(a) PLGA-PEGCOOH (−) and PLGA-PEG-biotin (+) nanoparticles incubated with the presence (+) or absence (−) of avidin-FITC and analyzed on a fluorescent plate reader. (b) Quantified extent of biotinylation for BSA (white) and TTC (gray) proteins for different NHS-PEG₄-biotin:protein molar ratios. (c) Schematic representation of overall protein conjugation to PLGA-PEG-COOH or PLGA-PEG-biotin polymer nanoparticles (as indicated). Avidin (Step 1) and biotinylated BSA-FITC (Step 2) were conjugated in sequence to the nanoparticle surface, with PBS washes to remove unbound protein at each step. (d) PLGA-PEG-COOH (−) and PLGA-PEG-biotin (+) nanoparticles incubated with the Captions presence (+) or absence (−) of avidin, subsequently incubated with biotinylated BSA-FITC (b-BSA-FITC) and analyzed on a fluorescent plate reader showing selective protein conjugation to nanoparticles. (e) Comparison of biotin binding proteins (BBP) as crosslinkers for protein conjugation to nanoparticles. Data is expressed as mean ± SEM for PLGA-PEG-COOH (white, gray) and PLGA-PEG-biotin (black) nanoparticles were prepared and incubated with avidin (left), streptavidin (middle), or neutravidin (right), and subsequently with biotinylated BSA-FITC. Avidin showed the highest overall protein conjugation, whereas neutravidin resulted in reduced non-specific interaction and the most specific protein conjugation.

Flow cytometry analysis of N18-RE-105 neuroblastoma cells (a), HepG2 liver (b), and b.End3 endothelial (c), cells following incubation with TTC-conjugated nanoparticles (green), BSA-conjugated nanoparticles (blue), or a PBS negative control (red). Other negative controls are shown for (a) for nanoparticles without neutravidin (purple), without TTC (orange), and blank nanoparticles (black), demonstrating that TTC-conjugated nanoparticles selectively target neurons.

Conceptual schematic (not to scale) illustrating (a) the cross-section of a biodegradable polymer nanoparticle that is functionalized with the tetanus toxin C fragment (TTC) and presumed to encapsulate a therapeutic substance, and (b) the conjugation system used to attach functional TTC to the nanoparticle utilizing PLGA-PEG-biotin functionalized biodegradable polymer, and biotinylated TTC with an avidin cross-linker, based on previously published structures [25–27]. Two of the four avidin subunits are shown for illustrative purposes. Proton NMR spectra of (c) PLGA-COOH, (d) PLGA-biotin, (e) PLGA-PEG-COOH, and (f) PLGA-PEG-biotin polymers dissolved in deuterated chloroform. Characteristic peaks are visible for PLGA (*) and PEG (#), but not for conjugated biotin.

Flow cytometry analysis of N18-RE-105 neuroblastoma cells incubated with fluorescent TTC (green), fluorescent BSA (blue), or PBS control (red), for NHS-FITC:protein conjugation ratios of 1:1 (a), 8:1 (b), and 24:1 (c). TTC remained functional following amine conjugation and preserved its ability to target neurons (a–c). Dynamic light scattering analysis of nanoparticle size following conjugation with neutravidin and biotinylated TTC (d–e). (d) Nanoparticles prepared with different molar ratios of PLGA-PEG-COOH to PLGA-PEG-biotin as indicated (control is 100% PLGA-PEG-Biotin), and (e) free biotin competition with TTC-biotin conjugation to nanoparticles at different concentrations of free biotin as indicated; control is without free biotin. These figures show that nanoparticle aggregation can be reduced by decreasing the number of biotins on the nanoparticle surface (d) or by free biotin competition during conjugation of the biotinylated protein (e). For all sizing experiments, results are expressed as mean±SD of 3 independent size measurements of one preparation of nanoparticles.