Purpose: To reduce the systemic exposure of a volatile anesthetic, isoflurane, by encapsulating it in a chitosan coated emulsion, Intralipid®, so isoflurane can be administered as a targeted immune modulating agent.
Methods: Chitosan coated, isoflurane loaded Intralipid® emulsion nanoplexes are formed by adding up to 100 µl of isoflurane to 1 ml of 20% Intralipid, vortexing for 15 seconds, followed by the addition of 4 ml chitosan (0.75 mg/ml; MW 5k) and vortexing for 15 seconds. Resulting nanoplexes are purified by centrifugation (18,500 x g, 10 minutes, 4°C). Nanoplexes are resuspended in water. An additional layer of low-viscosity alginate is added and purified by centrifugation, as described above. The nanoplexes are characterized by: size (dynamic light scattering); charge; recovery following centrifugation; encapsulation efficiency (GC-MS or FTIR), and effect (righting reflex). Righting reflex time was measured in CD-1 female mice that were administered a bolus tail vein injection (5 µl/g bw).
Results: Initial Intralipid emulsion is ~290 nm and negatively charged (-38 mV). Upon addition of chitosan and purification, the size increases to ~360 nm and becomes positively charged (+21 mV). Addition of the following alginate layer changes the surface charge back to negative (-46 mV), and the size increases to ~460 nm. A second layer of chitosan increases the size to ~870 nm with a positive charge (-29 mV). Isoflurane is stably entrapped in the emulsion for at least 2 h. The maximal time of lost consciousness is 75 seconds independent of formulation. The chitosan coated nanoplex emulsion has an increased lethal dose (100 vs 90 nl isoflurane/g bw for the non-coated nanoplex emulsion). Fitting the data with a sigmoidal curve, the chitosan coated nanoplex emulsion has an increased EC50 (85 vs 75 nl isoflurane / g bw for the non-coated nanoplex emulsion).
Conclusion: We have successfully developed a chitosan nanoplex that can entrap and deliver isoflurane to mice by i.v. injection. The chitosan coating increases the volume of isoflurane that can be administered prior to loss of consciousness, as well as decreases the total time of lost consciousness for an equivalent dose of non-coated isoflurane loaded Intralipid®. Future work involves increasing the layers of chitosan and alginate to continue to increase the amount of isoflurane that can be administered, as well as determining efficacy of this immune modulating drug delivery system in a murine model of influenza.
This work is supported by the Ruth L. Kirschstein National Research Service Award (NRSA) Institutional Research Training Grant 1T32GM099607 and the National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR001412. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Prince Bonsu– University at Buffalo, New York
Lisa Eagler– University at Buffalo
Sarah Reeves– University at Buffalo
Chris Russo– University at Buffalo
Blessing Hunsu– University at Buffalo
Charitie Hill– University at Buffalo
Paras Prasad– SUNY Distinguished Professor, Institute for Lasers, Photonics and Biophotonics, University at Buffalo, New York
Bruce Davidson– Research Associate Professor, Department of Anesthesiology, University at Buffalo, New York
Paul Knight– SUNY Distinguished Professor, Department of Anesthesiology, University at Buffalo, New York