Advanced Therapeutics

Osteosarcoma (OS) is the most prevalent primary bone malignancy in children and adolescents; however, therapeutic outcomes remain suboptimal due to tumor heterogeneity, chemoresistance, and inadequate immune activation. Doxorubicin (Dox), the standard therapy that induces immunogenic cell death, has its efficacy compromised by the immunosuppressive tumor microenvironment (TME). While interleukin-12 (IL-12) can activate and recruit various immune cells, making it an attractive combination partner, its systemic delivery is severely limited by dose-limiting toxicity. We have previously reported that intravenous injection of A3 collagen binding domain (CBD) of von Willebrand Factor preferentially accumulates into the TME of various tumor models enriched in collagen I and III. Furthermore, CBD-fused IL-12 (CBD-IL-12) demonstrated superior therapeutic effects against various cancer models compared to unmodified IL-12 due to its collagen-targeted delivery and the resulting tumor-localized inflammation. Given that the OS TME also exhibits higher collagen I and III expression compared to normal bone, we hypothesized that a CBD-IL-12 fusion protein could showcase potent anti-tumor efficacy in OS via tumor-specific accumulation. Here, we demonstrated that CBD-IL-12 exhibited 4-fold enhanced tumor accumulation compared to unmodified IL-12 and increased cytotoxic T cell infiltration by 2.2-fold within the immune-cold microenvironment in a mouse model of OS. The combination of CBD-IL-12 with Dox significantly prolonged median survival in two independent murine OS models. This coordinated approach utilizing Dox coupled with precision-targeted IL-12 immunotherapy represents a clinically translatable strategy that overcomes the inherent limitations of single-agent treatments for OS. HighlightO_LICollagen-targeted IL-12 increases tumor accumulation in osteosarcoma. C_LIO_LIThe collagen-targeted IL-12 synergizes with doxorubicin in osteosarcoma models. C_LIO_LICombination therapy enhances T cell differentiation and activates innate immunity. C_LI

Adoptive cell therapies used to treat advanced prostate cancer are being developed to target several tumor-associated antigens, including prostate-specific membrane antigen (PSMA). Chimeric antigen receptor (CAR) T cell therapy using the single chain variable fragment (scFv) derived from the humanized murine mAb clone, J591, as the antigen-binding domain has shown promising anti-tumor activity. However, it has also been associated with macrophage activation syndrome and other unwanted toxicities, highlighting the need for more specific and human-derived antigen-binders with optimized construct designs for improved safety and efficacy. Here, we optimize a human scFv-based PSMA-targeted CAR (hPSMA-CAR) with highly selective PSMA targeting. We further introduce a membrane-bound IL-12 (mbIL12) molecule, which enhances potency with increased T cell expansion, IFNy production and anti-tumor cell activity in vitro. Using two clinically-relevant bone-metastatic prostate cancer models, we show that mbIL12-engineered hPSMA-CAR T cells drive potent in vivo anti-tumor responses. In summary, we have developed a promising therapeutic that has potential to promote safe and effective treatment of advanced PSMA+ prostate cancer.

PurposeEndothelial cell (EC) activation, characterized by upregulation of adhesion molecules that drive monocyte recruitment, contributes to plaque progression while also providing an opportunity for targeted therapeutic delivery. Leveraging the cell membrane cloaking strategy, we recently developed a monocyte-mimetic nanoparticle (MoNP) platform that exploits the natural inflammatory tropism of monocytes for site-specific delivery to atherosclerotic vessels. Recognizing that integrin activation is a key determinant of monocyte adhesion to ECs, this study investigates whether pre-activating integrins on MoNP enhances their binding affinity and accumulation at atherosclerotic lesions. MethodsMouse bone marrow-derived monocytes were pretreated with CCL2 or Mn2{square} to activate membrane integrins. Isolated monocyte plasma membranes were cloaked onto fluorescently labeled polymeric cores to generate integrin-activated MoNPs (IA@MoNPs). The targeting capability of IA@MoNPs toward endothelial ligands, inflamed ECs, and atherosclerotic lesions was evaluated using in vitro and in vivo models. ResultsIA@MoNPs exhibited markedly enhanced binding to VCAM1, the primary endothelial ligand mediating integrin-dependent monocyte adhesion, and significantly increased uptake by ECs under atheroprone conditions compared to standard MoNPs. In vivo, IA@MoNPs demonstrated enhanced accumulation in atherosclerotic arteries without increasing nonspecific binding, and blocking {beta}1-integrins on IA@MoNPs abolished this targeting effect. Importantly, integrin activation on IA@MoNPs did not compromise circulatory stability or induce immune or organ toxicity. ConclusionIntegrin activation represents a simple yet effective strategy to enhance MoNP targeting to inflamed ECs and atherosclerotic lesions. This mechanism-driven approach improves targeting performance while maintaining specificity and safety, advancing the translational potential of the biomimetic nanomedicine platform for atherosclerosis.

