Oral siRNA Delivery to Treat Colorectal Liver Metastases - ACS Nano

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Oral siRNA Delivery to Treat Colorectal Liver Metastases Sung Hun Kang, Vishnu Revuri, Sang-Joon Lee, Sungpil Cho, InKyu Park, Kwang Jae Cho, Woo Kyun Bae, and Yong-kyu Lee ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b05547 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 14, 2017

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Oral siRNA Delivery to Treat Colorectal Liver Metastases Sung Hun Kang1, Vishnu Revuri8, Sang-Joon Lee2, 3, Sungpil Cho4, In-Kyu Park5 Kwang Jae Cho6, *, Woo Kyun Bae7, * and Yong-kyu Lee1,4,8, * 1

Department of Chemical and Biological Engineering, Korea National University of

Transportation, Chungju 380-702, Republic of Korea 2

Department of Biomedical Science, Chonnam National University Medical School, Gwangju,

Republic of Korea 3

Department of Health Administration, Gwangju Health University, Gwangju, South Korea.

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KB BioMed Inc., Chungju 380-702, Republic of Korea

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Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju,

Republic of Korea 6

Department of Otolaryngology, Head & Neck Surgery, The Catholic University of Korea,

College of Medicine Uijeongbu St. Mary’s Hospital, Kyunggi-Do 480-717, Republic of Korea 7

Department of Hematology-Oncology, Chonnam National University Hwasun Hospital,

Hwasun-gun, Jeollanamdo, Republic of Korea

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Department of Green Bio Engineering, Korea National University of Transportation, Chungju

380-702, Republic of Korea KEYWORDS: oral siRNA delivery. colorectal liver metastases. bile acids. cancer therapy. targeted oral delivery. gold nanoparticles

ABSTRACT

Convenient multiple dosing makes oral administration an ideal route for delivery of therapeutic siRNA. However, hostile GI environments and non-specific biological trafficking prevent from achieving appropriate bioavailability of siRNA. Here, an orally administered AuNP-siRNAglycol 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.


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Colorectal 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-flurouracil (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 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 micrometastases 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 non-specific uptake into biological systems, suggesting a need for the development of efficient delivery systems.8


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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 non-specific 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 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, an AuNPsiRNA (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


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chitosan-taurocholic acid (GT) through a charge-to-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 in facilitating targeting to CLM through the enterohepatic recycling process. In vivo biodistribution and therapeutic studies indicated that the NPs exhibited enhanced anti-cancer 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 nm ± 2 with a surface charge of -13 mV ± 1 (Figure S1B,C). In further analysis with both SEM and 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 ≥ 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


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analysis of AR conjugates demonstrated particle sizes from 18 nm to 22 nm with surface charges from -13 mV 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 4NPC 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 charge-to-charge interaction between AR conjugates and GT prepared with various TCA ratios. The synthesis of either ARGT or AR-Chitosan 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). AFM and DLS analysis revealed that the NPs attained spherical morphologies (Figure 1A) with mean diameters of 59 nm ± 10, 100 nm ± 3, 115 nm ± 2 corresponding to AR-Chitosan, AR-GT50 and AR-GT100, respectively (Table S3). Furthermore, the surface charge of AR-GT50 and AR-GT100 indicated zeta potential values of 29 mV ± 1 and 4 mV ± 1, 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)


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and in blood (0.002 mM) has the potential to pre-release 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 pre-release, we measured the release of siRNA from either AR or AR-GT100 under various GSH concentrations. AR demonstrated approximately 60% pre-release 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 pre-release 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 pre-release 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 AR-GT100 in different GI pH conditions compared to that of the bare AR-Chitosan control. As shown in Figure 1F–G, ARGT100 demonstrated stability without noticeable changes in particle size under near stomach pH (pH 2) and pH 5, while AR-Chitosan gradually reduced 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.


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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, 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 were not able to enter and degrade the siRNA. However, AR NPs which lacked the GT100 coating resulted in enhanced siRNA degradation.

Cellular interactions and uptake, migration, and carrier toxicity of AR-GT100. Oral delivery of siRNA has mostly depended on the concept that the tight inter-connections of the intestinal epithelial lining will be bypassed via passive paracellular or transcellular pathways.16 However, minimal transport of delivery vehicles across the intestinal epithelial lining and even inconsistent outcomes from current delivery strategies show that efficient approaches for PO siRNA delivery are required. Here, we tried to mimic the enterohepatic bile acids recycling process through the apical sodium bile acid transporter (ASBT) on the ileal regions of the small intestines, which have approximately 90% reabsorption efficiency of bile acids.27,28 We designed our siRNA delivery system with TCA ligands, which have a potential role in interacting with ASBT in the small intestine and ultimately may aid migration of NPs to the target site, the liver, through the enterohepatic recycling system.23,27,29 We expect this system to achieve enhanced PO bioavailability, reproducibility, and even therapeutic activity through enterohepatic recyclingmediated siRNA delivery. To confirm the cellular interaction and uptake of our PO siRNA delivery system, AR-GT100 was labeled with Rhodamine B (RB) and was used to study the cellular uptake profiles in human intestinal epithelial cell lines, Caco-2 and human liver hepatocellular carcinoma cell lines,


