Journal of Controlled Release

Efficient delivery of molecular therapies to the central nervous system (CNS) remains a major barrier to treating neurogenetic disorders such as Niemann Pick type C (NPC) disease. Focused ultrasound-mediated blood-brain barrier opening (FUS-BBBO) has emerged as a non-invasive strategy to enhance delivery of systemically administered therapeutics. In this study, we evaluated whether FUS-BBBO could enable delivery of lipid nanoparticle (LNP)-packaged modified mRNA (modRNA) to the cerebellum in an NPC mouse model. A pilot study in wild-type mice demonstrated successful FUS-mediated BBB opening, delivery of LNP-packaged GFP mRNA, and subsequent protein expression in the cerebellum. We then performed a controlled study in NPC mice comparing delivery of LNP-GFP and LNP-NPC modRNA using intravenous administration with and without FUS-BBBO. BBB opening was confirmed by contrast-enhanced MRI in FUS-treated animals. Quantitative PCR revealed the presence of GFP mRNA in the cerebellum following FUS-BBBO, whereas NPC mRNA was minimal or undetectable across groups. However, no GFP or NPC1 protein expression was detected in the cerebellum by western blot in any experimental group. Consistent with this, no therapeutic effect on Purkinje cell survival was observed. These results demonstrate that while FUS-BBBO reliably induces BBB opening and can facilitate limited delivery of LNP-packaged mRNA to the brain, this did not translate into detectable protein expression or therapeutic benefit in the NPC model under the conditions tested. This discrepancy between successful delivery in wild-type mice and lack of efficacy in diseased animals points to potential important biological and/or formulation-dependent barriers that must be addressed to enable effective CNS delivery of LNP-based mRNA therapies.

Intrathecal (IT) drug delivery, i.e., the infusion of substances directly into cerebrospinal fluid (CSF) by lumbar, ventricular, or cisternal access points, is one method that can be used to bypass the blood brain barrier (BBB), however, IT-administered substances also suffer from rapid turnover and poor tissue penetration. Although nanoparticles and colloids can circulate within the subarachnoid space to sustain the levels of encapsulated drug in CSF, their access to deep tissue regions remains incomplete. Here, we present a new method for enhancing CNS delivery of IT-administered nanoparticles. CSF Flow Enhancement (CFE) refers to the manipulation of CSF production, distribution, and clearance for therapeutic purposes. We tested the overarching hypothesis that infusion of hypertonic fluid adjacent to the choroid plexus would enhance fluid production and movement to improve the CNS delivery of IT-administered nanoparticles. Model polystyrene nanoparticles (100nm) were solubilized in aCSF of increasing tonicity (1-9X tonicity) and infused into the cisterna magna, after which tissues were removed to examine delivery to CNS tissues and peripheral organs. Our results demonstrate that an infusion of up to 4X hypertonic aCSF in 10uL is well tolerated and yields significant improvements in CNS localization of co-administered nanoparticles, more than doubling the delivery of nanoparticles to the ventral surfaces of the brain and sometimes dramatic (up to 10-fold) increases in delivery to specific tissue regions and surfaces of the CNS. Significantly, we provide early evidence that modulation of tonicity can define the parenchymal fate of IT administered colloids: while nanoparticles were not detected in the brain parenchyma of mice that received a standard infusion, parenchymal delivery was observed for the 2X condition, and extensive perivascular infiltration of nanoparticles was observed for the 4X condition. Lastly, we show that the delivery improvements achieved by CFE are generalizable across multiple sizes of polystyrene nanoparticle (20, 40, or 100nm). Collectively, this work describes a tonicity-based approach for achieving CFE by the intrathecal route, which we posit is a useful and potentially generalizable approach for improving CNS drug delivery.

Therapies to activate the STING immune response pathway represent promising potential anticancer treatments. However, the native STING activating molecule, 2',3'-cGAMP, is a poor drug candidate due to its susceptibility to nuclease degradation and its relatively poor cell uptake. In this study, we present a nanoscale delivery vehicle based on the bacteriophage MS2 virus-like particle that can both protect cGAMP and deliver it into cells to access and bind cytosolic STING. MS2-delivered cGAMP achieved greatly increased STING activation potency relative to both free cGAMP and a nuclease-resistant synthetic cGAMP analog. In an in vivo murine colon carcinoma model, MS2-cGAMP elicited significant and prolonged antitumor activity in a STING-dependent manner at 50-fold lower concentrations relative to free cGAMP and synthetic analogs. These results demonstrate that MS2 delivery of cGAMP can yield a highly potent STING agonist immunotherapy with in vivo anticancer activity.

