Cancer cell membrane-modified Soluplus® micelles for gemcitabine delivery to pancreatic cancer using a prodrug approach

Pancreatic cancer (PC) is one of the most lethal malignancies worldwide and its incidence is increasing. Chemotherapy is often associated to limited efficacy, poor targeting and systemic toxicity. In this work, the hydrophilic gemcitabine (GEM), widely used in PC treatment alone or in combination, was conjugated with vitamin E succinate (VES) and encapsulated in Soluplus® micelles. This prodrug approach facilitated encapsulation of the anticancer drug into the self-assembled copolymer micelles. Soluplus®/VES-GEM micelles were optimized regarding the ratio of the components and the preparation process. The micelles were small-sized (<80 nm), monodisperse, and highly stable, efficiently retaining the conjugate drug and showing significant antiproliferative activity against BxPC3 cell line.

Highlights

• Lipophilic conjugate of gemcitabine with vitamin E succinate (VES-GEM) was encapsulated in Soluplus® micelles.
• The micelles strongly retained the conjugate when exposed to dilution in different pH media.
• Soluplus®/VES-GEM micelles were hemocompatible but cytotoxic to pancreatic cancer cells.
• BxPC3 cancer cell membrane nanovesicles were produced following various protocols.
• The hydrophilic shell of micelles made cancer cell membrane coating difficult.

To improve biofunctionalization and targeting properties of prepared Soluplus®/VES-GEM micelles, biomimetic modification with PC cell membrane was further attempted by co-extruding PC cell membrane (BxPC3) nanovesicles with Soluplus®/VES-GEM micelles. Several protocols were attempted to prepare the BxPC3-modified Soluplus®/VES-GEM micelles and the outcomes were analyzed in detail. Overall, the results pave the way to innovative PC-targeted nanotherapies by maximizing GEM encapsulation in hydrophobic compartments with high stability and affinity. The results also highlight the need of higher resolution techniques to characterize cell membrane coating of nanocarriers bearing highly hydrophilic shells.

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Materials

Gemcitabine hydrochloride (GEM·HCl, C9H11F2N3O4·HCl) (MW 299.66 g/mol), Triton-X-100, poly(lactic-co-glycolic acid) nanoparticles (PLGA, 0.67 dL∕g carboxy-terminated 50:50, MW 24,000–38,000), bovine serum albumin (BSA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine labelled with Atto 488 (DOPE-Atto 488) and RPMI-1640 medium supplemented with 10 % of FBS and 1 % antibiotics were purchased from Sigma-Aldrich, St. Louis, MO, USA. Vitamin E succinate (VES) (MW 530.80 g/mol) was purchased from Santa Cruz Biotechnology Inc., Dallas, TX (USA); Soluplus® (polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer) (MW ∼ 115,000  g/mol) was purchased from BASF®, Ludwigshafen, Germany; sodium 1-heptanesulfonate (MW 220.26 g/mol) was purchased from Fluka Chemie, Buchs, Switzerland; di-sodium hydrogen phosphate di-hydrate (Na2HPO4·2H2O); ethanol absolute with purity > 99.9 %, methanol (HPLC grade) and sodium hydroxide (NaOH) were purchased from VWR Chemicals, Leuven, Belgium; sodium dihydrogen phosphate anhydrous AGR (NaH2PO4) and sodium chloride (NaCl) were purchased from Labkem, Barcelona, Spain; potassium chloride (KCl) and potassium di-hydrogen phosphate (KH2PO4) were from Panreac AppliChem, Barcelona, Spain. Ultrapure water was continuously obtained from Milli-Q® Benchtop Lab Water Purification System (Millipore Ibérica, Madrid, Spain). Hydrophilic polytetrafluoroethylene (PTFE) syringe filters (13 mm, 0.2 µm; 25 mm, 0.4 µm), tris hydrochloride (Tris-HCl) and magnesium chloride (MgCl2) were purchased from Scharlab S.L., Barcelona, Spain. EDTA-free Pierce Protease Inhibitor Tablets were obtained from Thermo Fisher™, Madrid, Spain. 4′,6-Diamidino-2-phenylindole (DAPI), Nile red and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) were purchased from Invitrogen™, Thermo Fisher™, Madrid, Spain. Nylon membrane discs (47 mm, 0.2 µm) were obtained from Pall Corporation, Waters, Ann Arbor, Michigan, USA. Phosphate buffered saline (PBS, 0.01 M, pH = 7.4) was prepared with 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4·2H2O, and 0.24 g/L KH2PO4. All additional reagents were of analytical or HPLC grade and used following the manufacturer’s instructions.

Miguel Pereira-Silva, Luis Diaz-Gomez, Bárbara Blanco-Fernandez, Alba Ferreirós, Francisco Veiga, Angel Concheiro, Ana Cláudia Paiva-Santos, Carmen Alvarez-Lorenzo, Cancer cell membrane-modified Soluplus® micelles for gemcitabine delivery to pancreatic cancer using a prodrug approach, International Journal of Pharmaceutics, Volume 662, 2024, 124529, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2024.124529.

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