Graduation Year

2026

Document Type

Thesis

Degree

M.S.

Degree Name

Master of Science (M.S.)

Degree Granting Department

Pharmacy

Major Professor

Qingyu Zhou, Ph.D.

Committee Member

Vijaykumar Sutariya, Ph.D.

Committee Member

Manas Biswal, Ph.D.

Keywords

KRAS, Nanoparticle Delivery, p5RHH Peptide, Pharmacokinetics, siRNA, ddPCR

Abstract

This study focuses on the development and validation of analytical methods for the detection of therapeutic KRAS-targeting siRNA in biological systems. Physicochemical characterization showed that p5RHH-formulated siRNA nanoparticles exhibit a controlled nanoscale size distribution, moderate polydispersity, and a negative zeta potential, suggesting colloidal stability and potential suitability for cellular uptake. Scanning electron microscopy confirmed a predominantly spherical morphology with a relatively uniform size distribution, consistent with dynamic light scattering measurements.

To enable accurate siRNA quantification, stem–loop reverse transcription (SL-RT) coupled with SYBR Green–based quantitative Polymerase Chain Reaction (qPCR) was optimized. Key parameters, including a primer concentration of 100 nM and a 10⁴-fold dilution of SL-RT products, were identified as suitable for subsequent droplet digital PCR (ddPCR) analysis. Application of these conditions in ddPCR enabled quantification of siRNA across multiple biological matrices, including plasma, tumor homogenates, and cell lysates. The ddPCR assays demonstrated high sensitivity (a low limit of quantification (LLOQ) of 1.5 pM), linear response (the coefficient of determination (R2) above 0.91), specificity, with some variability observed in complex biological matrices.

Application of the optimized ddPCR platform in vitro indicated cellular uptake of p5RHH-formulated siRNA in A549 non-small cell lung cancer cells, with peak intracellular concentrations (7.71 ± 1.89 nM; N = 3) at 2 hours post-treatment, followed by a gradual decline and an estimated terminal elimination half-life of 9.4 ± 5.7 hours (N = 3).

Overall, these results describe an analytical framework for siRNA quantification that enables pharmacokinetic and biodistribution assessments and may support further investigation of siRNA-based nanomedicine in oncologic applications.

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