Journal of Controlled Release

With the rapid advancement of mRNA vaccines, lipid nanoparticles (LNPs) have emerged as pivotal carriers and adjuvants for non-mRNA vaccine modalities, driven by their superior nucleic acid delivery efficiency and intrinsic self-adjuvanting properties. In this study, we systematically evaluated various formulation strategies combining LNPs and the CpG adjuvant within a varicella-zoster virus glycoprotein E (VZV-gE) subunit vaccine framework. We demonstrated that uniform nanoparticles formed by LNP-encapsulated CpG (LNP-CpG), when simply admixed with the gE antigen, elicited superior immunogenicity compared to alternative encapsulation configurations. Intramuscular administration of a two-dose (LNP-CpG)+gE regimen significantly augmented both humoral and cellular immune responses in mice, markedly outperforming the commercial vaccine Shingrix (administered at a 1/10 human dose). Crucially, the identical regimen induced robust, comparable immune profiles to a full human dose of Shingrix in rhesus macaques. Furthermore, LNP-CpG displayed broad-spectrum utility across diverse vaccine platforms, demonstrating efficacy against both respiratory (RSV) and neurotropic (HSV) pathogens, compatibility with multiple modalities, including subunit (VZV-gE, RSV-Pre-F), live-attenuated (LA-HSV), and inactivated (i-HSV) vaccines; and versatile implementation in a combined VZV+RSV formulation. Collectively, our findings position LNP-CpG as a versatile, safe, highly efficacious adjuvant platform with substantial clinical translational potential, offering a compelling paradigm for next-generation vaccine development.

Immunotherapy shows limited efficacy in brain tumours, where restricted immune access, antigenic heterogeneity and local immunosuppression constrain durable responses. Low-intensity pulsed ultrasound with microbubbles (LIPU+MB) transiently modulates the blood-brain barrier (BBB) and is widely assumed to enhance immunotherapy by facilitating drug and immune cell penetration into the central nervous system (CNS). However, whether increased anatomical access alone is sufficient to generate effective CNS immunity remains unclear. Here, using a transgenic mouse model with astrocyte-restricted antigen expression, we showed that BBB modulation alone is insufficient to generate functional T-cell immunity in the CNS. Although LIPU+MB enabled rapid T-cell entry, accumulation required prior T-cell activation and integrin-dependent mechanisms, indicating that entry remains governed by canonical immune processes. Moreover, T-cells failed to persist owing to insufficient activation of antigen-presenting cells (APCs) within the CNS. Systemic immune adjuvants (poly-ICLC and IL-2; PI) induced APC activation, promoted tissue-resident-memory-like differentiation and supported durable T-cell responses. LIPU+MB further enhanced these responses by increasing T-cell recruitment, resulting in greater accumulation than with PI alone. Mechanistically, antigen presentation by bone marrow-derived APCs was more critical than that by microglia for the accumulation and persistence of antigen-specifc T-cells in the CNS. In antigenically heterogeneous glioma models resistant to CAR T-cell therapy, combining PI with BBB modulation enhanced the efficacy of immunotherapy, which was mirrored by prolonged survival and endogenous tumour-specific T-cell responses, consistent with epitope spreading. Together, these findings define key limitations of LIPU+MB in enabling effective T-cell therapy and establish that BBB modulation must be coupled to systemic immune activation to support T-cell-mediated antitumour immunity in the CNS.

Lung-targeted delivery of mRNA with lipid nanoparticles (LNPs) demonstrates high potential for therapeutic applications in pulmonary disorders. However, progress in pulmonary mRNA therapeutics is constrained by the challenges of engineering lipids that are both safe and highly effective at targeting the lungs. To meet these critical needs, we designed triazine-based (TZ) ionizable lipids with cyanuric chloride as the linker between the cationic head and the lipophilic tail, which allows for easy derivatization capable of systemic mRNA delivery. Three TZ-based lipids were synthesized using the same ionizable headgroups while differing in the carbon tail length and evaluated for their in vitro and in vivo protein expression. Notably, all three lipids result in pulmonary expression after intravenous administration, but the TZ lipid containing a C14 tail does so without any indication of thrombosis, both in vitro and in vivo as compared to other formulations. Our findings highlight the effect of minor chemical modifications driving altered in vivo activity, thus enabling new opportunities for safe pulmonary delivery of mRNA for lung-related diseases.

