Poly(β-amino ester) (PBAE) nanoparticles have emerged as a promising non-viral delivery system due to their tunable chemical structures, biodegradability, and ability to transfect a wide range of cell types8,9. However, the structure–function relationships that govern their performance in immunologically cold tumors remain underexplored. To address this gap, we undertook a comprehensive and systematic evaluation of a library of over 100 structurally distinct PBAE polymers to identify formulations optimized for gene delivery across diverse cancer models. This study aims to not only uncover top-performing materials but also to provide mechanistic insights into how polymer architecture impacts intracellular trafficking, endosomal escape, and functional transgene expression in the tumor context. Using these insights, we aim to restore Signal 1 processing and presentation in a variety of cancer models, ultimately rejuvenating natural anti-tumor immunity.
Methods/Results The library encompassed four major architectural classes of PBAEs: linear, linear-lipophilic, branched, and branched-lipophilic. We began by screening these formulations using a GFP reporter plasmid in 4T1 triple-negative breast cancer cells, a more difficult-to-transfect and immunologically cold model. Transfection efficiency was quantified using flow cytometry, and lead candidates were identified based on the percentage of GFP-positive cells while maintaining high cell viability. Branched and branched-lipophilic polymers consistently outperformed linear variants in both transfection percentage and mean fluorescent intensity, while also reducing the dosage required. This observation aligns with previous reports suggesting that branching increases electrostatic interaction between oligonucleotide cargo, reducing the required dosage10. Additionally, the higher charge density of branched polymers enhances their buffering capacity in endosomes, which more significantly disrupts the Nernst equilibrium. This amplifies the electrochemical and osmotic gradients within endosomes, promoting more efficient endosomal swelling and escape. Added lipophilicity may also produce more tightly packed nanoparticles due to hydrophobic interactions but tends to increase toxicity.
Notably, a subset of branched PBAEs yielded over 50% GFP+ cells in 4T1 cells—an unprecedented efficiency in this context—while maintaining >85% viability. These formulations were further optimized through titration of DNA dosage and polymer:DNA weight ratios (w/w), and nanoparticle physicochemical properties were characterized using dynamic light scattering (DLS) to measure size distribution, zeta potential, and polydispersity index (PDI). Top candidates demonstrated hydrodynamic diameters in the 50–150 nm range with positive surface charges, favoring efficient uptake and endosomal trafficking. Structural integrity and molecular weight were verified via ^1H-NMR and gel permeation chromatography (GPC), respectively.
Following selection of top-performing formulations, we assessed their generalizability by testing across additional tumor models with differing degrees of immunogenicity, including B16-F10 melanoma, CT26 colorectal carcinoma, and MC38 colon adenocarcinoma. Similar trends in delivery efficiency were observed, with branched polymers maintaining high transfection efficiency in both epithelial and mesenchymal cancer types. These results suggest that polymer architecture, particularly branching, plays a central role in enhancing endosomal escape and nuclear delivery in diverse tumor environments.
To translate these materials toward functional immunotherapy applications, we next repurposed our lead formulations to deliver a panel of antigen processing machinery (APM) components. This panel, generated via Gibson Assembly, included critical proteins such as TAP1 and TAP2 (peptide transporters), Tapasin and ERp57 (peptide loading complex), Psmb8/LMP7 and Psmb9/LMP2 (immunoproteasome subunits), and master transcriptional regulators including NLRC5, IRF1, IRF2, BATF3, and CIITA. These components were selected based on their roles in stabilizing MHC-peptide complexes, promoting cross-presentation, and regulating transcriptional programs necessary for antigen display. Transfections were carried out using the optimized PBAEs in B16-F10 and 4T1 cells, followed by flow cytometric analysis of MHC class I, MHC class II, and PD-L1 surface expression. Delivery of APM constructs led to significant upregulation of MHC molecules—up to 10-fold increase in MHC-I and 100-fold in MHC-II—indicating successful functional rescue of antigen presentation machinery in B16-F10 cells. These effects were further validated by qPCR analysis, which confirmed transcriptional induction of multiple APM genes and transcription factors, verifying plasmid uptake and expression.
To assess whether enhanced MHC expression translated into improved antigen presentation quality, we utilized a B16-F10-OVA model and stained cells with the SIINFEKL-H-2Kb-specific antibody to detect presentation of the OVA-derived epitope. APM delivery led to a notable increase in SIINFEKL-MHC complexes, indicating that the improved antigen processing machinery not only restored surface MHC expression but also facilitated effective peptide loading. These findings support the hypothesis that even in the absence of co-delivered cytokines or costimulatory molecules, tumor cells can be made more immunogenic through targeted restoration of Signal 1—the antigen presentation signal necessary for T cell activation.
Future Directions The next phase of the study involves determining whether these modifications are sufficient to induce functional immune responses. To this end, we are conducting co-culture assays using tumor cells transfected with APM components and T cell clones specific for model antigens. These include PMEL17-specific CD8+ T cells for melanoma antigens, OT-1 CD8+ T cells for OVA, and OT-2 CD4+ T cells to assess MHC-II-dependent recognition. Tumor cells with restored antigen processing machinery will be evaluated for T cell activation markers (CD69, IFNγ), cytotoxicity (Granzyme B), and proliferation using multiparameter flow cytometry. Preliminary data suggest that APM restoration significantly enhances CD8+ T cell-mediated cytotoxicity and cytokine production, reinforcing the therapeutic relevance of targeting Signal 1. We will further validate these findings in vivo using flank tumor models in B16-F10 and orthotopic mammary tumors in 4T1-bearing mice. Tumor growth kinetics, immune infiltration (CD8+, CD4+, Tregs), and survival outcomes will be monitored to determine the therapeutic potential of this approach.
Conclusions In summary, this work demonstrates a rational strategy for engineering polymeric nanoparticles to overcome delivery challenges in solid tumors and to restore critical immune functions lost during tumor progression. By establishing the superior performance of branched PBAE polymers in enabling efficient gene delivery and endosomal escape, and by showing functional rescue of antigen presentation pathways, this research advances both non-viral delivery vehicles and cancer immunotherapy. The findings offer a path forward for enhancing tumor immunogenicity and may be synergistically combined with other immune-modulating agents to achieve full Signal 1/2/3 activation. These insights are relevant not only for immunoengineering and drug delivery fields within chemical engineering but also for broader efforts to design rational combination immunotherapies.
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