| Project/Area Number |
23K17212
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 90120:Biomaterials-related
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| Research Institution | Japan Advanced Institute of Science and Technology |
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Principal Investigator |
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| Project Period (FY) |
2023-04-01 – 2026-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2025: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2024: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
Fiscal Year 2023: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
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| Keywords | Nano-enhancer / Adjuvant / Anti-angiogenesis / Epigallocatechin gallate / Synergistic effect |
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| Outline of Research at the Start |
The drug-free green tea-based nanoparticle (NP) adjuvant will be developed for effective anti-angiogenic treatment without adverse effects. The NP will be formulated with desirable structure and stability for prolonged blood circulation to achieve tumor-specified accumulation. The combined effects of the NP/drugs will be investigated for their maximized synergy and minimized toxicity, elucidating their mechanisms. For an advantageous translation, a prediction of the synergistic effects of the NP with various anti-angiogenic drugs will be performed using an artificial intelligence-based tool.
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| Outline of Annual Research Achievements |
Drug-free poly(ethylene glycol)-epigallocatechin-3-O-gallate conjugate (PEG-EGCG) nanoparticles (NPs) with desirable structure (size < 200 nm, polydispersity index < 0.2) were successfully formulated via two self-assembly steps in aqueous solution: (1) complexation between oligomerized EGCG (OEGCG) and bovine serum albumin (BSA) to form the core and (2) complexation of the PEG-EGCG surrounding the pre-formed core to form an inert and hydrophilic PEG outer shell. The PEG-EGCG NPs exhibited great stability against extreme dilution (1000-fold) and pH change in gastrointestinal tract (pH 1.6 and 6.5).
The selected PEG-EGCG NPs were studied for their inhibitory effects on vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) proliferation, which is well-studied as the in vitro setup that replicates the representative angiogenesis mechanism. The PEG-EGCG NPs inhibited VEGF-induced HUVEC proliferation at much greater degree than EGCG itself. The PEG-EGCG NPs also showed a much lower cytotoxicity than that of EGCG on normal HUVEC and normal human renal proximal tubule epithelial cells (HRPTEC).
When treated in combination with sunitinib (SU), the PEG-EGCG NPs effectively amplified inhibitory effects on the VEGF-induced HUVEC proliferation of SU. For quantitative analysis of the combinational effects of SU and PEG-EGCG NPs, the combination index (CI) was determined by the Chou－Talalay method. The CI values of various PEG-EGCG NPs and SU combinations were lower than one, demonstrating a synergistic effect.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The research has been progressing smoothly. For the formulation of PEG-EGCG NPs, the NPs were initially formulated purely by using inter-molecular interaction of EGCGs without any addition of other molecules. The PEG-EGCG NPs were successfully formulated, however, the high concentrations of PEG-EGCG were required for the NP formulation and the NPs were not stable against extreme dilution. To improve the stability of the PEG-EGCG NPs against dilution, OEGCG and BSA (protein without therapeutic effect) were used to improve the inter-molecular interaction of EGCG core. By using two self-assembly steps as mentioned in the previous section, the PEG-EGCG NPs with great stability against extreme dilution up to 1000-fold were successfully formulated. Since more optimizations were required, the formulation of the PEG-EGCG NPs took slightly longer time than predicted.
Even with longer time on the optimization of the PEG-EGCG NPs, the in vitro studies for their inhibitory effects on VEGF-induced HUVEC proliferation and reduced cytotoxicity on normal cells were able to be proceeded as planned. Their inhibitory effects on the VEGF-induced HUVEC proliferation when treated in combination with SU were also proceeded and the NP-SU synergism has been confirmed. With the current progress, the PEG-EGCG NPs will be screened for the in vitro combinational effects with other anti-angiogenic drugs and their synergistically amplified efficacy and improved safety in the NP－drug combinations will be validated in vivo using human tumor-xenograft mouse models as planned in the following year.
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| Strategy for Future Research Activity |
The PEG-EGCG NPs will be screened for the combinational effects in varying combination ratios with other anti-angiogenic drugs (e.g. pazopanib, vatalanib etc.) on top of SU on in vitro models that simulate the angiogenic mechanism (VEGF-induced HUVEC proliferation). The synergies of the NP－drug will be evaluated via CI. The NP－drug combinations indicating the efficient synergism (CI < 1), will be quantitated in terms of gene and protein expression of putative molecular players in the anti-angiogenic signaling pathway, to gain mechanistic insights. The cytotoxicity reduction of drug in the NP－drug combinations will be analyzed in comparison to the drug alone on human normal cells.
The synergistically amplified efficacy and improved safety in the NP－drug combinations will be validated in vivo using human tumor-xenograft mouse models. The feasibility of the NP adjuvants will be investigated with both intravenous injection and oral administration added to the drug administration based on their standard routes. The synergistic amplification of efficacy will be quantitated via CI (target: CI < 1) and its mechanism will be elucidated through the quantitation of tumor microvascular density and tumor cell proliferation/apoptosis. Also, the dose-sparing effect of their amplified efficacy will be determined by dose reduction index (DRI), which represents the folds of dose reduction allowed in combination for a given degree of effects (target value: DRI > 1). Safety of the adjuvant treatment will be evaluated via histology, hematology and blood chemistry analysis.
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