Glioblastoma (GBM) remains one of the most lethal primary brain tumors, in part because the blood-brain barrier (BBB), restricts delivery of most systemically administered chemotherapeutics. Although focused ultrasound (fUS) can transiently increase BBB permeability, therapeutic efficacy remains limited by reliance on systemic drug exposure and heterogeneous intratumoral distribution. Here, we report a pressure-gated ultrasound-triggered drug delivery strategy that enables localized intravascular release of chemotherapy at the site of sonication. Freebase doxorubicin and afatinib were encapsulated within ultrasound-sensitive mPEG-PDLLA/PFOB microdroplets and administered systemically to N-TVA::Ink4a/Arflox/lox;Ptenlox/lox mice bearing genetically engineered glioblastomas. Animals received repeated transcranial focused ultrasound over a 30-day treatment period. Ultrasound-triggered release of the dual-drug formulation significantly extended survival compared with untreated controls, with median survival increased by over two weeks - approximately a 30% improvement. Furthermore, this survival improvement was reflected in histological analysis, showing decreased tumor burden and severity. These improvements were not found in any control groups, demonstrating that spatially and temporally controlled intravascular drug release can substantially improve therapeutic efficacy in an aggressive immunocompetent glioblastoma model. These findings support pressure-gated ultrasound-triggered chemotherapy as a promising activation-based strategy for overcoming BBB-associated delivery limitations and improving outcomes in malignant brain tumors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/735435v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1767043org.highwire.dtl.DTLVardef@c46048org.highwire.dtl.DTLVardef@8d3b44org.highwire.dtl.DTLVardef@2df0b8_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPressure-gated focused ultrasound enables localized release of doxorubicin and afatinib in glioblastoma. C_LIO_LIUltrasound-triggered chemotherapy significantly extends survival in a genetically engineered immunocompetent GBM model. C_LIO_LILocal activation outperforms systemic administration of identical drug combinations. C_LIO_LIThis strategy shifts focused ultrasound therapy from general BBB opening to spatially controlled drug activation. C_LI

Despite their therapeutic potential across a wide range of central nervous system (CNS) disorders, nucleic acid-based therapeutics are limited by inefficient delivery to deep brain regions at clinically viable doses. Transferrin receptor 1 (TfR1) has emerged as an attractive target for receptor-mediated transcytosis across the blood-brain barrier (BBB), enabling systemic delivery of biologics such as lysosomal enzymes and monoclonal antibodies. In this study, we demonstrated the translational potential of recently described TfR1-targeting camelid-derived single-domain antibodies (VHHs) for CNS delivery of siRNAs. When conjugated 1:1 to different tool siRNAs, these VHHs promote rapid and robust intracellular uptake, resulting in potent RNAi activity at low nanomolar concentrations in neural cells. Systemic administration of VHH-siRNA conjugates in wild-type mice, hTfR1 transgenic-mice and non-human primates revealed a favourable pharmacokinetic profile characterized by rapid TfR-dependent distributional clearance and efficient functional uptake in deep brain structures. This translated into durable target knockdown of 50-80% at both mRNA and protein levels and with ED50 below 1 mg/kg siRNA. Collectively, these findings establish our TfR1 targeting VHHs as a fit-for-purpose platform for the systemic delivery of therapeutic oligonucleotides to deep brain structures at clinically relevant doses, opening new avenues for the treatment of diverse CNS disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/726486v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@13668eorg.highwire.dtl.DTLVardef@1b1feeeorg.highwire.dtl.DTLVardef@d7be2dorg.highwire.dtl.DTLVardef@6b221_HPS_FORMAT_FIGEXP M_FIG C_FIG

