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Conversion of Chemical Drugs into Targeting Ligands on RNA Nanoparticles and Assessing Payload Stoichiometry for
Congcong Xu1, Hongran Yin1, Tesla Yudhistira1
1Center for RNA Nanobiotechnology and Nanomedicine; Division of Pharmaceutics and Pharmaceutical Chemistry; College of Pharmacy; and James Comprehensive Cancer Center. The Ohio State University, Columbus, Ohio, USA.
Abstract:
Active targeting-mediated nanodelivery takes advantages of ligand-receptor specificity to avoid non-specific distribution, holding great promise for the treatment of a spectrum of diseases. RNA nanoparticles have demonstrated rapid spontaneous tumor targeting and very little organ accumulation due to rapid renal clearance of non-tumor accumulated RNA nanoparticles. However available ligands for specific cells are limited, yet many chemical entities possess receptor targeting capability and remains unexplored. To provide specific tumor accumulation, a multivalent targeting strategy on RNA nanoparticles to control their in vivo fate is implemented. Methotrexate (MTX), a clinically approved chemotherapy was used as a tumor-targeting ligand through conjugation to our RNA nanoparticle with controlled conjugation of various copy numbers. As copies of conjugated MTX increased on the nanoparticle, the specific binding to overexpressed folate receptor was enhanced as demonstrated by flow cytometry analysis and confocal microscopy imaging. Increasing the amounts of conjugated MTX did not significantly change the nanoparticle size, Zeta potential, or cytokine induction. Increased amounts of conjugated MTX resulted in improved cell inhibition due to MTX release following cell internalization. However, increasing conjugated MTX to the RNA nanoparticles reduced the melting temperature of RNA nanoparticles and increased in vitro serum protein binding to the nanoparticles. Thus, in vivo biodistribution profiles of RNA nanoparticles revealed different behaviors based on MTX conjugation in cancer targeting and clearance. Increased copies of MTX changed the ability of nanoparticles to target tumors, accumulate in healthy organs, and rapidly clear through the urine. Nanoparticle design must be closely considered for optimized cancer targeting and therapy, providing the rationale for a proper design of RNA nanodelivery in cancer treatment.
Insights
This study enhanced RNA nanoparticle cancer targeting by conjugating methotrexate (MTX). Increased MTX copies improved tumor accumulation and cell inhibition, optimizing nanodelivery for cancer therapy.
Area of Science:
- Biomedical Engineering
- Nanotechnology
- Drug Delivery
Background:
- Active targeting nanodelivery utilizes ligand-receptor specificity for precise drug distribution, showing promise in disease treatment.
- RNA nanoparticles offer rapid tumor targeting and renal clearance, but ligand availability is limited.
- Exploring novel ligands for RNA nanoparticles is crucial for enhancing targeted cancer therapy.
Purpose of the Study:
- To investigate the impact of conjugating methotrexate (MTX), a chemotherapy drug, as a ligand onto RNA nanoparticles.
- To evaluate how varying MTX copy numbers on RNA nanoparticles influence their in vivo fate, tumor targeting, and therapeutic efficacy.
- To assess the safety and stability of MTX-conjugated RNA nanoparticles.
Main Methods:
- Conjugation of methotrexate (MTX) to RNA nanoparticles with controlled copy numbers.
- In vitro characterization including flow cytometry, confocal microscopy, size, Zeta potential, and cytokine induction assays.
- In vitro cytotoxicity assays and in vitro serum protein binding assessments.
- In vivo biodistribution studies in a cancer model.
Main Results:
- Increased MTX conjugation enhanced specific binding to folate receptors and improved cancer cell inhibition.
- Nanoparticle size, Zeta potential, and cytokine induction remained largely unaffected by MTX conjugation.
- Higher MTX loads reduced RNA nanoparticle stability (melting temperature) and increased in vitro serum protein binding.
- In vivo studies showed altered tumor targeting, organ accumulation, and urinary clearance based on MTX conjugation levels.
Conclusions:
- Multivalent MTX targeting on RNA nanoparticles can be optimized for enhanced cancer targeting and therapeutic efficacy.
- Nanoparticle design, including ligand conjugation strategy, is critical for balancing targeting, stability, and clearance.
- This approach provides a rationale for designing improved RNA nanodelivery systems for cancer treatment.
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