Research Article: Biotinylated Bilirubin Nanoparticles as a Tumor Microenvironment‐Responsive Drug Delivery System for Targeted Cancer Therapy

Date Published: April 24, 2018

Publisher: John Wiley and Sons Inc.

Author(s): Yonghyun Lee, Soyoung Lee, Sangyong Jon.


The tumor microenvironment (TME) plays a crucial role in tumorigenesis and cancer cell metastasis. Accordingly, a drug‐delivery system (DDS) that is capable of targeting tumor and releasing drugs in response to TME‐associated stimuli should lead to potent antitumor efficacy. Here, a cancer targeting, reactive oxygen species (ROS)‐responsive drug delivery vehicle as an example of a TME‐targeting DDS is reported. Tumor targeting is achieved using biotin as a ligand for “biotin transporter”–overexpressing malignant tumors, and bilirubin‐based nanoparticles (BRNPs) are used as a drug‐delivery carrier that enables ROS‐responsive drug release. Doxorubicin‐loaded, biotinylated BRNPs (Dox@bt‐BRNPs) with size of ≈100 nm are prepared by a one‐step self‐assembly process. Dox@bt‐BRNPs exhibit accelerated Dox‐release behavior in response to ROS and show specific binding as well as anticancer activity against biotin transporter–overexpressing HeLa cells in vitro. bt‐BRNPs labeled with cypate, near‐infrared dye, show much greater accumulation at tumor sites in HeLa tumor‐bearing mice than BRNPs lacking the biotin ligand. Finally, intravenous injection of Dox@bt‐BRNPs into HeLa tumor‐bearing mice results in greater antitumor efficacy compared with free Dox, bt‐BRNPs only, and Dox@BRNPs without causing any appreciable body weight loss. Collectively, these findings suggest that bt‐BRNPs hold potential as a new TME‐responsive DDS for effectively treating various tumors.

Partial Text

Synthesis of PEGylated Bilirubin (PEG‐BR) and Biotin‐PEGylated Bilirubin (bt‐PEG‐BR): PEGylated bilirubin was prepared as described previously.[[qv: 8b]] Biotin‐PEGylated bilirubin was prepared by dissolving (ZZ)‐bilirubin‐IX‐alpha (0.5 mmol) (Tokyo Chemical Industry, Tokyo, Japan) and of EDC (0.4 mmol) [1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide; Sigma‐Aldrich, St. Louis, MO, USA] in dimethyl sulfoxide (5 mL) (DMSO). After stirring for 10 min at room temperature, biotin‐mPEG3,400‐NH2 (0.2 mmol) (Nanocs, Boston, MA), and triethylamine (150 µL) were added to the mixture, and the reaction was allowed to proceed with stirring for 4 h at room temperature under a nitrogen atmosphere. Chloroform (450 mL) was added to the reaction mixture, which was then washed with 0.1 m HCl and brine using a separation funnel. The organic layer was collected and concentrated under vacuum. For removal of free bilirubin, 45 mL of methanol was added to the concentrated reaction mixture and the solution was centrifuged at 2000 × g for 10 min, after which the resulting precipitate was discarded and the supernatant was evaporated. Ether was then added to the concentrated reaction mixture, and the resulting precipitate was dissolved in chloroform for subsequent purification by column chromatography on silica using chloroform:methanol (85:15) as the mobile phase. The solvents were evaporated to yield PEG‐BR, which was subsequently subjected to 1H‐NMR and MALDI‐TOF (matrix‐assisted laser desorption/ionization‐time of flight) spectroscopy. 1H‐NMR spectra were obtained using an AVANCE400 system (Bruker Daltonics, Bremen, Germany); chemical shifts represent ppm downfield from tetramethylsilane. MALDI‐TOF spectra were obtained using an Autoflex III MALDI‐TOF system (Bruker).

The authors declare no conflict of interest.




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