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HepG2. As shown in Figure 2A-B, strong intracellular red fluorescence was observed in the cell lines treated with AR-GT100 compared to the cells treated with the control AR-Chitosan. In addition, the time-dependent measurements of cellular uptake of NPs demonstrated an approximately 1.6 to 2-fold enhancement in red intracellular fluorescence 6 hr after the cells were treated with AR-GT100 compared to the cells treated with AR-GT50 or AR-Chitosan (Figure 2C,D). This supports the potential role of TCA in the interaction and uptake of AR-GT NPs into the cells. Further investigation of the role of TCA in the uptake of AR-GT NPs was performed by pretreating cells with different concentrations of TCA (0.1 mg/mL, 0.5 mg/mL and 1 mg/mL) for 30 min, followed by addition of AR-GT100. Comparison of the fluorescence intensity indicated that serial increases in the concentration of TCA resulted in a serial reduction in the level of cellular uptake of AR-GT100, almost near that of the control, AR-Chitosan (Figure 2E,F). These competitive cellular uptake analyses suggest TCA-mediated cellular uptake of AR-GT100 NPs. Previous experiments raised a further question regarding whether ASBT, a cellular surface receptor known to have affinity for TCA, was involved in intracellular uptake of AR-GT100. To answer this question, the nanocarriers were conjugated with RB, and the fluorescence intensities from RB were used to analyze the importance of ASBT receptors in the uptake of the TCAconjugated nanocarriers. MDCK cell lines without or with enhanced expression of ASBT (MDCK or MDCK-ASBT) were treated with either AR-Chitosan/RB or AR-GT100/RB. ARGT100 treatment resulted in an intensified red fluorescence in MDCK-ASBT cells compared to the fluorescence observed in MDCK cells (Figure 3A,B). To further confirm the role of ASBT in the cellular uptake of AR-GT100, MDCK-ASBT cells were treated with a competitive substrate, TCA for 30 min before adding AR-GT100. The results demonstrated that pretreatment with TCA


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led to inhibition of the cellular uptake of AR-GT100 in MDCK-ASBT cells, nearly restoring the fluorescence to levels equivalent to those observed with AR-Chitosan, the experimental control (Figure 3C-D). These observations suggest the critical role of the ASBT receptor in the cellular interaction and uptake of AR-GT100. One of the major limitations of PO delivery is the poor bioavailability of the drugs or biomolecules after PO administration due to low migration of delivery systems from the intestinal apical site to the basolateral regions before entering into systemic circulation. Here, we tested the migration of AR-GT50 and AR-GT100 from the apical site to the basolateral site using a transwell membrane migration model consisting of a CaCo-2 cell monolayer cultured over the transwell membrane (Figure 4A). The migration results were compared to the degree of migration obtained from the AR-Chitosan control. As shown in Figure 4B and Figure S4, the AR-GT100, AR-GT50, and AR-Chitosan migration exhibited 76% ± 1, 52% ± 1, and 40% ± 1 transportation efficiency, respectively, after 24 hr of treatment. The enhanced migration of ARGT NPs in response to increasing TCA conjugation suggests the importance of TCA conjugation in the migration of AR-GT NPs across the intestinal epithelial layer. Understanding the systemic toxicity of carriers in the development of a delivery system is a critical step in determining future applications in clinical settings. We studied the in vitro toxicology profiles of our carriers in HepG2 and CaCo-2 cells and in the hepatic metastatic colon cancer cell line CT26. Our gold-based nanoparticle carriers showed negligible toxicity profiles with a minimum of 80% cell survival in all the tested cell lines (Figure 4C,D and Figure S5). These studies indicate that AuNP carriers, AR-Chitosan or AR-GT100 delivery systems are nontoxic and can be further applied in in vivo testing systems.


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In vitro therapeutic efficacy of AR-GT100 nanoparticles. In vitro western blot analysis was performed to evaluate the expression of proteins involved in the Akt and cancer apoptosis (Bax, caspase-9, PARP) pathways after treating the cells with Akt siRNA samples (free siRNA, ARChitosan and AR-GT100) for 12 hr (Figure 5A). In general, phosphorylation of Akt proteins at their activation loop (T308 for Akt1) and C-terminus (S473 for Akt1) results in enhanced cellular proliferation in cancer cells.30 As shown in Figure 5B, a remarkable reduction in the expression of Akt proteins was observed in the cells transfected with Akt siRNA. Reduction in the levels of Akt proteins can result in activation of the pro-apoptotic protein Bax by Bad and lead to cell apoptosis.31 Bax expression levels were increased after treating the cells with Akt siRNA. In fact, enhanced Bax expression was observed in the cells treated with Akt siRNA loaded AR-GT100 NPs (Figure 5B, lane 4). Moreover, the level of the pro-apoptotic protein caspase-9, which is activated by Bax, was also enhanced in the cells treated with AR-GT100 NPs. Furthermore, the levels of PARP were also reduced in AR-GT100-treated cells. These results indicated that transfection of cells with targeted Akt siRNA-loaded AR-GT100 NPs enhanced cancer cell apoptosis compared to other treatment groups. An annexin V/dead cell marker assay was performed to confirm the induction of apoptosis after transfecting HepG2 cells with Akt siRNA samples (free Akt siRNA, AR-Chitosan, ARGT100). As shown in Figure S6, the cells treated with AR-GT100 exhibited enhanced apoptosis activation compared to other groups. In fact, 67% of the cancer cells showed apoptosis activation, which was higher than the percentage observed in the AR-Chitosan (35%) and free Akt (24%) transfection groups. From these data we confirmed that the reduction in cell proliferation was predominantly due to activation of Akt siRNA-induced apoptosis.