This work describes a new approach for rapid and reproducible formulation of drug loaded biodegradable nanoparticles based on polyester copolymers, including poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) and poly(caprolactone)-poly(ethylene glycol) (PCL-PEG). The new approach, termed Solvent-free Nanoparticle Assembly Protocol (SNAP), carries several advantages over conventional polyester formulation strategies, including very rapid formulation (minutes) and the ability to use nanoparticles immediately without lengthy solvent evaporation or washing steps. Altering polyester molecular weight and concentration, alongside the introduction of specific functional groups yielded precise control of nanoparticle properties, including size, shape, surface charge, drug release and loading. We examined loading of multiple therapeutic compounds, including diclofenac, loperamide, bortezomib, CT179, panobinostat, docetaxel, methotrexate, and camptothecin. The SNAP protocol facilitated the rapid production of stable, drug-loaded nanoparticles with a narrow size distribution and generally good drug loading. Using Fluorescence Resonance Energy Transfer (FRET) and size exclusion chromatography (SEC) with a focus on the model agent Rhodamine B, we were able to carefully examine stability of the nanoparticle and assess the distribution of small molecules within the polymer as well as nanoparticle stability. In vivo evaluation of fluorescently labeled nanoparticles using real-time, intravital microscopy showed that, after direct administration to cerebrospinal fluid (CSF) via the intrathecal cisterna magna (IT-CM) route, the dynamic accumulation of nanoparticles within the perivascular space (PVS) depends on the size of the vessel that is imaged. Nanoparticles accumulated steadily within the PVS of large vessels, while accumulating more slowly and exhibiting clearance from medium-sized and smaller vessels over the course of several hours. In sum, these studies present a new platform for facile production of polyester nanoparticles, demonstrate their ability to encapsulate a variety of hydrophobic small molecules, and expand our knowledge on the development of nanocarriers for intrathecal administration. Taken together, these data open new opportunities for development safer and more effective nanoparticle-based therapies.

While engineered nanomaterials offer unprecedented precision in targeting tumor cells, their efficacy is often limited by rapid clearance from the bloodstream via the mononuclear phagocyte system (MPS). To overcome this limitation, a promising strategy known as MPS-cytoblockade has been developed. This approach involves administering antibodies against host erythrocytes. The resulting saturation of the MPS with erythrocyte clearance creates a critical window, allowing subsequently administered nanoparticles to evade immune surveillance and circulate for a significantly extended period. However, MPS-cytoblockade induces a transient reduction in hematocrit, which can lead to adverse effects. Here, we demonstrate that approaches to restore hematocrit, specifically through the administration of donor erythrocyte suspension or the hormone erythropoietin, effectively prevent this drop while maintaining the efficacy of the MPS-cytoblockade. Notably, these interventions do not compromise the prolonged circulation time of the nanoparticles or alter their biodistribution, preserving high accumulation in tumors. Our findings establish a viable strategy to mitigate a key side effect of MPS-cytoblockade, thereby enhancing its therapeutic potential and safety profile.

Activation of innate immunity enhances adaptive immune responses and underlies the mechanism of action of vaccine adjuvants. First-generation mRNA vaccines lack a dedicated adjuvant, relying instead on the self-adjuvanting properties of lipid nanoparticles (LNPs), and are thus expected to benefit from additional engagement of innate immune pathways not activated by LNPs. Among innate immune modulators, TLR5 agonists are particularly promising adjuvants due to their ability to induce a balanced Th1/Th2 immune response and their relatively favorable safety profile. Here we tested whether supplementing mRNA vaccines with mRNA encoding a TLR5 agonist could enhance immunization efficacy by induction of TLR5 signaling coordinated with vaccine antigen expression. We designed FL711, an mRNA encoding a derivative of Salmonella flagellin optimized for mammalian expression, functionally active in TLR5 signaling, deimmunized for pre-existing human T and B cell epitopes, and engineered for secretion to stimulate the TLR5 pathway in the local tissue microenvironment. We characterized FL711 in vitro and in vivo for functional and pharmacological parameters and assessed its adjuvant effect as a component of experimental anti-influenza and anti-SARS-CoV-2 mRNA vaccines. Supplementation with small amounts of FL711 mRNA (up to 30-fold less than antigen-encoding mRNA) significantly enhanced vaccine immunogenicity and protective efficacy, stimulating local NF-{kappa}B induction, boosting antibody production and T cell activation, and prolonging the durability of the response -- while enabling a marked reduction in mRNA dose per vaccine. These findings support the potential of FL711 as a broadly applicable mRNA-encoded adjuvant to improve the potency, durability, and dose efficiency of next-generation mRNA vaccines.

