Angiopep-2 Grafted PAMAM Dendrimers for the Targeted Delivery of Temozolomide: In Vitro and In Vivo Effects of PEGylation in the Management of Glioblastoma Multiforme.

The present study was aimed to synthesize, characterize, and evaluate the angiopep-2 grafted PAMAM dendrimers (Den, G 3.0 NH ₂ ) with and without PEGylation for the targeted and better delivery approach of temozolomide (TMZ) for the management of glioblastoma multiforme (GBM). Den-ANG and Den-PEG ₂ -ANG conjugates were synthesized and characterized by ¹ H NMR spectroscopy. The PEGylated (TMZ@Den-PEG ₂ -ANG) and non-PEGylated (TMZ@Den-ANG) drug loaded formulations were prepared and characterized for particle size, zeta potential, entrapment efficiency, and drug loading. An in vitro release study at physiological (pH 7.4) and acidic pH (pH 5.0) was performed. Preliminary toxicity studies were performed through hemolytic assay in human RBCs. MTT assay, cell uptake, and cell cycle analysis were performed to evaluate the in vitro efficacy against GBM cell lines (U87MG). Finally, the formulations were evaluated in vivo in a Sprague-Dawley rat model for pharmacokinetics and organ distribution analysis. The ¹ H NMR spectra confirmed the conjugation of angiopep-2 to both PAMAM and PEGylated PAMAM dendrimers, as the characteristic chemical shifts were observed in the range of 2.1 to 3.9 ppm. AFM results revealed that the surface of Den-ANG and Den-PEG ₂ -ANG conjugates were rough. The particle size and zeta potential of TMZ@Den-ANG were observed to be 229.0 ± 17.8 nm and 9.06 ± 0.4 mV, respectively, whereas the same for TMZ@Den-PEG ₂ -ANG were found to be 249.6 ± 12.9 nm and 10.9 ± 0.6 mV, respectively. The entrapment efficiency of TMZ@Den-ANG and TMZ@Den-PEG ₂ -ANG were calculated to be 63.27 ± 5.1% and 71.48 ± 4.3%, respectively. Moreover, TMZ@Den-PEG ₂ -ANG showed a better drug release profile with a controlled and sustained pattern at PBS pH 5.0 than at pH 7.4. The ex vivo hemolytic study revealed that TMZ@Den-PEG ₂ -ANG was biocompatible in nature as it showed 2.78 ± 0.1% hemolysis compared to 4.12 ± 0.2% hemolysis displayed by TMZ@Den-ANG. The outcomes of the MTT assay inferred that TMZ@Den-PEG ₂ -ANG possessed maximum cytotoxic effects against U87MG cells with IC ₅₀ values of 106.62 ± 11.43 μM (24 h) and 85.90 ± 9.12 μM (48 h). In the case of TMZ@Den-PEG ₂ -ANG, the IC ₅₀ values were reduced by 2.23-fold (24 h) and 1.36-fold (48 h) in comparison to pure TMZ. The cytotoxicity findings were further confirmed by significantly higher cellular uptake of TMZ@Den-PEG ₂ -ANG. Cell cycle analysis of the formulations suggested that the PEGylated formulation halts the cell cycle at G2/M phase with S-phase inhibition. In the in vivo studies, the half-life ( t _1/2 ) values of TMZ@Den-ANG and TMZ@Den-PEG ₂ -ANG were enhanced by 2.22 and 2.76 times, respectively, than the pure TMZ. After 4 h of administration, the brain uptake values of TMZ@Den-ANG and TMZ@Den-PEG ₂ -ANG were found to be 2.55 and 3.35 times, respectively, higher than that of pure TMZ. The outcomes of various in vitro and ex vivo experiments promoted the use of PEGylated nanocarriers for the management of GBM. Angiopep-2 grafted PEGylated PAMAM dendrimers can be potential and promising drug carriers for the targeted delivery of antiglioma drugs directly to the brain.