Targeted Interleukin-22 Gene Delivery in the Liver by Polymetformin

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Biological and Medical Applications of Materials and Interfaces

Targeted Interleukin-22 Gene Delivery in the Liver by Poly-Metformin and Penetratin-Based Hybrid Nanoparticles to Treat Non-Alcoholic Fatty Liver Disease Wenjing Zai, Wei Chen, Zimei Wu, Xin Jin, Jiajun Fan, Xuyao Zhang, Jingyun Luan, Shijie Tang, Xiaobin Mei, Qiang Hao, Hongrui Liu, and Dianwen Ju ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19717 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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ACS Applied Materials & Interfaces

Targeted Interleukin-22 Gene Delivery in the Liver by Poly-Metformin and Penetratin-Based Hybrid Nanoparticles to Treat Non-Alcoholic Fatty Liver Disease Wenjing Zai§,†, Wei Chen§,‡, Zimei Wu†, Xin Jin†, Jiajun Fan‡, Xuyao Zhang‡, Jingyun Luan‡, Shijie Tang‖, Xiaobin Mei‖, Qiang Hao‖, Hongrui Liu*,†, Dianwen Ju*,‡ †Department

of Pharmacology, School of Pharmacy, Fudan University, Shanghai,

201203, P. R. China. ‡Department

of Microbiological and Biochemical Pharmacy, School of Pharmacy,

Fudan University, Shanghai, 201203, P. R. China. ‖Changhai

§These

Hospital, Naval Military Medical University, Shanghai, 200433, China.

authors contributed equally to this work.

*Corresponding

author:

Hongrui Liu, Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, 201203, P. R. China; Tel: +86 21 51980043; Fax: +86 21 51980015; E-mail: [email protected]. Dianwen Ju, Department of Microbiological and Biochemical Pharmacy, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, China. Tel: +86 21 51980037; Fax: +86 21 5198 0036; E-mail: [email protected]

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ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is now a leading cause of chronic liver disease, while there is currently no available treatment strategy. Interleukin-22 (IL-22) has been recognized as a promising agent for alleviating NAFLD, but the efficacy of IL-22 is far from satisfactory because safe dose of IL-22 elicited limited improvement while higher concentration might induce serious side effects and off-target toxicities. Thus, targeted and sustained expression of IL-22 in the liver is necessary. To meet the challenge, we elaborately developed a novel poly-metformin carrier by conjugating biguanide to chitosan, termed chitosan-metformin (CM), which could exert advanced gene delivery efficiency and possess intrinsic therapeutic efficacy from metformin for NAFLD. CM accompanied with penetratin and DSPE-PEG2000 could self-assemble to form stable nanocomplexes with IL-22 gene via electrostatic interaction. This nanoparticle (CDPIA) exerted desirable particle size at ~100 nm, fine morphology and efficient cellular internalization. Furthermore, CDPIA also demonstrated a unique superiority in endosomal escape capacity and satisfactory biocompatibility as well as predominant liver accumulation. Most importantly, CDPIA distinctly alleviated hepatic steatosis, restored insulin sensitivity and improved metabolic syndrome in high fat diet (HFD)-fed mice model. This liver targeted delivery of IL-22 activated STAT3/Erk1/2 and Nrf2/SOD1 signaling transductions as well as modulated lipid metabolic-related gene expression. These findings altogether demonstrated that the poly-metformin and penetratin-based hybrid nanoparticles could be exploited as a novel safe and efficient strategy for the improvement of NAFLD. 2


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KEYWORDS: chitosan, metformin, penetratin, interleukin-22, non-alcoholic fatty liver disease.

3



INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) is now afflicting approximately onethird of the population worldwide, especially in western countries.1 This growing disease prevalence is commonly accompanied with wide array of metabolic syndromes, encompassing obesity, type 2 diabetes mellitus and increasing risk of cardiovascular diseases.2 The pathologies of NAFLD range from simple steatosis to non-alcoholic steatohepatitis (NASH), with the degree of which always extends to fibrosis, cirrhosis and even progresses to hepatocellular carcinoma.3 Although there have been certain progresses in clarifying the underlying mechanisms of NAFLD, identifying novel therapeutic targets and developing corresponding therapeutics, there are numerous unmet problems and no approved agents yet for this disease.4 Thus, exploring novel and more effective therapeutic antidotes for NAFLD is urgently needed. Interleukin-22 (IL-22), a novel cytokine that belongs to IL-10 family, possesses multiple biological functions such as promoting tissue repair as well as wound healing, activating innate host defense and maintaining homeostasis of commensal bacteria and so on.5, 6 Recent studies have also discovered a special action of IL-22 in relieving the progression of NAFLD. Administration of IL-22 relieved metabolic syndromes in obese mice, leading to reduction of body weight gain and epididymal fat-pad mass, improvement of glucose and insulin tolerance, as well as modulating lipogenesisrelated gene expression, respectively.7,

8

Furthermore, activation of JAK2/STAT3

signaling pathways might account for the protective action of IL-22 against fatty liver.9 However, the efficacy of IL-22 for NAFLD is still far from satisfactory because safe 4


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dose of IL-22 elicited limited improvement for the symptom of NAFLD while higher concentration of IL-22 might induce cachexia such as thymic atrophy and changes of renal proximal tubules thus reducing body weight.10,

11

Moreover, apart from its

unsatisfactory efficacy at safe dose as a metabolic modulating cytokine, there are still some other drawbacks for further development of IL-22 as a NAFLD-treatment antidote. The most important obstacle is the pleiotropic function of IL-22, which limits its therapeutic application via arising of off-target toxicities, especially autoimmune diseases like rheumatoid arthritis (RA), multiple sclerosis (MS) and psoriasis.12 The extensive tissue expression of its receptor (IL-22R), which counteracts its functional binding and the relative short half-life time of this cytokine further add to handicaps for its clinical utilities.13 It’s therefore important to optimize the distribution and metabolism of this molecular via structural or formulation modification, which could help reduce its side effects and improve its efficacy for NAFLD. Our group have previously reported a gene-based therapy by tethering IL-22 to apolipoprotein-I (ApoAI), which exhibited liver targeting property through binging with scavenger receptor class B type 1 (SR-B1) in the liver. Specially, this therapy gene significantly prolonged the circulation time of IL-22 and relieved its systemic toxicity when expressed in vivo.14 Evidences also suggested that ApoA-I could relieve the pathological process of NAFLD via reducing fatty acid and triglyceride deposition in liver.15-18 These promising properties promoted us to develop a high efficient carrier to deliver this IL-22-based gene in the liver for the treatment of NAFLD. The major hindrance of gene therapy for NAFLD is the safety and transfection 5



