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A robust microfluidic technology and a new lipid composition for fabrication of curcumin-loaded liposomes: Effect on the anticancer activity and safety of cisplatin Nobuhito Hamano, Roland Böttger, Suen Ern Lee, Yang Yang, Jayesh A. Kulkarni, Shell Ip, Pieter R Cullis, and Shyh-Dar Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.9b00583 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019
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Molecular Pharmaceutics
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A robust microfluidic technology and a new lipid
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composition for fabrication of curcumin-loaded
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liposomes: Effect on the anticancer activity and
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safety of cisplatin
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Nobuhito Hamano†, Roland Böttger†, Suen Ern Lee†, Yang Yang†, Jayesh A.
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Kulkarni‡, Shell Ip#, Pieter R. Cullis‡, Shyh-Dar Li†*
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†Faculty
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V6T 1Z3, Canada
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‡Department
of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC
of Biochemistry and Molecular Biology, University of British Columbia,
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Vancouver, British Columbia, Canada
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#Precision
NanoSystems Inc, Vancouver, BC, Canada
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KEYWORDS
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Lipid nanoparticles, curcumin, cisplatin, enhanced efficacy and reduced toxicity,
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microfluidics
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Corresponding Author
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* 5519-2405 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3.
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Tel: 604-827-0675. Fax: 604-822-3035. E-mail:
[email protected].
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ABSTRACT
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Curcumin exhibits potent anticancer activity via various mechanisms, but its in vivo
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efficacy has been hampered by the poor solubility. Nanotechnology has been
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employed to deliver curcumin, but most of the reported systems suffered from low drug
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loading capacity and poor stability. Here, we report the development and optimization
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of a liposomal formulation for curcumin (Lipo-Cur) using an automated microfluidic
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technology. Lipo-Cur exhibited a mean diameter of 120 nm with a low polydispersity
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index (100 mg/kg, daily doses for 5
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days) for significant activities 15, likely due to its low bioavailability caused by the
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poor aqueous solubility (0.4 μg/ml) 16.
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To overcome the solubility problem and increase the systemic exposure, various
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nanotechnology-based delivery systems have been developed for curcumin, such as
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liposomes, polymer-based micelles and nanoemulsions 17–22. Among these systems,
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liposomes are attractive due to its high biocompatibility and established safety
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profile. However, the loading capacity of the reported liposomes or nanoemulsions
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for hydrophobic compounds like curcumin was typically 0.3, Fig 2B). In particular, curcumin-
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containing liposomes composed of DMPC/Tween 80 (95/5) displayed a mean
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diameter of 120 nm with a single peak size distribution, while the empty
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DMPC/Tween 80 liposomes exhibited a larger size (~300 nm) with multiple peaks in
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the size distribution profile (Fig 2C, D). The data suggest that curcumin could interact
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with the DMPC/Tween 80 bilayer and stabilized the formulation to yield drug-
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containing particles with a decreased size and improved uniformity. On the other
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hand, when curcumin was loaded within the DPPC/Tween 80 composition, the
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formulation aggregation was promoted (i.e. size increased from ~500 nm to 3,000
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nm). The DSPC/Tween 80 particles with or without curcumin displayed large
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aggregates (>2,000 nm). It was reported previously that curcumin affected the
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function of membrane proteins by modifying the thickness and the elastic property of
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the host lipid bilayer 35 and acting like cholesterol with a significant impact on the
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membrane stability 36,37. The study performed by Ali et al. 38 demonstrated that the
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lipid bilayer composed of a longer chain phospholipid displayed increased packing
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due to the enhanced interaction between the long acyl chains compared to the
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bilayer prepared with a shorter chain lipid. In other words, liposomal bilayer
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consisting of DMPC would exhibit increased flexibility to accommodate curcumin
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compared to that composed of DPPC or DSPC, and the increased curcumin-DMPC
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interaction led to a stabilized formulation.
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3.1.3. Microfluidic conditions
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We then pursued to optimize the microfluidic conditions to produce optimal curcumin
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nanoparticles using the DMPC/Tween 80 formulation. We conducted studies
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investigating the effect of changing the total flow rate and flow ratio on the physical
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characteristics (size and PDI) of the prepared liposomal curcumin. We tested three
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different total flow rates: 17 mL/min, 11 mL/min and 5 mL/min, and three flow ratios
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(solvent:aqueous): 1:5, 1:3 and 1:1. It is noted that the D/L ratio and the final drug
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concentrations were fixed for each run. As shown in Table 2, regardless of the total
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flow rate, when the ratio of organic (ethanol) to aqueous (PBS) phase was
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decreased (from 1:1 to 1:3 to 1:5), both size and PDI of the resulting formulations
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decreased. At the highest flow ratio (1:1), the liposomal size ranged between 532
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and 767 nm and the PDI was >0.65. When the flow ratio was decreased to 1:3, the
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particle size decreased to approximately 220 nm with a PDI of ~0.25. A final
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decrease of the flow ratio to 1:5 yielded even smaller liposomes with a mean
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diameter of ~125 nm and a PDI of ~0.1. The total flow rate, on the other hand, was
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not a critical parameter determining the particle size and PDI. The results were
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consistent with the previous reports 23,24. At a high organic to aqueous ratio (1:1), the
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final ethanol concentration in the liposomal product was high (50%). Liposomes
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might not be properly formed in the presence of such a high concentration of
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ethanol, and therefore, displayed significant aggregation after dialysis. In contrast, at
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a low ratio of organic to aqueous flow (1:5), the amount of the organic solvent in the
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mixture was reduced to
