Oral Delivery of Nanoparticles Loaded With Ginger Active Compound, 6-Shogaol, Attenuates Ulcerative Colitis and Promotes Wound Healing in a Murine Model of Ulcerative Colitis

Journal of Crohn's and Colitis, 2018, 217–229 doi:10.1093/ecco-jcc/jjx115 Advance Access publication August 18, 2017 Original Article Original Article Oral Delivery of Nanoparticles Loaded With Ginger Active Compound, 6-Shogaol, Attenuates Ulcerative Colitis and Promotes Wound Healing in a Murine Model of Ulcerative Colitis Mingzhen Zhang,a,# Changlong Xu,a,c,# Dandan Liu,d Moon Kwon Han,a Lixin Wang,a,b Didier Merlina,b Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA bAtlanta Veterans Affairs Medical Center, Decatur, GA, USA cDepartment of Gastroenterology, 2nd Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Zhejiang, P. R. China dDepartment of Chemistry, Georgia State University, Atlanta, GA, USA a These authors contributed equally to this work. # Corresponding author: Mingzhen Zhang, Institute for Biomedical Sciences, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302, USA. Tel.: +1 [404] 413 3597; fax: +1 [404] 413 3580; email: Abstract Background and Aims: Oral drug delivery is the most attractive pathway for ulcerative colitis [UC] therapy, since it has many advantages. However, this strategy has encountered many challenges, including the instability of drugs in the gastrointestinal tract [GT], low targeting of disease tissues, and severe adverse effects. Nanoparticles capable of colitis tissue-targeted delivery and sitespecific drug release may offer a unique and therapeutically effective system that addresses these formidable challenges. Methods: We used a versatile single-step surface-functionalising technique to prepare PLGA/ PLA-PEG-FA nanoparticles loaded with the ginger active compound, 6-shogaol [NPs-PEG-FA/6shogaol]. The therapeutic efficacy of NPs-PEG-FA/6-shogaol was evaluated in the well-established mouse model of dextran sulphate sodium [DSS]-induced colitis. Results: NPs-PEG-FA exhibited very good biocompatibility both in vitro and in vivo. Subsequent cellular uptake experiments demonstrated that NPs-PEG-FA could undergo efficient receptormediated uptake by colon-26 cells and activated Raw 264.7 macrophage cells. In vivo, oral administration of NPs-PEG-FA/6-shogaol encapsulated in a hydrogel system [chitosan/alginate] significantly alleviated colitis symptoms and accelerated colitis wound repair in DSS-treated mice by regulating the expression levels of pro-inflammatory [TNF-α, IL-6, IL-1β, and iNOS] and antiinflammatory [Nrf-2 and HO-1] factors. Conclusions: Our study demonstrates a convenient, orally administered 6-shogaol drug delivery system that effectively targets colitis tissue, alleviates colitis symptoms, and accelerates colitis wound repair. This system may represent a promising therapeutic approach for treating inflammatory bowel disease [IBD]. Key Words: Ulcerative colitis; drug delivery system; therapy Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: 217 M. Zhang et al. 218 1. Introduction 2. Methods 2.1. Materials Lactide:glycolide [75:25] [PLGA], molecular weight 66 000–107 000, poly [vinyl alcohol] [PVA, 86–89% hydrolyzed, low molecular weight], 6-shogaol, and its analysis standard, phalloidin-FITC, were purchased from Sigma [St. Louis, MO, USA]. PLA-PEG and PLA-PEG FA [PEG MW 5000 and PLA MW 10000] were obtained from NSP-Functional polymers and copolymers [Winston-Salem; NC, USA]. Fluorescent lipophilic dyes, 1, 1’-dioctadecyl-3,3,3’,3’tetramethylindocar-bocyanine perchlorate [DiL] and 1,1’-dioctadecyl-3,3’,3’- tetramethylindotricarbocyanine iodide [DiR], were purchased from Promokine [Heidelberg, Germany]. FITC-Annexin V/propidium iodide [PI] apoptosis kit was purchased from BD biosciences [San Jose, CA, USA]. XenoLight RediJect Chemiluminescent Inflammation Probe was purchased from PerkinElmer [Waltham, MA, USA]. Lipocalin-2 duoset enzyme-linked immunosorbent assay [ELISA] kit was purchased from RandD Systems [Minneapolis, MN, USA]. FR Antibody [FL-257] was obtained from Santa Cruz [Dallas, TX, USA]. 2.2. Preparation of NPs To prepare ginger active compound 6-shogaol loaded NPs-PEG-FA nanoparticles, PLGA [75 mg] and 6-shogaol (6 mg, 25 µL dimethyl sulphoxide [DMSO]) were dissolved in 2 mL of dichloromethane [DCM]. An oil-in-water emulsion was formed by emulsifying the polymer solution in 4 mL of 2.5 % w/v aqueous polyvinyl alcohol [PVA] solution using probe sonicator at 50% amplitude for 2 min [Branson S-450; Danbury, CT, USA] over an ice bath; 25 mg of PLA-PEG/PLA-PEG-FA mix with different PLA-PEG-FA amounts of 10 mg [as NPs-PEG-FA-1], 15 mg [as NPs-PEG-FA-2], and 20 mg [as NPs-PEG-FA-3] were dissolved in DCM [200 µL] and added dropwise to the above emulsion with stirring. The emulsion was stirred overnight at room temperature, followed by 2 h of rotary evaporation under vacuum to remove the residual DCM. Nanoparticles were recovered by centrifugation [15 000 rpm, 15 min, at 4°C] and washed three times with de-ionised water. NPs suspension was then lyophilised to obtain a dry powder. DiL- or DiR-loaded nanoparticles involved the same procedures. 2.3. Characterisation of NPs The particle size and zeta potential of NPs-PEG and NPs-PEG-FA were determined using a Malvern Zetasiser Nano ZS90 Apparatus [Malvern Instruments, Malvern, UK] at room temperature. The morphology of NPs-PEG and NPs-PEG-FA was obtained using transmission electron microscopy [TEM]. High performance liquid chromatography [HPLC] analysis was performed to evaluate 6-shogaol loading efficiency using an Agilent® 1100 LC System [Agilent Technologies Inc.; CA, USA]. The system was optimised in order to reduce dead volume and improve performance including: increasing the UV scan rate; changing the injector needle seat; re-plumbing the system with red PEEKsil™ Tubing [SGE]; and using a semi-micro flow cell. The C18 column was from Phenomenex [Torrance, CA, USA]. All samples were dissolved in Inflammatory bowel diseases [IBDs], which include Crohn’s disease [CD] and ulcerative colitis [UC], are chronic debilitating inflammatory conditions.1,2 The conventional therapies currently used to treat IBD patients can fall into three groups: anti-inflammatory medications [such as aminosalicylates and corticosteroids], immunosuppressants, and antibiotics.3–5 These medications can temporarily induce and maintain remission, but 80% and 45% of CD and UC patients, respectively, will require at least one surgical intervention in their lifetimes.6,7 Conventional oral formations [eg pellets, capsules, or tablets] are of limited use in IBD. These approaches, which are generally designed to achieve systemic delivery of therapeutics, can have many drawbacks including inefficacy, ineffective control of drug release, therapeutic variation due to IBD symptoms [eg diarrhoea and a modified colonic environment], and limit targeting of inflamed areas. Furthermore, the enha (...truncated)