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Tissue uptake, biodistribution, and transport of AR-GT nanoparticles. Successful PO delivery of drugs/biologics leads to an enhancement in bioavailability. The TCA moieties in the AR-GT NPs were carefully considered for their ability to enhance bioavailability through active ASBT receptor-mediated transport of NPs into enterohepatic circulation, followed by NTCP receptor-mediated selective uptake in the liver.32,33 To confirm the functions of TCA moieties for successful delivery of AR-GT NPs to the liver, we examined the time-dependent uptake and biodistribution of AR-Chitosan and AR-GT100 after labeling the NPs with RB. As shown in Figure 6A, most of the AR-GT100 was observed in the ileal tissue compared to the duodenal tissue distribution observed for the majority of AR-Chitosan. The quantitation of relative fluorescence intensity (R.F.I) from the tested organs/tissues demonstrated an approximately 1.21.4 times enhancement of the AR-GT100 accumulation in both ileal tissue and the liver compared to the AR-Chitosan at either 6 hr or 12 hr, respectively (Figure 6B). In conclusion, we confirm that chitosan-TCA plays a crucial role in ileal tissue uptake and eventual transport of AR-GT100 NPs to the liver through the natural enterohepatic circulation system. Additionally, we also measured the gold deposition in both AR-Chitosan and AR-GT NPs using inductively coupled plasma mass spectrometry (ICP-MS) to quantitatively analyze the previous observation of the tissue distribution of AuNP, AR-Chitosan and AR-GT100. As shown in Figure 6C-I, AR-Chitosan NPs demonstrated a non-specific distribution in all segments of tissues including the small intestine (duodenum, jejunum, and ileum) possibly due to electrostatic interactions between the positively charged AR-Chitosan NPs and either negatively charged cell surfaces or the partially opened tight junctions in the GI tract. However, by following the signal of the gold component of AR-GT100 in the liver, AR-GT100 NPs were found to exhibit a specific absorption peak in ileal tissue 6 hr after PO administration and eventually were observed


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in kidney tissue (Figure 6F-H) during the 48 hr detection period. Additionally, dose variations of AR-GT100 also showed that accumulation depended on the concentration of AR-GT100 in the liver (Figure S7). Moreover, the total amount of AR-GT100 was 2-fold higher than that of ARChitosan (Figure S8). This confirms that targeted AR-GT100 had enhanced depositions as well as enhanced targeting efficacy compared to the non-targeted AR-Chitosan. To confirm uptake, targeting, and excretion of AR-GT100, we performed a TEM analysis with frozen intestine, liver, and kidney tissues after PO administration to mice (Figure 7). Intestinal uptake of AR-GT100 resulted in vesicular-based transport from the surface of the ileal tissue (Figure 7D). In the liver, AR-GT100 was identified in hepatocytes without any visible accumulation in the immune-active Kupffer cells (Figure 7D). Lastly, deposition of AR-GT100 was also confirmed in the kidney suggesting excretion through the kidney (Figure 7D). However, the animals treated with AR-Chitosan showed opening of tight junctions and the transport of nanoparticles through the tight junctions (Figure 7C). Additionally, no presence of vesicles was observed in the ileal regions of the AR and AR-Chitosan treated animals (Figure 7B,C). Furthermore, AR-Chitosan started to aggregate in the liver, which resulted in reduced uptake by the liver cells compared to control. These results clearly suggest a biological cycle of ARGT100, beginning with specific absorption in the ileum, followed by transport to the liver, and eventual excretion from the kidney.

Therapeutic efficacy of AR-GT100 in a CLM cancer mouse model. To study the therapeutic efficacy of AR-GT100 after PO delivery, an orthotopic CLM mouse model was developed using CT26 cancer cells with luciferase activity. Luciferase activity was monitored to determine the growth of the CLM. A significant reduction in the size of tumors was observed in the CLM mice


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treated with AR-GT100 (either 25 μg/kg or 100 μg/kg) compared to the mice treated with saline or AR-Chitosan (Figure 8A left). CLM mice treated with a high dose of AR-GT100 (100 μg/kg) exhibited a more significant reduction of tumor nodules in the liver compared to those treated with a low dose of AR-GT100 (25 μg/kg) or with AR-Chitosan or saline (Figure 8A right). Furthermore, a quantification analysis demonstrated an approximately 43-58% reduction in the number of tumor nodules after treatment with either AR-GT100 (25 μg/kg) or AR-GT100 (100 µg/kg) compared to the outcomes of treatment with AR-Chitosan, which were comparable to the saline-treated CLM (Figure 8B). The metastasis scores in the mice treated AR-GT100 (100 µg/kg) was 2 ± 1 which was 2-fold efficient than the control groups which had a metastasis score of 3 ± 1 (Table S4). Moreover, the survival analysis reflected 100% survival rates of the CLM mice treated with AR-GT100 (25 μg/kg and 100 μg/kg), while the mice treated with ARChitosan had a survival rate of only 30% over 2 weeks (Figure 8C). Previous reports have demonstrated that non-specific delivery of siRNA could result in immune activation.34 Because AR-Chitosan displayed non-specific biodistribution, they have resulted in enhanced animal death compared to the saline treated animals. From these studies, the therapeutic efficacy of ARGT100 and even a survival benefit was confirmed in the CLM mouse model.

Potential mechanism of AR-GT100 activity in CLM. The PO delivery of AR-GT100 was designed to work in CLM by inhibiting the AKT/phosphatidylinositol 3-kinase (AKT/PI3K) cell signaling pathway and eventually inducing cell apoptosis.35 To confirm the potential mechanism of AR-GT100 activity, we tested the expression of Akt and signaling proteins related to the AKT/PI3K pathway after PO delivery of AR-GT100 using an immunoblot analysis. We observed a significant reduction in p-AKT expression compared to the control groups, while