Mycobacterium tuberculosis (Mtb) infection kills more people worldwide than any other pathogen. While the Bacille Calmette-Guerin (BCG) vaccine for Mtb has been widely used for over a century, it provides insufficient protection to eradicate this disease. One of our labs has recently established that a protein antigen (H1) can be combined with a STING pathway agonist to achieve strong protection against Mtb in mice, with performance that exceeds that of the BCG vaccine. However, its reliance on a synthetic cyclic dinucleotide (CDN) with relatively poor cell uptake requires higher dosing levels, thus increasing costs. To increase the efficiency of this vaccine and provide a delivery strategy that could also be used in humans, the H1 Mtb antigen and CDN adjuvant were conjugated to genome-free MS2 viral capsids that included cationic mutations to increase cell uptake. Specifically, the H1 antigen was conjugated to the external surface of MS2 using a tyrosinase-mediated oxidative coupling reaction, and the native STING agonist cGAMP was coupled to internal cysteine residues through a reductively cleavable disulfide linker. The resulting MS2-H1 and MS2-cGAMP conjugates were then co-delivered for three doses of vaccination in mice before exposure to Mtb. The MS2-based vaccine platform was observed to have comparable efficacy to the original H1/CDN formulation, but its enhanced uptake properties enabled 57-fold less CDN and 3-fold less H1 antigen. Additionally, this vaccine elicited immune responses that have been previously demonstrated to correlate with protection. The ability of the capsid shells to protect the CDN cargo during transport allowed enzymatically produced, and thus readily accessible, cGAMP to be used instead of more costly CDNs that require many synthetic steps. This, combined with the reduced overall amount of CDN and H1 that was required, could lower the production costs of future vaccines substantially. Finally, the ability of the capsid-based carriers to bypass the membrane transporters for CDNs suggests that this enhanced vaccination platform is likely to exhibit improved human efficacy in future studies.

IntroductionImmune checkpoint inhibitors (ICIs) have only shown limited efficacy for patients with ovarian cancer (OC), partly due to the immune suppressive tumour microenvironment (TME) and platelet cloaking of the cancer cells. We tested a nanomedicine strategy to enhance {gamma}{delta} T cell immunotherapy by conjugating the PD-L1 inhibitor (BMS202) to nanodiamonds (NDs). MethodsPatient-derived ascites cells were exposed to activated platelets to model platelet cloaking and the immunosuppressive phenotype in metastatic OC. ND/BMS202 nanocomplexes were then applied to platelet-conditioned OC cells and co-cultured with expanded {gamma}{delta} T cells. Cytotoxicity and immune activation were assessed by quantifying Granzyme B, CD107a, {gamma}-H2AX, cleaved caspase-3, and cleaved caspase-8. ResultsND-mediated delivery of BMS202 significantly enhanced {gamma}{delta} T cell-mediated killing of platelet cloaked OC cells in a dose-dependent manner, with greater efficacy than free BMS202 This enhanced cytotoxicity was supported by increased degranulation (CD107a), Granzyme B release, tumour cell apoptosis (caspase cleavage) and DNA damage ({gamma}-H2AX staining). ConclusionsND-based delivery of the PD-L1 inhibitor (BMS202) enhances {gamma}{delta} T cell-mediated killing of platelet-cloaked metastatic OC cells. While our data support enhanced {gamma}{delta} T cell cytotoxicity following BMS202 delivery, direct evidence of PD-L1 target engagement or PD-1/PD-L1 binding inhibition was not demonstrated in this study. These findings nonetheless justify further validation in patient-derived organoid models to optimize this {gamma}{delta} T cell-based combination immunotherapy and advance its development as a precision therapeutic strategy for metastatic OC.

