Branched PEGylation enhances nanoparticle delivery to tumor-associated immune cells

Introduction: Barriers to nanoparticle drug delivery to the tumor microenvironment such as ECM deposition and clearance by the mononuclear phagocyte system have necessitated strategies for more effective tumor penetration. PEGylation has been widely used to both enhance accumulation at the tumor site and increase blood circulation time. Recent work has also shown that immune cells (e.g. macrophages, dendritic cells, neutrophils) play an important role in the ability of NPs to effectively target and spread within a tumor. However, it is unclear how PEG architecture such as PEG branching affects NP uptake in the tumor microenvironment. In this study, we evaluated the influence of PEG branching on nanoparticle biodistribution and uptake in tumor cells as well as tumor-infiltrating immune cells. We found that tumor-bearing mice treated with nanoparticles coated with branched PEG had greater nanoparticle accumulation in the tumor and a significantly higher fraction of immune cell uptake at the tumor site compared to nanoparticles coated with linear PEG or no PEG. These studies provide insight into PEG architecture as a design parameter in nanomedicine that can facilitate the design of more effective cancer therapies.

Materials and

Methods: 5 kDa linear PEG (PEG-L5) or 10 kDa 4-arm branched PEG (PEG-B10) were coated on carboxylate-modified 100 nm NIR fluorescent polystyrene nanoparticles via a carboxyl-amine linkage as described in our previous work.4 For in vivo studies on nanoparticle biodistribution, 5-week old athymic nude mice received a subcutaneous injection of 10^6 4T1 breast cancer cells above the right hip. Tumors were grown for 9 days until reaching a size >5 mm in diameter after which mice were intravenously injected in the tail vein with 100 ul of PEG-L5, PEG-B10, or non-PEGylated NPs at a concentration of 10^13 NPs/mL (10^12 NPs/mouse). IVIS was then used for live animal imaging immediately after injections as well as at 6, 24, 48, and 120 hours later. Mice were euthanized 24 hours post injection and the tumor, liver, and spleen from each mouse was harvested and imaged ex vivo using IVIS. Cells were isolated from harvested tissue and stained for relevant markers for tumor cells, neutrophils, macrophages, dendritic cells for quantification of nanoparticle uptake in specific cell types via flow cytometry.

Results, Conclusions, and Discussions: IVIS live animal imaging conducted 24 hours after NP administration found PEGylated NP avoid liver accumulation with the greatest NP retention observed for PEG-B10 NP. Ex vivo imaging further confirmed the greatest extent of tumor and spleen accumulation for PEG-B10 NP. Furthermore, flow cytometry analysis also revealed that branched PEGylation led to increased nanoparticle uptake in dendritic cells and macrophages within the tumor microenvironment. These results suggest branched PEGylation may be a useful tool for nanoparticle-based immunotherapies and are likely to have better distribution throughout the tumor due to enhanced uptake in tumor-associated macrophages. In summary, we found that branched PEG coatings enhance nanoparticle accumulation in 4T1 tumors via uptake in tumor-infiltrating immune cells. These findings demonstrate the importance in PEG coatings for enhanced tumor penetration which may enable their use in cancer immunotherapy.

Acknowledgements (Optional): This work was supported by the NCI-UMD Partnership for Integrative Cancer Research and NSF CAREER Award 2047794.

References:
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4. Cahn D, Duncan GA. "High-Density Branched PEGylation for Nanoparticle Drug Delivery". Cellular and Molecular Bioengineering. 15, 355–366, 2022.