Hydrogels have emerged as attractive vaccine delivery platforms because they enable controlled modulation of antigen availability. However, how different hydrogel environments affect the release and functionality of mRNA-loaded lipid nanoparticles (mRNA-LNPs) remains poorly understood. Here, we investigated the release, stability, cellular uptake, and transfection capability of LNPs released from four hydrogel systems representing distinct crosslinking mechanisms: covalently crosslinked poly(ethylene glycol) (PEG), ionically crosslinked alginate, thermoresponsive Poloxamer 407 (P407), and protein-based Matrigel/collagen hydrogels. All hydrogels enabled release of LNPs over days, with kinetics strongly depending on hydrogel composition and polymer concentration. LNPs were quantitatively recovered from all hydrogel types, except from Matrigel/collagen where incomplete matrix dissolution was the limiting step. Lower polymer concentrations generally accelerated nanoparticle release. PEG offered greatest tunability of release kinetics; at the same time the recovery of the LNP-incorporated fluorescent dye DiI was reduced to about 80 %, indicating partial dye leakage. Alginate hydrogels exhibited recovery of DiI below 50 % and broader particle size distributions after release, while P407 hydrogels largely preserved LNP characteristics. Although quantitative recovery from Matrigel/collagen hydrogels was limited, released LNPs remained readily available for cellular uptake. Notably, LNPs released from low- and intermediate-concentration Matrigel/collagen hydrogels achieved approximately 80-90 % of the eGFP expression compared to mRNA-LNP that were not embedded into a hydrogel. Importantly, cellular uptake and transfection experiments demonstrated that all investigated hydrogels released biologically active mRNA-LNPs capable of mediating protein expression. Moreover, our findings show that hydrogel composition is a critical determinant of mRNA-LNP release, stability, and functional delivery. This work provides design principles for the development of hydrogel-based mRNA delivery systems aimed at sustained antigen availability and prolonged vaccine responses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/741169v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@138d9eforg.highwire.dtl.DTLVardef@16c0edaorg.highwire.dtl.DTLVardef@1432dd1org.highwire.dtl.DTLVardef@17511b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Current tuberculosis (TB) treatment suffers from drawbacks such as long regimens, high pill burden and side effects leading to non-adherence and poor treatment outcomes. Dissolution-controlled drug depot formulation with high drug loading is a clinically successful drug delivery strategy. Such depots reduce the dosing frequency for treatments requiring daily administration, thereby improving treatment adherence and compliance. However, dissolution-controlled depots for first-line TB drugs have not been demonstrated due to their high solubility and high dose requirements. In this study, we overcame this challenge by developing injectable, extended-release, dissolution-controlled depots of nanocrystalline rifapentine (NCRPT), microcrystalline rifapentine (MCRPT) and amorphous rifapentine microparticles (ARPT) with more than 75% loading. Crystalline formulations resulted in much slower depot dissolution compared to amorphous formulations. A single intramuscular (IM) injection of NCRPT in mice resulted in therapeutic serum concentrations for over a week. We then demonstrated the efficacy of NCRPT in both pre-exposure prophylaxis and therapeutic models of mice TB. NCRPT administered at 60 mg/kg once every two weeks demonstrated excellent efficacy in a mouse model of TB infection. In each case, a [~] 4-log-fold reduction in lung bacterial load compared to untreated mice was observed. These results open new avenues for developing LAI formulations of TB drugs and could improve patient compliance and TB management.

