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

Abstract

PurposeIn 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 methodsChemically 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.ResultsstPEI 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%.ConclusionThe plasmids/stPEI/HSA NPs could be a promising tool for gene delivery and improved immunotherapy.

Polyethylenimine based magnetic nanoparticles mediated non-viral CRISPR/Cas9 system for genome editing

“…Furthermore, lanes 3 and 4 are used as the controls of pure plasmids T0 and T3. These results are in accordance with previously published data 37,38 .…”

<p>Synthesis and characterization of nanometer-sized liposomes for encapsulation and microRNA transfer to breast cancer cells</p>

“…There are multiple non-viral vectors, such as polyethylenimine (PEI) and polyamidoamine (PAMAM), which are currently under development for delivery of anti-cancer drugs and gene therapy 36,37. However, liposome-based methods offer several advantages to these approaches.…”

“…and can be fine-tuned to provide rapid or delayed release.38 Additionally, the inherent cytotoxicity of liposomes is reduced compared to PEI or PAMAM. The efficiency of payload delivery in PEI or PAMAM is directly dependent on the molecular weight of the polymers with higher molecular weights exhibiting greater efficiency.39 However, this increased efficiency comes at a price as the larger PEI or PAMAM polymers also have a higher cytotoxicity compared to liposomes which are considered more biologically inert 36,37,40. PEI and PAMAM are also less biodegradable, which is an additional contributing factor to in vivo toxicity.…”

Modification of primary amines to higher order amines reduces in vivo hematological and immunotoxicity of cationic nanocarriers through TLR4 and complement pathways

“…The PD-L1 gene was targeted in B16-F10 melanoma cells, which is an intriguing therapeutic target that could potentially reduce tumor-related immune suppression. [32] PD-L1 was first induced in melanoma cells through treatment with interferon-g at a concentration of 50 ng/mL. Treatment of PD-L1-induced cells with unmodified bPEI or chitosan nanoparticles yielded two observations.…”