Abstract Tumor-targeted delivery of CRISPR/Cas9 plasmid or siRNA to inhibit immune checkpoint protein expression in tumor cells has the potential to improve anticancer immune responses. Cationic solid lipid nanoparticles (cSLNs) are promising carrier for the delivery of negatively charged nucleic acids. cSLNs with high surface charge and low PEG density have been demonstrated to enhance transfection efficiency in vitro, but for unknown reasons fail to improve gene delivery in vivo. We prepared 9 kinds of cSLNs with varying surface charge and PEG density and detected the interactions of these cSLNs with blood cells. We found that increasing surface charge and/or reducing PEG density present a risk for systemic platelet aggregation and activation. Thrombosis was clearly detected in liver, lung and kidney from mice treated with cSLNs with high surface charge and low PEG density. Mechanistic studies revealed that the intrinsic coagulation pathway was responsible for cSLN-induced thrombus formation. Furthermore, pretreatment of recipient mice with heparin, a clinically-approved intrinsic coagulation inhibitor, was highly effective in preventing toxicity, prolonging the circulation time, improving tumor accumulation and enhancing the tumor cell uptake in tumor-bearing mice. These results indicate that the thrombotic toxicity of cSLNs is an important factor affecting their drug delivery efficiency in vivo, and that heparin treatment offers a useful strategy for improving cSLN-based cancer immunotherapies.