Kenneth Hulugalla

Nanoparticles (NPs) offer significant promise as drug delivery vehicles; however, their in vivo efficacy is often hindered by the formation of a protein corona (PC), which influences key physiological responses such as blood circulation time, biodistribution, cellular uptake, and intracellular localization. Understanding NP-PC interactions is crucial for optimizing NP design for biomedical applications. Traditional approaches have utilized hydrophilic polymer coatings like polyethylene glycol (PEG) to resist protein adsorption, but glycopolymer-coated nanoparticles have emerged as potential alternatives due to their biocompatibility and ability to reduce the adsorption of highly immunogenic proteins. In this study, we synthesized and characterized glycopolymer-based poly[2-(diisopropylamino)ethyl methacrylate-b-poly(methacrylamidoglucopyranose) (PDPA-b-PMAG) NPs as an alternative to PEGylated NPs. We characterized the polymers using a range of techniques to establish their molecular weight and chemical composition. PMAG and PEG-based NPs showed equivalent physicochemical properties with sizes of ∼100 nm, spherical morphology, and neutral surface charges. We next assessed the magnitude of protein adsorption on both NPs and catalogued the identity of the adsorbed proteins using mass spectrometry-based techniques. The PMAG NPs were found to adsorb fewer proteins in vitro as well as fewer immunogenic proteins such as Immunoglobulins and Complement proteins. Flow cytometry and confocal microscopy were employed to examine cellular uptake in RAW 264.7 macrophages and MDA-MB-231 tumor cells, where PMAG NPs showed higher uptake into tumor cells over macrophages. In vivo studies in BALB/c mice with orthotopic 4T1 breast cancer xenografts showed that PMAG NPs exhibited prolonged circulation times and enhanced tumor accumulation compared to PEGylated NPs. The biodistribution analysis also revealed greater selectivity for tumor tissue over the liver for PMAG NPs. These findings highlight the potential of glycopolymeric NPs to improve tumor targeting and reduce macrophage uptake compared to PEGylated NPs, offering significant advancements in cancer nanomedicine and immunotherapy.

Conventional inhibitors of immune checkpoints such as antiprogrammed death-1 and its ligand (anti-PD-1/PD-L1) and anticytotoxic T lymphocyte-associated protein 4 (anti-CTLA4) have revolutionized therapeutic approaches to cancer, establishing immunotherapy as the standard of care for many cancers. A significant number of cancers, however, remain refractory to the inhibition of these immune checkpoints, leading to the search for alternative immune checkpoints that are more relevant to those diseases. Tumor-associated macrophage (TAM)-mediated efferocytosis is an increasingly appreciated immune checkpoint with a profound impact on the phenotype of the tumor microenvironment (TME). TAMs perform their efferocytic function through the receptor MerTK, and MerTK activity correlates with tumor progression. To combat efferocytosis in the TME, we developed poly[[2-(diisopropylamino)ethyl methacrylate]-b-poly(methacrylamidomannose)] nanoparticles (PMAM NPs) capable of encapsulating and preferentially delivering UNC2025 (a MerTK inhibitor) to TAMs. The NPs had suitable physicochemical properties, such as a size of 130 nm and a neutral surface charge. The PMAM NPs encapsulated hydrophobic cargo and released them in a pH-dependent manner, showing suitability for cytosolic delivery. Moreover, the PMAM NPs showed 12-fold greater macrophage internalization than traditional PEGMA NPs. Macrophage internalization was shown to be dependent on the mannose receptor CD206, as the blockade of CD206 led to a significant decrease in PMAM NP internalization. Furthermore, PMAM NPs had a lower internalization than PEGMA NPs in 4T1 cancer cells that do not express CD206, further confirming macrophage selectivity. In vivo biodistribution studies showed that the PMAM NPs were capable of internalization by TAMs in the TME. Lastly, UNC2025-PMAM NPs significantly reduced tumor volume compared to free UNC2025, showing greater therapeutic efficacy in a model of triple-negative breast cancer. These glycopolymer-based, efferocytosis-blocking NPs have promise both as a class of standalone cancer immunotherapy and as an adjuvant to improve response rates to checkpoint immunotherapy.

Abstract Nanoparticle delivery to tumors remains inefficient, with current nanomedicines achieving only 0.7% injected dose per gram (ID/g) of tumor tissue due to uncontrolled protein corona formation that redirects nanoparticles away from target sites. We engineered biomimetic protein coronas to control nanoparticle-protein interactions and enhance tumor targeting. Competitive binding studies using NMR spectroscopy revealed that transferrin (Tf) and fibronectin (Fn) outcompete albumin (BSA) and immunoglobulin G (IgG) for 15 nm gold nanoparticle surfaces, establishing a binding hierarchy that enables predictable corona composition. Pre-coating nanoparticles with a four-protein combination (BSA+Tf+Fn+IgG) created coronas that selectively enhanced cancer cell uptake while reducing macrophage recognition in vitro. When administered to tumor-bearing mice, these engineered coronas achieved 13 ppm/g tumor accumulation—equivalent to 4% ID/g—representing 6.5-fold improvement over bare nanoparticles and 2.6-fold improvement over PEGylated formulations. Proteomics analysis of secondary coronas formed in human serum revealed that engineered nanoparticles selectively recruit transport and adhesion proteins while limiting immune recognition signatures. The pre-formed coronas maintained targeting protein retention and reduced complement binding compared to controls. Circular dichroism confirmed minimal protein structural perturbation, preserving receptor-binding functionality for active targeting. The strategy harnesses natural protein adsorption processes to create “smart” biological interfaces that simultaneously evade immune clearance and promote tumor cell recognition through multiple receptor pathways. This approach demonstrates the feasibility of treating the coron as a programmable interface, addressing delivery limitations that have hindered clinical translation of cancer nanomedicines. For Table of Contents Only

Abstract In addition to their central role in blood hemostasis, it is increasingly clear that platelets contribute to multiple steps in the metastatic cascade. Platelets are one of the most abundant cells with which tumor cells interact once they enter the circulation, and the interaction of platelets with tumor cells can improve tumor cell survival, arrest and adhesion at secondary sites, and extravasation. Therefore, targeting the interaction between platelets and circulating tumor cells could be an effective approach for reducing metastasis. Here, we repurpose the thromboxane A 2 -prostanoid receptor (TPr) inhibitor, ifetroban, to block platelet-tumor cell interactions and reduce metastasis in models of triple negative breast cancer (TNBC). We utilize in vitro co-culture models of platelets and tumor cell lines to assess the impact of ifetroban treatment on the adhesion of platelets to tumor cells. In each case, platelet-tumor cell adhesion was significantly increased when the TPr agonist U46619 was introduced, while pre-treatment with ifetroban (TPr antagonist), significantly reduced platelet-tumor cell adhesion. Further, we used a zebrafish model system to rapidly assess metastasis and platelet interactions in vivo, showing that ifetroban reduces metastasis of MDA-MB-231 xenografts without reducing platelet number in CD41 transgenic zebrafish embryos. Finally, we confirm that ifetroban significantly reduces both lung and liver metastasis in multiple murine models of TNBC (4T1 and MDA-MB-231). In these models, we observed that ifetroban reduces metastasis in the absence of a primary tumor and when TPr is deleted from tumor cells, further supporting the notion that ifetroban attenuates the supportive role of platelet TPr in the metastatic cascade. Based on the results of this study, ifetroban could be pursued as a clinical agent to reduce metastasis in TNBC patients. Graphical Abstract