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In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells
Published on: September 23, 2021
A Tumor-Selective Self-Assembling Network of Poly(amino acid) Induces Cascading Bystander Cytotoxicity through
Jinfeng Sun1, Yang Ma1,2, Jingshan Sun1
1State Key Laboratory of Polymer Science and Technology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, P. R. China.
Abstract:
Signal transduction is essential for tumor progression and therapeutic response. Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and therapeutic delivery. However, current EV-based strategies that rely on ex vivo manipulation encounter significant limitations, including nonspecific biodistribution, limited tumor accumulation, and diminished biological activity, underscoring the need for the localized production of therapeutic EVs within tumors. To address this, a rationally designed poly(D-amino acid) copolymer, methoxy poly(ethylene glycol)-block-poly(D-lysine10)-block-poly(O-phospho-D-tyrosine5-co-D-alanine25) (EG45-D-K-D-pYA), is developed, which undergoes alkaline phosphatase (ALP)-catalyzed self-assembly to form a tumor-localized, positively charged network. This assembly triggers the formation of localized microvesicles (MVs) and initiates a self-sustaining fission cascade, amplifying the cytotoxic signaling. Upon ALP catalysis, 88.3% of the phosphate group in EG45-D-K-D-pYA is hydrolyzed within 24 h, shifting zeta potential from -4.95 to +25.0 mV and increasing β-sheet content from 4.0% to 46.0%, promoting a morphological transition from nanoparticle to network. EG45-D-K-D-pYA disrupts tumor cell membranes, mitochondria, and nuclei, inducing potent tumor-selective cytotoxicity (IC50 = 0.58 μM at 24 h) and in situ generation of positively charged MVs (ζ = +32.9 mV). These MVs, enriched with membrane fragments, poly(amino acid)s, and mitochondrial and nuclear components, further mediate tumor-selective cell death and sustain cytotoxic propagation across four MV generations (P0-P4), with cytotoxicity rates of 53.13%, 28.71%, 36.35%, 24.69%, and 7.05%, respectively. EG45-D-K-D-pYA achieves 90.1% tumor growth inhibition and markedly suppresses metastases by forming network in vivo. This study presents a tumor-selective therapeutic approach that integrates enzyme-triggered self-assembly with MV-driven fission amplification to induce robust bystander cytotoxicity and sustained tumor suppression.
Insights
A novel poly(D-amino acid) copolymer self-assembles in tumors, creating localized microvesicles that amplify cytotoxic signaling. This approach effectively inhibits tumor growth and suppresses metastasis through sustained bystander effects.
Area of Science:
- Biomaterials Science
- Nanotechnology
- Oncology
Background:
- Extracellular vesicles (EVs) are crucial for intercellular communication and therapeutic delivery.
- Current EV strategies face limitations like poor tumor accumulation and activity.
- Localized EV production within tumors is needed for enhanced therapeutic efficacy.
Purpose of the Study:
- To develop a novel polymer for enzyme-triggered self-assembly and localized therapeutic EV generation within tumors.
- To investigate the self-assembly mechanism and the resulting microvesicle (MV) formation and amplification cascade.
- To evaluate the in situ cytotoxic effects and tumor suppression capabilities of the developed system.
Main Methods:
- Design and synthesis of a poly(D-amino acid) copolymer (EG45-D-K-D-pYA) responsive to alkaline phosphatase (ALP).
- Characterization of polymer self-assembly, including changes in zeta potential and β-sheet content upon ALP catalysis.
- Assessment of in situ MV generation, cytotoxicity, and tumor growth inhibition in vivo.
Main Results:
- EG45-D-K-D-pYA undergoes ALP-catalyzed self-assembly into a tumor-localized, positively charged network, triggering MV formation and a fission cascade.
- Polymer hydrolysis and assembly led to significant changes in charge and secondary structure, promoting nanoparticle-to-network transition.
- The system demonstrated potent tumor-selective cytotoxicity (IC50 = 0.58 μM), in situ MV generation (ζ = +32.9 mV), and sustained cytotoxicity across four MV generations.
- In vivo studies showed 90.1% tumor growth inhibition and significant suppression of metastases.
Conclusions:
- The developed poly(D-amino acid) copolymer enables enzyme-triggered, localized production of therapeutic microvesicles within tumors.
- This strategy amplifies cytotoxic signaling through a self-sustaining fission cascade, inducing robust bystander cytotoxicity.
- The approach offers a promising tumor-selective therapeutic strategy for sustained tumor suppression and metastasis inhibition.
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