Advanced Therapeutics

Nanoparticle albumin-bound paclitaxel (Abraxane) was among the first clinically approved nanomedicines and is a first-line chemotherapeutic. It enhances therapeutic efficacy and reduces side effects of paclitaxel. Despite its extensive clinical use, mechanisms of Abraxane's therapeutic effects are the subject of ongoing study. This work posits a new mechanism for Abraxane's effects in lung cancers and metastasis. Tracing the albumin component of Abraxane with radioisotopes or fluorophores, we find that Abraxane homes to the lungs. Abraxane's lung tropism is elevated by inflammation and is eliminated in complement protein C3 knockout mice, showing that complement pathway drives the uptake. Flow cytometry and histology show that neutrophils are the dominant cell type taking up Abraxane in the lungs. We demonstrate Abraxane's tropism to pulmonary neutrophils in mice and in human donor lungs. We note previously undocumented side effects associated with Abraxane's lung tropism, including pro-thrombotic and pro-inflammatory responses in acute lung inflammation. We show that Abraxane's efficacy against lung metastases is affected when lung uptake is eliminated by C3 knockout. Our results provide insight into why Abraxane is effective in lung cancer and metastases. These findings may be used to improve outcomes in patients receiving Abraxane and help design next-generation cancer nanomedicines.

Glioblastoma and medulloblastoma are characterized by an immunosuppressive tumor microenvironment, in which tumor-associated macrophages may impair T-cell function, in part through the expression of PD-L1 and CSF1R. Single-target therapies have shown inconsistent results in brain tumors, likely due to the complex interplay involving tumor-associated macrophages and immunosuppression. We developed two immunoliposomes functionalized with antibodies against PD-L1 and CSF1R to co-target these receptors on M2-like tumor-associated macrophages. TAM2Ms were generated by polarizing THP-1 monocytes with glioblastoma (A172, U87MG) and medulloblastoma (DAOY, ONS-76) tumor-conditioned medium together with IL-4 and IL-13. In co-culture with activated Jurkat T cells under tumor-conditioned medium-influenced conditions, combined PD-L1 and CSF1R immunoliposomes enhanced T-cell proliferation, reduced apoptosis, and improved migration toward tumor spheroids compared to single-target liposomal treatments or free antibodies. These benefits varied between glioblastoma and medulloblastoma in vitro models, reflecting distinct TAM2M phenotypes and microenvironment contexts. Dual immunoliposome targeting of PD-L1 and CSF1R may offer a promising strategy for reprogramming tumor-associated macrophages in brain tumor immunotherapy and warrants further evaluation in primary human macrophages and in vivo models to clarify efficacy and mechanisms.

Previously, we demonstrated the biosafety of polyethylene glycol-coated iron oxide nanoparticles (MAG@PEG) in a cellular model of viral oncogenesis, supporting their further preclinical evaluation. Herein, we evaluated the safety and biodistribution of MAG@PEG in tumor-bearing mice and assessed the influence of an external magnetic field on nanoparticle distribution. Animals were randomly assigned to three groups: G1, control (PBS); G2, MAG@PEG (10 mg/kg); and G3, MAG@PEG (10 mg/kg) with magnetic exposure for 1 h. Doses were administered intraperitoneally weekly for 1 month. Results showed that animal behavior and body weight were unaffected by MAG@PEG, while tumor growth was significantly reduced only in G2. Biodistribution studies based on tissue magnetization revealed higher magnetic iron levels in the stomach followed by the spleen, lungs, and tumors, particularly in G2. Histological analysis detected Prussian blue-positive staining mainly in the spleen and in lymphoid-associated structures in the lung, stomach and intestine, while a fraction of MAG@PEG remained within the peritoneal cavity. Serum biochemistry showed no evidence of nephrotoxicity. Furthermore, hemocompatibility studies using human blood samples demonstrated that MAG@PEG was non-hemolytic according to the ASTM F756 standard. Taken together, the biodistribution, histological, biochemical, and hemocompatibility findings provide a comprehensive preclinical assessment of the biosafety of MAG@PEG and identify the route of administration and magnetic targeting as key determinants of nanoparticle biodistribution.