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efficiency of gene delivery carriers associated with the chronic nature and the longlasting treatment time stage of this disease. Therefore, designing biocompatible nonviral carrier materials with intrinsic biological activity is of great superiority. Chitosan, a natural-derived cationic copolymer, has been widely applied as a promising non-viral vector for its favorable properties such as biocompatibility, biodegradability and low immunogenicity.19 However, the significant limitation for chitosan is its poor transfection efficiency.20-22 Previous studies incorporated biguanide to carrier materials so that the biguanide groups could simultaneously act as the cationic blocks to improve gene transfection efficiency and exert similar biological activity as metformin.23,

24

Synthesizing with amino and hydroxyl groups could introduce the biguanide groups to chitosan easily, which acted as a prominent nano-absorbent.25 However, whether this modification could improve the gene delivery capacity waits for further exploration. Furthermore, metformin is the first-line treatment drug for type 2 diabetes mellitus, which has also been demonstrated to possess benefits on NAFLD both in experimental studies and clinical trials.26, 27 Thus, we hypothesized that incorporating biguanide to chitosan (CM) might act simultaneously as the cationic head group and the therapeutic adjunct for NAFLD. As for liver-targeted gene therapy, the distribution of gene vectors depends on the suitable delivery system and the mode of administration. Typically, intravenous injection of nanoparticles with a diameter of 100~300 nm could predominantly accumulate in liver via passive targeting effects.28 In addition, optimized formulation with suitable size, morphology, prolonged circulation time and enhanced cellular 6


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internalization could further promote efficient uptake by hepatocyte.29 Cell-penetrating peptides (CPPs), which have been widely utilized to transport different kinds of cargos, could improve cellular internalization and promote endosomal escape of nanocomplexes as well as optimizing transfection efficiency of relative gene carriers.30 Previous study reported that penetratin, a 16-redidue peptide derived from antennapedia homeoprotein, showed distinct permeability and delivery capacity over other peptides thus had been applied as a cellular internalization helper widely.31, 32 Our group also reported that penetratin accompanied with PAMAM 3.0 dendrimers could generate liver-specific delivery and expression of IL-22 via improving cellular internalization and diminishing Kupffer cells uptake.33 Therefore, penetratin was chosen to construct hybrid nanoparticles with CM to overcome the shortcomings of CM, enhance transfection efficiency and improve liver-specific accumulation of nanoparticles. In addition, DSPE-PEG2000 was also utilized as a helper lipid in the formulation of nanoparticles to reduce non-specific interactions and help plasmid to escape from endosomes via destabilizing lysosomal membranes.33, 34 In the present study, we first constructed a novel poly-metformin based gene delivery carrier by conjugating biguanide groups to chitosan to improve gene transfection efficiency of chitosan and apply therapeutic activity of metformin. Penetratin was utilized to enhance cellular internalization and endosomal escape capacity of CM, while DSPE-PEG2000 functioned as a help lipid. The CM/penetratin/DSPE-PEG2000 mixture self-assembled with IL-22-based therapy gene to form nanocomplexes (CDPIA) easily by electrostatic interaction. This hybrid 7



nanoparticle exerted liver-targeted IL-22 gene delivery ability and satisfactory biocompatibility when injected intravenously. The following study demonstrated that this nanoparticle possessed potent therapeutic activity to ameliorate pathological damages as well as relieve metabolic syndromes of NAFLD, thus representing a promising strategy for the treatment of NAFLD.

MATERIALS AND METHODS Materials. The IL-22/ApoA-I harboring plasmid DNA (pVAX1-IL-22/ApoA-1, pIA) was constructed by our group 14. The Endo-Free Plasmid Maxi Kit was purchased from Omega (Norcross, GA). Chitosan (degree of deacetylation ≥ 95%, viscosity 100~200 mpa.s) was purchased from Aladdin (Shanghai, China). Dicyandiamide was obtained from China National Medicines Corporation (Shanghai, China). Penetratin and 5carboxyfluorescein (FAM)-labeled penetratin were both synthesized by GL Biochem (Shanghai, China). DSPE-PEG2000 were obtained from A.V.T. Pharmaceutical (Shanghai, China). Synthesis of biguanide modified chitosan (CM). The reaction was performed as previously described

25.

The resulting product was dialyzed using a dialysis tubing

(MWCO: 3.5 kDa) against 5 mM HCl for 12 h a time, then 5 mM HCl containing 1% NaCl for two times and then 1 mM HCl twice at room temperature in the dark to remove byproducts. The obtained solution was frozen at -80 ℃ and then lyophilized until a stable weight of product was obtained. Formulation and characterization of nanoparticles. CM, penetratin, DSPE8


