Increased poly(ethylene glycol) density decreases transfection efficacy of siRNA/poly(ethylene imine) complexes

Rheiner, Steven; Bae, Younsoo

doi:10.3934/bioeng.2016.4.454

Cited by 8 publications

(8 citation statements)

References 29 publications

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“…The purity of PNAs were confirmed by GPC as shown in Supplementary Information (Figure S1). These results are consistent with our previous observations (26–28, 30, 31, 46), and indicate that conjugation of fluorescent dyes to the core of PNAs induces minimal changes to the uniformity and size of the nanoparticles used in this study.…”

Section: Resultssupporting

confidence: 94%

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Development of Halofluorochromic Polymer Nanoassemblies for the Potential Detection of Liver Metastatic Colorectal Cancer Tumors Using Experimental and Computational Approaches

et al. 2017

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Purpose To develop polymer nanoassemblies (PNAs) modified with halofluorochromic dyes to allow for the detection of liver metastatic colorectal cancer (CRC) to improve therapeutic outcomes. Methods We combine experimental and computational approaches to evaluate macroscopic and microscopic PNA distributions in patient-derived xenograft primary and orthotropic liver metastatic CRC tumors. Halofluorochromic and non-halofluorochromic PNAs (hfPNAs and n-hfPNAs) were prepared from poly(ethylene glycol), fluorescent dyes (Nile blue, Alexa546, and IR820), and hydrophobic groups (palmitate), all of which were covalently tethered to a cationic polymer scaffold [poly(ethylene imine) or poly(lysine)] forming particles with an average diameter < 30 nm. Results Dye-conjugated PNAs showed no aggregation under opsonizing conditions for 24 h and displayed low tissue diffusion and cellular uptake. Both hfPNAs and n-hfPNAs accumulated in primary and liver metastatic CRC tumors within 12 h post intravenous injection. In comparison to n-hfPNAs, hfPNAs fluoresced strongly only in the acidic tumor microenvironment (pH<7.0) and distinguished small metastatic CRC tumors from healthy liver stroma. Computational simulations revealed that PNAs would steadily accumulate mainly in acidic (hypoxic) interstitium of metastatic tumors, independently of the vascularization degree of the tissue surrounding the lesions. Conclusion The combined experimental and computational data confirms that hfPNAs detecting acidic tumor tissue can be used to identify small liver metastatic CRC tumors with improved accuracy.

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Section: Resultssupporting

confidence: 94%

“…These PNAs were synthesized according to previously reported methods (26–28, 30, 31, 46). Briefly, scaffolds (PEI or PLL) were reacted with NHS-activated PEG first in a mixed reaction solvent (water:DMSO:pyridine = 2:1:1) at room temperature for 2~3 days.…”

Section: Methodsmentioning

confidence: 99%

Development of Halofluorochromic Polymer Nanoassemblies for the Potential Detection of Liver Metastatic Colorectal Cancer Tumors Using Experimental and Computational Approaches

et al. 2017

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“…To achieve this, the hydrophobic component would be required to interact near the surface of the PNA. PNAs exist as a single polymer, core/shell systems regardless of the presence of siRNA, as evidenced by our previous study (Rheiner and Bae, 2016), and empty PNAs should behave similarly to complexed PNAs. Hydrophobic moieties are housed in the core of the PNA and the PEG shell should prevent their interaction while the PNA maintains its secondary structure, or folded state.…”

Section: Discussionsupporting

confidence: 65%

“…These PNAs displayed siRNA transfection efficiency comparable to PEG-PEI formulations developed by other research groups (Fella et al, 2008; Liu et al, 2013) as well as commercially available transfection reagents such as RNAiMAX. However, we have recently observed that PEG-PEI alters siRNA transfection among product batches partially due to PEG compositions such as molecular weight, substitution rate, and PEI contaminants (Rheiner and Bae, 2016). PEG-PEI also appeared to substantially change siRNA transfection efficiency before and after conjugation of hydrophobic pendant groups, although the mechanism remained elusive (Rheiner et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…Our previous findings suggest that the addition of hydrophobic pendant groups to PNAs would influence intracellular luciferase expression by either directly interacting with cells or indirectly disrupting the protein synthesis process (Rheiner and Bae, 2016; Rheiner et al, 2015). Therefore, this study elucidates the false positive effect of the hydrophobically modified PEG-PEI PNA on siRNA transfection by using PNAs made from combinations of PEG, PEI, poly(L-lysine) (PLL), palmitate (PAL), and deoxycholate (DOC): P EG- P EI (2P), P EG- P EI- P AL (3P), P EG- P LL (2P′), P EG- P LL- P AL (3P′), and P EG- P EI- D OC (2PD) as shown in Figure 1.…”

Section: Introductionmentioning

confidence: 99%

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Polymer nanoassemblies with hydrophobic pendant groups in the core induce false positive siRNA transfection in luciferase reporter assays

Rheiner

Reichel

Rychahou

et al. 2017

International Journal of Pharmaceutics

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Poly(ethylene glycol)-conjugated polyethylenimine (PEG-PEI) is a widely studied cationic polymer used to develop non-viral vectors for siRNA therapy of genetic disorders including cancer. Cell lines stably expressing luciferase reporter protein typically evaluate the transfection efficacy of siRNA/PEG-PEI complexes, however recent findings revealed that PEG-PEI can reduce luciferase expression independent of siRNA. This study elucidates a cause of the false positive effect in luciferase assays by using polymer nanoassemblies (PNAs) made from PEG, PEI, poly-(L-lysine) (PLL), palmitate (PAL), and deoxycholate (DOC): PEG-PEI (2P), PEG-PEI-PAL (3P), PEG-PLL (2P′), PEG-PLL-PAL (3P′), and PEG-PEI-DOC (2PD). In vitro transfection and western blot assays of luciferase using a colorectal cancer cell line expressing luciferase (HT29/LUC) concluded that 2P and 2P′ caused no luciferase expression reduction while hydrophobically modified PNAs induced a 35-50% reduction (3P′< 2PD < 3P). Although cell viability remained stagnant, 3P triggered cellular stress responses including increased membrane porosity and decreased ATP and cellular protein concentrations. Raman spectroscopy suggested that hydrophobic groups influence PNA conformation changes, which may have caused over-ubiquitination and degradation of luciferase in the cells. These results indicate that hydrophobically modified PEG-PEI induces cellular distress causing over-ubiquitination of the luciferase protein, producing false positive siRNA transfection in the luciferase assay.

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Double-grafted chitosans as siRNA nanocarriers: effects of diisopropylethylamine substitution and labile-PEG coating

et al. 2022

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Increased poly(ethylene glycol) density decreases transfection efficacy of siRNA/poly(ethylene imine) complexes

Cited by 8 publications

References 29 publications

Development of Halofluorochromic Polymer Nanoassemblies for the Potential Detection of Liver Metastatic Colorectal Cancer Tumors Using Experimental and Computational Approaches

Development of Halofluorochromic Polymer Nanoassemblies for the Potential Detection of Liver Metastatic Colorectal Cancer Tumors Using Experimental and Computational Approaches

Polymer nanoassemblies with hydrophobic pendant groups in the core induce false positive siRNA transfection in luciferase reporter assays

Double-grafted chitosans as siRNA nanocarriers: effects of diisopropylethylamine substitution and labile-PEG coating

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