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  • br Corresponding author at Moo Amphoe

    2020-07-06


    Corresponding author at: 99 Moo 9, Amphoe Muang, Phitsanulok 65000, Thailand. E-mail address: [email protected] (W. Tiyaboonchai).
    Herbal drugs or traditional medicine have been used for a long time in Asia with impressive therapeutic activities. Recently, α-mangostin, obtained from the pericarp of the mangosteen (Garcinia mangostana Linn), shows potential antitumor effects in various kinds of cancers such as breast, colon, skin, lung, and blood [13–17]. The safety of α-man-gostin was confirmed with no detectable unwanted effect at oral dosage up to 80 mg/kg in animal model [18]. Furthermore, a bioavailability clinical trial conducted on 10 healthy adult participants detected no toxicity at the oral dose of approximately 61.5 mg/day [19]. The big-gest drawback of α-mangostin is the low water solubility of 0.2 ± 0.2 μg/mL [20], which leads to a low oral bioavailability in mice [21]. Another disadvantage of α-mangostin is its high hematotoxicity due to the strong surfactant-like action [22]. Over 50% of the human red blood A 61603 were lysed in its antitumor effective dose of 15 μg/mL [23].
    Therefore, to overcome the mentioned problems, both in the che-motherapy issues and α-mangostin itself, α-mangostin loaded cross-linked FNPs were developed for injectable cancer treatment, focus on breast and colon cancers. These particles were prepared using simple desolvation method, followed by characterizations including size, zeta potential, entrapment efficiency (EE%), drug loading capacity (DL%), morphology, drug crystallinity, and dissolution profiles. The impact of intravenous diluent on the FNP properties was also investigated. Additionally, in vitro hemolysis activity in red blood cells, cytotoxicity and DNA fragmentation in Caco-2 colorectal and MCF-7 breast cancer cell lines were studied. Finally, the physicochemical stabilities of FNPs were determined in storage conditions up to 6 months.
    2. Materials and methods
    Bombyx mori silkworm cocoons were collected from Bodin Thai Silk Khorat Co., Ltd, Nakhon Ratchasima, Thailand. Standardized α-man-gostin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and poly(ethylenimine) (branched PEI, 25 KDa) were bought from Sigma-Aldrich, Singapore. Sheep whole blood was supplied by Terumo Corporation, Thailand. Caco-2 (HTB37™) and MCF-7 (HTB22™) were purchased from American Type Culture Collection (ATCC), USA. Dulbecco’s modified Eagle’s medium (DMEM-F12), fetal bovine serum (FBS) and trypsin-EDTA 0.25% were bought from Gibco, USA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) was imported from Amresco, USA. Agarose gel (low electroendosmosis) was bought from Research Organics, Thailand. Loading dye and DNA marker (100 bp ladder) were supplied by RBC Bioscience, Thailand. Other chemicals and solvents used are of analytical grade or higher.
    The processes of silk degumming, fibroin extraction and purification were previously described [11,12]. Briefly, the cocoons were de-gummed in 0.5% (w/v) Na2CO3 at 100 °C for 1 h, followed by washing thrice with de-ionized (DI) water and air-drying. The degummed silk was then dissolved in heated (85–90 °C) mixture of CaCl2:H2O:Ca (NO3)2:EtOH (30:45:5:20 w/w/w/w), dialysed against DI water at room temperature, and lyophilized using freeze dryer (Heto PowerDry LL3000, Thermo Fisher, USA) at −55 °C. The obtained fibroin dry powder was stored at −20 °C for further use.
    2.2.2. Preparation of α-mangostin loaded FNPs α-Mangostin loaded FNPs were prepared by desolvation technique. One mL of fibroin aqueous solution (1% w/v) was injected into 0.5 mL of ethanol containing 1 mg of α-mangostin, with or without PEI (1% w/ v aqueous solution, pH 7.0) or different EDC content. The sponta-neously formed translucent mixture was stabilized at 4 °C for 24 h.  Colloids and Surfaces B: Biointerfaces 181 (2019) 705–713
    Then, particles were washed with DI water by centrifugation at 31,514
    72 h. Lactose was used at 2.5% w/w as a cryoprotectant. The products were stored at 2–8 °C for further characterization. In this study, four different formulations were prepared, namely α-mangostin-FNP, α-mangostin-EDClow-FNP, α-mangostin-EDChigh-FNP, and α-mangostin-PEI-FNP.
    The mean particle size and size distribution (PI) were determined by a dynamic light scattering (DLS) method using ZetaPALS® analyzer (Brookhaven Instrument Corporation, USA). The instrument was equipped with a 35 mW helium-neon laser diode operating at 632.8 nm. Samples were diluted with DI water and the measurement was per-formed at 25 °C at a fixed angle of 90°.
    The zeta potential was determined by phase analysis light scattering method using the same instrument. Samples were diluted with DI water and the measurement was performed at 14.8° to the incident light. The zeta potential was calculated from the electrophoresis mobility based on the Smoluchowski equation.