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based on the immunoblot analysis, Akt protein was not visibly reduced (Figure 8D). The quantitative analyses of pAKT proteins also show a 45% reduction in the pAKT proteins in the AR-GT100 treated animals compared to the control groups (Figure S9). This is possibly due to rapid compensation of the loss of Akt in the cells. Interestingly, we also observed consistent changes in the expression of apoptotic proteins after PO delivery of AR-GT100. Expression of pro-apoptotic proteins such as cleaved caspase-9 and Bax was enhanced, while expression of the anti-apoptotic protein Bcl-2 was reduced (Figure 8D, Figure S9). These immunoblot analyses confirmed the induction of cellular apoptotic signaling after PO delivery of AR-GT100. Moreover, detection of cleaved PARP proteins, which are only expressed when single-stranded DNA breaks occur in cells, further confirmed the definite initiation of apoptosis in cells in CLM, which was the result of PO administration of AR-GT100.36 Subsequent analysis of p-AKT using an immunohistochemistry assay brown staining was not detected in the liver tissue after treatment with AR-GT100. However, in control livers, a ubiquitous scattering of brown spots was observed in the tissues, indicating the abundant presence of p-AKT (Figure 8E top). Furthermore, H&E staining revealed negligible changes in the tissue morphology of AR-GT100treated mice. This confirms that AR-GT100 did not induce non-specific tissue toxicity (Figure 8E bottom). The results of these experiments combined with all the previous observations suggest that AR-GT100 acts in CLM through induction of cell apoptosis by regulating the AKT/PI3K pathway and does not induce any harmful effects to normal tissues.

DISCUSSION Abnormal activation of the AKT/PI3K (or PKB) pathway strongly affects cell proliferation, migration, and survival as well as therapeutic resistance in CLM. Strategies to interfere with


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abnormal activation of AKT/PI3K have traditionally been based on the development of inhibitors targeting key proteins such as AKT/PI3K or mTOR. However, several studies reported that delivery of drugs such as trastuzumab, tamoxifen, or doxorubicin undesirably increased the Akt activity in the cancer cells.36 Moreover, several potent AKT/PI3K inhibitors, such as DPIEL and wortmannin, also showed limitations for further application because of severe cellular toxicity due to a lack of selectivity and sensitivity at the therapeutic dose. Moreover, these drugs often lose their activity through biodegradation after PO administration. To overcome these intrinsic problems, an effective and targeted strategy to deliver molecular inhibitors of AKT/PI3K, such as siRNA, through PO administration is an immediate demand for the treatment of CLM. Here, we developed a targeted PO siRNA delivery system to inhibit the expression of Akt2 in CLM. In this system, we developed an AR-GT NP system by combining AR with GT. GT forms a layer over the AR complex via electrostatic interactions between the negatively charged AR and the positively charged GT. The glycol chitosan layer of GT functions as a protective shell during GI transit of AR-GT. Furthermore, the TCA moiety on the GT facilitates ASBT receptormediated absorption and transport of AR-GT from the ileum to the liver using the natural bile acid recycling system. Bile acids absorbed in the ileum are transported to the liver for recycling through a cascade of processes that starts with endocytosis via the interaction of bile acids with the ASBT receptor in the ileum, followed by escape from endosomal/lysosomal degradation through interaction with iBABT and eventual exocytosis via OST receptors to portal circulation to reach the liver.37 The formation of vesicular structures is a typical characteristic of endocytosis mediated by the interaction between bile acids and ASBT receptors. PO administration of ARGT also resulted in the formation of these vesicular structures in the ileal region of the small intestine, without opening of the tight junctions or leaky vasculature. Moreover, in vitro


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competitive uptake assays suggested the importance of TCA in the transport of AR-GT NPs to the target organ, the liver. Furthermore, tracking dye or gold in AR-GT NPs clearly demonstrated NP accumulation in the liver followed by arrival in the kidney for excretion. These results suggest that AR-GT NPs follow a series of events that include specific receptor-mediated uptake at the ileum, transportation to the liver without disintegration, and final arrival in the kidney. PO delivery of AR-GT NPs is anticipated to inhibit the expression of Akt2 protein in cells in CLM and subsequently downregulate AKT/PI3K signaling pathways. In addition to in vitro confirmation of these therapeutic properties, we also verified effective reduction of CLM nodules in the liver in vivo through PO delivery of Akt2 siRNA-loaded AR-GT NPs. Moreover, we also discovered the foundation of a possible working mechanism based on the observation of reduced expression of p-Akt and anti-apoptotic proteins such Bcl-2 and enhanced expression of pro-apoptotic proteins such as cleaved caspase 9 and Bax. Our AR-GT NPs delivery system demonstrated that actively targeted PO siRNA delivery systems have enhanced therapeutic efficacy compared to passively targeted PO drug delivery systems. Moreover, the developed ARGT NPs are biocompatible and non-toxic in biological systems.

CONCLUSIONS We have developed a targeted oral siRNA delivery system, AR-GT NPs to treat CLM. The PO administration of AKT siRNA loaded AR-GT NPs were able to effectively cross the intestinal epithelia and presented an enhanced accumulation in the liver. PO administration of AKT siRNA loaded AR-GT NPs resulted in not only reducing the expression of AKT proteins in the cancer tissues, thereby reducing the AKT/PI3K downstream signaling but also initiated the cancer


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apoptosis in orthotopic CLM animals. Moreover, the animals PO administered with AR-GT NPs displayed enhanced tumor reduction and tumor nodular growth with longer survival time and negligible toxicology profiles. This developed technology has a great importance in terms of patient compliance, therapeutic activity compared to other passively targeted gene therapies or drug delivery systems and we are looking forward to translate this technology towards clinical studies.

EXPERIMENTAL SECTION Materials Glycol chitosan (MW: 82 kDa), Gold (III) chloride hydrate, sodium dodecyl sulfate, HEPES (sodium salt, 99%), Diethyl pyrocarbonate (DEPC), Taurocholic acid sodium salt (TCA) Triethylamine (TEA), 4-Nitrophenyl chloroformate 96% (4-NPC), dimethyl sulfoxide (DMSO), Rhodamine B, Triton® X-100, HANK’S balanced salt solution and Thiazolyl Blue Tetrazolium Bromide was purchased from SIGMA-ALDRICH. Trisodium citrate dihydrate was purchased from Junsei (Tokyo, Japan). Akt2 siRNA were purchased from ST Pharm (Seoul, South Korea). MDCK, Caco-2, CT26 and HepG2 cell were obtained from Korean Cell Line Bank (Seoul, South Korea). Dulbecco’s Modified Eagle’s Medium (DMEM), Minimum Essential Medium (MEM), RPMI Medium 1640, Fetal bovine serum (FBS), Penicillin and 0.05% Trysin-EDTA were purchased from Gibco® by life technologies

TM

. Phosphate Buffered Saline (PBS) was

purchased from BioNieer (Daejeon, Korea).

