The aim of this study was to optimize the cationic PEGylated niosome-containing anti-cancer drugs and siRNA to enhance the therapeutic response. Therefore, various surfactant-based (tween-60) vesicles of doxorubicin (DOX; a chemotherapeutic drug) and quercetin (QC; a chemosensitizer) were prepared. To load siRNA on niosomes, 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) was used as a cationic lipid. The optimum formulation containing tween-60:cholesterol:DPPC:DOTAP:DSPE-PEG2000 at 49.5:5.5:15:25:5 demonstrated that the vesicle size and zeta potential were 52.8 ± 2.7 nm and +27.4 ± 2.3 mV, respectively. Entrapment efficiency (EE%) for DOX and QC was 86.4 ± 2.1% and 94.9 ± 3.9%, respectively. Moreover, the drug release during 6 h was 32.1 ± 1.6% and 30.5 ± 1.3% for DOX and QC, respectively denoted on the controlled release. The gel retardation assay demonstrated that siRNA could be successfully loaded into a cationic niosome:siRNA in a weight ratio 40:1. Additionally, noisome-encapsulated drugs had a higher toxicity against cancer cells when compared with un-encapsulated forms and the synergistic effects of co-delivery of siRNA and DOX with QC on gastric, prostate, breast cancer cells as well as human foreskin fibroblast as a normal cell line was shown. The results showed that the co-delivery of drugs and siRNA using cationic PEGylated niosomes exhibited an increased anti-cancer activity against the tumor cell death. It seems that cationic PEGylated niosomes have opened up a new avenue to enrich the armamentarium of therapeutic agents to fight cancer.

Doxorubicin is one of the most effective chemotherapeutic agents; however, it has various side effects, such as cardiotoxicity. Therefore, novel methods are needed to reduce its adverse effects. Quercetin is a natural flavonoid with many biological activities. Liposomes are lipid-based carriers widely used in medicine for drug delivery. In this study, liposomal doxorubicin with favorable characteristics was designed and synthesized by the thin-film method, and its physicochemical properties were investigated by different laboratory techniques. Then, the impact of the carrier, empty liposomes, free doxorubicin, liposomal doxorubicin, and quercetin were analyzed in animal models. To evaluate the interventions, measurements of cardiac enzymes, oxidative stress and antioxidant markers, and protein expression were performed, as well as histopathological studies. Additionally, cytotoxicity assay and cellular uptake were carried out on H9c2 cells. The mean size of the designed liposomes was 98.8 nm, and the encapsulation efficiency (EE%) was about 85%. The designed liposomes were anionic and pH-sensitive and had a controlled release pattern with excellent stability. Co-administration of liposomal doxorubicin with free quercetin to rats led to decreased weight loss, creatine kinase (CK-MB), lactate dehydrogenase (LDH), and malondialdehyde (MDA), while it increased the activity of glutathione peroxidase, catalase, and superoxide dismutase enzymes in their left ventricles. Additionally, it changed the expression of NOX1, Rac1, Rac1-GTP, SIRT3, and Bcl-2 proteins, and caused tissue injury and cell cytotoxicity. Our data showed that interventions can increase antioxidant capacity, reduce oxidative stress and apoptosis in heart tissue, and lead to fewer complications. Overall, the use of liposomal doxorubicin alone or the co-administration of free doxorubicin with free quercetin showed promising results.

Cationic liposomes have been investigated as non-viral vectors for gene delivery for more than a decade to overcome challenges associated with viral gene delivery. However, due to instability of liposomes, siRNA delivery is still a serious problem. In this study, we developed stealth PEGylated liposome formulations and focused on the effects of PEGylated liposomes on parameters related to size, zeta potential, polydispersity index, siRNA-loading efficiency and long-term stability of the siRNA-liposome complex. We were able to generate siRNA lipoplexes that could be very efficiently loaded, did not aggregate, could be stored at 4 °C for at least 6 months with only marginal release (1–5%) of siRNA and enhanced intracellular delivery of siRNA. Moreover, we could demonstrate that PEGylation positively contributed to all these parameters compared to liposomes, which were not PEGylated. The prepared lipoplex was successfully silenced J1P1 expression in MG-63 osteosarcoma cell line. In conclusion, our novel PEGylated liposomes have high potential for systemic delivery of siRNA and can improve in vivo stability of free siRNA and also siRNA lipoplexes. Graphical Abstract