Pulmonary delivery of lipid nanoparticles (LNPs) remains an area of significant interest, given the broad range of genetic disorders that could be addressed through localized administration of therapeutic nucleic acids to the lung. In this study, we investigated how incorporation of the clinically used lung surfactant cocktail Poractant alfa affects the in vitro and in vivo transfection performance of mRNA-loaded LNPs. The resulting lung surfactant-enhanced LNPs (Surf-LNPs) exhibited substantial improvements in particle assembly, yielding an order of magnitude higher particle concentration at equivalent input conditions compared to conventional (Onpattro-like) LNP formulations. In vitro, Surf-LNPs demonstrated several-fold increases in mRNA transfection efficiency and protein expression while maintaining excellent cytocompatibility. These enhancements are attributed to an elevated apparent pKa and the surface-active properties of surfactant protein B (SP-B), which promote more rapid and efficient endosomal escape relative to conventional LNPs. In vivo evaluation following intranasal administration further revealed enhanced mCherry expression in the lungs of mice treated with Surf-LNPs compared to conventional LNPs. Ultimately, these findings establish lung surfactant incorporation as a simple yet powerful formulation strategy to improve pulmonary gene delivery using LNPs, with the potential to significantly advance the translation of inhaled nucleic acid therapeutics.

Bispecific fusion proteins represent a unique strategy for developing precision therapeutics. By linking functional domains from distinct proteins, these biomolecules can engage multiple targets, enhancing both therapeutic efficacy and safety. Unlike bispecific antibodies, low-molecular-weight fusion proteins offer distinct advantages, including reduced immunogenicity and superior tissue penetration due to their relatively compact size and structure. Such a profile is particularly valuable in managing complex inflammatory diseases, where modulating multiple pathways is required to impart an effective anti-inflammatory effect. Among the various regulators of immune signaling, the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and interleukin-10 (IL-10) play imperative roles in immune suppression through their interactions with CD80/86 and IL-10R, respectively. While Fc-fused CTLA-4 is a clinically approved drug (e.g., Abatacept), the clinical development of IL-10 has been hampered by unpredictable immunostimulatory side effects. Here, we engineered a bispecific fusion protein linking the extracellular domain of CTLA-4 to IL-10. We successfully expressed the protein in E. coli as an N-terminal GST-tagged variant and refolded it from the inclusion bodies. Additionally, we achieved soluble expression of an Fc-tagged variant in mammalian CHO cells. Both origins demonstrated binding to their cognate receptors, CD80 and IL-10R. Finally, the fusion protein demonstrated a T cell-inhibitory effect by reducing Interferon-{gamma} (IFN{gamma}) secretion levels in an in vitro human Virus-Specific T cells (VSTs) model. This innovative protein engineering offers a promising strategy for addressing unmet clinical needs in autoimmune and inflammatory diseases.

Medulloblastoma (MB) is an aggressive central nervous system (CNS) malignancy that primarily affects children and frequently exhibits metastasis to the leptomeninges of the brain and spinal cord. We developed a {beta}-Cyclodextrin-poly({beta}-Amino Ester) nanoparticle system to deliver the histone deactylase inhibitor (HDACi) Panobinostat to MB by the intrathecal route. Various imaging methods were utilized to study nanoparticle and payload fate following infusion into the cerebrospinal fluid (CSF) of mice via cisterna magna or lumbar access points. Nanoparticles dramatically improved penetration of hydrophobic small molecules into distal regions of the spinal cord. Panobinostat-loaded nanoparticles were effective at treating patient-derived MB, activating pharmacodynamic targets, slowing growth of the primary tumor, decreasing incidence of metastasis at the time of death, and ultimately prolonging survival. These studies provide insight into the mechanisms mediating transport of colloids and therapeutic molecules in the subarachnoid space and highlight new approaches for treating metastatic disease in the CNS.

