(350t) Sonication Effectively Reduces Particle Diameter in Hemoglobin Nanoparticles Made By the Co-Precipitation Method

Purpose/Introduction: A variety of techniques have emerged for creating nano- and micro-particles. Protein based particles are especially appealing for biomedical applications due to their inherent biocompatibility. The method of co-precipitation, wherein a carbonate matrix is filled with a protein before being dissolved, creates a porous particle with substantial surface area, with high potential for drug or oxygen delivery applications. However, published methods using this technique produce particles with diameters in the submicron or micron range, generally regarded as incompatible with intravenous use due to rapid macrophage-mediated clearance. This study describes the use of ultrasound (US) sonication to achieve smaller hemoglobin nanoparticles (Hb-NPs) by the co-precipitation method which would be suitable for preclinical investigation as an oxygen therapeutic or drug delivery platform.

Experimental Design/Method: Hemoglobin was extracted and purified from expired human red blood cells and dissolved in a solution of manganese chloride. An equimolar amount of sodium carbonate was added to this solution, resulting in rapid precipitation of particles and particle aggregates. The entrapped hemoglobin was chemically cross-linked before dissolving the manganese carbonate particles with EDTA. Particles were washed into buffered saline and then sonicated with a 500 W probe sonicator for a variable duration and intensity to reduce overall size and break apart aggregates.

Major Findings/Results: We found that sonication effectively reduces particle size by an order of magnitude, reducing diameters from several micrometers to below 500 nm. A larger sonication probe tip (1/2”) was more effective at creating a monodisperse particle distribution than a smaller (1/16”) tip, even when the ratio of probe surface area to sample volume was fixed. Increased US intensity reduced diameter to a certain threshold size of about 250 nm, beyond which no significant difference was seen. Duration of sonication appeared to be a less important parameter than anticipated, with no difference seen with treatment greater than 5 minutes. Hb-NPs demonstrated a capacity to bind and release oxygen through stopped-flow deoxygenation kinetics as well as oxygen binding equilibria studies.

Summary of your interpretation of the data or conclusion: The reduction in particle diameter shown in these results opens the door to a wider variety of applications for nanoparticles made with the co-precipitation technique. These Hb-NPs exemplify one potential application, using the large surface area inherent to the method to facilitate oxygen transport by the hemoglobin protein.