Human Immunodeficiency Virus (HIV)-1 broadly neutralizing antibodies (bNAbs) have demonstrated clinical efficacy, but face manufacturing challenges associated with recombinant protein production and purification. Here, we present a ribonucleic acid (RNA)-encoded bNAb (RibobNAb) platform that enables in vivo antibody production of the clinically validated bNAb PGT121 via lipid nanoparticle (LNP) delivery, supporting rapid evaluation of Fc variants (LS, del294, LS-del294) in vitro and in vivo. We confirmed expression, sub-nanomolar HIV-1 Env binding, and potent neutralization across all RibobNAb variants in vitro. In mice, single RNA-LNP administrations yielded in vivo expression of all RibobNAb variants, with PGT121-LS exhibiting a prolonged half-life compared with PGT121. In non-human primates (NHPs), a single intravenous administration of PGT121-LS RNA-LNP was well tolerated without anti-drug antibody (ADA) formation over 180 days and resulted in PGT121-LS half-lives comparable to the reference protein. Single intramuscular administration showed RibobNAb expression but resulted in ADA development from Day 14 onwards and lower bioavailability. In vivo-expressed PGT121-LS RibobNAb retained identical antiviral functionality to PGT121-LS reference protein. An NHP pharmacokinetics model integrating RNA transfection and translation dynamics enabled allometric scaling and first-in-human dose prediction. We highlight RibobNAbs as an alternative to conventional purified protein antibodies for rapid development of bNAb-based therapeutic strategies.

BackgroundReliable tracking of extracellular vesicles (EVs), key biological nanocarriers in nanomedicine, remains a major technical challenge due to the limitations of conventional lipophilic dyes, including aggregation, micelle formation, and nonspecific background signals that compromise biodistribution analyses. MethodsHere, we present a fluorogenic labeling strategy based on Aco-600, a water-soluble probe exhibiting a "light-on" activation in hydrophobic environments. Medium/large EVs (m/lEVs) derived from murine BV2 microglial cells were labeled and intranasally administered to adult C57BL/6 mice. EV biodistribution and brain uptake were quantitatively assessed by ex vivo fluorescence imaging on brain cryosections at multiple time points (5-1440 min), focusing on the cortex and hippocampus. ResultsAco-600 labeling enabled high signal-to-noise detection with minimal background and no evidence of dye aggregation artifacts. Quantitative analysis revealed a consistent spatiotemporal distribution profile across brain regions, with peak signal intensity at 60 minutes post-administration, followed by progressive clearance. This approach provided reproducible and sensitive tracking of EV biodistribution following a clinically relevant intranasal delivery route. ConclusionsOur findings establish fluorogenic labeling as a robust and artifact-minimizing strategy for in vivo EV tracking. This method enhances the accuracy of biodistribution studies and supports the development of EV-based nanomedicine platforms, particularly for central nervous system delivery applications.

Traumatic brain injury (TBI)-induced neuroinflammation can evolve over weeks or months, contributing to ongoing secondary damage and worsening neurological recovery. RNA-based therapeutics hold great potential to regulate inflammatory signaling, but delivery to the brain remains challenging because of the blood brain barrier. Intranasal administration offers a direct non-invasive route for brain access but is often limited by low delivery efficiency. Here we constructed a combinatorial library of DNA-barcoded 103 lipid nanoparticles by varying lipid components with diverse headgroup chemistries and bioreactive moieties. A high throughput screening of these nanoparticles in vivo following intranasal administration led to the identification of top candidates with highest brain accumulation. Intranasal delivery of an antagomir targeting microRNA-9-5p loaded nanoparticle effectively reduced cerebral microRNA-9-5p levels, suppressed inflammatory markers, and improved neurological outcomes in TBI. These results demonstrate a systematic approach for optimizing intranasal lipid nanoparticle design and support the feasibility of RNA delivery to modulate neuroinflammation after brain injury.

Intratumoral (i.t.) delivery of nanoparticles (NPs) is widely used to achieve high local NP concentrations. However, the temporal fate of i.t.-injected NPs remains poorly understood. We present a quantitative approach using whole-body magnetic particle imaging (MPI) to track magnetic NPs (MNPs) following i.t. injection. Using fiducial-calibrated imaging, we quantified MNP mass over time in subcutaneous 4T1 breast tumors. Longitudinal imaging revealed progressive loss of i.t. MNP content and heterogeneous systemic redistribution across animals despite standardized delivery conditions. Ex vivo MPI confirmed off-target accumulation primarily in the liver and spleen, consistent with reticuloendothelial clearance pathways. Histological analysis demonstrated spatially heterogeneous i.t. MNP deposition, potentially associated with local vascular features and tumor microenvironmental heterogeneity that may influence i.t. MNP retention or MNP clearance from the tumor. These findings highlight the importance of quantitative longitudinal whole-body MPI for understanding the fate of MNPs for informing localized nanotherapy.

