Poster R6 - Microfluidic synthesis of short-wave infrared emitting nanoparticle contrast agents for biomedical imaging

Introduction: Fluorescent contrast agents are commonly used to highlight tissue structures and biomarker expression when used in combination with pre-clinical and clinical optical imaging systems. Most optical contrast agents emit light in the visible or near-infrared wavelength range ( < 1000 nm), which limits image resolution, contrast, and imaging depth because of photon scattering and tissue autofluorescence. We have developed nanoparticle-based contrast agents that overcome these limitations by emitting short-wave infrared light (1000 – 1700 nm wavelengths). These materials are synthesized from nanoscale rare-earth core elements encapsulated in a biocompatible protein or polymer shell. Surface ligands targeting specific biomarkers can be conjugated to the shell surface. Traditional small batch synthesis methods are suitable for performing individual in vitro or in vivo studies, but are challenging to scale up for large longitudinal studies while maintaining batch to batch consistency. We have developed a continuous flow microfluidic platform for nanoparticle synthesis which aims to deliver larger quantities of contrast agent with lower variability in physical and optical characteristics.

Materials and

Methods: A custom microfluidic platform using a micromixer chip and user-controlled pumps combined rare-earth nanoparticle cores in ethanol with human serum albumin in NaCl, followed by addition of glutaraldehyde for albumin cross-linking. These components were introduced into the micromixer chip at independently controlled flow rates varied from 375 ul/min to 3000 ul/min. Nanoparticles produced by the microfluidic system were characterized using dynamic light scattering (size, polydispersity) and imaging (optical brightness). Imaging was performed using a custom-built pre-clinical imaging system providing wide-field illumination at a wavelength of 980 nm and intensity of 200 mW/cm2. SWIR emissions from the contrast agent were imaged by an InGaAs camera with a 25 mm focal length lens (f/1.4) and two 1150 nm long-pass filters. Sample intensity levels were quantified by analysis of camera pixel values using ImageJ and Matlab software.

Results, Conclusions, and Discussions: Formation of albumin nanoparticles was achieved using the microfluidic platform with flow rate ratios (albumin solution flow rate: nanoparticle solution flow rate) ranging from 1:3 to 1:7 producing nanoparticles ranging in diameter from 147 nm to 113 nm. Five repeated synthesis runs at a fixed flow rate ratio of 1:4 produced nanoparticles with mean diameters of 131, 127, 125, 136, and 129 nm. Synthesis runs with input nanoparticle core solutions of 0.2, 0.4, and 0.8 mg/ml indicated that encapsulation was complete at concentrations up to 0.4 mg/ml, but some cores remained unencapsulated at 0.8 mg/ml, indicating an upper limit to the concentration of cores that can be reliably encapsulated. Imaging studies with nanoparticles synthesized using the microfluidic platform demonstrated high signal-to-background ratios in pure solutions and following administration to in vivo mouse models.

In conclusion, we have demonstrated that luminescent nanoparticle cores can be encapsulated in a biocompatible protein (albumin) shell using a continuous flow microfluidic platform. Preliminary studies demonstrated the ability to tune nanoparticle size by modification of component flow rates. The reproducibility of the approach was quantified. Our previous synthesis method produced 1.4 ml of contrast agent per batch, requiring several hours of hands-on bench work. Multiple batches would need to be synthesized and combined to generate enough material for a typical in vivo study. In contrast, the present microfluidic system produced nanoparticles at an output flow rate of 0.5 ml per minute with minimal user input.

In summary, SWIR-emitting contrast agents are gaining interest among researchers due to their ability to generate images with improved contrast and resolution, compared to visible / NIR agents. However, the range of available SWIR contrast agents remains limited and methods to synthesize these materials consistently and at high volume have not been explored. This presentation will present a new microfluidic method for continuous flow synthesis of a SWIR-emitting nanoparticle contrast agent, along with characterization methods and imaging data.

Acknowledgements (Optional): This research was funded by NIH/NIBIB award EB018378, NIH/NHLBI award U01HL150852, the Rutgers University ROI HealthAdvance program, and the NSF REU Summer program on Cellular Bioengineering at Rutgers University.