An empty self-assembled hyaluronic acid nanoparticle as a therapeutic agent for treatment of type 2 diabetes
- 주제(키워드) Diabetes , Inflammation , Hyaluronic acid
- 발행기관 아주대학교
- 지도교수 김욱
- 발행년도 2021
- 학위수여년월 2021. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030552
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Self-assembled hyaluronic acid nanoparticles (HA-NPs) have been extensively investigated for biomedical and pharmaceutical applications as the constituent of the drug carrier for the target-specific and long-acting delivery of various chemicals and biopharmaceuticals, owing to their biocompatibility and receptor-binding properties. Here, I report novel function of an empty HA-NP itself not bearing any drug, which could be a therapeutic agent, not as a drug carrier, for the treatment of type 2 diabetes (T2D) by breaking the links between adipose tissue inflammation and insulin resistance. In part Ⅰ, I show that a drug-free HA-NP itself has therapeutic effects on adipose tissue inflammation and insulin resistance. HA-NPs inhibited not only the receptor-mediated internalization of low-molecular-weight (LMW) free HA but also LMW free HA-induced pro-inflammatory gene expression in mouse primary bone marrow-derived macrophages (BMDMs) isolated from wild-type mice, but not in CD44-null (CD44-/-) BMDMs. An in vivo biodistribution study showed the distribution of HA-NPs and their co-localization with CD44 in adipose tissues including epididymal white adipose tissues (eWATs), but these were rarely observed in the eWATs of CD44-/- mice. In addition, CD44 expression and HA-NP accumulation in the eWATs were increased in mice with diet-induced obesity (DIO) compared to lean mice. Interestingly, treatment with HA-NPs in DIO mice suppressed adipose tissue inflammation as indicated by reduced macrophage content, the production of proinflammatory cytokines and NLRP3 inflammasome activity in eWATs, leading to improved insulin sensitivity and normalized blood glucose levels. Collectively, these results suggest that an empty HA-NP itself can be a therapeutic agent for the treatment of T2D. In part Ⅱ, I identify effect of a self-assembled HA-NP on macrophage polarization into pro-inflammatory M1 and anti-inflammatory M2 types using mouse primary BMDMs and peritoneal macrophages. HA-NP suppresses the M0 to M1 polarization and M2 to M1 repolarization induced by LPS, but not the M0 to M2 polarization and M1 to M2 repolarization induced by IL-4. Through the direct interaction with Toll-like receptor 4 (TLR4), HA-NPs effectively suppressed Myd88- and TRIF-dependent LPS-induced expressions of pro-inflammatory mediators by acting as a competitive inhibitor of LPS-TLR4 interactions in macrophages. The effect of HA-NP on LPS-induced TLR4 signaling is attributed to the presence of self-assembled hydrophilic HA shell, not free HA and hydrophobic core 5β-Cholanic acid. Thus, these results identify the potential of an empty HA-NP as an effective therapeutic agent against TLR4-mediated inflammatory disorders
more목차
Introduction 1
1.1. Inflammation 1
1.2. Macrophage 2
1.3. Self-assembled hyaluronic acid nanoparticle 3
Material and Methods 6
2.1. Materials 6
2.2. Synthesis and characterization of HA-NPs 6
2.3. Mice and treatment protocols 7
2.4. In vivo biodistribution of 235k-HA-CA NP 8
2.5. In vitro competitive inhibition assay 9
2.6. Analysis of metabolic parameters 10
2.7. Intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance tests (ITT) 10
2.8. Inflammasome characterization 10
2.9. Histological analysis 11
2.10. Cell culture and treatment 11
2.11. Quantitative real-time PCR analysis 12
2.12. Cell viability assay 15
2.13. RNA Sequencing 16
2.14. Binding kinetics using SPR 16
2.15. Evaluation of the cytokines secretion level by ELISA 16
2.16. Flow cytometry 17
2.17. Protein expression and western blot 18
2.18. Statistical analysis 19
Results 20
Self-assembled hyaluronic nanoparticles as a nanomedicine for treatment of T2D 20
3.1. Synthesis and characterization of self-assembled 235k-HA-CA NP 20
3.2. In vivo biodistribution of 235k-HA-CA NP 20
3.3. In vitro competitive inhibition assay of 235k-HA-CA NP 25
3.4. Self-assembled 235k-HA-CA NP reduce body weight and improve the impaired glucose tolerance and insulin resistance 28
3.5. Self-assembled 235k-HA-CA NP treatment reverses macrophage infiltration into eWATs of DIO mice 36
3.6. Self-assembled 235k-HA-CA NP suppress NLRP3 inflammasome activation in DIO mice 39
3.7. Self-assembled 235k-HA-CA NP mitigates adipose tissue fibrosis in DIO mice 42
3.8. Other self-assembled HA-NPs, 12k-HA-CA NP and 12k-HA-PCL NP have similar effects with 235k-HA-CA NP on adipose tissue inflammation and insulin resistance 45
Self-assembled HA-NPs a modulator of macrophage polariza-tion into M1 phenotype 58
4.1. Synthesis and characterization of self-assembled 12k-HA-CA NP 58
4.2. 12k-HA-CA NP as a competitive inhibitor of LPS-TLR4 interaction in macrophages 58
4.3. 12k-HA-CA NP suppresses LPS-induced macrophage polarization into M1 phenotype 66
4.4. 12k-HA-CA NP suppresses the canonical and non-canonical TLR4 signaling in macrophages 80
4.5. 12k-HA-CA NP suppresses LPS-induced NLRP3 inflammasome activation in macrophages 83
4.6. 12k-HA-CA NP dose not affect IL-4-induced macrophage polarization into M2 phenotype 86
Discussion 93
Reference 98
