Isolation methods commonly used to study the liposomal protein corona suffer from contamination issues

The liposomal protein corona has been the focus of numerous studies, but there is still no consensus regarding its extent and composition. Rather, the literature is full of conflicting reports on the matter. To elucidate whether there could be a methodological explanation for this, we here scrutinize the efficiency of three commonly used liposome isolation methods at isolating stealth liposomes from human plasma. Firstly, we show that size-exclusion chromatography (SEC) in its standard form is prone to isolating unbound protein material together with the liposomes, but also that the method may be optimized to mitigate this issue. Secondly, we demonstrate that SEC in combination with membrane ultrafiltration is no better at removing the unbound protein material than SEC alone. Thirdly, we show that centrifugation is not able to pellet the liposomes. Overall, our results suggest that previous research on the liposomal protein corona may have suffered from significant methodological problems, in particular related to contaminant proteins interfering with the analysis of the protein corona. We believe that the data presented here may help guide future research around this challenge to reach a converging understanding about the properties of the protein corona on liposomes. Statement of significance: Upon administration into the circulatory system, liposomal drug carriers encounter an environment rich in proteins. These proteins may adsorb to the liposomes to form what is known as the protein corona, potentially governing the interactions of the liposomes with tissues and cells. However, despite decades of intense research efforts, there is currently no clear understanding about the extent and composition of the liposomal protein corona, making it impossible to assess its mechanistic importance. Here we report that the methods commonly used to isolate liposomes from blood plasma or serum to study the protein corona are susceptible to protein contamination. This may be the underlying technical reason for the current confusion about the characteristics of the liposomal protein corona.