Edible plant derived extracellular vesicles (PDEVs) are emerging as biocompatible, orally deliverable nanocarriers with therapeutic potential; however, their phytometabolite cargo, gastrointestinal stability, and source specific biological functions remain poorly characterized. Here, PDEVs were isolated from four phytochemically distinct plant based foods (black carrot, ginger, garlic, and turmeric), selected for their diverse bioactivity, and characterized by transmission electron microscopy, nanoparticle tracking analysis, and zeta potential. Gastrointestinal stability was evaluated in simulated digestion model. Source specific phytometabolites were profiled by untargeted LC MS MS metabolomics. Functionally validated in ammonia stressed epithelial cells and steatotic hepatocytes. PDEVs exhibited characteristic cup shaped morphology with particle sizes ranging from 60 to 214 nm and zeta potentials of -6.0 to -49.0 mV. PDEVs retained colloidal stability, supporting their suitability for oral delivery. We identified 572 phytometabolites with distinct source specific signatures, including lignin and quercetin in carrot EVs, [6] gingerol and silymarin in ginger EVs, diosgenin in garlic EVs, and curcumin in turmeric EVs. These metabolites found associated to antioxidant, anti inflammatory, epithelial barrier, lipid metabolic, and apoptotic pathways. Functional validation demonstrated carrot EVs significantly enhanced epithelial barrier integrity by increasing claudin (>8-fold, p<0.05), occludin (>2-fold, p<0.05). Ginger EVs restored ZO 1 while suppressing cyclin D1 and MMP9(p<0.05). Garlic and turmeric EVs attenuated inflammatory signaling by reducing STAT3, AKT1, and TNF , whereas turmeric EVs additionally decreased caspase 3 and PTGS2(p<0.01). In steatotic hepatocytes, garlic EVs significantly reduced PNPLA3 (p<0.001) and SREBP 1c while increasing PPAR- (p=0.002). Hence, our results indicate that edible PDEVs are gastrointestinally stable, phytometabolite enriched nanocarriers with distinct source specific functional properties, supporting their potential as orally deliverable nutraceuticals for improving gut liver functions.

BackgroundCannabis derivatives have been reported to possess antinociceptive properties. However, oral delivery is limited by poor bioavailability, stability, and reliability of effects. Previously, we reported an analgesic effect of the aqueous complex {Delta}9-tetrahydrocannabinol/2-hydroxypropyl-{beta}-cyclodextrin (THC/HP{beta}CD) after intracerebroventricular administration in male rats. MethodsHere, we investigated the analgesic effects of the THC/HP{beta}CD complex after oral administration (0.3 and 3 mg/kg) by the tail flick test after both acute and chronic administration (15 days) in female rats. Locomotor activity and anxiety-like behavior were also evaluated at the same experimental conditions. Moreover, dopamine and glutamate content in the periaqueductal gray (PAG), a key area for the antinociceptive action of THC, were also measured by HPLC. ResultsAfter acute administration, the antinociceptive effect of the complex was seen at 3 but not 0.3 mg/kg THC, with a maximum effect observed at 30 min (MPE 60%). Similar results were obtained after 15 days of treatment, although partially reduced (max MPE 20%). Reductions in locomotor activity with the dose of 3 mg/kg and a slight biphasic effect of the two doses on anxiety-like behavior were also observed. Finally, neurochemical analyses revealed that the dose of 3 mg/kg significantly increased dopamine and glutamate content in the PAG, an effect no longer present after 15 days of treatment. ConclusionsOur results highlight the antinociceptive efficacy of the THC/HP{beta}CD complex also after oral administration, notably higher than that previously seen with other carriers, although with some degree of tolerance after chronic administration. From a translational point of view, these results are relevant for the development of THC-based oral formulations with analgesic properties for the treatment of pain in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/742765v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fff791org.highwire.dtl.DTLVardef@d672f4org.highwire.dtl.DTLVardef@1150b3forg.highwire.dtl.DTLVardef@956403_HPS_FORMAT_FIGEXP M_FIG C_FIG

Small interfering RNA (siRNA) enables precise post-transcriptional gene silencing for refractory diseases, yet its clinical translation remains limited by the lack of safe and efficient delivery vectors. Inspired by the dissymmetric alkyl chain architecture of natural membrane phospholipids, we designed and synthesized 34 novel ionizable lipids with dissymmetric hydrophobic tails and formulated them into lipid nanoparticles (LNPs). Through systematic physicochemical and biological assessments, we established clear structure-activity relationships and identified two lead LNPs (O14-LNP, H18a-LNP) with superior endosomal escape capacity, enhanced in vivo gene silencing potency, and favorable biosafety relative to the clinical benchmark MC3-LNP. In both streptozotocin-induced and spontaneous db/db type 2 diabetes (T2D) mouse models, lead LNPs delivering ferroptosis-related siRNAs effectively ameliorated glucose and lipid metabolic disorders, restored islet function, and alleviated hepatic steatosis. This study not only lays a theoretical foundation for the rational design of novel ionizable lipids, but also validates the therapeutic potential of siRNA therapy targeting ferroptosis, providing a versatile delivery platform and targeted therapeutic strategy for the treatment of T2D.

