*3.1. E*ff*ective rAAV Association to Carbon Dots and Release*

The reporter rAAV-*lacZ* gene vector was first formulated with the various CDs (CD-1 to CD-4) to examine the ability of these nanoparticles to associate with rAAV and release it over time (up to 10 days, the longest time point evaluated) using Cy labeling and fluorescent evaluation of the vectors in the systems and by measuring the rAAV concentrations in the culture medium via AAV titration ELISA.

Successful formulation of Cy3-labeled rAAV vectors with the different CDs was seen as revealed by the effective detection of live fluorescence in the samples after 24 h relative to the control conditions (CDs formulating unlabeled rAAV and CDs lacking rAAV), without visible difference between CDs or when using Cy3-labeled rAAV vectors in the absence of CD formulation (Figure 2A). Furthermore, all CDs were capable of releasing rAAV over a period of at least 10 days, with CD-2 allowing for the highest early vector release and a good maintenance of vector concentration over time (rAAV-*lacZ*/CD-2) relative to the other CDs (rAAV-*lacZ*/CD-1, rAAV-*lacZ*/CD-3, and rAAV-*lacZ*/CD-4) and versus free vector control (rAAV-*lacZ*) (Figure 2B).

**Figure 2.** Complexation and release of rAAV vectors from the carbon dots. The rAAV-*lacZ* vector was labeled with Cy3 and formulated with the various CDs (40 <sup>μ</sup>L rAAV, 8 <sup>×</sup> 10<sup>5</sup> transgene copies/40 <sup>μ</sup>L CD) and placed in culture over time. (**A**) Cy3-labeled rAAV formulated with the various CDs were examined under live fluorescence after 24 h (magnification ×10; scale bars: 100 μm; all representative data). Control conditions included CD formulations with unlabeled rAAV, CDs lacking rAAV, and absence of CDs. (**B**) rAAV release from the various CDs was monitored by measuring the rAAV concentrations in the culture medium at the denoted time points using an AAV titration ELISA. Free vector treatment was used as a control condition.
