The transport of peptides and nucleic acid using polyplexes as carrier system shows potential in the gene and DNA-based delivery systems to treat various conditions such as infection, inflammation, cancer, etc. The present study focused on the development of neuropeptide Y-loaded polyplexes from cationic polymer coated with PEG 2000 by application of microfluidics. The PEGylated polyplexes were statistically optimized by 3-Level factorial design with entrapment efficiency of 87.06±6.15% and in-vitro release of neuropeptide Y of 83.71±6.31% for 24 h. These polyplexes displayed 15.5% of α-helical content with no shift in the β-sheet content from Reed reference using circular dichroism spectroscopy. The FTIR and NMR studies confirmed the polymeric reaction with neuropeptide Y in PEGylated polyplexes by displaying the presence of PEG-NHS interaction with PEG-PEI grafting. The formulation PPN showed G0G1 cell arrest with an IC₅₀ value of 4.98±0.5 μg/mL and 54.46±6.33% apoptosis on the MCF-7 and IC₅₀ value of 6.23±2.11 μg/mL and 50.23±5.71% apoptosis on MDA-MD-231 cells by upregulating phosphor-p38 MAPK pathway. The PEGylated polyplexes were found to be stable for 6 months and displayed particle size 360.52 ± 8.35 nm at 5°C and 368.02 ± 7.36 nm at 25°C due to formation of steric shield by PEGylation on the structures of polyplexes. The animals study showed no toxicity on administration of formulation PPN via intravenous route and better t_1/2 (5.79 h) and AUC (84888.63 pg/mL*min) without mortality. Hence, PEGylated polyplexes emerge as a novel, effective and economical nanocarrier delivery system contributing towards long-term stability, less toxicity with significant apoptotic action on breast cancer cell lines.