Materials and Methods 115

PLGA (150 kDa, LA:GA=85:15, PLGA150) was purchased from Akina Inc. (IN,

USA). Paclitaxel (PTX) was a gift from Samyang Genex Corp (Seoul, Korea). Tannic acid (Pure) and albumin from human serum were purchased from Sigma-Aldrich (MO, USA). Dopamine HCl was purchased from Alfa-Aesar (MA, USA). Pierce BCA protein assay and CBQCA protein assay kit were purchased from Thermo Scientific (NY, USA). (3-(4,5- Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) (MTT) was purchased from Invitrogen (Eugene, OR, USA). All other materials were of analytical grade.

5.2.2 Preparation and Characterization of NPs

Conventional method. PLGA NP cores were prepared via single emulsion-solvent

evaporation method. Briefly 100 mg of PLGA and 10 mg of PTX were dissolved in an organic phase of DCM (10 mL) were sonicated into an aqueous phase of PVA (4%, 20 mL) using Vibra-Cell probe sonicator (Sonics, Newtown, CT, USA) with 45% amplitude at a pulse of 4-s on and 2-s off. Formed emulsion was dispersed into 30 mL of water and stirred for 5 hours to allow evaporation of DCM. NPs were collected by centrifugation at 25,000 rpm (33,000 xg rcf) and washed 3 times with water to remove any remaining traces of DCM and PVA. To coat NPs with TA, PLGA NPs were dispersed in 0.07 mg/mL of TA solution in bicine buffer (0.1 M, pH 7.4) at a concentration of 0.5 mg NP/mL. The suspension was shaken for three hours, after which the formed PLGA-TA NPs were collected by centrifugation and further washed with water. The pD coated NPs were prepared as described in section 2.2.4. To functionalize PLGA-TA or PLGA-PD NPs with a certain ligand (albumin or fluoresceinamine (FA)), they were further incubated in ligand solution in bicine buffer (0.1 M, pH 7.4) with specified concentration and time, followed by NPs collection and washing. The washed NPs were freeze-dried and stored in 4 ˚C until needed.

One-pot method. PLGA emulsion was prepared in a manner similar as in the

conventional method, replacing 4% PVA/water with 4% PVA in bicine buffer (0.1 M, pH 7.4). After DCM evaporation, TA was directly added to the continuous phase in a ratio of 1:1 (TA: NP), followed by addition of sodium periodate at a final concentration of 0.2 mg/mL. NP suspension was shaken for 1.5 hour to form PLGA-TA NPs, collected and conjugated with ligands in the same as detailed in the conventional method section.

5.2.3 NP Characterization

Size, zeta potential, and morphology. Different types of NPs were evaluated for

particle size and zeta potential as described in section 2.2.5. NP morphology was observed by transmission electron microscopy (TEM) using the method described in section 2.2.5, employing different stains.

TA-induced AgNO3 deposition. The presence of TA was detected by deposition of

Ag metal on TA surface. (Sileika et al., 2013). Briefly, PLGA or PLGA-TA NPs were incubated overnight in AgNO3 aqueous solution (17 mg/mL) with shaking and collected

by centrifugation. The NPs were washed once with water to remove excess AgNO3. Finally,

NPs were visualized with TEM using 1% phosphotungstic acid as a negative stain.

TA content. To examine the efficiency of TA coating on PLGA NP surfaces, TA

content was directly and indirectly quantified by bicinchoninic acid (BCA) assay. TA is a reducing agent and can reduce cupric ions in the assay reagent to cuprous, which form colored complexes with BCA (Marino et al., 2009). This reaction is sensitive and linear. To directly determine TA content, TA coated NPs of a known weight were incubated with BCA working reagent for an hour at 37 ˚C. A supernatant was separated from the suspension via centrifugation, and its absorbance was read at 562 nm using a SpectraMax M3 microplate reader (Molecular Device, Sunnyvale, CA). The amount of TA per NP sample was calculated after subtracting the background absorbance of uncoated PLGA NPs, using a calibration curve drawn with TA solutions of known concentrations. Alternatively, TA content of PLGA-TA NPs was determined indirectly by quantification of TA in bicine buffer after incubation of PLGA NPs and separation of NPs by centrifugation. The amount

of TA in the supernatant was measured using BCA assay and compared to control samples (100% TA, no PLGA NPs). The TA content of PLGA NPs (%w/w) was calculated as:

% %

Human Serum Albumin content. To determine the amount of albumin conjugated

to NPs surface, NPs were dispersed in water (10 mg/mL), and albumin content was determined via CBQCA fluorescence protein assay (Life Science, NY, USA) per vendor’s instruction. A calibration curve was constructed using known concentrations of albumin. Fluorescence of different samples was measured at Excitation/Emission of 465/550 nm using a SpectraMax M3 microplate reader (Molecular Device, Sunnyvale, CA). The amount of albumin per NP sample was calculated after subtracting the background fluorescence level of corresponding NPs before albumin conjugation.

Fluoresceinamine content. The amount of Fluoresceinamine (FA) conjugated to

different types of NPs was quantified indirectly by measuring the absorbance of FA in supernatant before and after NPs incubation. Briefly, 0.4 mg of NPs was dispersed in 1 mL of FA solution (15 ug/mL) in Tris buffer (10 mM, pH 8.5) for 40 minutes. Then, NPs were centrifuged, and the FA absorbance in the supernatant was measured at 497 nm using a SpectraMax M3 microplate reader (Molecular Device, Sunnyvale, CA) and compared to that of original FA solution. The amount of FA per NP weight was determined using a standard calibration curve of FA in the same buffer solution.

5.2.4 In vitro Cytotoxicity of TA coated NPs

To evaluate the cytotoxic effect of TA coated NPs. An in vitro cytotoxicity study was performed using NIH/3T3 fibroblast cell line (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle medium (DMEM) medium supplemented with 10% fetal calf serum and 100 units/ml of penicillin and 100 ug/ml of streptomycin. Cells were seeded in 96-well plate at a density of 10,000 cell/well (200 µL). Next day, concentrated NP suspension was added (20 uL) to a final concentration of 0.1, 0.01, 0.001,

or 0.0001 mg/mL. After 72 hours, mitochondrial activity of the cells was evaluated using the MTT assay. NP containing medium was replaced with that containing MTT solution (115 µL) and cells were incubated for 3.5 hours, after which 100 µL of stop solution (50 % DMSO, 20% SDS and 0.02% acetic acid) was added. Next day, wells absorbance was read using a SpectraMax M3 microplate reader at 529 nm. % viability of the cells was determined in comparison to control cells that did not receive NP treatment.