br Statistical analysis br Data
2.2.6. Statistical analysis
Data were represented as a mean of atleast three individual ex-periments with standard deviation. Student’s t-test (GraphPad Software, Inc.; San Diego, CA) was performed to establish significance between groups. A p value of less than 0.05 was considered statistically sig-nificance. The representation of *, ** or ##, *** or ### corresponds to p values < 0.05, 0.01 and 0.001, respectively.
3. Results and discussion
3.1. Synthesis and characterization of multifunctional dendrimer conjugate
In the NMR spectrum, the aromatic groups of PTX resulted in peaks at δ (ppm) 7–8.5 and aliphatic protons at δ (ppm) 1.3. The protons of PEG chains were observed at δ (ppm) 3.2–4.1 whereas the protons corresponding to the PAMAM dendrimer molecule were observed at δ (ppm) 1.5–3.5 as shown in Fig. 2A. In Fig. 2B, the peak at δ (ppm) 4.4 and 4.6 corresponds to the ring juncture protons of Biotin. This data confirms the successful conjugation of biotin to the dendrimer surface to form G4-PTX-PEG-Biotin. Attachment of fluorescein moiety was confirmed by the shift in MPP+ Iodide maxima as determined by r> S.V.K. Rompicharla et al.
Zeta potential values of dendrimer conjugates after each step of synthesis (Mean ± SD, n = 3).
Dendrimer conjugate Zeta potential (mV)
UV–Visible spectroscopy (Fig. S1).
In addition, from the GPC analysis relative mass of each conjugate was identified. From the mass value, the approximate number of mo-lecules anchored to each dendrimer molecule was calculated. It was observed that approximately 2.76 molecules of PTX, 10.9 molecules of PEG were attached on the dendrimer surface following the synthesis procedure mentioned above (Table S1). Further, characterization by TEM analysis illustrates that the synthesized conjugate was spherical in morphology and has size in nanometer range as depicted in Fig. 2C.
Furthermore, the HABA/Avidin assay revealed the degree of bioti-nylation of the dendrimer molecules. The number of biotin molecules attached to each dendrimer was calculated to be 20.98 (see Table S1). Also, a variation in the zeta potential as presented in Table 1 following each synthetic step supports the successful construction of the con-jugates.
3.2.1. Cellular uptake by confocal microscopy
The cellular internalization of F-G4-PEG and F-G4-PEG-Biotin by the A549 cells after 1 h and 4 h treatment was observed by confocal mi-croscopy. A bright green fluorescence was noticed in the cells treated with biotin anchored dendrimer conjugate in comparison to the cells treated with the non-targeted conjugate. The green fluorescence caused by F-G4-PEG-Biotin in cells saturated with free biotin was seen to be less in intensity than the cells without excess biotin. This suggests that the biotin targeted dendrimers internalize actively by the biotin re-ceptors present in higher concentration on the surface of the A549 cells. The less significant uptake occurring in the presence of free biotin also suggests that the dendrimer conjugates underwent charge based ad-sorptive endocytosis. The adsorptive endocytosis of positively charged moieties and the biotin receptor mediated uptake together significantly improved the cellular association of the synthesized dendrimer con-jugates. Further, the results of confocal microscopy corroborated with the flow cytometry analysis indicating a higher cellular uptake in cells which received F-G4-PEG-Biotin treatment compared to F-G4-PEG, in a time dependent manner. The images captured at 1 h and 4 h incubation were represented in Fig. 3.
3.2.2. Cellular uptake by flow cytometry
Quantitative assessment of cellular association was performed using flow cytometry. Similar to the microscopy studies, influence of free biotin on the uptake of targeted dendrimers was also studied. The sa-turation of the biotin vitamin transporters with addition of excess free biotin resulted in decreased intensity of the fluorescence in the cells treated with F-G4-PEG-Biotin. Flow cytometry data revealed a time International Journal of Pharmaceutics 557 (2019) 329–341
dependent increase in the internalization at the end of 1 h and 4 h treatment. An increase in the geometric mean fluorescence was ob-served with time resulting in 1.53 fold and 1.94 fold higher uptake at 1 h and 4 h respectively for biotin tagged dendrimer conjugate. The results also indicated that the Biotin tagged dendrimer conjugates were promptly internalized via the biotin receptors. The role of biotin re-ceptors in uptake of actively targeted dendrimer conjugates was un-derstood and the data was in accordance with the confocal microscopy results. A bar graph of geometric mean fluorescence was plotted from the values obtained at 1 h and 4 h and represented along with the his-tograms of events collected by flow cytometer in Fig. 4.