Stearyl polyethylenimine complexed with plasmids as the core of human serum albumin nanoparticles noncovalently bound to CRISPR/Cas9 plasmids or siRNA for disrupting or silencing PD-L1 expression for immunotherapy.
Purpose
In this study, a double emulsion method for complexing plasmids with stearyl poly-ethylenimine (stPEI) as the core to form human serum albumin (HSA) (plasmid/stPEI/HSA) nanoparticles (NPs) was developed for gene delivery by non-covalently binding onto plasmid/stPEI/HSA nanoparticles with CRISPR/Cas9 or siRNA, which disrupts or silences the expression of programmed cell death ligand-1 (PD-L1) for immunotherapy.


Materials and methods
Chemically synthesized stearyl-polyethyenimine (stPEI)/plasmids/HSA nanoparticles were maded by double emulsion method. They were characterized by dynamic light scattering (DLS), transmission electron microscope and also evaluated by in vitro study on CT 26 cells.


Results
stPEI was synthesized by an N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS) reaction, and we found that the degree of substitution was ~1.0 when the ratio of PEI to stearic acid was 1:7 in the reaction. Then, two sgRNA sequences were selected and evaluated for their ability to knock out PD-L1 by decreasing its expression by about 20%. Based on the trend of particle size/zeta potential values as a function of ratio, F25P1 containing 25 μg of plasmid/stPEI/HSA NPs noncovalently bound to 1 μg plasmids via charge-charge interactions was found to be optimal. Its particle size was about 202.7±4.5 nm, and zeta potential was 12.60±0.15 mV. In an in vitro study, these NPs showed little cytotoxicity but high cellular uptake. Moreover, they revealed the potential for transfection and PD-L1 knockout in an in vitro cell model. Furthermore, F25P1S0.5 containing 25 μg of plasmid/stPEI/HSA NPs noncovalently bound to 1 μg of plasmids and 0.5 μg siRNA was prepared to simultaneously deliver plasmids and siRNA. An in vitro study demonstrated that the siRNA did not interfere with the transfection of plasmids and showed a high-transfection efficiency with a synergistic effect on inhibition of PD-L1 expression by 21.95%.


Conclusion
The plasmids/stPEI/HSA NPs could be a promising tool for gene delivery and improved immunotherapy.
['Cheng|Wei-Jie|WJ|', 'Chen|Ling-Chun|LC|', 'Ho|Hsiu-O|HO|', 'Lin|Hong-Liang|HL|', 'Sheu|Ming-Thau|MT|']
D000818:Animals D060890:B7-H1 Antigen D000076987:CRISPR-Associated Protein 9 D064113:CRISPR-Cas Systems D016923:Cell Death D045744:Cell Line, Tumor D002470:Cell Survival D004705:Endocytosis D020868:Gene Silencing D006801:Humans D007167:Immunotherapy D051379:Mice D053758:Nanoparticles D010316:Particle Size D010957:Plasmids D011094:Polyethyleneimine D066244:Proton Magnetic Resonance Spectroscopy D034741:RNA, Small Interfering D000075462:Serum Albumin, Human D055672:Static Electricity D013229:Stearic Acids D014162:Transfection
stearyl polyethylenimine human serum albumin nanoparticles CRISPR Cas9 siRNA PD-L1 gene delivery