Determination of Fluorescent Dendrimer Concentrations Using Modified Bradford Assay

Polyamidoamine (PAMAM) dendrimers are a specific class of nanoparticle that can potentially be used for a variety of medical applications, including as delivery vehicles for chemotherapy and gene therapy. These dendrimers vary in size depending on their generation (G1, G2, G3…etc.) and have many features (size range, shape, solubility, bonding, etc.) similar to those of globular proteins. Thus, it is hypothesized that colorimetric assays typically used for protein quantitation should also be viable methods for measuring dendrimer concentration. The most widely-used methods for the colorimetric detection and quantitation of total protein involve either protein-dye binding chemistry or protein-copper chelation chemistry. Coomassie Blue G-250 dye binding (Bradford Assay) is used extensively because of its consistent, reliable results in protein quantitation. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Blue shifts from 465 nm to 595 nm when bound to proteins. For these studies, the Bradford Assay was studied in conjunction with generation 4 and 5 PAMAM dendrimers. Additionally, bovine serum albumin (BSA) protein standards were used as a reference. For both the G4 and G5 dendrimers and the BSA, a 2-fold serial dilution was performed and aliquots of each mixed with Coomassie Assay reagent. Absorbance of the samples was then measured within 5 minutes after mixing using a UV-VIS spectrophotometer. While the assay worked well for the BSA, producing expected results, there was no correlation between absorbance and concentration for the dendrimers. In an effort to identify conditions under which this assay would work for PAMAM dendrimers, we undertook a series of investigations to test the effects of pH on the reaction. Briefly, a fixed volume of sodium hydroxide at different concentrations (0-1 N) was added to a fixed volume of the Coomassie assay reagent. This “modified” reagent was then used in reactions with BSA and dendrimers at different concentrations. Results reveal that the Bradford Assay works well when the reagent is modified with sodium hydroxide concentrations from 500 mN to 1 N with the “best” results obtained using the 1 N base. The modified Bradford Assay yielded a logarithmic relationship with dendrimer concentration in the range of 0-3.28 milligrams per milliliter, that was similar in shape to the standard BSA protein calibration curve obtained using the same reagent. The resulting calibration curve obtained for each dendrimer type using the “modified” assay reagent was used to quantify the concentration of generation 4 and 5 dendrimers labeled with either NHS-fluorescein or Texas Red. The results form the basis for on-going studies to assess analytical methods for dendrimer quantitation. With such methods, it is proposed that work involving dendrimers as tracers and drug carriers may be more effectively pursued.