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PEG2000 and plasmid were separately dissolved in ddH2O. CM containing DSPEPEG2000 (weight ratio 10:1) were first mixed with penetratin and then mixed with plasmid at different weight radio. The mixtures were then vortexed for 60 s and stood at room temperature for 30 min to allow the formation of the complexes. The complexes were then applied to gel retardation assay. The particle size, polydisperse index (PDI) and zeta potential were determined via dynamic light scattering (DLS) (Malvern, Westborough, UK). The optimal construction of the complexes was selected as CM/DSPE-PEG2000/penetratin/pIA at the weight ratio of 8:0.8:14:1. CDPIA was used as abbreviation for this nanoparticle, and CM/DSPE-PEG2000/pIA, abbreviated as CMIA, was used as a control. Cell culture. HepG2 and Huh7 cells were purchased from Shanghai Cell Bank, Chinese Academy of Science (Shanghai, China). Both cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and maintained at 37℃ in a humidified atmosphere with 5% CO2. Cellular uptake and endosomal escape. For cellular uptake assay, the plasmid was pre-stained with TOTO-3 dye (Life Technologies, Karlsruhe, Germany), penetratin was conjugated with FAM at the C-terminal. Cells were incubated with naked plasmid, CMIA or CDPIA containing 5 μg of pIA in 1 mL DMEM with 10% FBS at 37℃ for 2 h. Subsequently, cells were rinsed with cold PBS for three times and nucleus were stained by Hoechst 33342, then imaged by confocal microscopy (Zeiss, Oberkochen, Germany). For endosomal escape analysis, cells were exposed to equivalent amount of CDPIA 9



nanoparticles for 0.5 h, 1 h, 4 h or 8 h. After incubation, cells were washed with cold PBS for three times and stained with LysoTracker Green dye for lysosome detection, nucleus were stained with Hoechst 33342. Then cells were visualized by confocal microscopy. In-vitro cytotoxicity assay. The cytotoxicity of CM was evaluated via 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Cells were seeded in 96-well plates then treated with different concentrations of CM for 24 h. After treatment, 0.5 mg/mL of MTT was added and incubated with cells for 4 h at 37℃. The formed formazan was then dissolved by dimethyl sulfoxide (DMSO) and determined by microplate reader at 570 nm. Biodistribution. C57BL/6 mice were administrated with TOTO-3 labeled CMIA or CDPIA complexes via tail vein. Mice were euthanized at indicated time points and major organs of the mice (heart, liver, spleen, lung, kidney and brain) were harvested. Fluorescence of each mice were visualized by IVIS Spectrum imaging system (Caliper life science, Inc., USA). The distribution of the nanocomplexes in liver and other organs were determined by confocal microscopy. Animal study. Eight- to ten-week-old C57BL/6 male mice (22-24 g) were obtained from Shanghai Slaccas Experimental Animal Co., Ltd. (Shanghai, China) and maintained in a standard environment with 12 h of dark/light cycle. After acclimated to laboratory condition for 1 week, mice were then fed with normal chow diet (TP23302, Trophic, Nantong, China) or high-fat diet (HFD) containing 60% calories fat (TP23300, Trophic, Nantong, China) for 17 weeks. Mice were first fed with chow diet or HFD for 10


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4 weeks, then intravenously (i.v.) treated with PBS, CM, CMIA or CDPIA containing 50 μg of pIA once a week for 12 weeks. All animal studies were performed according to protocols approved by the Ethics Committee of School of Pharmacy, Fudan University. Metabolic analysis. For insulin tolerance test (ITT), mice were fasted for 4 h, then intraperitoneally (i.p.) treated with 1 IU/kg of body weight of insulin (Beyotime, Nantong, China). For glucose tolerance test (GTT), mice were fasted for 6 h and then intraperitoneally injected with 1 g/kg of body weight glucose. Serum samples were taken at indicated time points and serum glucose was examined, respectively. Biochemistry analysis. For liver function analysis, the serum of mice was applied for alanine aminotransferase (ALT) and aspartate transaminase (AST) detection by commercial kits (Jiancheng Bioengineering, Nanjing, Shanghai). Liver lipid contents and serum levels of glucose and lipid were determined by analytical kit from Jiancheng Bioengineering (Nanjing, Shanghai). Histological analysis. For histological study, liver samples, adipose tissues and other major tissue organs were fixed with 4% paraformaldehyde, embedded with paraffin and then applied for hematoxylin and eosin (H&E) staining. For Oil Red O analysis, fixed liver samples were embedded in OTC, frozen in dry ice and then were subjected to Oil Red O staining. Quantitative real-time PCR (qPCR) examination. Total liver RNA was extracted by TRNzol reagent (Tiangen Biotech, Beijing, China) and cDNA was obtained by cDNA reverse transcription kit (Takara Biotechnology, Otsu, Japan), then the mRNA levels 11



were detected using SYBR green qPCR mix kit (Beyotime, Nantong, China). GAPDH were used as an internal control. Western blot analysis. The liver samples were homogenized and total protein extracted from liver were applied for BCA protein analysis (Novoprotein Scientific Inc., Shanghai, China). Equal amount of proteins was subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The aiming proteins were detected by special primary antibodies and then subjected to HRP-conjugated secondary antibodies. Then the blots were visualized by enhanced chemiluminescence system (Pierce, Rockford, IL., USA) and calculated via ChemiDoc software (Bio-Rad, USA). Statistical analysis. Data were presented as means ± standard deviations (S.D.) and difference were operated by Student’s t-test or one way of analysis (ANOVA). Value of P ≤ 0.05 were considered of significant difference.

RESULTS Preparation and characterization of CDPIA nanocomplexes. The designed structure of the hybrid nanoparticle was presented in Figure 1A. Chitosan-metformin (CM) was synthesized by introducing biguanide group to chitosan via reacting with active amino groups (Supporting Information, Figure S1). The sequence and preparation method of IL-22/ApoA-I (pIA) plasmid were consistent with previous report.14 The modified chitosan showed desirable condensation ability and retarded the migration of DNA (pIA) completely at the weight ratio of 1, as evidenced by agarose 12


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gel electrophoresis analysis (Figure 1B-C). To optimize the formulation of our gene delivery system, penetratin and DSPE-PEG2000 were incorporated with CM, and equal volume of CM/penetratin/DSPE-PEG2000 solution and plasmid were blended to allow self-assemble of CM/penetratin/DSPE-PEG2000/pIA (CDPIA) complexes. Suitable nanoparticle size and positive surface charge are both important factors to ensure efficient assess of targeting site and cellular uptake of gene carriers.35 Therefore, different weight ratio of CM and pIA was monitored to evaluate the formation of CDPIA complexes via detecting particle size, dispersed size distribution (PDI) and zeta potential values. The proportions of penetratin and DSPE-PEG2000 were determined according to the recommended concentrations in previous reports. An increased weight ratio of CM and pIA resulted in a significant decrease of particle size and PDI while led to a distinct increase of zeta potential (Figure 1D-F). The optimal formulation of CDPIA was chosen at the weight ratio of 8, where the particle size was approximately 100 nm and the zeta potential value was ~30 mV, accompanied with a relative homogenous size distribution and unimodal (Figure 1G). Transmission electronic microscopy (TEM) was further applied to observe the morphology of the nanocomplex, and the result revealed that this nanoparticle possessed a homogenous and spherical shape (Figure 1G). Moreover, the particle size and PDI remained stable when stored at room temperature for 1 d, 2 d, 7 d, 15 d, which confirmed its optimal construction (Figure 1H). The size of CDPIA was smaller than CMIA, indicating that penetratin helped to compact the structure of the nanoparticle. Taken together, these results presented that CDPIA possessed desirable particle 13



