we utilized an inverse electron-demand Diels-Alder cycloaddition between transcycloctene (TCO) and benzyltetrazine (Tz), one of the fastest and most efficient biorthogonal reactions reported to date. An N-hydroxysuccinimidyl derivative of TCO (TCO-NHS) was conjugated to the N-terminus of the tetrapeptide in pBAE-CRHR, which was then purified by precipitation with acetone:diethyl ether yielding pBAE-CRHR-TCO. BB4 was derivatized in solution with an excess of Tz-NHS to form Tz-BB4 and purified by reverse-phase HPLC (Figure ). pBAE-CRHR-TCO was then reacted with Tz-BB4 and purified by precipitation with acetone:diethyl ether to yield pBAE-CRHR-BB4. The reaction was monitored by HPLC-UV (Figure ), which showed disappearance of the Tz-BB4 peak at 6.5 min in the chromatogram and the polymer broad peak also shifted toward a higher retention time (6.2 min). Next, different ratios of BB4-targeted pBAE were mixed with pBAE-CRHR-Zw, ranging from 5% to 20%, and pGFP to form the polyplex (Figure ). These formulations were tested for transfection in HeLa cells, which express high levels of TfR1 .