Microbubble-enhanced focused ultrasound (MB-FUS) enables noninvasive blood-brain barrier (BBB) opening to improve the delivery of drugs and other therapeutics to the brain, supporting more effective treatment of brain disorders. The benefits of this rapidly advancing and highly versatile technology have been demonstrated in clinical trials, sparking a growing interest in expanding FUS applications that require higher intensity treatments, larger targeted brain volumes and larger therapeutics. However, in-depth safety profiling of such treatments has not been done and is limited in the clinic. Preclinical studies have been restricted in their readouts, focusing on acute imaging and simple behaviors. To address the need for more holistic safety profiling, we present a novel preclinical workflow for determining adverse effects of MB-FUS in rats, both acutely and long-term, by combining MRI, histology, and a custom motor task which provides fine-scaled readouts for complex learned behavior. Using this approach and taking advantage of our previous delineation of the relevant circuitry, we show dose- and target-dependent adverse effects in high dosing regimens. All prescribed acoustic doses opened the BBB; but while low doses had no overt adverse effects, high doses targeting the involved circuitry had severe effects on both behavior and brain tissue integrity. These effects persisted for weeks and recovered over differing time courses, with tissue disruptions and behavioral changes outlasting general performance deficits. Our results reinforce the need for multimodal, highly sensitive, and longitudinal readouts to holistically characterize adverse effects of MB-FUS, allowing for its safe use across a wide range of applications. SignificanceMicrobubble-enhanced focused ultrasound is emerging as a powerful noninvasive approach for delivering drugs, genes, and cell therapies through the blood-brain barrier, sparking broad interest in expanding clinical and preclinical applications. However, its effects on complex neurological function, especially for higher dosing regimens, remain poorly defined. To address this, we have established a novel multimodal preclinical strategy for defining functional safety limits and guiding clinical translation, integrating sensitive behavioral testing with MRI, histology and acoustic emissions analysis. We show dose- and target-dependent impairments in complex learned motor behavior and evidence of possible brain injury, with substantially different recovery time courses. Together, our findings demonstrate the importance of a multimodal, longitudinal approach for comprehensively characterizing treatment-related adverse effects and evaluating safety.

Activation of the STING pathway can induce potent antitumor immunity, but effective delivery of STING agonists to the tumor while limiting systemic exposure remains challenging. We previously developed MUSIC, an ultrasound-guided platform that uses microbubbles (MBs) to deliver the STING agonist 2'3'-cGAMP and locally activate antitumor immunity. However, the vascular confinement of MBs and the need for intratumoral administration limit the potential for systemic tumor targeting. To overcome these limitations, we developed SONATA (Systemic Oncotherapy using Nanobubbles for Acoustically-guided Tumor Activation), which employs nanobubbles (NBs) that are approximately 10-fold smaller than conventional MBs, enabling systemic administration and tumor extravasation. Following NB accumulation within tumors, ultrasound exposure triggers localized cGAMP release, facilitating delivery to targeted CD11b+ antigen-presenting cells (APCs) and STING activation with spatial and temporal control. NBs are composed of the same components as MBs, including phospholipid shells and a perfluorobutane core and are functionalized with anti-CD11b antibodies to target CD11b+ APCs and spermine-modified dextran to stably load cGAMP through nanocomplex formation. Upon ultrasound activation, SONATA induced phosphorylation of STING, TBK1, and IRF3 and increased IFN-{beta} production in bone marrow-derived macrophages. In an orthotopic breast cancer model, intravenously administered SONATA combined with tumor-localized ultrasound significantly inhibited tumor growth compared with controls. Furthermore, SONATA synergized with immune checkpoint blockade prolonged the median survival of tumor-bearing mice. Collectively, these findings establish SONATA as a systemically administered immunotherapy platform that enables ultrasound-guided, spatially controlled STING activation.

