*3.3. E*ff*ective rAAV-Mediated SOX9 and TGF-*β *Overexpression in hMSCs upon Vector Delivery via Carbon Dots*

In light of the efficacy and safety of CD-2, the therapeutic rAAV-FLAG-h*sox9* and rAAV-hTGF-β were next formulated independently with these nanoparticles (rAAV-FLAG-h*sox9*/CD-2 and rAAV-hTGF-β/CD-2, respectively) to determine the ability of the system to promote the overexpression of each candidate gene (SOX9, TGF-β) in hMSCs over time (up to 21 days, the longest time point evaluated) relative to control conditions (CD-2 lacking rAAV, i.e., -/CD-2, CD-2 formulating rAAV-*lacZ*, i.e., rAAV-*lacZ*/CD-2) using immunocytochemical detection of each transgene product. Therapeutic (SOX9, TGF-β) rAAV vectors without CD-2 were not included, as controls because they have been characterized in similar culture conditions in earlier studies [44,45], and in light of the quantitative estimation of the β-gal activities in the cells on day 10, significantly increased activities with rAAV-*lacZ*/CD-2 versus free rAAV-*lacZ* vector treatment were revealed (Figure 3B).

An immunocytochemical analysis of SOX9 expression in the cells revealed that administration of rAAV-FLAG-h*sox9* to hMSCs via CD-2 led to significantly higher levels of SOX9 expression relative to all other conditions after 21 days (65-, 43.3-, and 1.8-fold difference using rAAV-FLAG-h*sox9*/CD-2 versus -/CD-2, rAAV-*lacZ*/CD-2, and rAAV-hTGF-β/CD-2, respectively; always *P* ≤ 0.001) (Figure 5A and Table 2). An evaluation of TGF-β expression using immunocytochemistry also showed that delivery of rAAV-hTGF-β to hMSCs via CD-2 led to significantly higher levels of TGF-β expression relative to all other conditions after 21 days (10.3-, 6.8-, and 9.4-fold difference using rAAV-hTGF-β/CD-2 versus -/CD-2, rAAV-*lacZ*/CD-2, and rAAV-FLAG-h*sox9*/CD-2, respectively; always *P* ≤ 0.001) (Figure 5B and Table 2). This result was corroborated by an estimation of the levels of TGF-β production in the cells using ELISA, with up to 2.8-, 2.8-, and 3.8-fold higher TGF-β secretion levels when using rAAV-hTGF-β/CD-2 after 5, 7, and 21 days, respectively, versus all other conditions (always *P* ≤ 0.001) (Figure 5B).

**Figure 5.** Detection of therapeutic (SOX9, TGF-β) gene overexpression in hMSCs transduced with the rAAV/CD-2 system. The rAAV-FLAG-h*sox9*, rAAV-hTGF-β, and rAAV-*lacZ* vectors (40 μL each vector, 8 <sup>×</sup> 105 transgene copies) were formulated with CD-2 (40 <sup>μ</sup>L), and the resulting rAAV/CD systems (80 <sup>μ</sup>L, i.e., 8 <sup>×</sup> <sup>10</sup><sup>5</sup> transgene copies) were incubated with hMSCs (10,000 cells in 48-well plates; MOI <sup>=</sup> 80) for up to 21 days. SOX9 (**A**) and TGF-β (**B**) expression was examined using immunocytochemistry (A, B; magnification ×20; scale bars: 50 μm; all representative data) and using specific (TGF-β) ELISA (B). rAAV-*lacZ*/CD-2 and CD-2 lacking rAAV were used as controls.

**Table 2.** Histomorphometric analyses in hMSCs transduced with the rAAV/CD-2 systems.


Values are given as mean ± SD. All parameters are in % of positively (SOX9+, TGF-β+, type-II+/-I+/-X<sup>+</sup> collagen) stained cells to the total cell numbers. Statistically significant relative to <sup>a</sup> -/CD-2, <sup>b</sup> rAAV-*lacZ*/CD-2 and <sup>c</sup> rAAV-FLAG-h*sox9*.
