Prohibitin-targeted gene/drug delivery nanoplatform for the treatment of obesity and obesity-induced metabolic syndrome Juhyeong Hong

다국어 초록 (Multilingual Abstract)

Obesity by excessive fat accumulation represents a significant public health concern, carrying substantial economic and societal implications, and is linked to metabolic disorders like type 2 diabetes mellites (T2DM) and cancer. Existing pharmaceutical interventions for obesity, targeting either the gastrointestinal tract or the central nervous system, have demonstrated restricted effectiveness in reducing obesity, while also being associated with significant adverse effects. For example, Glucagon-like peptide-1 receptor (GLP-1R) agonists are a major group of drugs that targets incretin hormone action for the suppression of appetite, and its receptors are widely distributed in nerves, islets, heart, lung, skin, and other organs to mediate. However, several case reports have linked the use of these drugs with occurrence of acute kidney injury, primarily through hemodynamic derangement due to nausea, vomiting, and diarrhea. Therefore, a novel therapeutic delivery targeting adipocytes and normalizing excess fat transport and accumulation is necessary to maximize efficacy and reduce side effects for long-term treatment. To overcome this limitation, adipocyte targeting drug delivery system based with prohibitin binding peptide (PBP) were utilized to deliver therapeutic gene and drug for modulating obese lipid bearing white adipocytes into energy expending adipocytes. Prohibitin (PHB) is a protein that has been the subject of research in the context of adipocytes (fat cells) due to its potential roles in adipocyte differentiation, mitochondrial function, and its impact on various metabolic processes. Furthermore, PHB is also overexpressed in the vasculature of fatty adipose tissue which facilitates PBP based delivery system to be internalized into target tissue avoiding off-target effects. In part 1, development of a prohibitin targeting oliopeptpolex (POP), a complex with genetic materials and cationic oligopeptide consisited with nona- arginine and prohibitin binding peptide(PBP9R), mediated internalization of therapeutic gene (sh(FABP4/5)) into prohibitin-rich obese white adipocytes. Successfully internalized genetic materials in POP could alter lipid metabolism or differentiate cell types of adipocytes by generating multiple shRNAs targeting fatty acid binding protein 4 (FABP4) and fatty acid binding protein 4 (FABP5). FABP4 and FABP5 are an adipokine that coordinates lipid transport in mature adipocyte and its inhibition in obesity model showed weight loss and normalized insulin response. Prohibitin binding peptide (PBP) successfully delivered genetic materials in obese white adipocyte in vitro and in vivo and expressed desired genes. Furthermore, Furthermore, dual gene silencing efficiently alleviated obesity, improved insulin sensitivity, and restored hepatic metabolism in high fat diet- induced T2DM model. Furthermore, we verified therapeutic effects of POP and further development in non-invasive formulation as microneedle-based transdermal delivery system and oral administrable lipid nanoparticles. The development of a dissolvable hyaluronic acid-based microneedle patch (SLMN) for precise POP delivery is a noteworthy achievement, providing efficient gene silencing with minimal pain and improved storage stability. In vitro and in vivo experiments have shown its superior efficacy over existing treatments. Furthermore, As POP based adipocyte targeting based therapeutics could make synergistic effects if dietary lipids absorption are decreased. To address this, oral administration formulation (csBiNP@cLOP) of POP were developed with co-loading of siFABP2 which acts only in enterocytes to reduce uptake of dietary fatty acids in GI tracts. csBiNP@cLOP showed enhanced the bioavailability of POP, and syntergistic anti-obesity effects in T2DM model with siFABP2 by down regulating dietary lipid absorption and brown adipogenesis simultaneously. In part 2, PBP showed targeting effects not only in obese white adipocytes but also macrophages in obese adipose tissue in T2DM model and fatty liver in NASH model. To modulate obese tissue environments simultaneously, we challenged to selectively deliver inducers of heme oxygenase-1 (HO-1), a strong enzyme showing protective response against reactive oxygen species (ROS) and stress associated with inflammation, to desired cells. PBP was conjugated on the surface of Hemin- or CoPP-loaded poly(lactide-co-glycolide) nanoparticles (PBP- NPs). PBP-NPs efficiently differentiated lipid storing white adipocytes into energy- generating brown adipocytes in T2DM and NASH models. In addition, PBP-NPs were found to target prohibitin overexpressed fatty liver in NASH model and inhibit hepatic uptake of circulating lipids. Furthermore, PBP-NPs switched phenotypes of inflammatory macrophages in damaged organs and lowered inflammation. Taken together, dual-targeted induction of HO-1 in fatty adipose and liver tissues was proven to be a promising therapeutic strategy to ameliorate obesity, insulin resistance, and steatohepatitis by lowering lipids and cytokines. Taken together, prohibitin binding peptide (PBP) based therapeutic gene and drug delivery were able to modulate obese lipid bearing white adipocytes, hepatocytes and also inflammatory macrophages simultaneously into anti-diabetic and anti-inflammatory types for the treatments of obesity-related metabolic syndrome. In conclusion, prohibitin-targeted gene/drug delivery nanoplatform addresses various aspects, such as avoiding off-target effects, inducing brown adipogenesis, reducing systemic inflammation, decreasing lipid accumulation, normalizing hepatic lipid metabolism, and inhibiting inflammatory macrophage activation. The use of prohibitin-targeted gene/drug delivery nanoplatform with sh(FABP4/5) and HO-1 inducers has shown efficiency in downregulating disease- causing molecular targets in injured organs, presenting a promising innovative therapeutic approach for treating obesity and obesity-related metabolic syndrome.

목차 (Table of Contents)

• 1. Introduction 1
• Part 1. sh(FABP4/5) gene delivery for the treatment of obesity and obesity related metabolic syndrome 8
• Chapter 1. Adipocyte targeted delivery of sh(FABP4/5) via subcutaneous injection for the treatment of obesity and obesity related metabolic syndrome 8
• 1. Introduction 8
• 2. Materials and Methods 11
• 3. Results and Discussions 17
• 4. Conclusion 33
• Chapter 2. Transdermal delivery of sh(FABP4/5) via self-locking microneedle for the treatment of obesity and obesity related metabolic syndrome 35
• 1. Introduction 35
• 2. Materials and Methods 39
• 3. Results and Discussions 47
• 4. Conclusion 69
• Chapter 3. Oral administration of sh(FABP4/5) and siFABP2 for simultaneous modulation of adipose tissue and gut for the treatment of obesity and obesity related metabolic syndrome 72
• 1. Introduction 72
• 2. Materials and Methods 76
• 3. Results and Discussions 82
• 4. Conclusion 113
• Part 2. Heme oxygenase-1 induing drug delivery for the treatment of obesity and obesity related metabolic syndrome 115
• Chapter 4. Adipose tissue targeting heme oxygenase 1 up-regulation for the treatment of obesity and obesity related metabolic syndrome 115
• 1. Introduction 115
• 2. Materials and Methods 118
• 3. Results and Discussions 124
• 4. Conclusion 142
• Chapter 5. Fatty liver and adipose tissue targeting heme oxygenase 1 up-regulation for the treatment of non-alcoholic fatty liver disease 144
• 1. Introduction 44
• 2. Materials and Methods 147
• 3. Results and Discussions 152
• 4. Conclusion 165
• Conclusion 167
• References 171
• 국문요지 182
• 감사의 글 184