Synthesis of gold nanoparticles Gold nanoparticles (AuNP) were synthesized following the previously reported protocol.24 All the glassware were rinsed with aquaregia (HNO3: H2SO4 - 1:3). In a typical synthesis, HAuCl4 (2


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mL, 10 mg/mL) were added into the distilled water heated at 98℃ and sustained at the same temperatures for 30 min. Later, trisodium citrate hydrate (74 mg) was added to the above solution and stirred for 30 min. Finally, the synthesized AuNP were cooled down by stirring them in room temperature for 30 min and stored at 4oC for further use. The synthesis and concentration of AuNP were confirmed using UV-Vis spectrophotometer (OPTIZEN IV, Mecasys Co.Ltd, South Korea).38 The physical morphology of AuNP were characterized using Scanning electron microscopy (JSM-7610F, JEOL, Akishima, Japan) and transmission electron microscopy (TEM; JEM-2100F, JEOL, Akishima, Japan). The average particle size and surface charge of AuNP were also characterized using Zetasizer (Nano-s90, Malvern, UK) and Zeta compact (CAD instruments, France).

Synthesis of AuNP conjugated siRNA Citrate-stabilized AuNP were initially treated with 0.1% diethylpyrocarbonate(DEPC) for 12 hr followed by sterilizing the samples by heating them at 121oC for 60 min. Thiolated siRNA (1000 nm) were further incubated with RNase-free AuNP (10nM) for 20 min at room temperature. Later NaCl solution was added to the above samples to attain a final concentration of 0.05mM NaCl. The samples were then incubated at room temperature for 20 min followed by centrifugation (15000 x g for 15 min at 4oC). The pellet was then washed three times and then resuspended in RNase free water. The synthesis of AuNP-siRNA was then characterized using UV-Vis spectrophotometer. Average particle size, surface charge and physical morphology of AuNP-siRNA was characterized using DLS, zeta compact and TEM analysis respectively. Quantitative analysis of siRNA conjugation to AuNP were characterized using FITC labelled


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siRNA where the FITC fluorescence was used to quantify the amount of siRNA conjugated to AuNP.

Synthesis of glycol chitosan conjugation with TCA Different concentrations of TCA was dissolved in 10 mL of dd H2O and stirred at 4oC for 15 min. Later, 4-NPC and TEA (TCA:4-NPC:TEA- 1:3:3) dissolved in DMSO was added to the above solution and stirred at 4oC for 60 min. The activated TCA solution was added to the glycol chitosan (Glycol chitosan: TCA- 1:50 or 1:100) solutions and stirred at room temperatures for 24 hr. Later, the solutions were dialyzed in dd H2O (Membrane filter MWCO: 1 kDa) for 24 hr by replacing the media every 4 hr. Finally, the samples were freeze dried and the lyophilized samples were stored at 4oC for further use. 1H NMR spectroscopic (AVANCE 400FT-NMR, Bruker, USA) studies were used to confirm the synthesis of glycol chitosan-TCA conjugates (GT). The peaks from ethyl protons at 0.7-0.85 ppm and methyl protons at 2.9 ppm from TCA and the proton peaks at 3.3-3.7 ppm and 4.3 ppm confirms the synthesis of GT. The number of TCA conjugated to glycol chitosan was quantified using TNBSA assay following manufacturer’s protocol.

Synthesis of AuNP-siRNA coated glycol chitosan or TCA modified glycol chitosan AuNP-siRNA was added drop wise into Chitosan, GT 50 or GT 100 solutions and then incubated at 4oC for 60 min. The samples were then centrifuged (15,000 x g for 15 min at 4oC) to remove the free unbound siRNA. The samples were lyophilized and stored at 4oC for further use. Synthesis of AR-Chitosan or AR-GT NPs were confirmed using UV-Vis spectrophotometer. The physical morphology of the NPs was studied using Atomic force microscopy (Multimode N3-


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AM, Bruker, USA). Average particle size (DLS) and surface charge of the NPs were also characterized.

In vitro glutathione mediated siRNA release The concentration of glutathione (GSH) is substantively higher in cytoplasm (1-10 mM) compared to that of the blood plasma or the extracellular environments. It was therefore anticipated to study if the GT coating over the AuNP-siRNA conjugates were able to prevent the non-specific release of siRNA in the extracellular environments. FITC-labelled thiolated siRNA were used for the synthesis of AuNP-siRNA or AR-GT100 NPs. In brief, NPs containing 1µg of siRNA were incubated in 1mL of PBS containing different concentrations of GSH (2 µM, 1 mM and 10 mM) for 60 min. Later the samples were centrifuged at 15000 x g for 15 min at 4oC. The fluorescence from the free FITC-siRNA in the supernatants were used to analyze GSH mediated siRNA release from the NPs.

In vitro RNase mediated siRNA degradation FITC-labelled thioloated siRNA were used for the synthesis of AuNP-siRNA or AR-GT100 NPs. In brief, NPs containing 1µg of siRNA were incubated in 1mL of PBS containing RNase (1unit/ug of siRNA) for 60 min. Later the samples were centrifuged at 15000 x g for 15 min at 4oC. The fluorescence from the free FITC-siRNA in the supernatants were used to analyze RNase mediated siRNA degradation from the NPs.