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.

The predominance of lipophilic molecules in drug pipelines poses a fundamental delivery challenge in the aqueous physiological environment. Inspired by the endogenous lipid-transport machinery of lipid droplets and lipoproteins, adiposome was developed as a biocompatible platform for hydrophobic drug delivery. Here, docetaxel (DTX) was selected as a model hydrophobic chemotherapeutic agent to evaluate the therapeutic versatility of adiposome. Non-targeted DTX-loaded adiposomes (DTX-Ad) reduced systemic toxicity by avoiding the allergenic excipients Tween 80 and ethanol used in commercial DTX formulations. The cytotoxic mechanism and intracellular responses of DTX-Ad were comprehensively characterized. To boost antitumor efficacy, three tumor-targeting DTX-Ad formulations were subsequently engineered. Lung-targeting DTX-adiposomes (Lung@DTX-Ad), mediated by AAM-B1-28-CRGDK fusion peptide/NRP1 axis, enhanced antitumor efficacy in a lung metastasis model. BCMA-targeting DTX-adiposomes (BCMA@DTX-Ad), conferred by biotin-avidin-mediated antibody conjugation, achieved sustained suppression in a multiple myeloma model. Liver-targeting DTX-adiposomes (Liver@DTX-Ad), enabled by the ApoE/LDLR axis, outperformed both commercial DTX injection and sorafenib in hepatocellular carcinoma models. Taken together, these results demonstrate adiposome as a promising and broadly applicable drug delivery platform, with considerable potential for clinical translation across diverse malignancies.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks effective targeted therapies and is frequently associated with chemotherapy resistance and immunosuppression. Inducible nitric oxide synthase (iNOS) is overexpressed in breast cancer and has been strongly correlated with poor clinical outcomes, owing to its role in promoting tumor progression, invasiveness, and resistance to therapy. Although highly selective iNOS inhibitors such as N-(3-(Aminomethyl)benzyl)acetamidine (1400W) exhibit considerable therapeutic promise, their clinical translation has been hindered by unfavorable pharmacokinetic properties. To overcome these limitations, we developed a pH-responsive nanoscale formulation based on Schiff base conjugation between 1400W and oxidized PEGylated alginate (OPA), in combination with ionic interactions. The resulting nanoparticles (NPs) exhibited efficient release of 1400W under acidic conditions and effectively suppressed nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. While the NPs alone did not induce significant cytotoxicity, they synergistically enhanced the anticancer efficacy of paclitaxel (PTX) in MDA-MB-231 TNBC cells, significantly inhibiting cell viability and migration. In addition, the NP-PTX combination markedly reduced endothelial tube formation in HUVECs, compared to PTX alone indicating potentiation of the anti-angiogenic activity of PTX. In conclusion, the pH-responsive NPs enables effective modulation of NO signaling and enhances the therapeutic activity of PTX in TNBC cells. These findings support the potential of iNOS-targeted nanomedicine as an adjuvant strategy for TNBC treatment and warrant further investigation using in vivo models to evaluate pharmacokinetics, tumor accumulation, and antitumor efficacy of the NPs.

