(A) lipid nanoparticle platform for tumor-associated macrophage reprogramming in cancer

다국어 초록 (Multilingual Abstract)

Tumor-associated macrophages (TAMs) predominantly exhibit an immunosuppressive M2-like phenotype within the tumor microenvironment (TME) that hinders cancer immunotherapy, necessitating effective reprogramming strategies. Here, I developed a biomimetic macrophage-targeting lipid nanoparticle (Mac LNP) platform co-loaded with CpG oligodeoxynucleotides (CpG-ODN) and STAT3 siRNA to induce potent M2-to-M1 repolarization. By incorporating triglyceride and phosphatidylserine to mimic chylomicrons and apoptotic bodies, optimized Mac LNP formulation achieved selective uptake in macrophages over non-target cells. I demonstrated that the simultaneous delivery of CpG-ODN and STAT3 siRNA synergistically activated Toll-lie receptor 9 (TLR9) while silencing the M2-maintaining transcription factor, effectively induce M2-to-M1 reprogramming of macrophages in vitro. Furthermore, intratumoral administration of these dual-loaded Mac LNPs in a 4T1 subcutaneous breast cancer model significantly inhibited tumor growth and remodeled the immune microenvironment by increasing the M1/M2 macrophage ratio and promoting cytotoxic CD8+ T cell infiltration. These findings establish Mac LNP platform as a potent therapeutic strategy for achieving macrophage-specific targeting and modulating innate immunity to enhance antitumor responses.

국문 초록 (Abstract)

종양 미세환경 내 종양 연관 대식세포는 주로 면역 억제성 M2 표현형을 나타내어 암 면역치료를 저해하므로, 이를 효과적으로 재프로그래밍하는 전략이 필수적이다. 이에 본 연구에서는 M2-to-M1 재분극화를 강력하게 유도하기 위해 CpG-ODN과 STAT3 siRNA를 동시에 탑재한 대식세포 표적 지질 나노입자(Mac LNP) 플랫폼을 개발하였다. 킬로미크론과 세포사멸체를 모방하기위해 중성지방과 phosphatidylserine을 도입하여 최적화한 Mac LNP 제형은 비표적 세포 대비 대식세포에서 선택적인 유전자 전달을 보임을 확인하였다. CpG-ODN과 STAT3 siRNA의 동시 전달은 Toll-like receptor 9을 활성화하는 동시에 M2 유지 전사인자를 억제하는 시너지 효과를 통해 in vitro 상에서 대식세포가 M1 표현형으로 강력하게 분극화하는 것을 확인하였다. 또한, 4T1 유방암 모델에 Mac LNP를 종양 내 투여했을 때, M1 대식세포 비율을 증가시키고 세포독성 CD8+T세포 침윤을 촉진함으로써 종양 성장을 유의하게 억제하고 면역 미세환경을 리모델링하였다. 이러한 결과는 본 연구의 Mac LNP 플랫폼이 대식세포 특이적 전달을 확인하고 대식세포를 리프로그래밍하여 항종양 효과를 강화할 수 있는 치료 전략임을 입증한다.

목차 (Table of Contents)

• Abstract vii
• 1. Introduction １
• 2. Materials and Methods ６
• 2.1. Materials ６
• 2.2. Preparation of LNPs ７
• 2.3. Characterization of LNPs ８
• 2.4. Cell culture ９
• 2.5. In vitro evaluation of gene delivery of LNPs １０
• 2.6. Quantitative real-time PCR (qRT-PCR) １０
• 2.7. Western blot analysis １１
• 2.8. Expression and secretion of M1 marker of RAW264.7 macrophage cells １２
• 2.9. Flow cytometry analysis １２
• 2.10. In vivo antitumor efficacy １３
• 2.11. In vivo histological analysis １４
• 2.12. In vivo analysis of immune cells in tumor after treatment of LNPs１４
• 2.13. Analysis of tumor macrophage delivery of Mac LNP containing EGFP mRNA by flow cytometry １５
• 2.14. Statistical analysis １６
• 3. Results １８
• 3.1. Formulation and optimization of macrophage targeting LNPs １８
• 3.2. Characterization of macrophage targeting LNPs ２８
• 3.3. In Vitro analysis of M1 reprogramming induced by CpG+STAT3 siRNA loaded Mac LNPs ３２
• 3.4. Anti-tumor therapeutic effect of Mac LNPs on 4T1 mouse model ４１
• 3.5. In vivo analysis of apoptosis induction and antitumor immune response ４６
• 4. Discussion ５０
• References ５３
• 논문요약 ６０