In vitro stability of the AR-GT100 nanoparticles


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Previous reports have suggested that chitosan NPs are susceptible to aggregation in the neutral or basic pH environments18. We studied if the conjugation of TCA to the chitosan prevented the destabilization/ aggregation of the AR-GT100 NPs. In this prospects, AR-Chitosan and ARGT100 NPs were incubated in PBS with different pH condition (pH 2, pH 5 and pH 7) and the change in the average particle size of the NPs were analyzed for 5 days.

In vitro cellular uptake study The carboxyl groups of Rhodamine B were conjugated to the amine groups of glycol chitosan using EDC-NHS chemistry. The Rhodamine B conjugated Glycol chitosan, GT 50 or GT 100 were used synthesize AR-Chitosan, AR-GT50 and AR-GT100 and further used for cellular uptake and cellular permeability studies. Cells (3 x 105 cells/well) were seeded in 12-well cell culture plates and incubated for 24 hrs. The cells were treated with different NPs, incubated for 4 hr and later washed with PBS three times. The cellular uptake of the NPs was visualized using laser scanning confocal microscopy (DE/LSM-510, Carl Zeiss, Germany). For quantitative analysis of the cellular uptake of these NPs, the sample treated cells were washed with PBS three times at different time points, followed by incubating the samples with 1 mL lysis buffer (0.3% Triton® X-100 in PBS) at 37 ℃ for 1 hr. The fluorescence from these solutions were used to quantify the cellular uptake of these NPs.

In vitro competitive cellular uptake assay Free TCA were used as a competitive inhibitor to study the cellular uptake of AR-GT100 NPs. Cells (Caco-2, HepG2, MDCK and MDCK-ASBT cell lines; 5x104 cells/well) were seeded in a


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96 well plate for 24 hr in a 37oC incubator supplemented with 5% CO2. Prior to the incubation of Rhodamine B conjugated AR-GT100 NPs, the cells were pretreated with different concentrations of TCA (20µL; 0.1, 0.5 and 1 mg/mL) for 30 min. Later the cells were incubated with ARGT100 NPs and incubated in a 37oC incubator for 4hr. The cells were then washed with PBS three times and then 100 µL of lysis buffer were added to each sample. The cells were collected and the fluorescence intensities from these cells were used to quantify the free TCA competitive cellular uptake assay. Rhodamine B conjugated AR-Chitosan were used as control.

In vitro transepithelial permeability study The Rhodamine-B conjugated glycol chitosan, GT-50 or GT-100 were used to study the transepithelial permeability of the NPs. Caco-2 cells cultured in MEM media supplemented with 15% FBS solutions were seeded over the polycarbonate transwell cell culture inserts (3.0 µm pore size). The cells were incubated in 37oC incubator supplemented with 5% CO2 for seven days by replacing the cells with the fresh media every day in both apical and basolateral regions. After attaining the monolayers, the cells were pre-incubated with HBSS buffer supplemented with 0.05% BSA and 10 mM MES (pH 6.0) in the apical compartment and HBSS buffer supplemented with 0.05% BSA and 10 mM HEPES (pH 7.4) in the basolateral compartments of the transwell plate respectively. Rhodamine-B conjugated NPs (AR-Chitosan, AR-GT50 or ARGT100) were then added in the apical chambers of the transwell plate and the media from the basolateral compartments were collected at different time points (10 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr, 12 hr and 24 hr). The fluorescence intensities from the media collected in the basolateral compartments were used for comparing the trans-epithelial permeability of the NPs


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In vitro cytotoxicity assay MTT assay was used to quantify the toxicity of the NPs. Cells (HepG2, Caco-2 and CT 26 cell lines; 5 x 104 cells/well) were seeded in a 96 well plate and incubated at 37oC equipped with 5% CO2 for 24 hr. Later, the cells were treated with different concentrations NPs (Au concentration: 0.5, 1 and 5 nM) for 24 hr or 48 hr. Finally, the MTT assay was performed following the manufacturers protocol. The percentage of cell survival was quantified using the following equation: Cell viability (%) = (absorbance of sample cells/absorbance of control cells) x 100

In vitro western blot analysis HepG2 cells (4x105 cells/well) were seeded in a 6-well plate and incubated for 24 hr in 37oC incubator supplemented with 5% CO2. Later, the cells were replenished with fresh media and incubated with the samples (PBS, AKT siRNA, AR-Chitosan andAR-GT100) for 12 hr. After 12 hr, the cells were washed with PBS and lysed with RIPA buffer containing 1X protease inhibitor cocktail mix. After quantifying the total protein by BCA protein assay kit, the proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto membranes (Millipore, Bedford, MA, USA). After blocking the membranes with TBS buffer containing 5% non-fat dry milk, primary antibodies of AKT, Caspase9, PARP (Merck, USA), Bax and B-actin (Santa cruz biotechnology, USA) with 1:1000 dilutions in TBS buffer were added and incubated at 4℃ overnight. After washing the membranes with TBS buffer containing 0.05% Tween 20, anti-rabbit or anti-mouse-conjugated alkaline phosphatase secondary antibodies (1:5000 dilutions) were applied for 1 hr at room


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temperature. The developed protein bands were visualized using the Chemidoc image analyzer (LAS-Amersham 600UV, GE, USA).

In vitro apoptosis assay In vitro apoptosis assay were measured using Muse® AnnexinV Dead Cell Kit (Millipore Corporation, USA). HepG2 cells (4x104 cells/well) were seeded in a 96 well plate and incubated for 24 hr. Later, the cells were treated with PBS, siRNA, AR-Chitosan and AR-GT100 nanocomplex for 12 hr. The cells were then washed twice with PBS, resuspended in 100 µL of trypsin-EDTA, and incubated with 100 µL of Muse® AnnexinV at room temperature for 20 min. The percentage of apoptosis induction was quantified using MUSE (Millipore Corporation, USA). The experiment was repeated three times.

