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  • br Synthesis of multifunctional PAMAM dendrimer br Synthesis


    2.2.1. Synthesis of multifunctional PAMAM dendrimer Synthesis of fluorescently tagged G4 PAMAM dendrimer. Fluorescently labelled G4 PAMAM dendrimer was prepared following an earlier reported procedure with a small modification (Biswas et al., 2012). To the DMF solution of G4 dendrimer (100 mg), Fluorescein NHS was added at a mole ratio of 1:1 dropwise under inert atmosphere. The solution was stirred in dark conditions for 8 h at normal room temperature. Following the reaction, DMF was removed using rotary evaporator and resulting product was subjected to dialysis to remove unconjugated fluorescein. After dialyzing for 48 h, the product (F-G4) was freeze dried and stored until further use. Synthesis of G4-PTX. The OH group at 2′ position of paclitaxel (PTX) was modified to its hemisuccinate form using succinate linker (succinic anhydride) prior to reacting with amine groups of G4 PAMAM dendrimer. In brief, 25 mg of PTX was dissolved in dichloromethane and reacted with succinic anhydride (4.4 mg) for 3 days under stirring in presence of dry pyridine. The product was collected by ethyl acetate extraction and dried to yield PTX-2′-hemisuccinate as white solid. The hemisuccinate –COOH group was reacted with DCC/NHS for 8 h to form NHS ester of PTX which can easily attach with –NH2 surface groups of dendrimer molecules.
    The activated PTX NHS ester was added to 50 mg of G4 dendrimer in DMF at a mole ratio of 1:4 and stirred overnight. Following the re-action, DMF was evaporated and the product was dialyzed using cel-lulose ester membrane of MWCO 12–14 kDa against water to remove impurities. Further, a white fluffy product of G4-PTX was collected by freeze drying.
    The schematic representation of synthesis of the multifunctional PAMAM dendrimer is shown in Fig. 1. Synthesis of G4-PTX-PEG. The G4-PTX construct was further attached with PEG using mPEG SCM ester (2 kDa). G4-PTX was dissolved in DMF, and mPEG SCM was added drop-wise at a mole
    Fig. 1. Schematic illustration of synthesis of G4 dendrimer conjugates.
    S.V.K. Rompicharla et al.
    ratio of G4-PTX: mPEG-SCM as 1:12. The reaction mixture was stirred overnight in presence of DIPEA (20 µl). The solvent was removed using rotary evaporator. The product was subjected to dialysis using regenerated GSK-3 dialysis membranes of MW cutoff 12–14 kDa with frequent water changes for 48 h. After dialysis, the product was freeze dried to obtain a solid white product of G4-PTX-PEG. The fluorescently tagged dendrimer conjugates were also prepared in the same manner. The GSK-3 PEGylation of G4-PTX was confirmed by measuring the zeta potential changes, and by 1NMR and GPC analysis. Biotinylation of G4-PTX-PEG. Biotinylation of G4-PTX-PEG conjugate was performed by activating the carboxy group of biotin molecule using EDC/NHS (3 mol equivalent of biotin) in DMF and attaching it with amine group of G4-PTX-PEG at a mole ratio of biotin: G4-PTX-PEG as 30:1. After reaction, the solvent was removed using rotary evaporator and the product was subjected to dialysis against water for 2 days. The final G4-PTX-PEG-Biotin construct was obtained after lyophilization.
    2.2.2. Characterization of multifunctional conjugate
    Characterization of the PAMAM dendrimer conjugates G4-PTX, G4-
    PTX-PEG, and G4-PTX-PEG-Biotin was performed by 1H NMR (300 MHz, Bruker, USA) and zeta potential measurements by using dynamic light scattering instrument (Nano ZS, Malvern Instruments, UK). To determine the relative molecular weights of synthesized con-jugates, gel permeation chromatography (GPC) analysis was performed. Zorbax GF-250 (9.4 mm ID × 25 cm × 4–4.5 µm) size exclusion analy-tical column was used to run the samples in a HPLC system (Agilent 1100 series). Tris buffer (50 mM) and KCl (100 mM) was used as eluting phase in isocratic mode at a flow rate of 1 ml/min. A calibration curve was plotted with the standard molecular weight compounds prior to analyzing the samples. Further, to determine the morphology of the conjugate G4-PTX-PEG-Biotin, TEM analysis was performed.
    Biotin attachment to the dendrimer was quantitatively estimated using HABA/Avidin assay. This assay is based on the association of the dye HABA (4′-hydroxyazobenzene-2-carboxylic acid) to avidin and the ability of biotin molecule to displace HABA in stoichiometric fractions. HABA binds to avidin to produce a yellow-orange colored complex which absorbs at 500 nm. Biotin in the sample, if present, would dis-place the HABA dye and cause the absorbance to decrease. As per the manufacturer’s protocol, to 900 µl of HABA/Avidin reagent, 100 µl of sample containing biotin dissolved in deionized water was added. The absorbance of the test solution was recorded at 500 nm. Further, the biotinylation degree was calculated from the absorbance values ob-tained.