Comparative Analysis of the Physicochemical and Biological Characteristics of Freeze-Dried PEGylated Cationic Solid Lipid Nanoparticles

Abstract

Cationic solid-lipid nanoparticles (cSLNs) have become a promising tool for gene and RNA therapies. PEGylation (PEG) is crucial in enhancing particle stability and protection. We evaluated the impact of PEG on the physicochemical and biological characteristics of cholesteryl-oleate cSLNs (CO-cSLNs). Several parameters were analyzed, including the particle size, polydispersity index, zeta potential, shape, stability, cytotoxicity, and loading efficiency. Five different formulations with specific PEGs were developed and compared in both suspended and freeze-dried states. Small, homogeneous, and cationic suspended nanoparticles were obtained, with the Gelucire 50/13 (PEG-32 hydrogenated palm glycerides; Gelucire) and DSPE-mPEG2000 (1,2-distearoyl-phosphatidylethanolamine-methyl-polyethyleneglycol conjungate-2000; DSPE) formulations exhibiting the smallest particle size (~170 nm). Monodisperse populations of freeze-dried nanoparticles were also achieved, with particle sizes ranging from 200 to 300 nm and Z potential values of 30-35 mV. Notably, Gelucire again produced the smallest particle size (211.1 ± 22.4), while the DSPE and Myrj S100 (polyoxyethylene (100) stearate; PEG-100 Stearate) formulations had similar particle sizes to CO-cSLNs (~235 nm). The obtained PEGylated nanoparticles showed suitable properties: they were nontoxic, had acceptable morphology, were capable of forming SLNplexes, and were stable in both suspended and lyophilized states. These PEG-cSLNs are a potential resource for in vivo assays and have the advantage of employing cost-effective PEGs. Optimizing the lyophilization process and standardizing parameters are also recommended to maintain nanoparticle integrity.

Keywords: PEG; SLNplexes; cSLNs; cationic solid lipid nanoparticles; freeze-drying; gene therapy; lyophilization; morphology; poly(ethylene glycol); stability.

Figure 1

Comparative analysis of the (A) particle size distribution (PSD), (B) polydispersity index (PdI), and (C) zeta potential (ZP; C) between PEGylated and non-PEGylated nanoparticles. CO represents cholesteryl oleate, corresponding to the formulation without PEG. Statistical analysis was performed in triplicate. Bars without an asterisk (*) are not significant (ns), * p = 0.01–0.05, ** p = 0.001–0.01 and *** p < 0.001.

Figure 2

Comparative analysis of the (A) particle size distribution (PSD), (B) polydispersity index (PdI), and (C) zeta potential (ZP) of PEGylated and non-PEGylated nanoparticles with two different protocols. CO represents cholesteryl oleate, corresponding to the formulation without PEG. Statistical analysis was performed in triplicate. Bars without an asterisk (*) are not significant (ns), * p = 0.01–0.05 and *** p < 0.001.

Figure 3

Comparative analysis of the (A) particle size distribution (PSD), (B) polydispersity index (PdI), and (C) zeta potential (Z) of suspended (Susp.) and freeze-dried (Lyo.) PEGylated and non-PEGylated nanoparticles. CO represents cholesteryl oleate, corresponding to the formulation without PEG. Statistical analysis was performed in triplicate. Not significant (ns), * p = 0.01–0.05, ** p = 0.001–0.01 and *** p < 0.001.

Figure 4

TEM images and morphological analysis of suspended and freeze-dried PEGylated and non-PEGylated nanoparticles. Abbreviations: Susp., nanoparticles suspended in an aqueous solution; Lyo., lyophilized nanoparticles; CO, cholesteryl oleate corresponding to the formulation without PEG. Gray arrows indicate particle size (PS).

Figure 4

TEM images and morphological analysis of suspended and freeze-dried PEGylated and non-PEGylated nanoparticles. Abbreviations: Susp., nanoparticles suspended in an aqueous solution; Lyo., lyophilized nanoparticles; CO, cholesteryl oleate corresponding to the formulation without PEG. Gray arrows indicate particle size (PS).

Figure 5

Polydispersity index (PdI) of the four PEGylated nanoparticles over a 30-day period at different temperatures (4 °C and 25 °C). Statistical analysis was performed in triplicate. Not significant (ns), * p = 0.01–0.05, ** p = 0.001–0.01 and *** p < 0.001.

Figure 6

Particle size distribution (PSD) of the four PEGylated nanoparticles over a 30-day period at different temperatures (4 °C and 25 °C). Statistical analysis was performed in triplicate. Statistical analysis was performed in triplicate. Not significant (ns), * p = 0.01–0.05, ** p = 0.001–0.01 and *** p < 0.001.

Figure 7

Zeta potential (ZP) of the four PEGylated nanoparticles over a 30-day period at different temperatures (4 °C and 25 °C). Statistical analysis was performed in triplicate. Not significant (ns), * p = 0.01–0.05, ** p = 0.001–0.01 and *** p < 0.001.

Figure 8

Polydispersity index (PdI) of the five PEGylated nanoparticles over one year at different temperatures (4 °C and 25 °C). Statistical analysis was performed in triplicate. Not significant (ns), * p = 0.01–0.05 and ** p = 0.001–0.01.

Figure 9

Particle size distribution (PSD) of the five PEGylated nanoparticles over one year at different temperatures (4 °C and 25 °C). Statistical analysis was performed in triplicate. Not significant (ns), * p = 0.01–0.05, ** p = 0.001–0.01 and *** p < 0.001.

Figure 10

Zeta potential (ZP) of the five PEGylated nanoparticles over one year at different temperatures (4 °C and 25 °C). Statistical analysis was performed in triplicate. Not significant (ns), * p = 0.01–0.05, ** p = 0.001–0.01 and *** p < 0.001.

Figure 11

Cell viability of HEK293T cells after 48 h of incubation with two different volumes (10 μL and 20 μL) of PEG-cSLNs in suspended and lyophilized states using flow cytometry. Abbreviations: PC, positive control; COS, cholesteryl oleate suspended; COL, cholesteryl oleate lyophilized; BS, Brij S100 suspended; BL, Brij S100 lyophilized; DS, DSPE suspended; DL, DSPE lyophilized; GS, Gelucire suspended; GL, Gelucire lyophilized; M52S, Myrj 52 suspended; M52L, Myrj 52 lyophilized; MS100S, Myrj S100 suspended; MS100L, Myrj S100 lyophilized.

Figure 12

Agarose gel electrophoresis of the five different PEG-cSLNs–DNA complexes in suspended and lyophilized states with increasing amounts of plasmid DNA. Image (A) corresponds to 500 ng of DNA, image (B) to 750 ng, image (C) to 1000 ng, and image (D) to 2000 ng. Lanes 1 and 9: DNA ladder; lanes 2 and 10: MHC luciferase plasmid; lanes 3 and 4: cholesteryl oleate suspended and lyophilized; lanes 5 and 6: DSPE suspended and lyophilized; lanes 7 and 8: Myrj S100 suspended and lyophilized; lanes 11 and 12: Gelucire suspended and lyophilized; lanes 13 and 14: Myrj 52 suspended and lyophilized; lanes 15 and 16: Brij S100 suspended and lyophilized.