In vivo oral absorption and biodistribution study Rhodamine B conjugated AR-Chitosan or AR-GT100 were used for the qualitative analysis of the carrier distribution in vivo. Seven week old female Balb/c mice (average body weight of 2025 g) were purchased from Orient Bio INC. (Seoul, Korea), housed in a metal cage and were gained access to food and water. The mice were randomly divided into 2 groups: AR-Chitosan and AR-GT100. At predetermined time points (0.5 hr, 12 hr and 24 hr), the animals were sacrificed and the organs (stomach, duodenum, jejunum, ileum, liver, spleen, heart, kidneys and lungs) were harvested. The organs were washed three times in PBS and later fixed using 4% paraformaldehyde. Finally, the fluorescence from the harvested organs were analyzed using Kodak in vivo multimodal imaging system (4000MN PRO, Kodak, USA). For quantitative analysis, the harvested organs were homogenized using the lysis buffer and rhodamine B


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fluorescence from each organ were used to determine the accumulation of the NPs in different organs. Inductively coupled plasma mass spectrometry (ICP-MS) was also used to quantify the biodistribution of the NPs (AR-Chitosan and AR-GT100). Seven week old female Balb/c mice (average body weight of 20-25 g) were purchased from Orient Bio INC. (Seoul, Korea). The mice were fasted for 5-6 hr before orally administering different NPs. Each orally administered NP formulation contained Au concentrations of 20 mg/kg. Pristine AuNP were used as control. The animals were sacrificed at different time points (0.5, 2, 4, 6, 12, 24 and 48 hr) and their organs were collected and stored at -20oC. The tissues were later defrosted and PBS was added to homogenize each tissue. Later, 5-fold excess aquaregia was added to each homogenized samples and sonicated for 2 hr. The sonicated samples were diluted 100 fold with double distilled water and were further used for ICP-MS analysis. Results were expressed as the amount of gold accumulated in each tissue. For analyzing the oral absorption mechanism, the female Balb/c mice, N=3 (seven weeks old, Orient Bio INC, Seoul, South Korea) was orally administered with AR-GT100 NPs. At predetermined time points (30 min post injection for ileum and liver; 24 hr post injection for kidneys), the animals were sacrificed and the organs were collected. The harvested tissue (less than 1mm block) was fixed in 4% paraformaldehyde solutions for 24 hr. The tissues were washed with saline and then dehydrated. The tissues were then embedded in paraffin and semithin sections from the paraffin blocks were then used to examine the tissues under TEM.

In vivo suppression of tumor growth and hepatic metastases cancer therapy study


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All animal experiments were performed under the guidelines of the committee-Chonnam National University Medical School Research Institutional Animal Care and all the experiment protocols were approved by the committee. Five-Six-week-old Balb/c mice were obtained from Jungang Lab Animal, Inc. (Seoul, Korea), housed in a metal cages with free access to water and food. Orthotopic CLM animal model was established following the protocol described earlier.39 In brief, firefly luciferase-expressing CT26 cells (1 x 105 cells/mouse) were injected into the spleen of the mice to induce CLM. Splenectomy was perform after 10 min post-injection of CT26 cells. The mice skin as well as the peritoneum were sutured and were used for studying the tumor suppression growth. Mice were randomly assigned to four groups (N=4): saline (control), AR-Chtiosan (siRNA concentration: 25 µg/kg), AR-GT100 (siRNA concentration: 25 µg/kg) and AR-GT100 (siRNA concentration: 100 µg/kg). Each group was administered with NP via oral gavage every consecutive day for 16 days. Progression of the tumor growth was monitored by IVIS200 Imaging system (Xenogen Alameda, CA, USA), which included an optical CCD camera mounted on a light-tight specimen chamber. On the last day, the animals were sacrificed, the liver images and nodular number were collected. The metastasis score in the mice treated with PBS or AR-GT100 was assigned following the protocol described earlier.39 Animals were given score 0 (no gross metastasis), 1 (2 cm2 area of tumor), or 4 (complete infiltration) by analyzing the nodules and nodular regions in the harvested liver. Orthotopic CLM mice were used to study the survival analysis. The mice were randomly assigned to four groups (N=6, same group with in vivo therapy study). Each group was administered with NP via oral gavage every consecutive day for 16 days. Mice were monitored every week for survival analysis.


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For In vivo western blot analysis, the harvested liver tissue was homogenized using RIPA buffer containing 1X protease inhibitor cocktail mix. The cell lysate was centrifuged and the supernatant proteins were separated with 10 or 12% SDS-PAGE. The protocol for western blot analysis was followed similar as described earlier in the In vitro western blot analysis. The primary antibodies for Akt, Phospho-Akt (p-Akt), PARP, Caspase9, Bcl-2 and Bax (Cell Signaling Technology®, Massachusetts, USA) were used for in vivo western blot analysis. For Immunohistochemical analysis, the harvested liver tissue was fixed in 10% paraformaldehyde solutions for 24 hr. The tissues were washed with saline, dehydrated and then embedded in paraffin. Semi-thin sections from the paraffin blocks were obtained and stained with H&E or Phospho-Akt antibody (Ser473)(D9E) XP® Rabbit mAb for Immunohistochemical analysis. The stained tissues were visualized by light microscopy through a 20 x objective lens.

Statistical Analysis Data are presented as mean ± standard deviation of results obtained from three independent trials unless otherwise indicated. Analysis of variance (ANOVA) (OriginPro8) was utilized to determine statistical significance between three or more groups, respectively. P-values < 0.05 considered statistically significant.

FIGURES


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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


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absorption spectra indicate the increase in the particle size of the NPs (E) Effect of GSH concentration on the release of siRNA from the 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.