BackgroundExcessive accumulation of fibrillar collagen causes pathological scarring and fibrosis. A promising anti-fibrotic strategy targets the extracellular assembly of collagen fibrils rather than intracellular synthesis pathways. We previously developed a chimeric monoclonal antibody targeting the C-terminal telopeptide of the 2(I) chain of human collagen I that effectively disrupts fibrillogenesis. This study details the engineering of a humanized antibody variant optimized for therapeutic application, augmented with a collagen-binding peptide (CBP) to enhance targeted retention in fibrotic tissues. MethodsA humanized ACA was engineered by in silico homology modeling, complementarity-determining region grafting, and sequence optimization to eliminate chemical liabilities. Variants were expressed in mammalian cells and evaluated for binding kinetics and specificity. To improve spatial localization, the CBP was fused to the antibody. The lead variant was assessed for in vitro cytotoxicity, matrix retention, and in vivo efficacy using a rabbit model of post-traumatic knee arthrofibrosis. ResultsThe humanized ACA variants maintained high specificity and affinity for the 2Ct target domain. Fusing the CBP to the C-terminus of the light chain (C-cbpACA) successfully enhanced matrix retention without compromising target engagement or causing cellular toxicity. In the rabbit arthrofibrosis model, intra-articular C-cbpACA delivery significantly reduced flexion contracture and decreased total collagen deposition in the joint capsule compared to untreated controls. ConclusionWe successfully engineered a clinically viable, humanized, and matrix-targeted anti-fibrotic antibody that specifically inhibited extracellular collagen assembly and exhibited enhanced localization within fibrotic tissues. This construct represents a promising therapeutic strategy for mitigating pathological scarring and improving post-traumatic functional outcomes.

Pancreatic ductal adenocarcinoma (PDAC) remains difficult to treat with nucleic acid therapeutics because efficient intratumoral delivery is limited and off-target liver accumulation is common. Here, we developed a structure-activity map for intraperitoneally administered mRNA lipid nanoparticles (mRNA-LNPs) to identify formulation features that improve delivery to pancreatic tumors while reducing liver expression. A full-factorial library of 48 mRNA-LNP formulations was generated by varying ionizable lipid, sterol, phospholipid, and PEG-lipid components. Formulations were characterized for size, polydispersity, zeta potential, and encapsulation, then evaluated in an orthotopic KPC8060 pancreatic tumor model after intraperitoneal administration of firefly luciferase mRNA-loaded LNPs. Biodistribution was assessed by Rhodamine B fluorescence and functional delivery by luciferase expression 12 h after dosing. Lipid composition strongly influenced both physicochemical properties and in vivo performance. G0-C14-based formulations produced the smallest and most homogeneous particles, whereas FTT5-containing formulations were generally larger. Across the 48-formulation library, mRNA expression and nanoparticle biodistribution varied significantly among tumor, pancreas, liver, and spleen. Statistical, decision-tree, and predictive modeling analyses identified composition rules associated with organ-selective delivery. High tumor expression was associated primarily with G0-C14 combined with DSPC and {beta}-sitosterol, whereas liver expression was favored by C12-200 or DLin-MC3-DMA with DOPE and DSPE-PEG. Notably, a G0-C14/DSPC/DSPE-PEG formulation emerged as a lead candidate, producing a greater than 6-fold increase in tumor luciferase signal relative to the library median while reducing liver exposure by approximately 60%. Histopathology showed no treatment-related liver or lung toxicity. These findings define actionable formulation rules for tuning intraperitoneal mRNA-LNP delivery in PDAC and support further development of tumor-selective mRNA therapeutics for pancreatic cancer.

Clot resistance to pharmacological thrombolysis remains a critical challenge in ischemic stroke (IS) management. Thrombus heterogeneity, particularly the presence of thrombolysis-resistant domains composed of dense fibrin and non-fibrin components, including neutrophil extracellular traps (NETs), significantly limits the efficacy of recombinant tissue-type plasminogen activator (r-tPA) and its variant, Tenecteplase (TNK). Consequently, novel therapeutic strategies are urgently required. Emerging evidence suggests that co-administration of deoxyribonuclease I (DNase I) with r-tPA can degrade DNA fibers and enhance clot lysis. In this study, we optimized a previously developed theranostic agent--iron oxide microparticles coated with polydopamine--by dual-grafting both r-tPA and DNase to target resistant thrombi. Using functional ultrasound imaging (fUS) during the acute phase of IS, we demonstrated accelerated reperfusion with this dual-functionalized platform in a r-tPA resistant IS model. Furthermore, MRI analysis confirmed a significant reduction in lesion volume at 24 hours, correlating with improved functional recovery five days post-ischemia.

