Nano and Micro Technologies
Makena Andersen (they/them/theirs)
Research Associate, Undergraduate Researcher
Lewis & Clark College
Pahoa, Hawaii, United States
Qi Wang
Ph.D. Student
FAMU-FSU College of Engineering, United States
Jamel Ali
Professor
FAMU-FSU College of Engineering, Florida, United States
Amisha Martin
Research Associate
FAMU-FSU College of Engineering, United States
Astrid Daugherty (she/her/hers)
Research Associate
FAMU-FSU College of Engineering
Tallahassee, Florida, United States
Izabela Kowalik, UG
Research Associate
National High Magnetic Field Lab
Land O' Lakes, Florida, United States
Magnetic micromotors are small scale devices which convert magnetic forces and torques into rotational and translational motion. Micromotors of various designs and compositions have been investigated for their capacity to be used in biomedical applications, such as active targeted drug delivery, nano surgery, biopsy, and localized diagnosis1. As mature erythrocytes (red blood cells, RBCs) have lost their nuclei and most of their organelles, they become naturally versatile cargo-carriers. Their immunosuppressive properties2 prevent them from being removed by immune cells during their circulation in the bloodstream. These characteristics make erythrocytes an ideal drug carrier. Based on a reported method of hypotonic treatment to fabricate erythrocyte micromotors,1 here we measured the osmolarity of suspensions with different ionic strength to control the hypotonic treatment process more precisely. More importantly, we also simplified the motor driven force to a single magnetic field. We then measured the step-out frequency of the RBC micromotor by applying a frequency sweep at a fixed uniform rotating field strength.
Defibrinated bovine blood (Carolina Biological Supply Company, Item#: 828514) was separated by centrifuging then washed with PBS three times. RBCs were resuspended in PBS-water solutions with various osmolarities, then iron oxide nanoparticles (MNPs) were added to the suspension. The cells were incubated at 4°C overnight, then centrifuged and washed with PBS three times to remove excess MNPs. The RBC micromotors were examined using bright field (BF) and scanning electron microscopy (SEM). To characterize the RBC micromotor, a magnetic field generator was used to apply a uniform magnetic field (10mT). For SEM preparation, RBC micromotors were fixed by 1% glutaraldehyde, then dehydrated by washing with increasing concentration ethanol. Fiji (ImageJ) and MATLAB were used to analyze the step out frequency of micromotors.
Characterization of hypotonic solutions: Solutions were prepared by mixing PBS and DI water in 3:1, 2:1, 1.5:1 and 1:1 ratios. The osmolarity of prepared solutions as well as plasma were measured by using freezing point osmometer (Osmomat 3000 basic), as shown in Figure 1.a. The osmolarity of plasma is approximately 300 mOsmol/kg. The osmotic pressure to plasma is similar to that of 1X PBS, thus 1X PBS is considered to be an isotonic solution for erythrocytes. In other tested salt solutions, RBCs were in varying states of hypotonicity.
Characterization of RBC micromotors: RBC micromotors were first characterized by SEM (Figure 1.b). Then, we performed experiments to characterize micromotor kinematics. When an external rotating magnetic field is applied to free-floating magnetic material, the magnetic torque induces rotation of the material. However, when the frequency of the rotating magnetic field exceeds a critical value, the magnetic substance movement transition from synchronous to asynchronous. This critical value is known as the 'step-out frequency.' The step-out frequency of our RBC micromotor was observed to be 4Hz (Figure 1.c). From 1Hz to 4Hz, the rotating frequency of micromotors increases linearly with external field frequency; when magnetic field frequency exceeds 4 Hz, the frequency of the motor reaches its maximum value, at the 10mT; when external field frequency larger than 6Hz, motor almost stopped rotating. These results indicate that RBC micromotors actuated at 10mT should be driven at frequencies at or below 4Hz to avoid non-linear behavior.
Conclusion: Plasma was observed to have an osmotic pressure to close to 1X PBS (~300 mOsmol/kg), which indicates that 1X PBS is an isotonic solution. Lower concentrations of PBS created hypotonic environments that can facilitate update of magnetic particles. Through examination of a range of applied external field frequencies, several kinematic properties were revealed. The step out frequency, which is crucial for guiding movement using magnetic fields, was observed to be 4Hz. These results will aid future investigations aimed at using magnetic erythrocyte-based micromotors for in vitro and in vivo applications.
This work was funded by the National Science Foundation (No. EES-2000202 and EES- 2219558) and supported by the NSF FAMU CREST Center award (No. EES-1735968). This research work was also supported by The Grainger Foundation Frontiers of Engineering Grant under the National Academy of Sciences Award Number: 2000013181. This material is based upon work supported by the Air Force Office of Scientific Research under award number FA9550-22-1-0247. All the work was performed at the National High Magnetic Field Laboratory, which is supported by National Science Foundation Cooperative Agreement No. DMR-1644779 and the State of Florida.
(1) Zhiguang Wu, Tianlong Li, Jinxing Li, Wei Gao, Tailin Xu, Caleb Christianson, Weiwei Gao, Michael Galarnyk, Qiang He, Liangfang Zhang, and Joseph Wang. ACS Nano 2014 8 (12), 12041-12048 DOI: 10.1021/nn506200x
(2) Gao C, Lin Z, Wang D, Wu Z, Xie H, He Q. Red Blood Cell-Mimicking Micromotor for Active Photodynamic Cancer Therapy. ACS Appl Mater Interfaces. 2019 Jul 3;11(26):23392-23400. doi: 10.1021/acsami.9b07979. Epub 2019 Jun 18. PMID: 31252507.