The cornea is the outermost layer of the eye, most vulnerable to injury including from chemical exposure, either environmental, industrial or chemical weapons. For example, exposure of the eye to sulfur mustard (SM) can cause mustard gas keratopathy, resulting in devastating consequences for vision. Available treatments for corneal mustard keratopathy are either ineffective, or if used for a prolonged time, have deleterious side effects. Here, we aimed to harness the anti-inflammatory and anti-angiogenic peptide angiotensin-(1-7), known as Ang-(1-7), that is derived from the renin-angiotensin system (RAS). We designed and developed a protein-like polymer (PLP) displaying a brush of norleucine modified Ang (1-7) peptides (NLE Ang (1-7) PLP), which is a more potent derivative of Ang-(1-7), for administration as an eye drop. In a mouse model of nitrogen mustard (NM)-induced corneal injury, topical treatment and intraperitoneal (i.p.) injection of NLE Ang (1-7) PLP persistently minimized: (i) corneal haze; (ii) edema; (iii), expression of pro-inflammatory and pro-angiogenic genes; and (iv) neovascularization and conjunctivalization as evidenced after a prolonged treatment and recovery period. Collectively, our observations have demonstrated the efficacy of this novel polymer proteomimetic based eye drop treatment in reducing chemical agent-induced corneal injury as well as indicating its potential application in other ocular surface diseases associated with inflammation and angiogenesis.

Clinical studies have revealed that glioblastoma (GBM) is refractory to immunotherapy, such as immune checkpoint inhibitors (ICIs), due to the immunosuppressive tumor microenvironment (TME) primarily intensified by intratumoral upregulation of transforming growth factor-{beta} (TGF{beta}). Primary GBM tumor bulks are surgically removed in clinic, but invasive tumor cells infiltrated into surrounding normal brain tissue cannot be eradicated by systemic therapy due to the integral blood-brain barrier (BBB), leading to near-inevitable tumor recurrence. To address these translational challenges, we have engineered a TGF{beta}-inhibiting protein nanocage comprising human heavy-chain ferritin protein and TGF{beta} receptor II ectodomain that enables BBB penetration and multivalent TGF{beta} entrapment, respectively. We demonstrate that the nanocage platform, denoted T{beta}R2-HFt, following systemic administration, traverses the BBB, and extensively modulates immunosuppressive TME, both stromal and immune components, thereby potentiating the therapeutic efficacy of a clinically used ICI in multiple mouse glioma models. Notably, a majority of the glioma-bearing animals that received surgical tumor resection, followed by treatment with T{beta}R2-HFt and the ICI, survived the tumor and resisted tumor rechallenge, providing a potential means to control GBM in a durable manner.

Protein replacement therapy is highly effective for treating many protein-deficiency disorders, but its long-term clinical benefit is often limited by anti-drug antibody responses that neutralize therapeutic proteins, compromising safety and efficacy. To address these unwanted immune responses, a lipid nanoparticle-based immunotherapy platform has been developed to induce antigen-specific tolerance. Mice treated with antigen associated with either double-chain phosphatidylserine (PS) or lysophosphatidylserine (LysoPS), a single-acyl-chain phospholipid, showed reduced antibody titers following subcutaneous pre-exposure. However, only LysoPS nanoparticles induced tolerance after oral administration, suggesting structural differences between PS and LysoPS can impact tolerogenic potential, depending on the site of delivery. As an apoptotic "eat-me" signal, PS is recognized by phagocytic receptors such as TIM-4 and is associated with LC3-associated phagocytosis. These nanoparticles have been hypothesized to promote apoptotic mimicry to regulate immunity. However, the contribution of TIM-4 to oral tolerance remains unclear. Here, we investigated the role of TIM-4 in LysoPS-mediated oral tolerance using a knockout mouse model. Our data showed that TIM-4-/- mice induced tolerance to ovalbumin encapsulated in LysoPS comparable to that of wild-type mice. We also found that LysoPS nanoparticles did not follow the same surface engagement and intracellular trafficking pathways as PS nanoparticles. These results reveal that, despite sharing the same headgroup, differences in acyl chain number critically shape nanoparticle-immune cell interactions and provide a mechanistic framework that may explain the unique ability of LysoPS nanoparticles to induce oral tolerance.