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size, homogenous shape and storage stability, which laid the foundation for its further research as a therapeutic nanoparticle for gene delivery. Cellular uptake and intracellular localization of CDPIA. Successful gene transfection relies on efficient cellular internalization of nanoparticles.28 To monitor cellular uptake of CDPIA, we employed TOTO-3 red fluorescence dyed plasmid and FAM-labeled penetratin for visualization of intracellular distribution and localization of plasmid and gene carriers. HepG2 and Huh7 cells were incubated with naked pIA, CMIA and CDPIA for 2 h, then intracellular fluorescence was detected via confocal microscopy. The results showed that, in comparation with untreated control groups, cells incubated with naked pIA and CMIA only displayed minimal TOTO-3 fluorescence signal. Whereas cells that subjected to CDPIA exhibited much more red fluorescence internalization, indicating efficient cellular uptake of nanoparticles (Figure 2A, C). In addition, CDPIA-treated cells presented both accumulation of TOTO-3 labeled plasmid and FAM-labeled penetratin and a desirable co-localization of TOTO-3 and FAM were observed (Figure 2B, D). These results confirmed that the complexes were intact at this time point and penetratin could help benefit further intracellular trafficking of the plasmid.31 Besides, the co-localization of plasmid and carrier

vectors

also

suggested

that

the

nanoparticles

might

prevent

endosomal/lysosomal entrapment and nucleolytic degradation during cellular internalization. Altogether, our data indicated that CDPIA could be internalized by hepatocytes successfully with the help of penetratin, which formed the basic rationale for utilizing 14


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this nanoparticle for NAFLD gene therapy. Endosomal escape ability and cytotoxicity of CDPIA. Previous studies have reported that efficient endosomal/lysosomal escape is a key factor that dominates subsequent gene expression in addition to cellular uptake.36 After the internalization of CDPIA was confirmed, we further evaluated the subcellular localization of the gene cargos. Cells were first incubated with TOTO-3 labeled CDPIA complexes for various time periods, then stained with lysosomal specific LysoTracker Green dye and Hoechst 33342 to visualize the transportation of CDPIA in subcellular organelles and co-localization of CDPIA cargos with endosomes/lysosomes and nucleus. The red fluorescence of CDPIA was clearly observed in the cytoplasm of cells at 0.5 h and accumulated in bulk till 4 h, then the signals weakened at 8 h. Comparatively, some red nanoparticles colocalized with green endosomes/lysosome at the beginning, while the unappreciable colocalization gradually diminished over time. Some nanoparticles even translocated to the nucleus successfully at 8 h (Figure 3A-B). These results substantiated the effective endosomal escape capacity of CDPIA, which represented the basis of efficient gene transfection. We further evaluated the cytotoxicity of CM in HepG2 and Huh7 cells. No significant cytotoxicity was observed after 24 h of incubation even at the maximal concentration of 1000 μg/mL (Figure 3C-D). Altogether,

these

results

indicated

that

CDPIA

could

escape

from

endosomes/lysosomes successfully and elicited no toxicity in vitro. Liver-targeted biodistribution of CDPIA. To validate the liver-targeting capacity of CDPIA, we investigated the relative biodistribution of TOTO-3 labeled CDPIA in 15



comparation with CMIA. Both nanoparticles were intravenously injected in C57BL/6 mice via tail vein, major organs of the mice were collected at 6 h, 12 h and 24 h postinjection, and fluorescence signals were visualized via IVIS Spectrum imaging system. Both nanoparticles accumulated majorly in the liver and the fluorescence intensities increased over time while diminished at 24 h. The liver-specific accumulation of CDPIA was much higher than that of CMIA, which was consistent with in vitro cellular uptake results (Figure 4A-B). The organ samples at 12 h of injection were then frozen and liver-specific gene delivery was further confirmed via confocal microscopy. As expected, relative red fluorescence signal of CDPIA in the liver was 3-time higher than CMIA (Figure 4C-D). The improved CDPIA internalization probably arose from more desirable nanoparticle size, morphology and penetratin-mediated membrane translocation. In addition, the TOTO-3 fluorescence signal was preferentially observed in liver compared with other organs in both CMIA- and CDPIA- treated mice, which was consistent with the results observed by imaging system (Figure 4E). These results validated that CDPIA possessed an appreciable liver targeting capacity and enhanced internalization, which formed the premise for efficient liverspecific protein expression. CDPIA administration inhibited HFD-induced hepatic steatosis in mice. To investigate the therapeutic effects of CDPIA on high fat diet (HFD)-induced hepatic steatosis, we fed C57BL/6 mice with HFD for 4 weeks and then treated them with PBS, CM, CMIA or CDPIA during HFD feeding for 12 weeks. Mice fed with chow diet were employed as normal control (Figure 5A). The body weight of HFD mice was more than 16


