• 2019-10
  • 2020-07
  • 2020-08
  • br Ethyl hydroxy camptothecin SN is an active metabolite of


    7-Ethyl-10-hydroxy camptothecin (SN38) is an active metabolite of irinotecan (CPT-11), a chemotherapeutic agent that induces cell death primarily by inhibiting topoisomerase I [1]. It is not used clinically due to poor solubility and high toxicity, despite its high potency. Polymeric nanoparticles (NPs) can potentially address these issues [2–4]. For ex-amples, NPs composed of PLGA and docetaxel (BIND-014) are in phase 2 clinical trials for the treatment of metastatic prostate cancer [5]. NPs have been shown to increase drug stability and circulation time in vivo [6,7]. In addition, tumor targeting by the enhanced permeability and retention (EPR) effect could markedly enhance therapeutic index and
    reduce drug toxicity of the drug in nanocarriers [8,9].
    In a previous study, poly (ethylene oxide)-poly (butylene oxide) (PEO-PBO) has been shown to form nanocarriers for delivery of pacli-taxel by our group. PEO-PBO NPs showed Marizomib superior biocompatibility and antitumor efficacy compared to PEG-PDLLA NPs [10,11]. It was re-ported that LA-SN38 could form NPs (SNPs) by self-assembly [12–14]. Herein, NPs based on PEO-PBO were developed for encapsulation of linoleic Marizomib conjugated SN38 (LA-SN38). The characterization of NPs, in vitro release study, cell uptake experiments and cytotoxicity had been carried out. In addition, in vivo experiment including the phar-macokinetics, biodistribution and antitumor activity were performed.
    Corresponding authors.
    1 Authors contributed equally to this work.
    2. Materials and methods
    Linoleic acid SN38 conjugate (LA-SN38) was kindly synthesized and provided by Dr. Hangxiang Wang at Zhejiang University. Poly(oxy-ethylene)-b-poly(oxybutylene)(PEO45-PBO20) was obtained from Advanced Polymer Materials Inc. (Montreal, Canada). DiR was pur-chased from Caliper Life Sciences (Hopkinton, MA). Cell Counting Kit CCK8 was purchased from Sigma-Aldrich (St. Louis, MO). Penicillin-streptomycin, RPMI1640, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and 0.25% (w/v) trypsin with 0.03% (w/v) EDTA solution were purchased from Hyclone. Acetonitrile (HPLC grade) was obtained from Hangzhou Hede Chemical Co., Ltd. (Hangzhou, China).
    EBNPs were prepared through a titration hydration method as re-ported previously [10]. Briefly, 40 mg PEO45-PBO20 and 3.6 mg LA-SN38 were dissolved in 6.0 mL acetonitrile as the organic phase. The organic phase was dripped into a 10 mL water phase (H2O/acetonitrile, v/v = 5:5) with stirring and stirring was continued for 10 min. Then the organic solvent was removed under reduced pressure. Finally, the so-lution was concentrated into 4.0 mL.
    2.3. EBNPs characterization
    Particle size and zeta potential (ξ) for the EBNPs were determined by Zetasizer Nano ZS90 (Malvern, Worcestershire, UK). Experiments were performed in triplicate with a 0.5 mg/mL EBNPs at 25 ℃. The morphology of EBNPs was investigated through negative staining with phosphotungstic acid (1.0% w/v) and imaged by transmission electron microscopy (TEM).
    2.4. LA-SN38 encapsulation efficiency and in vitro release studies
    To determine the encapsulation efficiency (EE) and the loading capacity (LC) of LA-SN38 in EBNPs, acetonitrile was added to EBNPs to disrupt the NP structure and release LA-SN38 prior to high-performance liquid chromatography (HPLC) analysis. EE% and LC% were respec-tively defined as the ratio between the amount of LA-SN38 detected by HPLC and amount of LA-SN38 added or the weight of EBNPs.
    The in vitro release property of LA-SN38 from EBNPs was in-vestigated by a dialysis method [15]. Briefly, 1.0 mL of EBNPs solution (1 mg SN-38 equivalent) diluted with phosphate buttered saline (PBS) (pH 7.4) to 10.0 mL. Then, 9.0 mL of solution was transferred into a dialysis bag (MWCO = 14 kDa). The dialysis bag was end-sealed and immersed into 50 mL dialysis buffer (PBS (pH7.4) containing 0.2% Tween 80) at 37℃ and shaken at the speed of 100 rpm. Aliquots of 1.0 mL were withdrawn from release medium and replaced with fresh dialysis buffer at regular time intervals (0, 1, 2, 4, 8, 12, 24, 36, 48 h). The rest of the solution (1.0 mL) was used to calculate the initial con-centration. The samples were filtered, and the concentration of LA-SN38 was measured by the HPLC method described above. The release kinetics was calculated by a linear calibration curve of LA-SN38.  Colloids and Surfaces B: Biointerfaces 181 (2019) 822–829
    2.5. Cellular uptake of EBNPs
    Human colon carcinoma HCT-116 and HT-29 cells were maintained in RPMI 1640 and DMEM supplemented with 1% penicillin, 1% streptomycin, and 10% FBS, respectively. RAW264.7 murine macro-phage cells were cultured in RPMI 1640 culture medium as above ex-cept that it was supplemented with 20% (v/v) FBS. All the cells were maintained in a humid atmosphere at 37 °C with 5% CO2.