furthermore, organic solute transporter peptides (OST α/β) on the basolateral membrane of intestinal 10418
DOI: 10.1021/acsnano.7b05547 ACS Nano 2017, 11, 10417−10429
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ACS Nano
to-charge interaction between AR conjugates and GT prepared with various TCA ratios. The synthesis of either AR-GT or ARchitosan NPs was confirmed through observation of the red shift of UV−vis peaks in AR-GT or AR-chitosan spectra compared to those in the AR and AuNP UV−vis spectrum analysis (Figure 1D). Atomic force microscopy (AFM) and dynamic light scattering (DLS) analysis revealed that the NPs attained spherical morphologies (Figure 1A) with mean diameters of 59 ± 10, 100 ± 3, and 115 ± 2 nm, corresponding to AR-chitosan, AR-GT50, and AR-GT100, respectively (Table S3). Furthermore, the surface charge of AR-GT50 and ARGT100 indicated zeta potential values of 29 ± 1 and 4 ± 1 mV, respectively (Figure 1C). AR-GT100 NPs were designed to release their siRNA through cleavage of the disulfide bond between the siRNA and the AuNP in the intracellular environment via the enzymatic action of glutathione (GSH). However, the ubiquitous presence of GSH in the extracellular space (∼1 mM) and in blood (0.002 mM) has the potential to prerelease the siRNA before the NPs reach the intracellular GSH (10 mM) of the target site, which will impair the desired therapeutic effect. To confirm whether the GT100 coating over AR contributes to the prevention of siRNA prerelease, we measured the release of siRNA from either AR or AR-GT100 under various GSH concentrations. AR demonstrated approximately 60% prerelease of siRNA under a very low GSH concentration of 0.002 mM, which is equivalent to the concentration of GSH in the blood (Figure 1E). However, AR-GT100 prevented siRNA prerelease under the concentration of GSH in both the blood and the extracellular space and showed slightly enhanced release of siRNA at the intracellular GSH concentration compared to the release of siRNA at the GSH concentrations found in both blood and the extracellular space (20% vs 0− 12%) (Figure 1E). These results suggest that the AR coating over GT100 can prevent prerelease of siRNA and maintain therapeutic-siRNA efficacy until AR-GT100 reaches the target sites. Given that the ileum of the small intestine is the designated initial binding site of AR-GT100, AR-GT100 should maintain its stability under various GI pH environments (Figure 1, pH profiles of the GI tract). Here, we studied the stability of ARGT100 in different GI pH conditions compared to that of the bare AR-chitosan control. As shown in Figure 1F,G, AR-GT100 demonstrated stability without noticeable changes in particle size under near stomach pH (pH 2) and pH 5, while ARchitosan gradually decreased in size, with a 25% size reduction in acidic pH. Interestingly, the particle size of AR-GT100 NPs increased at pH 7 (Figure 1H). Deprotonation of chitosan on AR-GT100 at pH 7 could contribute to an increase in the overall negative charge of GT100 and disrupt the interaction between AR and GT100. Therefore, this observation suggests an increased chance of siRNA release from AR-GT100 NPs at the ileum surface. Later, we studied the ability of AR-GT100 to prevent the degradation of siRNA from the biological enzymes. As shown in Figure S3, after addition of RNase into the AR, an increase in the FITC fluorescence was observed, while no change in the fluorescence intensities was observed in AR-GT100 samples. Because GT100 completely protected the AR complex, RNase was not able to enter and degrade the siRNA. However, AR NPs which lacked the GT100 coating resulted in enhanced siRNA degradation.
