Purpose: Tissue engineering needs efficient, biocompatible non-viral gene delivery to mesenchymal stem cells (MSCs), which are inherently refractory to transfection. Existing vectors have limitations: PEI is cytotoxic and linear poly(L-lysine) (L-PLL) has poor transfection efficiency.
Hypothesis: If star-shaped poly(L-lysine) polypeptides (star-PLLs) are varied in arm number and arm length, then their pDNA condensation, nanomedicine stability, cellular uptake, and transfection efficiency in MSCs will differ systematically. A star-PLL with 64 poly(L-lysine) arms and 5 L-lysine subunits per arm will act as an efficient, low-cytotoxicity vector for MSC transfection.
Aims: Synthesize and characterize three architectural variants of star-PLL: 16-star-PLL, 32-star-PLL, and 64-star-PLL. - Assess their ability to self-assemble with pDNA into stable, cationic nanomedicines and define optimal N/P ratios. - Evaluate intracellular uptake, uptake mechanism, transfection efficiency, and cytotoxicity in MSCs. - Test delivery of therapeutic transgenes (pBMP-2 and pVEGF) and compare performance against PEI and linear PLL.
Delivery system:
Component: Vector class; Details: Star-shaped poly(L-lysine) polypeptides (star-PLLs) synthesized by NCA ring-opening polymerization from PPI dendrimer cores
Component: Variants; Details: 16-star-PLL: G3 PPI core, 16 arms, 40 L-lysine subunits/arm, MW 134,000 g/mol
Details: 32-star-PLL: G4 PPI core, 32 arms, 40 subunits/arm, MW 318,000 g/mol
Details: 64-star-PLL: G5 PPI core, 64 arms, 5 subunits/arm, MW 94,000 g/mol
Component: Payload; Details: Plasmid DNA: pGLuc (Gaussia luciferase), pGFP, pBMP-2, pVEGF
Component: Complexation; Details: Electrostatic self-assembly in molecular-grade water; N/P ratios 2–10
Component: Targeting ligand; Details: None
Component: Controls; Details: PEI-pDNA N/P 7; L-PLL-pDNA mass ratio 2:1; naked pDNA
Component: Key feature; Details: Biodegradable polypeptide architecture; high arm density for pDNA condensation and endosomal buffering
Approach: In vitro only: Rat MSCs (passage 5) cultured in DMEM + 10% FBS. No in vivo studies. - Transfection formats: 6-well, 96-well, and 12-well plates; 4 h incubation in serum-free OptiMEM, then replaced with complete media. - Doses: pDNA 0.06–5 µg depending on assay; typically 1 µg for star-PLL and L-PLL, 2 µg for PEI. - Group structure: n = 3 independent repeats, each run in triplicate. Controls included PEI-pDNA, L-PLL-pDNA, and untreated cells. - Disease context: Bone tissue engineering; therapeutic transgenes BMP-2 and VEGF.
Key methods: Physicochemical characterization: Nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), zeta potential, circular dichroism (CD), acid-base titration for buffering capacity. - Stability: DNase I and heparan sulfate challenge; storage at 2–8°C and lyophilization. - Uptake and transfection: High-content screening (HCS) with Cy3-labeled pDNA; flow cytometry for GFP; luciferase assay for pGLuc. - Therapeutic protein production: ELISA for BMP-2 and VEGF. - Cytotoxicity: MTT assay; Live/Dead imaging. - Uptake mechanism: Energy depletion (4°C, 20°C); endocytosis inhibitors genistein, methyl-β-cyclodextrin (mβ-CD), chlorpromazine; live-cell imaging.
Key results: Transfection efficiency (pGFP, day 7): 64-star-PLL = 24.6 ± 0.7%; 32-star-PLL = 22.3 ± 15.1%; 16-star-PLL = 2.0 ± 0.2%; L-PLL = 2.5 ± 0.5%. - Luciferase expression: 64-star-PLL-pGLuc (1 µg, N/P 5) reached 5.5 × 10⁶ ± 7.7 × 10⁵ RLU at day 7—comparable to PEI-pGLuc (2 µg, N/P 7) and ~1000-fold higher than L-PLL. - Uptake (HCS, Cy3 spots/cell): 32-star-PLL at N/P 10 = 20.73 ± 1.5; 16-star-PLL at N/P 20 = 16.55 ± 3.0; 64-star-PLL at N/P 5 = 13.93 ± 8.6—all higher than PEI. - Cytotoxicity: At 24 h, 64-star-PLL retained 57 ± 6.8% metabolic activity vs PEI 35 ± 7%; PEI fell to 19 ± 11% at 168 h, while star-PLLs recovered at 72 and 168 h. - Uptake mechanism: Energy-dependent; 9-fold reduction at 20°C and 13-fold at 4°C. Genistein and mβ-CD reduced transfection; chlorpromazine had no effect → clathrin-independent uptake. - Therapeutic protein production (14 days): 64-star-PLL-pBMP-2 = 184.30 ng cumulative vs PEI-pBMP-2 = 133.95 ng and L-PLL-pBMP-2 = 0.41 ng. 64-star-PLL-pVEGF = 8.18 ng vs PEI-pVEGF = 7.91 ng and L-PLL-pVEGF = 0.07 ng. - Stability: Star-PLL-pDNA nanomedicines stable for 14 days at 2–8°C and after lyophilization with sucrose; resistant to DNase I and heparan sulfate.
Interpretation: The authors claim that star-PLLs, especially the 64-arm/5-subunit variant, represent a versatile, biocompatible, non-viral polypeptide nanomedicine platform for efficient nucleic acid delivery to MSCs, achieving transgene expression comparable to PEI at lower pDNA doses and with significantly less cytotoxicity. They propose this platform has significant potential for tissue engineering applications.
Limitations: In vitro only: No in vivo validation or animal disease models were performed. - Single MSC source: Only rat MSCs were tested; human MSC data are absent. - No targeting ligand: Delivery relies on nonspecific electrostatic interactions. - Moderate transfection efficiency: Peak GFP transfection was ~25%, which is within tissue-engineering norms but still below viral vector efficiency. - Uptake mechanism data are shown primarily for the 64-star-PLL; other variants are stated to be similar but not fully presented. - Long-term safety and biodistribution were not assessed. - Just Accepted manuscript: The final peer-reviewed version may differ in minor details.