Glioblastoma (GBM) presents significant therapeutic challenges due to its aggressive nature, complex microenvironment and the limitations of conventional drug delivery systems. In this study, hybrid nanoparticles were developed by combining synthetic liposomes with macrophage-derived extracellular vesicles (EVs) to harness the strengths of both platforms. Two distinct liposomal formulations, DPPC:Chol:DSPE-mPEG2000 (F1) and DPPC:DPPS:Chol:DSPE-mPEG2000 (F2), were used as the basis for the synthesis. EVs derived from J774 macrophages were integrated with F1 and F2 to create hybrid nanoparticles (H-F1 and H-F2). Doxorubicin (DOX) was encapsulated using a pH gradient and a remote loading procedure. The mean particle size of H-F1-DOX and H-F2-DOX was 158.2 {+/-} 1 nm and 162.8 {+/-} 9 nm, respectively. The polydispersity index (PDI) was 0.130 {+/-} 0.012 and 0.084 {+/-} 0.033, while the zeta potential values were -14.9 {+/-} 0.7 mV and -26.7 {+/-} 3.1 mV, respectively. H-F2-DOX exhibited the highest encapsulation efficiency (EE%), reaching 76.5{+/-}3.4%. The encapsulated hybrids remained stable up to one week, at +5{degrees}C. The release of DOX from H-F2-DOX in DMEM supplemented with 10% serum showed pH sensitivity, with total DOX release of 64.9 {+/-} 5.3% at pH 7.4 and 90.7 {+/-} 6.5% at pH 5.5. The cell viability assay demonstrated that all formulations exhibited strong cytotoxic effects against GBM cells under normoxic conditions, with H-F2-DOX showing the most potent effect under hypoxia-mimetic conditions.

Extrahepatic delivery of lipid nanoparticles (LNPs) to non-phagocytic cells is a major challenge, with the leading strategy involving surface functionalization with target-specific monoclonal antibody (mAb) ligands. We investigate the stability of mAb-conjugated LNPs using two anchoring systems: the commonly used DSPE-PEG2kDa-maleimide and a block copolymer, PCL5kDa-b-PEG2kDa -maleimide, with the hypothesis that conjugation to a 150,000 Da antibody could overwhelm the relatively small ~600 Da aliphatic anchor on the PEG-lipid in vivo. Shedding of the mAB would compromise targeting. Conjugation integrity following IV injection was assessed by tagging LNPs and mAbs with metal ion tracers that could be quantified by ICP-MS. Results show that DSPE-PEG-mAb rapidly (within 1h) dissociates from LNPs in blood, leading to accelerated LNP clearance. In contrast, mAbs conjugated using PCL-b-PEG remained stably associated with the LNP over the 24h circulation and clearance of the construct. Results are connected to a thermodynamic model that reproduces experimental findings for PEG-anchor(-mAb) shedding in vitro and in vivo. This study identifies anchoring strength as a critical, unconsidered parameter for in vivo performance when conjugating mAbs to LNPs for extrahepatic delivery.

Manganese neurotoxicity, arising from environmental overexposure or inherited transporter disorders due to pathogenic variants in SLC30A10 and SLC39A14, leads to manganism, a debilitating Parkinsonian movement disorder. Alhtough chelation therapy can partially reverse neuropathology, current clinical practice relies on intravenous CaNa2EDTA, which is burdensome and poorly suited for long-term use. Consequently, there remains a significant unmet need for more effective, orally bioavailable chelators. This study aimed to establish and validate a pipeline for identifying and assessing novel ligands that attenuate manganese neurotoxicity and support preclinical translational development. Based on the structural features of manganese-based MRI contrast agents, we selected two chelators, N-picolyl-N,N',N'-trans-1,2-cyclohexylenediaminetriacetic acid (H3PyC3A) and ethylenediaminetetraacetic acid-benzothiazole aniline (H4EDTA-BTA), and their methyl ester derivatives, Me3PyC3A and Me4EDTA-BTA. These were evaluated in vivo using zebrafish (slc39a14U801/U801) and mouse (Slc30a10KO/KO) models of manganese overload. H3PyC3A and Me3PyC3A demonstrated greater manganese-mobilizing efficacy than CaNa2EDTA, improving locomotor behavior in slc39a14U801/U801 zebrafish. In Slc30a10KO/KO mice, intravenous administration confirmed selective in vivo chelation of excess manganese over physiological concentrations of zinc and copper. Although oral bioavailability was low (<1%), long-term oral administration of H3PyC3A modestly reduced liver and brain Mn accumulation, suggesting an added benefit of oral administration via gastrointestinal chelation. This integrated in vitro to in vivo pipeline provides a robust and scaleable approach for the development of next-generation Mn chelators. Slc39a14U801 loss-of-function zebrafish enable high throughput identification of candidate compounds while Slc30a10KO/KO mice offer a clinically relevant disease model for pharmacokinetic profiling and proof-of-concept validation.