Lipid nanoparticles (LNPs) formulated with neutral helper lipids efficiently deliver RNA to the liver in pre-clinical models and humans but achieving clinically relevant delivery to other tissues remains a major challenge. To reduce liver uptake, targeting strategies often range from active targeting relying on antibodies to quasi-active targeting by employing permanently charged helper lipids which influence biodistribution after administration. In this study, we present an alternative approach based on varying ionizable lipids and stabilizers, along with optimizing formulation parameters for targeted delivery of circular RNA via a passive targeting approach. We generated a library of 216 LNP formulations and evaluated their performance in vitro in Jurkat cells and human primary T cells. The lead LNPs showcasing activity in both Jurkat and T cells were explored for their efficacy in vivo via multiple routes of administrations. Our results show that both the identity of stabilizer and ionizable lipid had effects on decreasing hepatic vs. splenic delivery while enhancing splenic accumulation. In line with this improved tissue tropism, spleen-tropic LNPs induced distinct transcriptomic remodeling in vivo compared with conventional, FDA-approved SM-102 LNPs. These findings demonstrate that extrahepatic targeting of LNPs can be achieved without altering charge of the LNPs and further reveal that hepatic de-targeting efficiency could be influenced by the immune status of the recipient.

Despite a few clinical successes, the efficacy of cancer nanomedicines remains limited by rapid clearance by the mononuclear phagocytic system and poor permeation across the abnormal tumor vasculature. We previously showed that methyl palmitate nanoparticles (MPN) can safely and reversibly inhibit the phagocytic activity of immune cells for several hours, thereby improving tumor accumulation and the efficacy of systemically administered nanomedicines. Here, we demonstrate that, on a shorter time scale, MPN can induce vasodilation, introducing an additional mechanism to enhance the accumulation of therapeutic agents within the malignant tissue. Upon internalization by macrophages and endothelial cells, MPN trigger the release of endogenous nitric oxide (NO), a key mediator of vasodilation, in a concentration-, and time-dependent manner. Following MPN administration, raster-scanning optoacoustic mesoscopy (RSOM) revealed vasodilation across multiple tissues, with the strongest effect observed in tumors. To assess enhanced tumor accumulation, we injected 70 kDa fluorescent dextran and demonstrated via histology a markedly increased fluorescence signal exclusively in MPN-treated tumors compared to controls 24 hours later. In addition, positron emission tomography (PET) imaging of 89Zr-labeled clinical iron oxide nanoparticles (Feraheme) showed significantly greater tumor accumulation after a 15-minute MPN pretreatment. Finally, general serum biochemistry panels and histological analyses of major organs in healthy mice revealed no toxicity following either single or repeated MPN dosing. Overall, this study demonstrates that MPN-induced vasodilation occurring within minutes enhances intra-tumoral deposition of macromolecules and small nanoparticles. Together with their longer-term effects on phagocytosis inhibition, these findings indicate that MPN can improve therapeutic delivery through complementary, time-dependent mechanisms that increase tumor perfusion and vascular permeability.

Inflammatory bowel diseases (IBDs) are frequently accompanied by anxiety and depression, largely driven by perturbed gut-brain axis signaling. However, current oral therapies remain constrained by the spatial and functional separation between intestinal inflammation and central nervous system dysfunction. Here, we present a comprehensive gut-brain dual region integrated therapeutic strategy based on functionalized Bifidobacterium longum hydrogel (INPs@BL@Gel), in which baicalin and tyrosine are coordinated with Fe(III) to form infinite coordination polymers (ICPs), coated with inulin, assembled onto Bifidobacterium longum (BL), and subsequently encapsulated within a pH- and matrix metalloproteinase-responsive silk fibroin-gelatin hydrogel. INPs@BL@Gel exhibits high drug-loading, effective gastric protection, inflammation-triggered release, and long-term intestinal colonization. Within the inflamed intestine, BL and components synergistically suppress inflammatory responses, restore gut microbiota homeostasis, and promote intestinal barrier repair through multi-target integrated therapy. Importantly, BL combined with components markedly enhances the production of beneficial neuroactive metabolites such as homovanillic acid and short-chain fatty acids, which integrated regulate neuroinflammation, preserve synaptic function, and facilitate blood-brain barrier repair via the gut-brain axis. In vivo studies demonstrate that INPs@BL@Gel not only exert potent therapeutic efficacy against colitis and effectively alleviate associated depression, but also reshape the gut microbiota and restore barrier integrity, achieving an remarkable comprehensive therapeutic effect. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/710940v1_fig1a.gif" ALT="Figure 11"> View larger version (59K): org.highwire.dtl.DTLVardef@1ceb3ceorg.highwire.dtl.DTLVardef@17ed1b4org.highwire.dtl.DTLVardef@f98f8corg.highwire.dtl.DTLVardef@3f6a46_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO (a) Schematic diagram of the design and preparation of functionalized Bifidobacterium longum hydrogel. (b) Exploration of the mechanism of INPs@BL@Gel in treating colitis-associated anxiety and depression through a dual-site multi-target synergistic strategy. C_FIG

