Development and in vitro evaluation of liposomes and immunoliposomes containing 5-fluorouracil and R-phycoerythrin as a potential phototheranostic system for colorectal cancer

5-Fluorouracil (5-FU) is the first-line drug for the treatment of colorectal cancer (CRC), which is considered the third most prevalent type of cancer in the world. R-phycoerythrin (R-PE) is a phycobiliprotein isolated from red algae such as Solieria filiformis , with fluorescent properties, photodynamic activity and potential for cancer treatment. However, 5-FU toxicity promotes several side effects and R-PE low stability hampers its clinical use. Thus, the present work aimed to develop co-encapsulated liposomes system for co-delivery of 5-FU and R-PE as theranostic nanosystems for CRC, as well as immunoliposomes targeted with the anti-EGFR monoclonal antibody, cetuximab, as a strategy for targeted delivery to EGFR-positive CRC. To the best of our knowledge, this is the first study to report the development and in vitro evaluation of liposomes and immunoliposomes co-encapsulating 5-FU and R-PE. Thus, liposomes containing 25 mg or 50 mg of soybean phosphatidylcholine (SPC), diesterolphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC) with cholesterol (Chol) and 1,2-distearol- sn -glycero-3-phosphoethanolamine- N -[amino(polyethylene glycol)-2000] (DSPE-PEG 2000) were prepared and characterized. Among the liposomes, those containing HSPC lipid at 50 mg showed a low polydispersity index (PDI) (0.100 ± 0.022), small size (103.43 ± 1.31 nm), and slightly negative zeta potential (−12.23 ± 0.35 mV). The encapsulation efficiency (EE%) was 94 ± 2.4% for R-PE and 42 ± 2.8% for 5-FU. Regarding the stability study, the liposomes maintained vesicle size, PDI and zeta potential values in a stable range. From the choice of the 50 mg HSPC liposome, the immunoliposomes were developed. The selected immunoliposomes, composed HSPC/DOPE/Chol/DSPE-PEG-Mal in a ratio of 64:10:22.2:3.7, were named HSPC IM 07. This formulation presented low PDI (0.185 ± 0.01), small vesicle size (99.45 ± 1.81 nm), negative zeta potential (−14.8 ± 0.81 mV) and antibody conjugation efficiency of 34.4%. Topographical AFM analysis showed that HSPC-IM-R-PE presented significantly higher surface roughness and viscoelastic contrast, indicating successful antibody anchoring. For cell viability in the HCT-116 CCR cell line, the IC 50 values for immunoliposomes were higher than those for liposomes. Also, for phototoxicity experiments it was found a reduction in IC 50 for all groups tested. The internalization of R-PE was verified, highlighting a greater internalization in the immunoliposome within 24 h. Thus, the HSPC 50 formulation containing R-PE and 5-FU, functionalized with cetuximab, is a promising alternative for the development of co-encapsulation delivery systems as a phototheranostic nanocarriers.