(T1230-06-35) Lectin Conjugated Liposome Nanocarriers for Targeted Bladder Cancer Therapy

Purpose: Glycosylation is one of the key regulatory mechanisms on cells related to several pathophysiological processes. Alteration on glycosylation is often associated with diseases like oncogenic transformation [1,2]. N-glycan branching is one of the most-widely occurring cancer-associated changes in glycosylation on cells. An increased abundance of high mannose N-glycans in cancer cells has now been described in several studies [3,4]. Lectins are carbohydrate-binding proteins that have been investigated in the oncology realm as diagnostic and therapeutic agents. A high-mannose N-glycan specific lectin, cyanovirin-N homologue (Lectin), derived from the cyanobacterium Cyanothece7424 were engineered for cancer cell targeting [5]. Liposome nanoparticles (LP NPs), composed of self-assembling phospholipid bilayers, are effective in drug delivery due to their biodegradability, biocompatibility, and ease of surface modification. In this study, we aim to develop lectin-modified, drug-loaded liposome nanoparticles for targeted treatment of bladder cancer.

Methods: The expression of high-mannose N-glycans on bladder cancer cell lines was first evaluated by Alexa Fluor 647-labeled lectin (Lectin-AF647). Lectin was modified at its C-terminus with azide (Lectin-N₃) via sortase-mediated ligation. LP NPs were prepared by microfluidics using egg phosphatidylcholine (eggPC), cholesterol, and DSPE-PEG-DBCO at different ratios and lipid concentrations. Lectin-N₃ was conjugated to the LP NPs via copper-free click chemistry at DBCO: N₃ molar ratios of 10:1, 20:1, and 40:1 to generate Lectin-LP10, 20, and 40, respectively. Coumarin-6 was loaded as a fluorescent tracer to evaluate cellular uptake in T24 bladder cancer cells by flow cytometry.

Results: T24 bladder cancer cells exhibited the highest expression of high-mannose N-glycans, as determined by Lectin-AF647 binding. DBCO-functionalized LP NPs (DBCO-LP) were then prepared by microfluidic mixing of eggPC, cholesterol, and DSPE-PEG-DBCO at two different molar ratios (56.5:38.5:5 and 88.5:10:1.5) and lipid concentrations (3.54 mM or 8.29 mM). The optimal formulation was obtained at a lipid concentration of 3.54 mM, a flow rate of 5 ml/min, and a molar ratio of 88.5:10:1.5, resulting in NPs with a mean diameter of ~45.8 nm and a minimum polydispersity index (PDI) of 0.21 (Figure 1). Subsequently, Lectin-N₃ was conjugated to DBCO-LP in phosphate buffer (1 mM, pH 7.4) at varying DBCO: N₃ molar ratios to generate Lectin-LP10, Lectin-LP20, and Lectin-LP40, respectively. Coumarin-6 loaded DBCO-LP and Lectin-LP nanoparticles were incubated with T24 cells to assess cellular uptake. At a coumarin-6 concentration of 40 ng/mL, Lectin-LP40 demonstrated significantly higher uptake by T24 cells compared to DBCO-LP, indicating effective high mannose N glycan-mediated targeting ability Figure 2).

Conclusion: Lectin-modified liposome nanoparticles demonstrated effective targeting of bladder cancer cells with high expression of high-mannose N-glycans. This platform showed high potential the development of targeted bladder cancer treatment.

References: [1] Ladenson RP, Schwartz SO, and Ivy AC. Am. J. Med. Sci.1949 217(2):194-7.
[2] Hakomori SI, Murakami WT. Proc. Natl. Acad. Sci. 1968 59(1):254-61.
[3] Powers S, Holst M, Wuhrer AS, Mehta RR, Drake. Biomol. Eng. 2015 5: 2554–72.
[4] Takayama H, Ohta M, Iwashita Y, Uchida H, Shitomi Y, Yada K, Inomata M. BMC Cancer. 2020 20(1):192.
[5] Kurhade SE, Weiner JD, Gao FP, Farrell MP. Angew. Chem. Int. Ed. 2021 60(22):12313 –18.

Acknowledgements: This study was supported by the National Institutes of Health (P20GM113117 and UL1TR002366).


Figure 1. Hydrodynamic size and polydispersity index (PDI) of liposomes prepared with eggPC: Cholesterol: DSPE-PEG-DBCO at molar ratios of (A) 56.5:38.5:5 at a total lipid concentration of 3.54 mM, (B) 88.5:10:1.5 at a lipid concentration of 8.29 mM, and (C) 88.5:10:1.5 at a lipid concentration of 3.54 mM.




Figure 2. Mean fluorescence intensity (MFI) after incubating T24 cells with coumarin-6 loaded Lectin-LP nanoparticles at various concentrations (5, 10, 20, 40 ng/mL). DBCO-LP refers to liposome nanoparticles containing the DBCO group without Lectin modification, while Lectin-LP 10, 20, and 40 represent molar ratios of DBCO to Lectin-N3 at 10:1, 20:1, and 40:1, respectively.