Interleukin-4 (IL-4) is a key immunoregulatory cytokine that promotes type 2 inflammation, drives macrophage polarization toward an anti-inflammatory M2 phenotype, and supports tissue repair. However, clinical translation of IL-4 therapies to modulate the immune response is limited by the need for precise control over its delivery to avoid immune dysregulation. Here, we report an affinity-based strategy to modulate IL-4 delivery and bioactivity using engineered affibody proteins. A yeast surface display library was screened via magnetic- and fluorescence-activated cell sorting to identify two IL-4-specific affibodies with moderate binding affinities (dissociation constants, KD = 459 and 141 nM). Circular dichroism confirmed expected alpha-helical folding, and biolayer interferometry characterized the kinetics of IL-4 binding. Structural modeling using AlphaFold3 and RosettaDock and molecular dynamics simulations using GROMACS predicted distinct binding sites for each IL-4-specific affibody on the IL-4 protein and suggested potential interference with receptor complex formation. Bioactivity studies using murine bone marrow-derived macrophages demonstrated that IL-4 complexed with affibodies maintained Ym1 gene expression but significantly reduced Ym1 protein levels, indicating partial inhibition of IL-4 signaling. To enable controlled cytokine delivery via affinity interactions, affibodies were conjugated to polyethylene glycol maleimide (PEG-mal) hydrogels, which were loaded with IL-4. Affibody-conjugated hydrogels achieved high IL-4 loading efficiency (>90%) and exhibited sustained release over 7 days. Increasing affibody-to-IL-4 ratios significantly reduced both the rate and total amount of cytokine release. Overall, this work establishes IL-4-specific affibodies as versatile tools for tuning cytokine presentation and modulating bioactivity and provides a promising approach for regulating inflammatory responses and advancing cytokine-based therapies with improved temporal control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/723637v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@12bdb14org.highwire.dtl.DTLVardef@3c09eeorg.highwire.dtl.DTLVardef@1b00934org.highwire.dtl.DTLVardef@2c4840_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transforming growth factor-beta (TGF-{beta}), a potent promoter of extracellular matrix deposition and suppressor of infiltrating immunity, has arisen as an attractive target for improving outcomes in tissue fibrosis and cancer immune therapy. Despite the promise of TGF-{beta} inhibitors for attenuating the progression of fibrotic disorders or as adjuncts for cancer immunotherapy, current systemically administered inhibitors that target the ligand or receptors have significant on-target liabilities, including cardiotoxicity and development of pre-malignant cutaneous squamous lesions. Recently, an engineered mini monomer of TGF-{beta} (mmTGF-{beta}), which potently and specifically inhibits TGF-{beta} activity, was shown to strongly synergize with checkpoint inhibitors to suppress cancer progression in an aggressive model of melanoma when genetically delivered using an engineered form of vaccinia virus that preferentially infects cancer cells. Despite these promising results, however, a significant fraction of the mmTGF-{beta} was found to misfold, likely due to mispairing of the cysteines that comprise its cystine knot. Here, we demonstrate that inclusion of a modified form of the TGF-{beta} pro-domain that lacks its dimerization motif, the bowtie knot, dramatically improves both the folding and inhibitory activity upon secretion by mammalian cells, thus overcoming one of the major limitations of genetically delivering mmTGF-{beta}. Furthermore, we show that fusion of mmTGF-{beta} to a CD44 binding domain enhances the inhibitory potential of mmTGF-{beta} on immune cells, and on other cell types which express CD44, by more than 30-fold compared to cells negative for CD44. Together, these modifications provide a framework for further enhancing the efficacy and safety of mmTGF-{beta} for cancer immune therapy, and possibly also tissue fibrosis, when delivered genetically using vaccinia, or other related approaches.