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20% higher than that of chow diet-fed mice, indicating the successful establishment of NAFLD mouse model.37 The applied dosage of pIA (50 μg plasmid each mice) was chosen according to previous published studies.14, 24, 33 CDPIA treatment significantly improved liver coloration and morphology and reversed liver overweight, in comparation with PBS, CM and CMIA treatment (Figure 5B-C). Histological analysis demonstrated that HFD feeding led to an excessive accumulation of lipid drops in the liver, which was completely rescued by weekly CDPIA injection and partly relieved by CMIA treatment (Figure 5D). In consistent with histological observations, Oil Red O staining assay also demonstrated that CDPIA administration distinctly alleviated dietinduced hepatic steatosis, while CMIA only exhibited moderate alleviation (Figure 5EF). Specifically, CM treatment also elicited minimal improvement, indicating the intrinsic biological activity of poly-metformin. Similarly, triglycerides (TG) and nonesterified fatty acids (NEFA) contents in the liver, the indication of hepatic lipid accumulation, were markedly alleviated by CDPIA injection (Figure 5G-H). These results altogether suggested that CDPIA could efficiently protect mice from diet-induced hepatic steatosis, the effects of which were better than CM and CMIA, demonstrating the combinational protective role of CM, IL-22 and penetratin. CDPIA restored insulin sensitivity and reduced lipid accumulation. We further examined the effects of CDPIA in the modulation of metabolic syndrome-related to NAFLD in HFD-fed obese mice. The body weight determination revealed that CM slightly reduced body weight gain of HFD-fed mice, which might provide combinational therapeutic efficacy with IL-22-based gene therapy. Furthermore, 17



CDPIA administration profoundly reduced diet-induced overweight as well as lowered fasting blood glucose, in comparation with CMIA and CM-treated groups (Figure 6AC). Considering that insulin tolerance and glucose intolerance were major metabolic complications in NAFLD, we then performed insulin tolerance test (ITT) and glucose tolerance test (GTT). The ITT and GTT results demonstrated that CDPIA injection significantly improved insulin sensitivity and glucose tolerance in mice fed with HFD, estimating the area under the curve of ITT and GTT also presented an improvement in insulin action and glucose homeostasis (Figure 6D-E, G-H). Apart from metabolic syndromes, CDPIA also protected obesity-related liver injury, as reflected by serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) examination, respectively (Figure 6F, I). In addition, epididymal fat-pad mass was markedly attenuated and the size of adipocytes was obviously reduced by CDPIA treatment, while CM and CMIA only achieved marginally reduction (Figure 6J-L). Serum level of TG was also reduced by CDPIA administration, while no reduction was observed in serum levels of cholesterol (TC), high-density lipoprotein (HDL-c) and low-density lipoprotein (LDL-c) (Figure 6M, Supporting information, Figure S2). Collectively, these results demonstrated that CDPIA reversed metabolic abnormality and reduced whole body lipid accumulation in HFD-fed mice models. CDPIA activated STAT3/Erk1/2 and Nrf2/SOD1 signaling pathways and regulated hepatic lipid metabolism. To elucidate the underlying mechanisms of therapeutic efficacy of CDPIA, we first determined the biological activity of our nanoparticles by detecting the phosphorylation of STAT3 and its substrate proteins. 18


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Administration of CDPIA significantly stimulated STAT3 phosphorylation as well as Erk1/2 phosphorylation in the liver compared with PBS-, CM- and CMIA- treated mice. The expression of downstream signal transductions of STAT3 such as Bcl-2 and cyclin D1 were also upregulated in mice injected with CDPIA, which confirmed the activation of STAT3 signaling pathways (Figure 7A-B). These results indicated that IL-22 could be expressed successfully and could exert biological activities via stimulating target genes in vivo. Nrf2/SOD1 cascade is another related signaling pathway which performs as an antioxidant-response element (ARE) to counteract hepatic lipid peroxidationinduced oxidative stress.38,

39

As expected, our results demonstrated that expression

levels of Nrf2, and its downstream factor SOD1 were evidently upregulated by CDPIA administration, which indicated the participation of antioxidant modulation mechanisms in its protective action (Figure 7C-D). Previous studies reported that IL22 could exert transcriptional regulation on lipogenesis-related gene expression 9, 10. We further confirmed that CDPIA administration prominently promoted the expression of genes related to fatty-acid β-oxidation (Acox1 and Cpt1a) in comparation with PBS-, CM- and CMIA-treated groups, as well as induced expression of gene involved in lipid transportation (Acc1), whereas didn’t induce lipid de novo synthesis-related genes (CD36 and FAS) (Figure 7E). Taken together, these results indicated that CDPIA alleviated diet-induced fatty liver via activating STAT3/Erk1/2 and Nrf2/SOD signaling transductions, as well as modulating hepatic lipid metabolism-related genes including Acox1, Cpt1a and Acc1 expression. 19



Long-term CDPIA injection did not manifest systemic toxicity in mice. Considering relative long-term treatment period of NAFLD, the histopathology analysis of major organs of mice took place. After 12-week intravenous administration, neither CM-, CMIA- nor CDPIA-treated mice showed obvious histological abnormalities in major organs. (Figure 8A). Examination of serum blood urea nitrogen (BUN) and creatinine (CRE) for kidney toxicity didn’t show obvious toxic difference in hematological markers (Figure 8B-C). The relative normal level of serum lactate dehydrogenase (LDH) levels after long-term treatment further confirmed the low toxicity and appreciable biocompatibility of our nanoparticles (Figure 8D). Taken together, these results suggested that CDPIA showed appreciable safety and biocompatibility, thus could be utilized as a safe and efficient liver-targeting treatment strategy for alleviating diet-induced fatty liver.

DISCUSSION Previous studies have demonstrated that IL-22 possesses plenty benefits in alleviating obesity-related metabolic syndrome and hepatic steatosis thus exhibits as a promising therapeutic agent for NAFLD, which is a worldwide challenge with no pharmacological therapeutics clinically.1, 7 IL-22 elicits multiple effects in obese mice including reducing body weight gain, restoring glucose tolerance and insulin sensitivity as well as relieving hepatic lipid accumulation.8 Its protective actions are associated with suppression of oxidative and endoplasmic reticulum (ER) stress via activating JAK2/STAT3 signaling pathways, upregulating antioxidant-related proteins and 20