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 ARChitosan 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 < 0.0001)


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Figure 3. ASBT receptor mediated cellular uptake of AR-GT100. (A-B) Comparison of the uptake of Rhodamine B-conjugated AR-Chitosan and AR-GT100 NPs between MDCK and MDCK-ASBT cells. (C-D) Quantitative analysis of cellular uptake profiles in MDCK and MDCK-ASBT cell lines treated with AR-Chitosan and AR-GT100. The cells were pretreated with TCA(20μL, 1mg/mL) to study the uptake of AR-GT100 + TCA. (mean ± SD; N=5)


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Figure 4. Transepithelial permeability and toxicity of AR-GT nanoparticles. (A) Growth of Caco-2 monolayer on apical compartment of transwell culture plate. (B) Cumulative transepithelial permeability from apical to basolateral compartment in the intestinal Caco-2 cell cultured transwell plates treated with AR-Chitosan, AR-GT50 and AR-GT100 NPs (mean ± SD; N=5). (C-D) Cell viability assay to measure carrier-based toxicity of NPs incubated for 48hr in both Caco-2 and HepG2 cell lines.


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Figure 5. In vitro therapeutic mechanism of AR-GT100. (A) Mechanism of Apoptosis induction in the cancer cells by targeting Akt siRNA. (B) Western blot of different proteins expressed after treating the cells with Akt-siRNA loaded NPs for 12 hr. BAX, CAspase-9 and PARP expression indicates the progression of cancer apoptosis in the cells after treating with AR-GT100 NPs.


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Figure 6. In vivo biodistribution of AR-GT nanoparticles. (A) Representative ex vivo fluorescence images of major organs (stomach, duodenum, jejunum, ileum, liver, heart, lung, kidney and spleen) of the animals treated AR-Chitosan and AR-GT100 for 0.5 hr. (B) Measurement of fluorescence intensities from the tissues (stomach, duodenum, jejunum, ileum, liver, heart, lung, kidney and spleen) at 6 and 12 hr after oral administration of AR-Chitosan and AR-GT100 (P-value < 0.0001). (C-I) ICP-MS analysis to measure the accumulation of Au ions in different organs (stomach, duodenum, jejunum, ileum, liver, kidney and spleen) after oral delivery of AuNP, AR-Chitosan and AR-GT100 at 0.5, 2, 4, 6, 12, 24 and 48 hr.


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Figure 7. Intestinal uptake, liver targeting and elimination of AR, AR-Chitosan and ARGT100. In vivo mechanism of AR-GT100 transport. Tissue TEM images obtained from organs ileum, liver and kidneys after the oral delivery of A) Saline, B) AR, C) AR-Chitosan and D) AR-


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GT100 nanoparticles. Non-specific and suboptimal distribution of AR was observed in ileum, liver and kidneys Vesicular structures were observed in the ileum of AR-GT100 treated mice (Yellow arrows) while transport of NPs through the tight junctions were observed in the animals treated with AR-Chitosan (Red arrows). Tissue TEM images from liver indicates the entry of nanoparticles in the liver cells but not in the immune active Kupffer cells in AR-GT100 treated mice (Yellow arrows) while, no uptake into any specified cells was observed in AR-Chitosan treated animals (Red arrows). Moreover, enhanced number of aggregations were observed. Tissue TEM images from kidney determines the presence of NPs (AR-Chitosan and AR-GT100) in kidneys depicting their elimination from the body.


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Figure 8. Therapeutic efficacy of AR-GT100 in a CLM cancer mouse model. (A) The progression of CLM after oral delivery of AR-Chitosan and AR-GT100 (25 µg/kg or 100 µg/kg). (B) The number of cancer nodules present in the liver after oral administration of AR-Chitosan and AR-GT100. (N=4) (C) Kaplan-Meier survival analysis of the mice after oral administration of AR-Chitosan and AR-GT100 (25 µg/kg and 100 µg/kg). (N=6) (D) Immunoblot analysis of different proteins involved in PI3K/AKT signaling and the cancer apoptotic pathway. (E)


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Immunohistochemistry of p-AKT (top) and H&E (bottom) staining in the liver tissue treated with saline (control) and AR-GT100 (100 µg/kg). (Images were taken at 20x magnification) ASSOCIATED CONTENT Supporting Information. Table S1. Conjugation efficacy of siRNA to AuNP (AR). Table S2. Characterization of TCA-modified glycol chitosan (GT). Table S3. Particles size analysis of nanoparticles. Table S4. In vivo metastasis score of the mice treated with AR-GT100 NPs. Figure S1. Synthesis and characterization of gold nanoparticles (AuNP) Figure S2. 1H-NMR spectra of TCA-conjugated glycol chitosan (GT) Figure S3. Stability of siRNA in the presence of biological enzymes. Figure S4. Time-dependent Caco-2 cell transwell permeability study Figure S5. In vitro cellular toxicity Figure S6. In vitro Apoptosis analysis Figure S7. Measurement of dose-dependent Au accumulation in the liver Figure S8. Accumulation of AuNP, AR-Chitosan and AR-GT100 accumulation in the liver Figure S9. In vivo mechanism of AR-GT100 transport. AUTHOR INFORMATION


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Corresponding Author Kwang Jae Cho: [email protected] Woo Kyun Bae: [email protected] Yong-kyu Lee: [email protected] ACKNOWLEDGMENT This research was supported by The Leading Human Resource Training Program of Regional Neo industry through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF-2016H1D5A1910188, NRF2014R1A2A2A03004802 and NRF-2015R1D1A1A09060567). REFERENCES (1)

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Oral delivery of AKT-2 siRNA loaded AR-GT100 NPs can treat colorectal liver metastases cancer. 26x14mm (300 x 300 DPI)


Oral siRNA Delivery to Treat Colorectal Liver Metastases - ACS Nano

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