Synthesis and Characterization of Poly (β-amino Ester) and Applied PEGylated and Non-PEGylated Poly (β-amino ester)Plasmid DNA Nanoparticles for Efficient Gene Delivery

Purpose: Polymeric gene delivery vectors require detailed physicochemical characterization to ensure reproducible formulation and efficient transfection. PBAE-447 is a promising biodegradable cationic polymer, but its simple nanoparticles can degrade or aggregate in aqueous/physiological conditions. This study characterizes PBAE-447 and evaluates PEGylation and lyophilization to improve stability and transfection.

Hypothesis: Characterizing key PBAE-447 parameters—solubility, swelling, buffering capacity, and cytotoxicity—will enable optimized nanoparticle formulation. PEGylation and lyophilization will improve colloidal stability of PBAE-447/pDNA nanoparticles while maintaining or enhancing transfection efficiency compared with PEI.

Aims: Synthesize and characterize PBAE-447 by GPC and ¹H NMR. - Evaluate PBAE-447 aqueous solubility, swelling behavior, proton-buffering capacity, and cytotoxicity. - Formulate PBAE-447/pGFP plasmid DNA nanoparticles and compare as-prepared, lyophilized, and PEGylated formulations. - Assess transfection efficiency in HEK-293, BEAS-2B, and A549 cells and compare with PEI.

Delivery system: Polymer: PBAE-447 = 1-(3-aminopropyl)-4-methylpiperazine end-modified poly(1,4-butanediol diacrylate-co-4-amino-1-butanol). Synthesized via Michael addition from B4 and S4 at 1:1 molar ratio, end-capped with E7. - PEGylation: Methoxy polyethylene glycol-succinimidyl succinate (mPEG-SS), 5 kDa. - Nanoparticle type: PBAE-447/pDNA polyplexes formed by electrostatic self-assembly in 25 mM sodium acetate buffer (pH 5.0). - Payload: Plasmid DNA encoding green fluorescent protein (pGFP). - Formulation: PBAE-447/pDNA weight ratio 60; pDNA concentration 0.06 µg/µL; polymer 3.6 µg/µL. - Lyophilization: Sucrose (30 mg/mL in 25 mM NaAc) added 1:1 v/v; frozen at −80 °C; lyophilized 2 days; stored at 4 °C. - Targeting ligand: None. - Size/zeta: As-prepared ~111.1 nm, zeta ~+9.51 mV; PEGylated in FBS ~184 nm, in NaCl ~175.1 nm. Abstract reports 184 nm and +7.42 mV.

Approach: In vitro only. Cell lines: HEK-293 (human embryonic kidney), BEAS-2B (bronchial epithelial), A549 (lung adenocarcinoma epithelial). - Cytotoxicity: MTT assay at PBAE-447 concentrations 10–100 µg/mL; PEI 25 kDa as control. - Transfection: 96-well plates, 12,500 cells/well; 4 h nanoparticle incubation; GFP expression assessed after 48 h by fluorescence microscopy and ImageJ. - Confocal microscopy: uptake/localization after 2, 4, 6, and 8 h incubation with PEGylated PBAE-447/pDNA. - Stability: nanoparticle size and zeta potential measured in 10% FBS and 300 mM NaCl after 4 h; lyophilized nanoparticles stored 4 months at 4 °C. - Hemagglutination: 2% mouse erythrocytes incubated with PEGylated PBAE-447/pDNA. - n = 3 independent experiments unless noted.

Key methods: GPC for molecular weight and PDI. - ¹H NMR for polymer structure. - Absorbance at 620 nm for solubility. - Acid–base titration for proton-buffering capacity. - Gravimetric swelling degree in DMSO, DCM, THF, EtOAc. - MTT assay for cytotoxicity. - DLS for hydrodynamic size and zeta potential. - Fluorescence microscopy/ImageJ for GFP transfection. - Confocal microscopy for cellular uptake and intracellular localization. - Hemagglutination assay for blood compatibility.

Key results: Polymer characterization: Mn 5,354; Mw 9,575; MP 4,934; PDI 1.7. Complete end-capping confirmed by ¹H NMR. - Solubility/buffering/swelling: PBAE-447 completely soluble at 10 µg/µL in NaAc; buffering capacity required ~0.013 mmol HCl to change pH from 7.4 to 5.1; swelling order DMSO > DCM > THF > EtOAc, equilibrium ~2 h except DMSO ~5 h. - Cytotoxicity: At 100 µg/mL, PBAE-447 viability was 86 ± 4% vs. PEI 14 ± 5%. Cell viability remained >75% across HEK-293, BEAS-2B, and A549. - Nanoparticle stability: Non-PEGylated PBAE-447/pDNA aggregated in 10% FBS to ~567 nm after 4 h. PEGylated nanoparticles remained ~184 nm in FBS and ~175.1 nm in NaCl. Lyophilized nanoparticles retained size (~129.9 nm) and zeta (~+9.45 mV) after 4 months at 4 °C. - Transfection: A549 cells showed highest efficiency, above 66% and up to 72% with as-prepared nanoparticles; HEK-293 ~56%; BEAS-2B lower. Lyophilized and PEGylated nanoparticles showed no significant difference from as-prepared. PEI was slightly lower overall except in BEAS-2B. - Confocal uptake: 4 h incubation gave efficient cytoplasmic and nuclear localization; 2 h showed low uptake, while 6–8 h caused high cytotoxicity. - Hemagglutination: PEGylated PBAE-447/pDNA caused no red blood cell disruption.

Interpretation: PBAE-447 is biodegradable, cytocompatible, and has favorable buffering and swelling properties for gene delivery. PEGylation improves colloidal stability in serum/ionic conditions, and lyophilization maintains nanoparticle properties for at least 4 months. PBAE-447/pDNA nanoparticles outperform PEI in A549 and HEK-293 cells and show potential as stable nonviral vectors for gene therapy and gene editing applications.

Limitations: In vitro only: no in vivo animal, biodistribution, or efficacy data. - Limited cell panel: only HEK-293, BEAS-2B, and A549 cells. - No targeting ligand: delivery specificity not addressed. - Transfection quantification: semi-quantitative ImageJ analysis of fluorescence images. - PEI comparison: PEI may not have been fully optimized for each cell line. - Long-term safety: no chronic toxicity, immunogenicity, or repeat-dose evaluation. - Mechanism: endosomal escape and intracellular trafficking not directly measured beyond confocal localization. - Clinical translation: no pharmacokinetics, scale-up, or GMP manufacturing assessment.