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modulating lipogenesis-related gene expression.9 However, further utility of IL-22 as a specific antidote for NAFLD treatment needs careful optimization, in consideration of its pleiotropic functions which often leads to unsatisfactory complications especially autoimmune diseases, as well as off-target toxicities like cachexia.10, 11 To overcome the drawbacks of IL-22 and improve its therapeutic efficacy for liver disease, we have previously designed a fusion gene by tethering IL-22 to ApoA-I (pIA), thus endowed hepatic tropism of IL-22 and prolonged its half-life time in vivo.14 Recent studies indicated that ApoA-I also possessed therapeutic benefits for NAFLD. ApoA-I could relieve hepatic steatosis effectively via modulating lipid transportation, alleviating inflammation and oxidative stress as well as diminishing ER stress in hepatocytes.16, 17 To achieve better efficiency in the treatment of NAFLD and to avoid systemic side effects of IL-22, we elaborately designed a novel gene delivery platform to deliver this IL-22/ApoA-I therapeutic gene for its specific expression in liver. The major obstacle of non-viral gene delivery vectors might be their unsatisfactory transfection efficiency. Moreover, considering the long-lasting therapy period for this chronic disease, the safety of gene carriers arises to be another significant concern. In these regards, chitosan, a nature-derived cationic copolymer of saccharide, was applied in the present study. Since it possessed multiple advantages over other synthesized polymers, including low toxicity, high biocompatibility and well biodegradability.38, 39 However, the relative insufficient transfection efficiency of chitosan, due to its inefficient cellular internalization and poor endosomal escape capacity, limited its further application.21, 22 Herein, we developed for the first time a poly-metformin by 21



conjugating biguanide to amino groups of chitosan, which functioned as a cationic head group to promote gene transfection. Besides, as indicated by previous studies, modification of metformin to polymers could retain its original biological activity.23, 24 As the first-line treatment drug for type 2 diabetes mellitus in clinic, metformin has displayed therapeutic benefits for NAFLD both in experimental and clinical studies.26, 27 Thus,

metformin-modified chitosan (CM) could also exert intrinsic pharmacological

effects for NAFLD. Consistent with these studies, our data demonstrated that systemic administration of CM in mice fed with HFD could slightly alleviate obesity-related fatty liver and metabolic disorders. The relative low efficacy of CM might account for the relatively low dose and administration frequency of metformin. In addition, high doses of CM treatment didn’t lead to cytotoxicity in HepG2 or Huh7 cells and long-term CM administration didn’t manifest systemic toxicity in vivo as well. These results indicated that the CM could function as a safe and versatile gene deliver material with improved transfection efficiency and intrinsic biological activities for NAFLD. To further ensure efficient cellular uptake and gene transfection efficiency of the therapeutic IL-22/ApoA-I gene, penetratin, a kind of 16-residue cell-penetrating peptide, was employed in this gene delivery system. This penetratin has been widely utilized to promote cellular internalization via membrane translocation without triggering phospholipid bilayer perturbation and has been proven with minimal toxicity.29,

32, 41

The subsequent endosomal escape and intracellular DNA release

capacities further guaranteed its sufficient gene transfection efficiency.20 Previous studies reported that noncovalently combination of penetratin with a cationic dendrimer 22


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could form condense nanoparticles with plasmids easily by vortex.31 Moreover, we have previously reported that a hybrid nanoparticle consisting of penetratin and PAMAM 3.0 could exert preferential liver accumulation and high transfection efficiency.33 In the present study, we combined CM with penetratin by physical mixing and the mixture self-assembled into nanocomplexes with DNA (pIA) via simple electrostatic interaction. In addition, DSPE-PEG2000, a cationic lipid, was also introduced into our formulation as a help lipid, which could further reduce non-specific interactions, promote endosomal release of plasmids and thus increase transfection efficiency.34 The optimal formulation of our gene delivery system (CDPIA) was determined by desirable particle size and homogenous size distribution. The achieved nanocomplex demonstrated significant improved cellular internalization and escaped from endosomes/lysosomes sufficiently when incubated with cells in vitro. Furthermore, CDPIA could preferentially and efficiently accumulate in the liver comparing with other organs and groups when injected intravenously in mice. The prominent liver-specific biodistribution of CDPIA might be attributed to its desirable particle size and welldesigned formulation, which promoted passive targeting effects in the liver. Besides, the enhanced cellular internalization, improved transfection efficiency and prolonged circulation time contributed to elevated liver accumulation of nanoparticles. In addition, the systemic localization of liver, the high endocytosis feature of hepatocytes and the appropriate mode of administration also promised the liver-specific distribution.29, 42 Encouraged by those results above, we further evaluated the protective effects of CDPIA for NAFLD in mice fed with HFD. Systemic administration of CDPIA 23



significantly alleviated HFD-induced hepatic steatosis as well as decreased liver triglyceride and NEFA content in mice, in comparation with CM and CMIA treatment. In addition, CDPIA injection once a week profoundly reduced HFD-induced body weight gain and epididymal fat-pat mass. Glucose tolerance and insulin sensitivity were distinctly restored by CDPIA treatment. Furthermore, serum ALT and AST levels were reduced, indicating alleviation of hepatocellular injury. Specifically, CM injection exerted minimal therapeutic action, which might arise from intrinsic biological activity of poly-metformin. CMIA administration displayed moderate protective efficacy, indicating that IL-22 could be expressed in vivo efficiently and exert pharmacological function normally. The therapeutic action of CMIA might be achieved by the combinational pharmacological efficiency of poly-metformin and IL-22 gene. Meanwhile, the protective effects of CDPIA was much better than CMIA. This might be attributed to penetratin, which formed more compact nanoparticles of smaller particle size with CM thus reducing phagocytosis by reticuloendothelial system (RES) in liver. Furthermore, penetratin also significantly improved cellular internalization and liver-specific accumulation of nanocomplexes as well as enhanced gene transfection efficiency in mice. These observations altogether demonstrated that CDPIA possessed remarkable protective activities in the pathogenesis of obesity-related fatty liver due to the combinational benefits of poly-metformin, penetratin and local secretion of IL-22 in liver. These results indicated that our nanoparticle-based gene delivery system could be utilized as a superior agent for the treatment of NAFLD.

