In the present work, we demonstrated for the first time a simple method for the fabrication of drug loaded Microbubbles (MBs) by a single step via coaxial electrohydrodynamic atomization (CEHDA). As a proof of concept, a therapeutic agent (tissue plasminogen activator, tPA) and two types of shell materials (phospholipid and bovine serum albumin, BSA) were selected to produce tPA-entrapped MBs. Investigation using fluorescein isothiocyanate (FITC) labelled tPA revealed that the tPA-loaded MBs were successfully fabricated in a one-step procedure and the tPA was located in the shell layer for both the BSA and lipid MBs. By optimization of the operating conditions in terms of voltage, core/shell flow rate ratio as well as tPA volume ratio, minimum bubble sizes for tPA-BSA and tPA-lipid MBs were obtained. The fabricated tPA-BSA MBs was 41 mu m in mean diameter while similar to 41% of the tPA-lipid MBs ranged from 3 to 6 mu m and similar to 36% of them ranged from 6 to 9 mu m under optimal operating conditions. Sensitivity analysis on the effects of key process parameters was also performed to guide design and manipulation of bubble sizes. The investigation of gas phase showed that the usage of sulphur hexafluoride (SF6) as the core can enhance the stability of tPA-lipid bubbles. The presented one-step method displayed great flexibility for producing tPA-loaded MBs and thus can potentially serve as a new tool to generate/engineer tPA-bubbles for applications in ischemic stroke therapy. (C) 2016 Elsevier B.V. All rights reserved.