Glial cells play essential roles in maintaining neural tissue homeostasis and limiting the spread of injury-induced neuroinflammation, yet their dysfunction can also exacerbate neurodegenerative, neuroimmune, and neurodevelopmental disorders. Therapeutic targeting of specific glial cell populations would open up new treatment possibilities, but currently available viral and non-viral delivery vehicles are limited by carrier toxicity as well as poor delivery efficiency and specificity. To work towards new glia-targeted, non-viral delivery vehicles, we performed an in silico analysis on neural cell specific RNA-Seq datasets in mice to identify surface molecules differentially enriched on neurons, astrocytes, and microglia. We identified Slc2a5 (Glut5) as a microglia-enriched transporter and validated its microglia specific expression in healthy murine neural tissue using RNA in situ hybridization. By employing a sequential thiol-ene and enzymatic catalyzed reaction scheme, we synthesized polyelectrolytic dendrimers with multivalent presentation of fructose, the natural ligand for Glut5, and formulated nano-sized polyionic complexes (PICs) loaded with protein or nucleic acid cargo by mixing oppositely charged dendrimers. Fructose functionalized PICs lacking cationic surface charge enabled fructose-dependent intracellular protein delivery to Glut5-expressing human breast cancer cells (MCF-7) and mouse fibroblasts, while restricting uptake by Glut5-negative mouse astrocytes, confirming transporter-dependent selectivity in vitro. Fructose functionalized PICs resulted in minimal neuroinflammation when injected into mouse striatum at 1 mg/ml but elicited concentration-dependent neural tissue toxicity at higher doses. Fructose-PICs failed to achieve microglia specific intracellular delivery of functional proteins when injected into healthy neural tissue but aided in directing protein delivery to microglia and astrocytes at ischemic stroke lesions. Together, our findings demonstrate proof-of-principle for using in silico RNA-seq screening workflows to identify cell specific surfaceome targets and incorporating that insight into a modular biomaterial platform for targeted drug delivery.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. This clinical burden is predominantly attributable to atherothrombotic events resulting from platelet hyperactivation, a process integral to the pathophysiology of atherosclerosis. Cilostazol (CLZ) is a clinically established phosphodiesterase-IIIA inhibitor with antiplatelet and vasodilatory activity. However, its extremely low aqueous solubility results in dissolution-limited and variable oral absorption, while dose-dependent adverse effects and cardiovascular safety restrictions may constrain its clinical use. Lipid-polymer hybrid nanoparticles (LPHNPs) represent a highly promising drug delivery system to address these biopharmaceutical limitations. However, their interactions with blood components remain poorly understood, with surface charge among the most controversial physicochemical parameters implicated in platelet toxicity. Here, we applied an integrated ex vivo and in silico screening strategy to design LPHNPs based on poly(lactic acid) and poly(ethylene glycol)-modified lipids displaying positive (amine), nominally neutral (methoxy), and negative (carboxyl) surface charges to systematically identify safe boundaries at the nano-human platelet interface and evaluate their suitability as CLZ carriers. Positively charged nanoparticles precipitated immediately during self-assembly due to uncontrolled ionic pairing and were therefore structurally unviable. Neutral nanoparticles maintained a stable size (~113 nm hydrodynamic diameter) but unexpectedly induced spontaneous platelet aggregation, limiting their applicability. In contrast, negatively charged nanoparticles exhibited exceptional colloidal stability (hydrodynamic size of ~120 nm and zeta potential of -48 mV), achieved significant inhibition of ADP-induced platelet aggregation, and adhered to the platelet surface without inducing activation or membrane disruption. Steered molecular dynamics simulations, performed under identical non-equilibrium pulling conditions, showed the highest interfacial resistance to CLZ extraction for the negatively charged model, qualitatively consistent with the sustained CLZ release profile observed in vitro. These findings highlight the importance of precise surface charge tuning in the development of safe cardiovascular nanomedicines based on LPHNPs, emphasizing the need for comprehensive platelet compatibility testing before further biological evaluation.

