*3.2. E*ff*ective rAAV-Mediated Reporter lacZ Overexpression in hMSCs upon Delivery Assistance by Carbon Dots*

The reporter rAAV-*lacZ* gene vector was next formulated with the various CDs to determine the ability of the systems to promote the safe genetic modification of hMSCs over time (up to 10 days, the longest time point evaluated) relative to control conditions (CDs lacking rAAV, i.e., -/CD; free rAAV, i.e., rAAV-*lacZ*; absence of both CDs and rAAV, i.e., -) by monitoring *lacZ* expression using X-Gal staining and via quantitative detection of the β-gal activities in the cells using a Beta-Glo® Assay and by evaluating their viability using the Cell Proliferation Reagent WST-1.

A preliminary, histomorphometric analysis of the early X-Gal staining intensities in the cells on day 1 revealed that the CD-2, CD-3, and CD-4 formulations of rAAV-*lacZ* were capable of promoting *lacZ* expression in the hMSCs without significant difference relative to free vector administration (*P* ≥ 0.050) (Figure 3A). The staining intensities in the cells treated with rAAV-*lacZ*/CD-2, rAAV-*lacZ*/CD-3, and rAAV-*lacZ*/CD-4 increased on day 10, especially when using CD-2 (1.4-fold increase versus day 1; *P* = 0.060), again without difference compared with free rAAV-*lacZ* treatment (*P* ≥ 0.050) (Figure 3B). In marked contrast, delivery of rAAV-*lacZ* in the cells via CD-1 promoted a significant reduction of the staining intensities in hMSCs relative to free vector administration (102.9- and 32.5-fold decrease on days 1 and 10, respectively; always *P* ≤ 0.040) (Figure 3A,B). Overall, these results were supported by a comprehensive, quantitative estimation of the β-gal activities in the cells using the Beta-Glo® Assay, even showing increased activities when providing rAAV-*lacZ* via CD-2, CD-3, or CD-4 versus free vector treatment (up to 2.9- and 2.3-fold difference on days 1 and 10, respectively; always *P* ≤ 0.050) and with reduced activities when using CD-1 (19- and 15.8-fold difference versus free vector administration; always *P* ≤ 0.020) (Figure 3A,B).

CD-guided delivery of rAAV-*lacZ* to hMSCs using either CD-1 or CD-2 was safe, as revealed by the results of a WST-1 assay, with 100% cell viability preserved on day 1, without significant difference relative to the corresponding control conditions (-, -/CD-1, -CD-2, and free vector administration; always *P* ≥ 0.180) (Figure 4A). In contrast, CD-3 and CD-4 had significantly detrimental effects on cell viability (<32%; always *P* ≤ 0.010 versus all other conditions). Similar observations were noted on day 10, with 100% viability using CD-1 and CD-2, as noted in the corresponding control conditions (always *P* ≥ 0.050), and about 25-30% viability using CD-3 or CD-4 (always *P* ≤ 0.040 versus all other conditions) (Figure 4B).

**Figure 3.** *Cont*.

**Figure 3.** Detection of reporter (*lacZ*) gene overexpression in hMSCs transduced with the rAAV/CD systems. The rAAV-*lacZ* vector (20 <sup>μ</sup>L, 4 <sup>×</sup> 105 transgene copies) was formulated with the various CDs (CD-1 to CD-4; 20 <sup>μ</sup>L), and the resulting rAAV/CD systems (40 <sup>μ</sup>L, i.e., 4 <sup>×</sup> 105 transgene copies) were incubated with hMSCs (3000 cells in 96-well plates; MOI = 133) for up to 10 days. Expression of *lacZ* was examined using X-Gal staining (top panel: magnification ×4; scale bars: 500 μm; all representative data) with corresponding histomorphometric analyses (bottom left panel) and using quantitative estimation of the β-gal activities using the Beta-Glo® Assay System (bottom right panel) after one (**A**) and 10 days (**B**). Control conditions included CDs lacking rAAV (-/CD), free rAAV (rAAV-*lacZ*), and absence of both CD and rAAV (-). Statistically significant relative to <sup>a</sup> -, <sup>b</sup> -/CD and <sup>c</sup> rAAV-*lacZ*.

**Figure 4.** Cell viability in hMSCs transduced with the rAAV/CD systems. The rAAV-*lacZ* vector (20 μL, <sup>4</sup> <sup>×</sup> 105 transgene copies) was formulated with the various CDs (CD-1 to CD-4; 20 <sup>μ</sup>L) and the resulting rAAV/CD systems (40 <sup>μ</sup>L, i.e., 4 <sup>×</sup> 10<sup>5</sup> transgene copies) were incubated with hMSCs (3,000 cells in 96-well plates; MOI = 133) for up to 10 days. Cell viability was examined after one (**A**) and 10 days (**B**) using the Cell Proliferation Reagent WST-1. Control conditions included CDs lacking rAAV (-/CD), free rAAV (rAAV-*lacZ*), and absence of both CD and rAAV (-). Statistically significant relative to <sup>a</sup> - and <sup>b</sup> rAAV-*lacZ*.
