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Pharmaceutics 2020, 12, 1102 4 of 17 collected and redispersed in 20 mL of n-hexane. This mixture was centrifuged at 1000 g for 5 min to remove large particles. Finally, the supernatant containing fine UCNPs was harvested and placed in storage at 4–8 ◦C for further experiments. 2.3. Synthesis of Core-Shell UCNPs for Red Emission The core-shell upconverting nanoparticles were prepared by slight modification of a procedure described elsewhere [37]. Briefly, 1 mmol of Lu(CH3CO2)3 in 2 mL of methanol was added to the round bottom flask containing 8 mL of OA and 30 mL of ODE. Methanol was evaporated at 110 ◦C under vacuum for 10 min, and the temperature was maintained at 150 ◦C for 1 h under argon flow to obtain a clear solution. After cooling, 225 mg of the as-synthesized core UCNPs in n-hexane was added to the solution and heated to 110 ◦C for 30 min under vacuum to remove n-hexane. The temperature was decreased to 50 ◦C prior to the slow addition of 4 mmol NH4F and 2.5 mmol NaOH in methanol. After a 30 min incubation period at 50 ◦C, methanol was evaporated using the process described above. Next, the reaction vessel was heated to 310 ◦C at a rate of 10–15 ◦C /min and maintained at this temperature for 90 min under an argon atmosphere. The mixture was cooled to room temperature, and an equal volume of acetone was added to precipitate UCNPs. The resulting suspension was centrifuged at 6554 g for 10 min. The pellet was collected and redispersed in 20 mL of hexane. This mixture was centrifuged at 1000 g for 5 min to remove large particles. Finally, the supernatant containing fine UCNPs was harvested and placed in storage at 4–8 ◦C for further studies. 2.4. Synthesis of Core@shell Upconverting Nanoparticles (UCNPs) for Green Emission For the UCNP core structure, 3 mmol each of Y(CH3CO2)3, Yb(CH3CO2)3, Er(CH3CO2)3, and Nd(CH3CO2)3 [by molar ratio, 68.5% Y, 30% Yb, 0.5% Er, 1% Nd] was dissolved in 5 mL methanol. The core synthesis process is the same as that described above. All remaining steps were performed according to the same procedure used for the synthesis of the regular core UCNP structure described above. For the UCNP shell structure, 1 mmol each of Y(CH3CO2)3 and Nd(CH3CO2)3 [by molar ratio, 80% Y, 20% Nd] was dissolved in 2 mL methanol. All remaining steps are the same as those used for the synthesis of the regular shell UCNP structure described above [35,38]. 2.5. Surface Modification of UCNPs Amine-functionalized UCNPs (UCNPs@SiO2-NH2) were prepared through a series of steps described elsewhere [39]. A portion (25 mL) of UCNP solution (2 mg/mL in hexane) was mixed with 1200 mg of IGEPAL® CO-520 in an ultrasound bath. A 200 μL aliquot of ammonium hydroxide solution (30%) was added to the mixture and gently shaken. The reaction started by the addition of TEOS (150 μL, 0.7 mmol) under stirring and continued overnight at room temperature. The resulting UCNPs@SiO2 was purified by centrifugation with ethanol (3 × 8500 rpm, 10 min). Amino groups on UCNPs@SiO2 were functionalized with APTES (750 μL, 3.4 mmol) in 25 mL of ethanol. This mixture was stirred overnight at room temperature. The UCNPs@SiO2-NH2 nanoparticle fraction was centrifuged (3 × 8500 rpm, 10 min), harvested, and redispersed in 25 mL distilled water (DW). 2.6. Preparation of UCNPs@SiO2-NH2@FA/PEG/TCPP UCNPs@SiO2-NH2 was conjugated with TCPP, mPEG2k-SC-NHS, and FA/PEG5k-NHS using a slight modification of the procedure described elsewhere [40,41]. To synthesize TCPP-NHS, TCPP (0.2 mmol), N-dicyclohexylcarbodiimide (DCC, 0.3 mmol), N-hydroxysuccinimide (NHS, 0.3 mmol), and triethylamine (TEA, 0.3 mmol) were mixed in 5.3 mL dimethyl sulfoxide (DMSO), and the reaction was allowed to continue in the dark for 24 h [2,42]. After removing the precipitate of DCU, the product was stored at −80 ◦C. UCNPs@SiO2-NH2 (10 mg), FA/PEG5k-NHS (0.1 mg), and TCPP-NHS (1 mg) were mixed in PBS (pH 8.0). After 1 h, mPEG2k-SC (2 mg) was added to the resulting solution and stirred for 12 h. The mixture was centrifuged (8500 rpm, 20 min) and redispersed in DW.PDF Image | Red LED Erbium Upconverting Nanoparticles v Cervical Cancer
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