24


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It has been well demonstrated that STAT3-mediated signaling pathways functions as the major transductions in the regulatory activities of IL-22 for tissue protection. Evidences indicate that activation of STAT3 in the liver could alleviate hepatic steatosis and reduce lipid content in hepatocytes.43 Subsequent Erk1/2 transactivation following STAT3 phosphorylation and downstream substrates such as Bcl-2 and Cyclin D1 promote cellular survival and proliferation.14 In this work, CDPIA administration induced significant activation of STAT3/Erk1/2 as well as promoted substrates Bcl-2 and Cyclin D1 expression, which might underlie the treatment mechanisms for CDPIA. On the other hand, IL-22 could also alleviate oxidative stress initiated by lipids or inflammatory cytokines via STAT3-mediated expression of antioxidant proteins.8 As exhibited by our results, Nrf2, an important redox-sensitive transduction, and the substrate antioxidant-related enzyme SOD1, were drastically upregulated in CDPIAtreated obese mice. The expression levels of STAT3 downstream transductions presented that CDPIA induced the most sufficient activation of IL-22-related signaling cascade compared with CM- and CMIA-groups. This phenomenon further confirmed that CDPIA could realize better local delivery of therapy gene and possess higher transfection efficiency than other groups. In addition, previous studies indicate that multiple lipid metabolism-related genes could be modulated by IL-22 thus attenuate hepatic steatosis and metabolic syndrome.7, 9 Consistent with those results, CDPIA administration also promoted expression of lipid transportation- (Acc1) and fatty-acid β-oxidation- (Acox1 and Cpt1a) related genes in mice fed with HFD. These results suggested that STAT3/Erk1/2 and Nrf2/SOD1 signaling transactivation as well as lipid 25



metabolism-related gene regulation were likely the underlying protective mechanisms of CDPIA therapy.

CONCLUSION In conclusion, the present study developed a liver-targeting gene delivery nanoparticle to realize specific expression of IL-22 gene, overcome off-target effects of IL-22 and improve its therapeutic efficacy for NAFLD. Inspired by duel-function of metformin, which could simultaneously perform as a cationic heap group for gene encapsulation and as a therapeutic adjunct for NAFLD, a novel versatile gene carrier with low cytotoxicity, satisfactory biocompatibility and intrinsic pharmacological activity was designed by conjugating biguanide to chitosan. Furthermore, penetratin was applied in the nanoparticle to endow sufficient cellular internalization and endosomal escape capacity to chitosan-metformin (CM) whereas DSPE-PEG2000 functioned as a help lipid. The self-assembled nanocomplex (CDPIA) displayed desirable particle size and homogenous distribution, as well as liver preferential accumulation when intravenously administrated to mice. Systemic administration of CDPIA distinctly alleviated hepatic steatosis, alleviated body overweight and restored insulin sensitivity as well as relieved body fat accumulation in HFD-fed mice. The related protective mechanisms concerned with activating STAT3/Erk1/2 cascade and Nrf2/SOD1 signaling transductions by IL-22 specially expressed in the liver, as well as modulating lipid metabolism-related gene expression (Scheme 1). Collectively, our poly-metformin and penetratin-based hybrid nanoparticle system has been 26


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demonstrated as a promising therapeutic strategy to be utilized for promoting clinical application of IL-22 and alleviating obesity-related fatty liver.

ABBREVIATIONS ANOVA: one way of analysis; ALT: alanine aminotransferase; ApoA-I: apolipoprotein-I; ARE: antioxidant-response element; AST: aspartate transaminase; BUN: urea nitrogen; CM: chitosan-metformin; CPPs: cell-penetrating peptides; CRE: creatinine; DLS: dynamic light scattering; DMEM: Dulbecco’s Modified Eagle Medium; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; FAM: 5carboxyfluorescein; FBS: fetal bovine serum; GTT: glucose tolerance test; H&E: hematoxylin and eosin; HFD: high fat diet; IL-22: interleukin-22; ITT: insulin tolerance test; LDH: lactate dehydrogenase; MS: multiple sclerosis; MTT: 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAFLD: non-alcoholic fatty liver disease; NEFA: non-esterified fatty acids; PDI: polydisperse index; PVDF: polyvinylidene difluoride; RA: rheumatoid arthritis; RES: reticuloendothelial system; S.D.: standard deviations; SR-B1: scavenger receptor class B type 1; TEM: transmission electronic microscopy; TG: triglycerides.

ASSOCIATIED CONTENT Supplementary information Figure S1 showed the synthesis route of chitosan-metformin. Figure S2 showed the serum levels of CHOL, HDL-c and LDL-c. 27



AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. ORCID Wenjing Zai: 0000-0003-2550-331X Wei Chen: 0000-0002-7614-4436 Hongrui Liu: 0000-0003-3975-8196 Dianwen Ju: 0000-0002-3377-741X Notes The authors declare no competing financial interest.

ACKNOWLEGEMENTS This work was supported by grants from the National Natural Science Foundation of China (No. 81773620, 81573332, 31872746, 81871485), National Key Basic Research Program of China (No. 2015CB931800), the Scientific Research Projects of Shanghai Municipal Commission of Health and Family Planning (201740140), and National Major Scientific and Technological Special Project for “Significant New Drugs Development” (2018ZX09301044-006).

AUTHOR CONTRIBUTIONS 28


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Liu H and Ju D contributed to the design of the research. Zai W and Chen W conducted the major part of the experiments and drafted the manuscript. Wu Z and Jin X performed the formulation and characterization study. Fan J, Zhang X and Luan J contributed to animal experiments. Tang S, Mei X and Hao Q contributed to pathological examinations of tissue sections. All authors read and approved the final version of the manuscript.