A broad-spectrum antiviral is required to enable rapid intervention against current, newly emerging and mutating viruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), Sindbis virus (SINV) and chikungunya virus (CHIKV) all engage negatively charged moieties on host cells, such as heparan sulfate (HS) and sialic acid, to facilitate attachment for entry and infection. We designed HS-mimetic nanoparticles in the form of polymer vesicles (polymersomes) by blending membrane-forming and sulfonated copolymers. These biocompatible polymersomes successfully inhibited host cell infection of HS-dependent SARS-CoV-2 and HS-independent IAV in vitro with sub-nanomolar potency. Multivalent interaction of several virus ligands with a single polymersome as quantified by fluorescence correlation spectroscopy likely contributes to their potency. The sulfonated polymersomes also functioned in vivo as demonstrated by a reduction in the spread of SINV, an alphavirus model for CHIKV, in zebrafish embryos, which led to a significantly increased survival. Hence, our polymersomes could form the basis for an urgently required multifunctional broad-spectrum antiviral nanomedicine.

Targeted alpha therapy with 225Ac is a promising modality for cancer treatment, as shown in recent preclinical and clinical studies. Preclinical imaging of 225Ac is essential to fully understand the biokinetics of developmental 225Ac radiopharmaceuticals. Although single photon emission computed tomography (SPECT) is typically used to achieve this in vivo, conventional preclinical scanners struggle to provide high contrast and accurate quantification for 225Ac imaging due to the very low injected activities (below 1 kBq/g), the unfavorable gamma-ray branching ratios (<26%), and its complex decay chain with several gamma-ray emissions. In this work, we employ a compact CZT-based Compton camera to achieve quantitative tomography of 225Ac daughters in live mice at 18.5 kBq (0.5 Ci) injected activities. Three mice bearing 22Rv1 prostate cancer subcutaneous xenografts were scanned in a single bed position for 30 minutes following intravenous administration of 18.5 kBq of the Anti-CD46 225Ac-Macropa-PEG4-YS5 conjugate. We successfully visualized the in vivo biodistributions of the 225Ac daughters 221Fr and 213Bi, distinguishing between the tumor xenograft and the central organs. Quantification of the tumor uptake from the images revealed an activity as low as 1.1 kBq. In vivo activity quantification computed from the images was compared to ex vivo biodistribution measurements using a gamma counter after harvesting the tissue, showing strong agreement.

Biological systems such as extracellular vesicles (EVs) are highly sophisticated and functionally diverse. Despite growing recognition of EVs' roles in health and disease through mediating intercellular communication, their inherent heterogeneity and challenges in purity and yield hinder their mechanistic research and clinical translation. To address this gap, we sought to translate essential EV properties into a controllable and simplified platform by engineering biomimetic synthetic vesicles (SVs) incorporating defined proteins and lipids. Using microfluidics, we generated SVs loaded with bulk membrane and cytosolic proteins from cancer cells, as well as individual organotropism-associated integrins. Bulk membrane protein incorporation into SVs reduced surface charge, modified lipid profiles, and enhanced cellular uptake, while integrin incorporation altered biodistribution. To investigate protein loading, we profiled input materials and SVs by mass spectrometry, identifying features linked to high incorporation, such as low molecular weight, positive charge, high isoelectric point, and predicted stability, globularity, lipidation, and amphipathic helices. This work presents a synthetic platform for dissecting the roles of EV-associated features and cargo, while providing insights into protein properties relevant to the optimization of delivery systems.

For regenerative medicine applications, transplanted cells are difficult to retain in a specific place to maximize the localized release of cytokines, which can positively modulate immune responses, angiogenesis and tissue regeneration. Here, we developed a hollow electrospun (ESP) scaffold with one open and one closed end to enclose a hydrogel laden with human mesenchymal stem cells (hMSCs); we termed this combination the hydrocup. hMSCs remained viable for 28 days in vitro and released functional cytokines similar to hMSCs in transplanted but non-enclosed hydrogels. The hydrocups remained intact and fixed in place for 6 weeks after subcutaneous implantation in rats, demonstrating in vivo compatibility. These results demonstrate that the hydrocup is the first, to our knowledge, cell-laden hydrogel delivery scaffold. This innovation could be developed for a wide variety of cell-based cytokine secretion or drug-laden hydrogel applications.