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable efficacy in hematological malignancies, yet its efficacy in solid tumours remains limited by poor persistence and progressive exhaustion within the tumour microenvironment. These barriers may be particularly pronounced in emerging in vivo CAR-T therapies, in which transient transgene expression and insufficient control over T-cell differentiation restrict the generation of durable antitumour immunity. Here, we report a primary lymphoid tissue-targeting lipid nanoparticle (pLNP), that directs in vivo CAR-T programming to the thymus and lymphoid tissues, thereby increasing the proportion of stem-like CAR-T cells and promoting durable, exhaustion-resistant antitumour responses. After antibody conjugation, pLNP enabled in vivo CAR expression in developing T cells, generating CAR-T cells enriched in naive and stem cell-like memory phenotypes with prolonged persistence. To reinforce this, we co-administered interleukin-7 (IL-7) mRNA, which increased stem-like CAR-T populations, favoured progenitor exhausted T (Tpex) cells over terminally exhausted states, and enhanced cytotoxic function without overt inflammatory amplification. This stemness-promoting strategy also improved responsiveness to immune checkpoint blockade, producing synergistic antitumour effects with anti-PD-1 therapy, reducing LNP dose requirements, and inducing durable tumour regression with prolonged survival in both subcutaneous and orthotopic DLL3-positive small-cell lung cancer models. Similar enhancement of in vivo CAR-T efficacy was also observed in aged mice with thymic involution. Together, these findings illustrated that primary lymphoid tissue-directed in vivo CAR-T programming is a potential strategy to overcome insufficient persistence and progressive exhaustion in solid tumours.

Sex differences influence distinct inflammatory responses after spinal cord injury (SCI), yet their impact on immune-modulating nanotherapeutics remains unclear. Here, we investigated the sex-dependent effects of drug-free poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) following SCI. Systemic NP administration enhanced locomotor recovery in both sexes and eliminated the functional gap observed in controls. Mechanistically, NPs engaged distinct immune pathways between sexes. Females accumulated more NPs in the spleen, leading to reduced monocyte-derived macrophage infiltration, whereas males showed greater NP accumulation at the lesion and attenuated microglial activation. Transcriptomic analysis showed preferential modulation of eicosanoid-related pathways in females and NF-{kappa}B-linked signaling in males. These sex-specific, yet convergent NPs-induced immunomodulatory effects reduced fibrotic scarring and enhanced remyelination, with females showing greater Schwann cell-mediated repair and males exhibiting marked suppression of microglial activation. Collectively, these findings demonstrate that NPs promote comparable functional recovery in both sexes through distinct, sex-influenced immune mechanisms and establish a translational framework for sex-informed immune targeting and nanotherapeutic design in SCI and other inflammation-mediated diseases. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/709912v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@5af2ecorg.highwire.dtl.DTLVardef@1027071org.highwire.dtl.DTLVardef@12449ceorg.highwire.dtl.DTLVardef@169a327_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ionizable lipid nanoparticles (iLNPs) are powerful platforms for mRNA-based vaccines and immunotherapies; however, their intrinsic liver tropism compromises both safety and efficacy. Off-target hepatic protein expression from delivered mRNA raises safety concerns, and hepatic clearance limits efficient iLNP delivery to target organs. In this study, we address these challenges in mouse models by stealth-coating the liver sinusoidal endothelial (LSE) wall, the primary gateway for nanoparticle entry into the liver. Specifically, oligocations conjugated with two-armed PEG (2-arm-PEG-oligocations), a clinically relevant material used in oligonucleotide delivery trials, were employed to transiently anchor PEG to the LSE wall with balanced affinity, ensuring robust coating followed by gradual biliary clearance. This approach reduced hepatic protein expression from iLNPs, subsequently administered either systemically or locally, by more than tenfold. Importantly, the strategy preserved iLNP accumulation in the spleen, a key target organ for vaccines, effectively redirecting iLNPs from the liver to the spleen. Consequently, in vaccine applications, pre-injection of the 2-arm-PEG-oligocation preserved or even enhanced vaccination efficacy while minimizing concerns associated with antigen expression in the liver. In applications involving cytokine mRNA therapy, specifically intratumoral interleukin-12 (IL-12) mRNA administration, systemic pre-injection of the 2-arm-PEG-oligocation successfully reduced off-target hepatic IL-12 expression and subsequent systemic IL-12 exposure, while maintaining antitumor efficacy. Collectively, these results demonstrate that LSE-wall stealth coating is a generalizable strategy to improve both the safety and efficacy of iLNP-based mRNA vaccines and immunotherapies.

