ABSTRACT
Lipid-polymer nanoparticles offer a promising strategy for improving gene nanomedicines by combining the benefits of biocompatibility and stability associated with the individual systems. However, research to date has focused on poly-lactic-co-glycolic acid (PLGA) and resulted in inefficient transfection. In this study, biocompatible Eudragit constructs E100 and RS100 were formulated as lipid-polymer nanoparticles loaded with pDNA expressing red fluorescent protein (RFP) as a model therapeutic. Using a facile nanoprecipitation technique, a core-shell structure stabilised by lipid-polyethylene glycol (PEG) surfactant was produced and displayed resistance to ultracentrifugation. Both cationic polymers E100 (pH-sensitive dissolution at 5) and RS100 (pH-insensitive dissolution) produced 150-200â¯nm sized particles with a small positive surface charge (+3-5â¯mV) and high pDNA encapsulation efficiencies (EE) of 75-90â¯%. The dissolution properties of the Eudragit polymers significantly impacted the biological performance in human embryonic kidney cells (HEK293T). Nanoparticles composed of polymer RS100 resulted in consistently high cell viability (80-100â¯%), whereas polymer E100 demonstrated dose-dependent behaviour (20-90â¯% cell viability). The low dissolution of polymer RS100 over the full pH range and the resulting nanoparticles failed to induce RFP expression in HEK293T cells. In contrast, polymer E100-constructed nanoparticles resulted in reproducible and gradually increasing RFP expression of 26-42â¯% at 48-72â¯h. Intraperitoneal (IP) injection of the polymer E100-based nanoparticles in C57BL/6 mice resulted in targeted RFP expression in mouse testes with favourable biocompatibility one-week post-administration. These findings predicate Eudragit based lipid-polymer nanoparticles as a novel and effective carrier for nucleic acids, which could facilitate pre-clinical evaluation and translation of gene nanomedicines.
ABSTRACT
Nanoparticles have gained considerable attention as one of the pollutants released into the environment through consumer products. This study describes the sub-chronic and generational effects of TiO2 (rutile) nanoparticles on earthworms over a 252-day duration, with exposure ranging from 0.1 to 1000 mg kg-1. Results indicate that sub-chronic exposure (28 days) of TiO2 nanoparticles did not cause notable adverse effects on the weight, reproduction, and tissue accumulation in parent earthworms. However, the F1 generation displayed remarkable growth and maturity retardation during their early developmental stages, even at lower nano-TiO2 (rutile). Significant impacts on the reproduction of the F1 generation were observed solely at the highest concentration (1000 mg kg-1), which is predicted to be below the highest exposure scenario. Moreover, long-term (252 days) exposure resulted in considerable bioaccumulation of Ti metal in the F1 generation of E. fetida. This study uncovers the negative effects of TiO2 rutile nanoparticles on earthworms across two generations, with pronounced effects on the growth, maturity, and bioaccumulation in the F1 generation compared to the parent generation. These findings suggest the potential induction of toxic effects by TiO2 rutile nanoparticles, emphasizing the sensitivity of juvenile parameters over adult parameters in toxicity assessments. Furthermore, the study highlights the urgent need for comprehensive evaluations of the longer-term toxicity of nanoparticles on terrestrial organisms. Implementing multigenerational studies will contribute significantly to a better understanding of nanoparticle ecotoxicity on environmental organisms.
ABSTRACT
Lipid/polymer hybrid nanoparticles loaded with red fluorescent protein (RFP) encoded plasmid DNA (pDNA) was formulated using poly-lactic-co-glycolic acid (PLGA), cationic lipid DC-cholesterol and surfactant mPEG2000- DSPE. A lipid/ polymer ratio of 1: 10 at 1 mg/mL surfactant concentration was found to be optimal for producing nanoparticles with diameters of 100-120 nm that remained stable upon ultracentrifugation. The production of lipid/ polymer hybrid nanoparticles was investigated using microfluidics with a toroidal mixer design. Our results showed that the flow parameters significantly influenced the physicochemical characteristics of nanoparticles and loading of pDNA was only achieved at flow rate ratio (FRR) of 3: 1. The pDNA associated with nanoparticles was demonstrated to be structurally intact using gel electrophoresis, and the encapsulation efficiency (EE) was measured to be â¼65%. The prepared hybrid nanoparticles resulted in 20% of transfection efficacy in human embryonic kidney cells (HEK293T). This study demonstrated the potential of microfluidics in the development of hybrid nanoparticles for pDNA delivery, thus facilitating the clinical translation of DNA therapeutics.
