The prevalence of anti-polyethylene glycol (PEG) antibodies poses a significant challenge for the clinical translation of PEGylated liposomes, leading to accelerated blood clearance (ABC) phenomenon and diminishing therapeutic effectiveness. To address this limitation, we designed an asymmetrical branched PEG derivative (mPEG2,5K-DSPE) and prepared mPEG2,5K-DSPE-modified liposomes, which included 1,1'-dioctadecyl-3,3,3,3'-tetramethylindo dicarbocyanine iodide liposomes (P2,5K-DiR) and mitomycin C lipid prodrug (MSC) liposomes (P2,5K-MSC) to minimize the binding of anti-PEG antibodies. Cellular binding assays revealed that the anti-PEG antibodies binding rate for P2,5K-DiR was only 0.91%, significantly lower than that of conventional linear P2K-DiR and P5K-DiR liposomes (99.1% and 79.9%). Pharmacokinetic studies further demonstrated that branched PEG-modified DiR and MSC liposomes effectively eliminated the ABC phenomenon: upon repeated administration, the ABCindex values for P2,5K-DiR and P2,5K-MSC were 0.820 and 1.153, respectively, markedly superior to those of linear mPEG2K-DSPE (0.080 and 0.099) and mPEG5K-DSPE (0.075 and 0.066). In vivo anti-tumor experiments, the tumor growth inhibition rates of P2,5K-DiR and P2,5K-MSC against B16 cells reached 94.6% and 93.89%, respectively, far exceeding those of the linear mPEG2K-DSPE groups (22.47% and 43.8%) and the mPEG5K-DSPE groups (37.8% and 59.17%). More importantly, the tumor inhibition index (TIindex) of the branched PEG groups reached 190.51 and 193.25, while the traditional linear PEG2K groups displayed much lower values (14.53 and 20.2) and the PEG5K group showed TIindex values of 17.98 and 27.46. These findings demonstrate that replacing the conventional linear PEG2K or PEG5K with the branched PEG2,5K can significantly enhance drug accumulation in tumor tissues while avoiding the non-selective systemic damage associated with traditional linear PEG. This study provides an innovative solution to overcome the immunogenicity challenges of PEGylated formulations, highlighting its significant potential for clinical translation.