Extracellular vesicles (EVs) are versatile therapeutic candidates due to biological roles in intercellular communication and amenability to bioengineering. Compared with lipid nanoparticles (LNPs), native or surface-modified EVs may have favorable immunogenicity and biodistribution profiles. However, when administered intravenously (IV), EVs are rapidly cleared and accumulate mostly in the liver and spleen. With the goal of modifying EV biodistribution, we engineered EVs to display the human endogenous retrovirus (HERV) envelope glycoprotein Syncytin-1, an SLC1A5-binding fusogenic viral protein essential for syncytiotrophoblast formation in pregnancy. Here, we comprehensively characterize engineered Syncytin-1+ EVs, examine their interactions with cells in vitro, and assay biodistribution, immunogenicity, and pharmacokinetics ex vivo and in vivo in non-human primates. IV-administered Syncytin-1+ EVs are well tolerated, persist in the blood stream, and have altered organ biodistribution compared with unmodified EVs, suggesting therapeutic potential of Syncytin-1+ EVs at specific sites.

Dexamethasone (DEX), a synthetic glucocorticoid widely prescribed for allergic and inflammatory diseases, is known to induce adverse effects, particularly skeletal muscle atrophy. DEX-induced atrophy exacerbates sarcopenia and has a more pronounced impact on aged skeletal muscle than on young skeletal muscle. To address this unmet clinical need, we introduce an electroceutical approach that counteracts DEX-induced muscle atrophy and enhances functional recovery in aging muscle. When applied to both young and aged human-derived skeletal muscle cells (skMCs) exhibiting DEX-induced atrophy, electroceutical treatment promoted recovery of myotube diameter and upregulated hypertrophy-related gene expression. Furthermore, in a preclinical study, young and aged mice treated with DEX to induce muscle atrophy exhibited significant muscle recovery following electroceutical treatment. This effect was evident from the restored cross-sectional area (CSA) of type IIA muscle fibers and the upregulation of hypertrophy-related genes. This study highlights electroceuticals as a pioneering non-pharmacological strategy complementary to glucocorticoid therapies, potentially transforming clinical outcomes and quality of life, particularly for older populations vulnerable to muscle wasting.

Curcumin is a naturally occurring polyphenol that demonstrates considerable anti-cancer activity, however the aqueous insolubility, rapid metabolism and relatively low bioavailability are limiting to its clinical application. As such, a curcumin-magnesium (Cur-Mg) coordination complex was synthesized and subsequently encapsulated within DNA hydrogels (Cur-Mg-Hgel). The Cur-Mg complex was fully characterized using UV-Vis spectroscopy, FTIR and X-ray diffraction (XRD). UV-Vis, FTIR and XRD all support the formation of a coordination complex and suggest a decreased level of crystallinity compared to free curcumin. DNA hydrogels were formed and characterized using atomic force microscopy, rheology and swelling kinetic studies. In vitro cytotoxicity studies utilizing an MTT assay demonstrate dose dependent inhibition of HeLa cell proliferation and a slightly better retention of RPE-1 viability at low concentrations (suggesting some difference in sensitivity) though significant cell death is seen at higher concentrations and both cells. Intracellular production of ROS was measured using the DCFH-DA assay and is seen to increase when HeLa cells are treated with Cur-Mg-Hgel in comparison to un-treated controls. Annexin V/PI staining demonstrates primarily late or early apoptotic activity with minimal necrosis following treatment with Cur-Mg-Hgel. The evidence presented strongly supports the notion that Cur-Mg-Hgel is a ROS-modulating, pro-apoptotic Hydrogel suitable for cancer treatment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/724072v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@18727aeorg.highwire.dtl.DTLVardef@3e20adorg.highwire.dtl.DTLVardef@d3703eorg.highwire.dtl.DTLVardef@16e260e_HPS_FORMAT_FIGEXP M_FIG C_FIG