RESULTADOS DE LA ETNOEDUCACION EN LA COMUNIDAD DE SAN BASILIO DE PALENQUE

Human serum albumin, lyophilized powder ≥ 97%, 8% aqueous glutaraldehyde, RPMI 1640, porcine trypsin - 1X Solution, fetal bovine serum, streptomycin and amphotericin, were obtained from Sigma Aldrich (St. Louis, MO). Ethanol, 200-proof, ACS/USP grade was obtained from Pharmco-AAPER (CT, USA), Phosphate buffer (pH 7.2) and a Mini-Pump Variable Flow peristaltic pump were obtained from VWR International (Radnor, PA). An AccuSpin Micro centrifuge was obtained from Fisher Scientific (Pittsburg, PA). Water was distilled, deionized, and then further purified with a Barnstead Nanopure system. Camptothecin (S) (+) was obtained from VWR and BACPT was obtained from Dekk-Tec, Inc. (New Orleans, LA). Cells: “CL” Human Sarcoma, cultured from a patient; Brain Dekk-Tec. Inc., 725 Topaz St, New Orleans, LA)

5.3.2 Synthesis of Unloaded HSAPs

The HSAPs were prepared using a conventional desolvation technique.6, 15, 82, 81, 83 The standard method for preparation included addition of 20 mg of solid HSA to 1 mL of water . After dissolving, the pH was around 7. A small stir bar was added to the solution, and 3 mL of 100% ethanol was added at a rate of 1 mL/min using a peristaltic pump. Upon addition of the ethanol, the solution became turbid. Finally, 4.7 μL of 8% glutaraldehyde (aq.) was added to crosslink the HSA within the particles. After 24 h of stirring at room temperature, the particles were centrifuged at 13,000 rpm (16,060 g) for 30 minutes to 1 hour. The supernatant was

washed twice with water with intervening centrifugation steps. The washed particles were ultrasonicated in water and stored at 4 °C.

5.3.3 Synthesis of BACPT-HSAPs

The procedure for preparing the drug-loaded particles followed section 5.3.2 with a modification to the ethanol addition step and final volume. To create the BACPT loaded HSAPs, a stock solution of the BACPT was first created: 1 mg BACPT/mL ethanol. Then, an aliquot of the drug stock solution was added to more ethanol. The BACPT/EtOH solution was added to an HSA sample solution drop-wise. The overall parameters used for preparing HSAPs loaded with varying amounts of BACPT are provided in Table 5.1 (all samples contained 4.7 µL of 8% glutaraldehyde; causing 40% theoretical crosslinking). The washes for the drug- loaded particles were done with a solution of 1:3 water:ethanol.

Table 5.1. Preparation Parameters for all BACPT-loaded HSAPs Sample Preparation Type Drug Stock Solution (mL) EtOH (~mL) Drug Added (mg) BACPT-HSAP-1 0.01 3.99 0.01 BACPT-HSAP-2 0.1 3.9 0.1 BACPT-HSAP-3 0.25 3.75 0.25 BACPT-HSAP-4 0.5 3.5 0.5 BACPT-HSAP-5 1 3.0 1 5.3.4 Synthesis of CPT-HSAPs

The procedure for preparing the drug-loaded particles followed section 5.3.2 with a

stock solution of the CPT was first created: 1 mg CPT/mL in 2:3 chloroform:ethanol. Then, an aliquot of the drug stock solution was added to more ethanol. The CPT/EtOH solution was added to an HSA sample solution drop-wise. The overall parameters used for preparing HSAPs loaded with varying amounts of CPT are provided in Table 5.2 (all samples contained 4.7 µL of 8% glutaraldehyde; causing 40% theoretical crosslinking). The washes for the drug-loaded particles were done with a solution of 1:3 water:ethanol.

Table 5.2. Preparation Parameters for all CPT-loaded HSAPs Sample Preparation Type Drug Stock Solution (mL) EtOH (~mL) Drug Added (mg) CPT-HSAP-1 0.1 3.9 0.1 CPT-HSAP-2 0.25 3.75 0.25 CPT-HSAP-3 0.5 3.5 0.5 5.3.5 Absorbance

Absorbance measurements of the BACPT-HSAPs were performed with a Cary 60 absorbance spectrometer (Agilent Technologies).

5.3.6 HPLC

The HPLC system used was an Agilent 1100 instrument (Palo Alto, CA) with a diode array absorbance detector. The column was an Agilent Eclipse XDB-C18 column (150 X 4.6 mm, particle size 5μm). The injection volume was 200 μL, and the absorbance wavelength used for quantitation of BACPT was 227 nm and for CPT it was 254 nm. The mobile phase was 60% acetonitrile: 40% water at a flow rate of 1 mL/minute.

5.3.7 Fluorescence

Fluorescence was measured with a Cary Eclipse fluorometer with excitation for BACPT at 403 nm and emission scanned from 413-600 nm. For CPT, the excitation wavelength was 358 nm and the emission was scanned from 368-550 nm. Excitation and emission slits were 2.5 nm. 5.3.8 Dynamic Light Scattering

The completed HSAPs were characterized by measuring the diameter, mobility, and zeta potential using a Mobius dynamic light scattering (DLS) instrument (Wyatt Technology Corporation, Santa Barbara, CA). Samples were diluted with water prior to analysis. 5.3.9 Microscopy

Particles were imaged using a field emission-scanning electron microscope FESEM, LEO 1530VP (LEO Elektronenmikroskopie GmbH, Oberkochen, Germany). HSAPs were sputtered with a thin film of gold or silver to facilitate imaging. To check cell vitality after the addition of nanoparticles to the cancer cells, a compound microscope was used; Meiji Techno, TC5300, Compound - 100x on large view, 200x in zoom view, (Meiji Techno America, San Jose, CA) 5.3.10 Cell Cultures

Cell vitality studies were performed by two different methods.

CELL METHOD 1. For visual cytotoxicity studies without washing off unloaded-HSAPs, CPT-HSAPs or BACPT-HSAPs: On a 12-well cell plate, to the each well, 1 mL of sterile RPMI media was added, then, 1-drop aliquots of diluted (with RPMI) or undiluted harvested cell solutions were placed into every well. The well plates were incubated at 37°C for 24 hours to facilitate cell growth. After the 24 hour period, addition of the nanoparticles was accomplished by adding 0.5 ml of the particle solution being tested; either unloaded-HSAPs or a particular concentration of CPT-HSAPs or BACPT-HSAPs. The control was a well containing only cells

and no nanoparticles. After the plates were incubated again with the particles at 37°C for 24 hours, the cells were visually checked for blebbing and necrosis on the compound microscope; with the nanoparticles still present.

CELL METHOD 2. For visual cytotoxicity studies involving field of view differences after washing the wells of nanoparticles and dead cells: the well plates were prepared as they were in cell method 1, but after the initial incubation period, only 0.1 mL of particle solution being tested was added. A control of cells only was prepared a well. After 24 hours of incubation, the wells were removed of all particles and dead cells by pipetting off all media in which they were incubated. The wells were washed twice with 1 mL of fresh RPMI. Any remaining cells were viable and stuck to the well plate. Dead cells were washed away. A visual inspection on the compound microscope showed differences in the live cell population of the control and the treated wells.