enterocytes aid transport of bile acids into enterohepatic circulation for recycling of bile acids in the liver.20−23 Inspired by enterohepatic recycling of bile acids, we employed a dual padlocked system to protect Akt2-siRNA from GI degradation and to target it to CLM. In this regard, a AuNP−siRNA (AR) complex was prepared by conjugation of gold nanoparticles (AuNP) with thiolated siRNA. The AR complex was further modified by wrapping the AR complex with glycol chitosan−taurocholic acid (GT) through a chargeto-charge interaction between negatively charged AR and positively charged GT conjugates to generate AR-GT nanoparticles (AR-GT NPs). Taurocholic acid (TCA) on the surface of the AR-GT NPs plays a critical role in both protecting the siRNA from GI degradation and facilitating targeting to CLM through the enterohepatic recycling process. In vivo biodistribution and therapeutic studies indicated that the NPs exhibited enhanced anticancer activity with reduced toxicity in a CLM animal model. We further anticipate translational application of AR-GT NPs to treat CLM.
RESULTS Synthesis and Characterization of AR-GT Nanoparticles. AR-GT NPs for PO delivery of therapeutic Akt2 siRNA were prepared by wrapping Akt2 siRNA−AuNP with GT. First, AuNP were synthesized by reacting HAuCl4 with trisodium citrate.24 The progression of the chemical reaction was monitored by the color changes of the HAuCl4 solution from yellow to wine red. The appearance of a UV absorption peak at 520 nm indicated the successful synthesis of AuNP (Figure S1A). The zeta potential analysis revealed that the AuNP had an average size of 18 ± 2 nm with a surface charge of −13 ± 1 mV (Figure S1B,C). In further analysis with both scanning electron microscopy (SEM) and transmission electron microscopy (TEM), AuNP showed a uniform morphological distribution (Figure S1E,F). Second, gold−thiol interactions between AuNP and thiolated Akt2 siRNA facilitated the synthesis of AR conjugates. Compared to pristine polymeric nanoparticles, AuNPs have higher surface area, which could facilitate enhanced loading of siRNA.25 The AR conjugates with different molar ratios of thiolated siRNA-FITC to AuNP confirmed the achievement of saturated conjugation of siRNA to the AuNP at a molar feed ratio of ≥1:10 (AuNP:siRNA) (Table S1). The siRNA layer on the dark core of the AuNP was observed in the AR conjugates using TEM analysis (Figure 1A). Further analysis of AR conjugates demonstrated particle sizes from 18 to 22 nm with surface charges from −13 to −28 mV depending on the molar feed ratio of siRNA to AuNP (Figure 1B). Lastly, AR-GT was prepared via a charge-to-charge interaction between AR conjugates and GT. Before initiating the interaction between AR and GT, GT was synthesized by reacting the amine functional groups of glycol chitosan (chitosan) with the hydroxyl groups of TCA activated by 4nitrophenyl chloroformate 96% (4-NPC) treatment. The successful synthesis of GT was confirmed by 1H NMR analysis, indicating proton peaks at 0.8 ppm from the methyl carbon at the C18 position and 2.9 ppm from the ethyl protons at the C25 position of TCA (Figure S2) in the GT spectra.26 Further, the degree of TCA conjugation to chitosan was analyzed using a TNBSA assay. The results revealed that a higher TCA feed ratio resulted in a reduced number of free amine groups on chitosan as well as a positive chitosan surface potential (Table S2), which suggested increased chitosan conjugation of TCA with GT. Subsequently, AR-GT was prepared through charge10419
DOI: 10.1021/acsnano.7b05547 ACS Nano 2017, 11, 10417−10429
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ACS Nano
Figure 2. Contribution of taurocholic acid (TCA) in the cellular uptake of AR-GT nanoparticles. (A, B) Confocal images depicting the uptake of rhodamine B-conjugated AR-chitosan and AR-GT100 nanoparticles in Caco-2 and HepG2 cells. (C, D) Quantitative cellular uptake profiles of AR-chitosan, AR-GT50, and AR-GT100 in Caco-2 and HepG2 cells. (E, F) Competitive cellular uptake assay of AR-GT100 in Caco-2 and HepG2 cells pretreated with different concentrations of TCA (mean ± SD; N = 5; P-value