The blood-brain barrier (BBB) is a highly selective cell layer that restricts the diffusion of diverse chemical entities into the central nervous system (CNS) from systemic circulation. Macromolecular therapeutics including oligonucleotides, peptides, and monoclonal antibodies exhibit only minimal brain distribution after systemic dosing due to exclusion by the BBB. Receptor-mediated transcytosis (RMT) has evolved to transport vital cargo across the BBB through a specialized vesicular transport pathway. Transferrin receptor 1 (TfR1) shuttles transferrin, its natural ligand, across the BBB, as well as TfR1-binding IgG antibodies and conjugates. Here, we describe a novel monovalent TfR1-binding VHH-Fc for the delivery of oligonucleotide cargo, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) across the BBB in rodents and non-human primates (NHPs), supporting the translational potential of the VHH-antisense RMT platform for the treatment of neurological disorders. We explore the role of binding affinity, conjugation site, drug-antibody ratio (DAR), and conjugation chemistry, and determine that binding affinity, DAR and conjugation site are major determinants of RMT capacity and brain activity of siRNAs delivered across the BBB. Graphical Abstract / Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/744307v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d1d648org.highwire.dtl.DTLVardef@4b22d3org.highwire.dtl.DTLVardef@db8b6borg.highwire.dtl.DTLVardef@19e5ac3_HPS_FORMAT_FIGEXP M_FIG C_FIG - Anti-TfR1 (-TfR1) VHH ligands formatted as heterodimeric, 2-chain monovalent VHH-Fc were engineered for conjugation to siRNA and ASO. - Systematic in vivo evaluation of VHH clones spanning a range of TfR1 binding affinities revealed a relationship between TfR1 binding affinity and the CNS activity of intravenously dosed VHH-Fc-siRNA conjugates. - By optimizing TfR1 binding affinity, conjugation site, and conjugation chemistry, we identified VHH-Fc-siRNA molecules that efficiently cross the BBB via receptor-mediated transcytosis and reduce target mRNA across CNS tissues, including deeper brain regions, after intravenous (IV) or subcutaneous (SC) dosing in mice and non-human primates (NHPs).

Macrophages are highly dynamic cells that maintain tissue homeostasis by regulating both initiation and resolution of inflammation. During efferocytosis, macrophages recognize the eat me signal, phosphatidylserine (PS), exposed at the surface of apoptotic cells, leading to the resolution of inflammation and acquisition of a pro-resolving phenotype. Inspired by this endogenous mechanism, PS-based biomaterials have demonstrated immunomodulatory potential. However, the molecular mechanisms underlying PS-mediated macrophage reprogramming remain poorly understood. Here, submicron PS-exposing polymeric particles (PSPs; [~]300 nm) were developed to improve the suitability of PSP formulations for future systemic administration while preserving their immunomodulatory activity. PSPs were efficiently internalized by macrophages through both actin- and dynamin-dependent pathways. PSP treatment significantly reduced IL-6 and IL-12p70 production in LPS-stimulated macrophages, whereas induction of the classical anti-inflammatory M2 marker CD206 was limited. Transcriptomic analysis revealed coordinated attenuation of inflammatory signaling pathways, including downregulation of Myd88, Nfkb1, Rel, and Irf8, together with activation of NRF2-associated antioxidant pathways characterized by increased expression of Nfe2l2, Hmox1, Prdx1, Gclm, and Gclc. Activation of antioxidant-associated genes together with reduced Irf8 expression suggests that PSP promotes inflammatory resolution through coordinated redox adaptation and selective attenuation of inflammatory signaling. Collectively, these findings provide mechanistic insight into PS-mediated macrophage reprogramming and support the future development of systemically administered therapies for chronic inflammatory diseases, including vascular inflammatory disorders. HighlightsO_LISubmicron PSPs retain immunomodulatory activity of apoptotic cell-mimicking biomaterials. C_LIO_LIPSPs are rapidly internalized through actin- and dynamin-dependent pathways. C_LIO_LIPSPs attenuate inflammatory signaling and selectively suppress IL-6 and IL-12p70 production. C_LIO_LIPSPs induce NRF2-associated antioxidant and glutathione responses. C_LIO_LITranscriptomics reveals an early redox-adaptive macrophage program. C_LI