Mesaconate (MES, an immunosuppressive metabolite) but not itaconate is upregulated in stool of Crohns Disease (CD) patients, albeit below therapeutic levels.. However, colonic MES level is difficult to maintain because of its high absorption rate, limiting its therapeutic potential for CD. Herein, we developed two strategies that elevate MES level and suppress inflammation: EcN-CAD, an engineered probiotic Escherichia coli Nissle 1917 (EcN) that overexpresses cis-aconitate decarboxylase (CAD, produces itaconic acid that is converted to MES); and MES+Zn, a Zinc-MES ionically crosslinked formulation. Mechanistically, MES modulates T cell function by downregulating glycolytic and mitochondrial activity and decrease ROR{gamma}t expression. In preclinical SAMP1/YitFC dexamethasone-withdrawal model and TNFDARE model, MES+Zn decreased gut permeability significantly compared to controls and reduced myeloperoxidase (MPO, an inflammatory marker) 1000-fold compared to controls. EcN-CAD probiotics decreased ROR{gamma}t expression in CD4+ and CD8+ T cells, gut permeability and inflammation in preventive SAMP1/YitFC model, and SAMP1/YitFC dexamethasone-withdrawal model. Overall, MES rewires T cell responses and technologies that enhance MES levels can be effective in gut-related inflammatory diseases.

The immunosuppressive tumor microenvironment (TME) poses formidable challenges for cancer immunotherapies. Among the complex TME landscape, the cytokine TGF-{beta}1 is an attractive soluble target for reinvigoration of anti-tumor immunity due to its pivotal function in immune cell suppression, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT) promotion, while immune checkpoint receptors exemplified by PD-1 and CD24 are promising surface targets. Though adaptive immune checkpoint inhibition is well established in clinical practice, TGF-{beta} blockade or innate immune checkpoint blockade is yet to be harnessed for tumor treatment. Here, leveraging CD24 overexpression on multiple tumor tissues and its unique recycling kinetics, we designed an innate Immune Checkpoint TArgeting Chimera (ICTAC) G01, a bifunctional CD24 antibody-TGF-{beta}1 receptor fusion protein that disrupts TGF-{beta}1 signaling by TGF-{beta}1 degradation instead of simple blockade and simultaneously inhibits the "don't eat me" CD24 signaling. We demonstrated that G01 effectively suppressed TGF-{beta}1-Smad signaling, reversed the EMT, and attenuated fibrotic collagen deposition. Additionally, it antagonized CD24 signaling to unleash robust antibody-dependent effector activity, further reprogramming the immunologically cold TME toward inflammatory phenotype. By selectively targeting malignant cells while sparing CD24-expressing B cells, G01 demonstrated a favorable safety profile. Notably, in patient-derived tumor fragments (PDTFs), G01 also inflamed the TME, underscoring its translational potential. Together, these results establish ICTAC as an effective strategy for tumor-specific receptor-mediated extracellular protein depletion, which supports its application for reversing TGF-{beta}1; and innate immune checkpoint-mediated tumor immunosuppression by concurrent targeting of both surface and soluble tumor-promoting factors.

Cationic lipid nanoparticles have demonstrated unique potential for extrahepatic mRNA delivery, particularly enabling selective targeting of the pulmonary endothelium. However, their translational development has been hampered by reports of infusion-related immune reactions and innate immune system activation, most notably transient complement activation. Here, we present a case study illustrating the discovery and translational advancement of a selected cationic LNP into non-human primates (NHPs) for initial pharmacokinetic assessment and evaluation of potential immunostimulatory side effects. We show surface charge dependent organ-selective expression of reporter mRNAs from different LNPs in vivo. An mRNA encoding the Tie2 agonist COMP-Angl, was formulated with LNP002, and respective pharmacokinetic and pharmacodynamic readouts were analyzed in two independent non-human primate studies. Notably, dose-dependent transient complement activation could be abrogated by extending the infusion time. Finally, we identified the blood-borne pharmacodynamic biomarker PDGFB for LNP002/mRNA-76 treatment reflecting activated Tie2-signalling in healthy pulmonary endothelium in vivo supported by single cell sequencing and cluster-alignment of downstream effector genes with the same spatial profile as the delivered mRNA.

