Tracking Dye Encapsulated Pathogen-like Particles (PLPs) using Ultrasound and Photoacoustic Imaging

Introduction:: Particulate vaccines, also known as pathogen-like particles (PLPs), have shown a more robust immune response compared to traditional, soluble vaccine delivery methods. This is mainly due to the modular design of PLPs, which allows for customization of size, shape, adjuvants, and surface molecule organization. However, the mechanism and characteristics of vaccine particle migration and delivery to the lymph nodes are still not well understood. Currently, fluorescent/bioluminescent in vivo imaging (IVIS) and MRI are the main non-invasive methods for in vivo imaging. However, these imaging methods are limited in their spatiotemporal resolution, low sensitivity, and imaging depth. Here, we aim to develop an ultrasound-guided photoacoustic (US/PA) imaging method to track dye encapsulated PLPs coated with CpG, a model oligodeoxynucleotide adjuvant known to boost immune response.


Materials and Methods:: IR780 dye was encapsulated in the poly (lactide-coglycolic acid) (PLGA)-PLPs nanoparticles in the oil phase during the double emulsion synthesis. To mimic vaccine particles and compare differences in lymph node trafficking, the IR780-PLPs was then coated with CpG adjuvant using EDC/NHS chemistry (Fig. A). All US/PA datasets were acquired using the Vevo LAZR (20 MHz, 680 - 950 nm wavelength). First, the particles with no dye (PLPs), uncoated particles (IR780-PLPs) and coated particles (CpG-IR780-PLPs) in a tube phantom were US/PA imaged (Q-Switch Nd:YAG pumped laser, 780 nm, 5 frames/sec, 7 ns pulse duration). Then, the IR780-PLGA-PLP NPs were injected intramuscularly in BALB/c mice in vivo (30 µL at 1 mg/ml). Photostability of the IR780-PLGA-PLPs was tested in vivo using US/PA imaging (1,500 laser pulses). Multi-wavelength, three-dimensional US/PA images were acquired using the US/PA imaging and a translational motor stage (step size 0.152 mm). The inguinal lymph node and injection site were imaged at multiple time points up to 24 hours post-injection.


Results, Conclusions, and Discussions:: Results:
IR780-PLPs had a hydrodynamic diameter of ~250 nm and the dye were successfully encapsulated inside the PLPs (Fig. B). The PA signal was higher in the IR780-PLPs and CpG-IR780-PLPs compared to particles with no dye encapsulated, demonstrating the ability to detect the dye encapsulated and coated particles using US/PA imaging (Figs. C-D). The IR780-PLPs showed excellent photostability and resistance to photobleaching, indicated by a minimal change in PA signal with more than 300 secs (5 frames/sec) of laser irradiation. These findings also ensure that true IR780-PLPs migration was observed in vivo in the non-photobleached treatment groups, which had a different signal profile compared to the photobleached control. (D). For in vivo tracking studies, the IR780-PLPs showed a 3.75-fold decrease in average PA intensity at the injection site at 2 hours and a 16.3-fold-decrease at 24 hours, which indicate a steady transport of nanoparticles away from the injection site (Figs. F-G). When monitoring the inguinal lymph node, there was a presence of IR780-PLPs, with a 1.5-fold enhancement in average PA intensity at 2 hours and a 6-fold enhancement at 24 hours (Figs. E and G). These preliminary results demonstrate qualitative and quantitative imaging of IR780-PLPs trafficking from the injection site to the lymph node.

Conclusion:
Our results indicate the feasibility of US/PA imaging to monitor the trafficking of PLGA-PLPs to the lymph node in vivo. By tracking and monitoring these vaccine particles, we can gain insights on the transport kinetics of the particles, routes of delivery, efficiencies of varying adjuvants, and vaccines physical properties, which can improve PLP design in translation.


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