Encapsulation Of Anticancer Drugs Within Multifunctional Poly (Amidoamine) Dendrimers For Targeted Cnacer Therapy

We report here a general approach to using multifunctional poly(amidoamine) (PAMAM) dendrimer-based platform to encapsulate anticancer drugs for targeted cancer therapy. In this approach, amine-terminated generation 5 (G5) PAMAM dendrimers were sequentially modified with fluorescein isothiocyanate (FI) and folic acid (FA) via covalent conjugation, followed by an acetylation reaction to neutralize the remaining amines of the dendrimer surfaces. The main content of this thesis include three parts:(1) G5.NH2 dendrimers (52.0 mg,0.002 mmol) were dissolved into DMSO (5 mL). Then, FI (3.9 mg,0.01 mmol) in DMSO solution (5 mL) was dropwise added into the dendrimer solution under vigorous magnetic stirring at room temperature. The reaction was stopped after 24 h. The reaction mixture was dialyzed against phosphate buffered saline (PBS) buffer (3 times,4 liters) and water (3 times,4 liters) through a 10,000 MWCO membrane for 3 d to remove the excess of reactants, followed by lyophilization to get G5.NH2-FI dendrimers.(2)To synthesize both FA-and Fl-modified G5 dendrimers (G5.NH2-FI-FA), FA (2.55 mg, 0.00577 mmol) dissolved in DMSO (5 mL) was mixed with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (22.1 mg,0.1154 mmol) under magnetic stirring at room temperature for 3 h. The resulting solution was added dropwise to a solution of G5.NH2-FI (35.0 mg,0.00115 mmol) in DMSO (5 mL) under vigorous stirring at room temperature. The reaction was stopped after 3 days and the reaction mixture was dialyzed against PBS buffer (3 times,4 liters) and water (3 times,4 liters) through a 10,000 MWCO membrane, followed by lyophilization to get the G5.NH2-FI-FA product. (3)To neutralize the remaining amine groups of G5.NH2-FI-FA dendrimers, G5.NH2-FI-FA dendrimer (26.3 mg,0.00087 mmol) dissolved in 5 mL DMSO was mixed with triethylamine (52,74 mg,0.52128 mmol). Then acetic anhydride (44.35 mg,0.4344 mmol) in 5 mL DMSO was added dropwise into the dendrimer/triethylamine mixture solution while stirring at room temperature. The reaction was stopped after 24 h. The reaction mixture was dialyzed extensively against PBS buffer (3 times,4 liters) and water (3 times,4 liters) through a 10,000 MWCO membrane. Lyophilization of the dialysis liquid gave the product of G5.NHAc-FI-FA.The synthesized multifunctional dendrimers (G5.NHAc-FI-FA) were then used to complex anticancer drugs,2-methoxyestradiol (2-ME) or Doxorubicin (DOX) for targeted delivery of the drugs to cancer cells overexpressing highaffinity folic acid receptors (FAR). The method of encapsulation is as follow:G5.NHAc-FI-FA dendrimers (10 mg) were dissolved in 1.5 mL water. The drug with 10 molar equivalents of dendrimers was dissolved in 300μL methanol, and then mixed with the 1.5-mL dendrimer aqueous solution. The mixture solution was vigorously stirred overnight to allow the evaporation of the methanol solvent. The dendrimer/drug mixture solution was centrifuged (7,000 rpm for 10 min) to remove the precipitates related to non-complexed free drug, which is insoluble in water. The precipitate was collected and dissolved into 1 mL methanol for high-performance liquid chromatography (HPLC) analysis. The supernatant was lyophilized for 3 days to obtain the dendrimer/drug complex.Results and conclusion:We show that the formed G5.NHAc-FI-FA/drug complexes with each dendrimer encapsulating approximately 3.7 2-ME or 1.0 DOX molecules are water soluble and stable. In vitro release studies show that the anticancer drugs (2-ME or DOX) complexed with the multifunctional dendrimers can be released in a sustained manner. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in conjunction with cell morphology observation demonstrates that the G5.NHAc-FI-FA/drug complexes can specifically target and display specific therapeutic efficacy to cancer cells overexpressing high highaffinity FAR. Findings from this study suggest that multifunctional dendrimers may be used as a general drug carrier to encapsulate various cancer drugs for targeted therapy of different types of cancer.