Regulatory T cells (Tregs) are key mediators of immune resolution and tissue repair following acute kidney injury (AKI), yet strategies to selectively enrich Tregs within the renal microenvironment remain limited. Interleukin-2 (IL-2) is essential for Treg survival and function, but systemic IL-2 delivery is constrained by a short half-life, narrow therapeutic window, and risk of off-target activation of cytotoxic T cells. Effective Treg-based immunomodulation may therefore require both localized cytokine signaling and targeted recruitment of precursor immune populations. Here, we present a sequential, biomaterial-based immunomodulatory strategy that integrates immune cell recruitment with sustained cytokine delivery. An injectable granular hydrogel physically encapsulating the chemokine CCL21 was used to initially recruit CD4+ T cells to the injured kidney. Subsequently, supramolecular guest-host interactions were leveraged to deliver IL-2 modified with the hydrophobic "guest" moiety adamantane, enabling prolonged local cytokine release over 28 days. In healthy mice, we observed early CD4+ T cell recruitment, followed by a pronounced increase in Treg frequency, consistent with immune reprogramming driven by our sequential delivery system. In a murine model of bilateral ischemia-reperfusion (BiAKI), this injectable biomaterial induced a local increase in the proportion of FOXP3-expressing Tregs and a concomitant decrease in the levels of AKI markers. This two-step immunomodulatory approach minimizes acute immune activation while promoting long-term enrichment of Tregs within the renal microenvironment. Together, these findings demonstrate that granular hydrogels with supramolecular host capability can orchestrate immune recruitment and differentiation in a spatiotemporally controlled manner, providing a versatile immunomodulatory platform for applicability in kidney disease.

Solid tumors are known to be refractory to treatment due to a poorly perfused microenvironment that blocks immune cell infiltration and effective drug delivery. Histotripsy is a non-invasive focused ultrasound modality that generates cavitation microbubbles to mechanically disrupt solid tumors, resulting in decreased hypoxia and stimulation of tumor-targeted adaptive immune responses. In this study, we aim to investigate whether post-histotripsy hypoxia reduction and cytotoxic immune cell infiltration are associated with normalization of tumor vasculature and enhanced perfusion. Structural analysis of both treated and contralateral off-target tumors following partial histotripsy revealed significant vascular remodeling, enhanced vascular integrity, and decreased vascular leakage indicative of vascular normalization in multiple in vivo models of melanoma, hepatocellular carcinoma, and pancreatic adenocarcinoma. These effects appeared antigen-specific, as no remodeling was observed in contralateral off-target tumors of discordant pathology. Mechanistically, CXCR3+CD8+ T cells played a role in mediating these vascular changes, as genetic knockout of CD8 and pharmacological antagonism of CXCR3 impaired both remodeling and normalization. Additionally, significant changes in endothelial CXCR4 and Angiopoietin-1 and -2 expressions were observed following histotripsy, indicating their potential involvement in promoting vascular normalization and remodeling. Significant vascular remodeling was accompanied by enhanced tumor perfusion and intratumoral delivery of chemotherapeutic agents and therapeutic monoclonal antibodies. These findings suggest that histotripsy not only creates a more favorable microenvironment for immunotherapy, but also optimizes drug delivery into solid tumors. Combinatorial approaches using histotripsy with chemotherapy or immunotherapy may be a novel therapeutic strategy to overcome current limitations of conventional solid tumor therapies.