29



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FIGURE LEGENDS Figure 1. Construction and characterization of CDPIA nanocomplexes. (A) Schematic illustration of CDPIA. (B) Agarose gel electrophoresis retardation analysis of CM complexed with pIA at various weight ratio. (C) Ethidium bromide exclusion assay of CMIA complexes. The data were presented as means ± S.D. (n = 3). (D) Determination of particle size, (E) zeta potential and (F) PDI values of CDPIA complexes at different weight ratio via dynamic light scattering. (G) TEM image of freshly prepared CDPIA and size distribution at the weight ratio of 8. (H) Determination of particle size and PDI values of CDPIA at the weight ratio of 8 at room temperature at 0 d, 1 d, 2 d, 7 d and 15 d. Figure 2. Cellular uptake and intracellular localization of CDPIA. (A) HepG2 and (C) Huh7 cells were incubated with PBS, naked pIA, CMIA and CDPIA containing equivalent amount of pIA (5 μg) for 2 h, then cellular uptake of TOTO-3 and FAMlabeled nanoparticles were determined via confocal microscopy. (B) Fluorescence colocalization of TOTO-3 and FAM in HepG2 and (D) Huh7 cells was determined by ImageJ software. Relative fluorescence intensities of both cells were also calculated. Dara were presented as means ± S.D. (n = 3). *** for P ≤ 0.001 compared with CMIA group. Figure 3. Endosomal escape ability and cytotoxicity analysis of CDPIA. (A) Cells were incubated with CDPIA (containing 5 μg pIA) for 0.5 h, 1 h, 4 h or 8 h, then intracellular

TOTO-3

labeled

nanoparticles,

LysoTracker

Green

stained

endosomes/lysosomes and nucleus stained by Hoechst 33342 were visualized by 37



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confocal microscopy. (B) Fluorescence co-localization ratios were calculated by ImageJ software. (C) Cytotoxicity analysis of CM at different concentrations were evaluated in HepG2 and (D) Huh7 cells. * for P ≤ 0.05 compared with normal control. Figure 4. Biodistribution studies. (A) C57BL/6 mice were intravenously injected with CMIA or CDPIA containing equivalent amount of pIA (50 μg). Major organs including heart, liver, spleen, lung, kidney and brain were obtained at 6 h, 12 h or 24 h, then visualized via imaging system. (B) Total radiant efficiency in each organ were quantitated and presented as means ± S.D. (n = 8). (C) Liver sections were frozen at 80 ℃, sliced and stained by Hoechst 33342, then visualized by confocal microscopy. (D) Relative red fluorescence intensities were determined by ImageJ software and presented as means ± S.D. (n = 3). (E) Fluorescence of TOTO-3 in each organ were visualized by confocal microscopy. * for P ≤ 0.05, ** for P ≤ 0.01 compared with CMIA group. Figure 5. CDPIA administration inhibited HFD-induced hepatic steatosis in mice. (A) Schematic presentation of experimental outline to evaluate the effects of CM, CMIA and CDPIA on NAFLD in HFD-fed mice. (B) Representative images of liver gross morphology. (C) Liver weights were presented as means ± S.D. (n = 8). (D) Representative H&E-stained and (E) Oil Red O-stained liver sections were presented (magnification: 100×, 400×). (F) Quantification of Oil Red O staining index (n = 8). (G) Hepatic triglyceride (n = 8) and (H) NEFA concentrations were evaluated (n = 8). ##

for P ≤ 0.01 compared with chow diet-fed mice, * for P ≤ 0.05 and

compared with HFD-fed mice. 38


**

for P ≤ 0.01

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Figure 6. CDPIA restored insulin sensitivity and reduced lipid accumulation. (A) Whole body weight, (B) fasted body weight and (C) fasted serum glucose were presented (n = 8). (D) ITT and (G) GTT were performed after 16-week HFD-feeding. (E) Areas under the curve (AUC) of ITT and (H) GTT were evaluated by ImageJ software and presented as means ± S.D. (n = 4). (F) Serum ALT and (I) AST levels were determined (n = 8) by commercial available kit. (J) H&E staining of adipose cell in epididymal fat tissue (magnification: 100×, 400×). (K) Relative adipose cell diameters were calculated (n = 8). (L) Epididymal fat tissue weight (n = 8). (M) Serum TG levels were determined (n = 8).

#

for P ≤ 0.05,

##

for P ≤ 0.01 and

###

for P ≤

0.001compared with chow diet-fed mice, * for P ≤ 0.05 and ** for P ≤ 0.01and ** for P ≤ 0.001 compared with HFD-fed mice. Figure 7. CDPIA activated STAT3/Erk1/2, Nrf2/SOD1 signaling pathways and regulated hepatic lipid metabolism. (A) Western blot analysis of indicated proteins in liver extracts were presented. (B) Quantification of expression levels of p-Stat3Y705, p-Erk-T202/Y204, Bcl-2 and Cyclin D1 were presented (n = 4). (C) The expression of Nrf2 and SOD1 proteins were determined and (D) relative expression levels were evaluated by ImageJ software. Data presented as means ± S.D. (n = 4). (E) Relative mRNA expression levels of Acox1, Cpt1a, Acc1, CD36 and FAS in the liver (n = 4). * for P ≤ 0.05 and ** for P ≤ 0.01and ** for P ≤ 0.001 compared with HFD-fed mice.

39



Figure 8. Long-term CDPIA injection did not manifest systemic toxicity in mice. (A) H&E staining of major organs (heart, spleen, lung, kidney, brain, muscle and intestine) after 16-week HFD feeding and 12-week treatment were presented. (B) Serum BNU (n = 8), (C) creatinine (n = 8) and (D) LDH levels were determined and presented as means ± S.D. (n = 8).

Table of Contents. Schematic illustration of gene therapy design and relative mechanisms of CDPIA.

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Figure 1. Construction and characterization of CDPIA nanocomplexes.



Figure 2. Cellular uptake and intracellular localization of CDPIA.


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Figure 3. Endosomal escape ability and cytotoxicity analysis of CDPIA.



Figure 4. Biodistribution studies.


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Figure 5. CDPIA administration inhibited HFD-induced hepatic steatosis in mice.



Figure 6. CDPIA restored insulin sensitivity and reduced lipid accumulation.


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Figure 7. CDPIA activated STAT3/Erk1/2, Nrf2/SOD1 signaling pathways and regulated hepatic lipid metabolism.



Figure 8. Long-term CDPIA injection did not manifest systemic toxicity in mice.


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Scheme 1. Schematic illustration of gene therapy design and relative mechanisms of CDPIA.


Targeted Interleukin-22 Gene Delivery in the Liver by Polymetformin

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