Near-Infrared Fluorescent Highly Branched Poly(β-Amino Ester)s Nanoparticles for Gene Delivery: In vivo Biodistribution and Safety Evaluation

Hongzhen Zhang,1,2,* Jiahao Liu,1,2,* Rui Huang,1– 3,* Ailin Hu,2 Rui Miao,1– 3 Guang Chen,4 Haonan Li,5 Wenxin Wang,1– 3 Zhonglei He1,2,41School of Public Health, Anhui University of Science and Technology, Huainan, 232001, People’s Republic of China; 2Institute of Precision Medicine (AUST-IPM), Anhui University of Science and Technology, Huainan, 232001, People’s Republic of China; 3School of Medicine, Anhui University of Science and Technology, Huainan, 232001, People’s Republic of China; 4Zhejiang Key Laboratory of New Drug Development for Central Nervous System Diseases, School of Medicine, Taizhou University, Zhejiang, 318000, People’s Republic of China; 5ACROBiosystems Inc, Beijing, 100176, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhonglei He, Institute of Precision Medicine (AUST-IPM), Anhui University of Science and Technology, Huainan, 232001, People’s Republic of China, Email [email protected] Wenxin Wang, Institute of Precision Medicine (AUST-IPM), Anhui University of Science and Technology, Huainan, 232001, People’s Republic of China, Email [email protected]: Cationic highly branched poly(β-amino ester)s (HPAEs) represent a promising class of nonviral gene-delivery polymers; however, their in vivo distribution and biological fate remain challenging to monitor. Here, we explored covalent conjugation of indocyanine green (ICG) as a strategy to impart near-infrared-I (NIR-I) fluorescence to HPAEs while maintaining their DNA complexation capacity and gene-delivery performance.Methods: HPAE was modified with increasing feed amounts of ICG-N-hydroxysuccinimide (ICG-NHS), generating a series of fluorescent polymers designated HPAE-0, HPAE-1, HPAE-3, HPAE-5, HPAE-7, and HPAE-9, where the numerical suffixes indicate ICG-NHS feed volumes rather than substitution ratios. The resulting conjugates and their DNA nanoparticles were characterized by spectroscopic and chromatographic analyses, DNA-binding assays, dynamic light scattering, zeta-potential measurements, transmission electron microscopy, and optical-stability evaluation. In vitro gene-delivery activity and cytocompatibility were assessed in multiple cell models, while systemic distribution, biocompatibility, and tissue responses were evaluated in healthy BALB/c mice following administration.Results: Increasing ICG-NHS feed resulted in tunable incorporation of fluorescent moieties into the HPAE backbone. Among the tested formulations, HPAE-3 exhibited an apparent amine substitution degree of 18.98% ± 0.54% and a fluorescence emission maximum at approximately 834 nm. HPAE-3-based nanoparticles displayed favorable physicochemical properties, including hydrodynamic diameters of approximately 170– 290 nm, low-to-moderate dispersity (PDI, 0.18– 0.40), positive surface potentials (+23 to +40 mV), and efficient DNA condensation at polymer/DNA ratios ≥ 20:1. Importantly, ICG incorporation at this level preserved reporter-gene expression in HEK293T, RAW264.7, and MLE-12 cells while maintaining acceptable cytocompatibility. Following systemic administration in mice, HPAE-3 mediated luciferase reporter-gene expression predominantly in the liver, spleen, and lungs. HPAE-3-associated NIR-I fluorescence was most evident in the liver and lungs at 6 h, with a weaker signal in the spleen, and declined thereafter; by 72 h, residual ex vivo fluorescence was detected predominantly in the liver. No apparent acute tissue damage, significant alterations in serum biochemical parameters, or deviations in body-weight profiles were observed compared with control groups.Conclusion: HPAE-3 achieved a balanced integration of NIR-I fluorescence, DNA-delivery capability, nanoparticle stability, and preliminary in vivo tolerability. These results establish ICG-labeled HPAE as a potential platform for noninvasive visualization of polymer-mediated gene delivery and provide a foundation for further investigation of its biodistribution, intracellular fate, and therapeutic applications.Keywords: hyperbranched polymers, indocyanine green, nanoparticles, gene delivery, in vivo fluorescence imaging