Recent efforts to reconstruct critical-sized craniomaxillofacial defects using biomaterials have increasingly sought strategies to accelerate proangiogenic processes immediately after surgical implantation to improve host-graft integration. A class of mineralized collagen scaffolds has recently been identified to promote osteogenesis of bone marrow and adipose-derived mesenchymal stem cells both in vivo and in vitro, motivating efforts to identify strategies to promote osteogenic-angiogenic coupling in order to support regenerative healing. Here, we report the first-time use of a glycoengineering approach to generate a population of engineered ASCs (eASCs) from human adipose-derived stem cells (hASCs). This effort inserts an azido sugar onto the cell membrane that can be selectively targeted using DBCO-tagged biomolecules to selectively boost eASCs activity. We report the effects of azido sugar labeling conditions (concentration and incubation time) on eASCs labeling efficiency. We show proliferation and pro-regenerative gene expression activity of eASCs in mineralized collagen scaffolds are largely similar to conventional hASCs. And we demonstrate that molecular cargo functionalized with dibenzocyclooctyne (DBCO) can be conjugated to azido groups on eASCs via click chemistry. Together, this work shows metabolically labeled eASCs have the potential to be used for selective delivery of activity-inducing bioactive cues to enhance regenerative potential.

The current treatment regimen for neuroblastoma involves immunotherapy, including a monoclonal antibody that recognizes disialoganglioside (GD2), expressed at high levels on neuroblastoma. GD2 is not present on most normal tissues except for nerves. Thus, anti-GD2 antibody treatment causes substantial, dose-limiting neuropathic pain. B7-H3 is overexpressed on multiple tumor types, including neuroblastoma, and is largely absent on nerves and other normal tissues. We designed a bispecific antibody (bsAb) that requires simultaneous binding of these two tumor antigens to achieve tight binding of tumor cells. Our preclinical research shows that, compared with a monospecific anti-GD2 antibody, the GD2xB7-H3 bsAb has improved tumor specificity, comparable antitumor efficacy, and reduced nerve binding and pain-associated toxicity. Since this bsAb does not bind to nerves, it may permit more tolerable and sustained treatment schedules than are currently feasible with monospecific anti-GD2 antibodies. In addition, its enhanced tumor specificity may support future development as a targeted delivery platform for antibody-drug conjugates or other payload-based therapies, potentially improving both efficacy and quality of life for patients with neuroblastoma.

The delivery of small-molecule therapeutics across cellular membranes remains a fundamental challenge in drug development, particularly for compounds whose physicochemical properties limit intracellular bioavailability. Inspired by the low toxicity, biodegradability, and synthetic accessibility of triethanolamine-derived lipids, we investigated a series of C18 aminotriester lipids as excipients for the codelivery of anticancer therapeutics. Four structurally related triester lipids bearing saturated, cis-monounsaturated, trans-monounsaturated, and di-unsaturated C18 tails were synthesized through straightforward esterification reactions with triethanolamine. These lipids were evaluated for their ability to enhance the antiproliferative activity of several anticancer agents, including paclitaxel, doxorubicin, amonafide, homoharringtonine, camptothecin, and SN-38. Across multiple drug classes, the aminotriester lipids exhibited minimal intrinsic cytotoxicity and generally produced limited enhancement of drug potency. However, the fully saturated tris-stearate aminotriester lipid selectively enhanced the antiproliferative activity of amonafide in HCT-116 colorectal cancer cells, particularly at lower concentrations. In contrast, unsaturated lipid analogues displayed negligible enhancement effects, and the activity of the tris-stearate lipid was substantially attenuated in A549 lung adenocarcinoma cells, weakly observed in HT-29 colorectal cancer cells, and absent in CT26 murine colorectal cancer cells. Lactate dehydrogenase release assays indicated that these effects were not associated with membrane lysis or nonspecific cytotoxicity. Further, fluorescence microscopy time course experiments suggest improved membrane trafficking of amonafide when codelivered stoichiometrically with fully saturated C18 aminotriester lipids. Collectively, these findings demonstrate that saturated triethanolamine-based aminotriester lipids may function as selective and biocompatible codelivery excipients for certain small-molecule therapeutics. The observed dependence on both lipid saturation state and cellular context suggests that membrane organization and lipid packing may play important roles in mediating these effects. These results establish a foundation for further mechanistic investigations into aminotriester lipid-assisted small-molecule delivery systems.