Chronic pain is frequently accompanied by negative affect and motivational deficits due to dysregulated mesocorticolimbic dopamine and kappa opioid receptor (KOR) signalling. Although intracranial KOR antagonism prevents pain-induced negative affect in preclinical models, systemic KOR antagonists can produce adverse off-target effects in the periphery, thereby limiting its clinical utility. Consistent with this, we found that systemic administration of KOR antagonist norbinaltorphimine (NorBNI), exacerbated motivational deficits in rats with persistent inflammatory pain. We hypothesized that maximizing central and minimizing peripheral KOR antagonism could overcome these limitations. To test this, we engineered an intranasal liposomal NorBNI formulation incorporated into an in-situ forming mucoadhesive hydrogel to enable selective nose-to-brain delivery (Nor-BNILV-HG). We characterized its physicochemical properties and functional efficacy in rats with inflammatory pain produced by Complete Freund's Adjuvant (CFA). NorBNI-loaded liposomes exhibited high drug entrapment efficiency, nanometric size, and suitable surface charge for intranasal administration. The selected thermosensitive hydrogel demonstrated appropriate gelation properties and sustained drug release. Intranasal administration of NorBNI-LV-HG produced negligible systemic NorBNI levels compared with intraperitoneal delivery. In vivo microdialysis showed that NorBNI-LV-HG prevented KOR agonist-induced reductions in nucleus accumbens (NAc) dopamine release, confirming functional central KOR blockade. Behaviourally, intranasal NorBNI-LV-HG attenuated pain-induced impairments in sucrose motivation. Importantly, unlike systemic NorBNI, repeated intranasal NorBNI-LV-HG did not alter mechanical nociceptive thresholds in pain-naive animals, suggesting this strategy mitigates unwanted peripheral nociceptive effects. Together, these findings demonstrate that intranasal NorBNI-LV-HG achieves functional brain KOR antagonism while minimizing systemic exposure and off-target effects. Selective nose-to-brain delivery of KOR antagonists therefore represents a promising therapeutic strategy to prevent and potentially reverse the affective and motivational consequences of pain and may overcome key translational barriers associated with systemic KOR treatments.

The outer blood-retinal barrier (OBRB) is the primary interface through which systemically circulating drugs reach the retina. A tool that measures delivery across this barrier would support the development of targeted therapies as alternatives to repeated intravitreal injection, and the screening of drugs that reach the retina as an off-target toxicity. Such a tool should deliver drugs fluidically through a vascular compartment, measure transport across the retinal pigment epithelium (RPE), and display disease phenotypes relevant to efficacy. Here we adapt the SiM platform, which places epithelium and endothelium in direct juxtaposition across a permeable, optically transparent silicon nitride nanomembrane. ARPE-19 and human umbilical vein endothelial cells (HUVECs) were used as development cell sources. ARPE-19 monocultures reached a transepithelial electrical resistance of 68 {+/-} 26 {Omega} cm2 by 28 days, and ARPE-19 + HUVEC co-cultures reached a small-molecule permeability of 6.34 {+/-} 1.3 x 10-4 cm min-1 within 14 days, a state reported elsewhere only after longer culture. The barriers developed an intervening basement membrane. Drugs perfused through the basal vascular channel crossed into an open apical well, where sampling and mass spectrometry showed transport correlating with lipophilicity, as reported in vivo. The device also displayed two clinically relevant phenotypes. Digoxin at a clinically toxic concentration reduced viability in the co-barrier by about half and doubled permeability. In a vascularized configuration, VEGF drove endothelial invasion of the RPE layer, as seen in neovascular AMD. The SiM-OBRB therefore satisfies basic design criteria for measurement of drug bioavailability, toxicity, and efficacy.

Fluorescence imaging in the second near-infrared window (NIR-II, 950-1700 nm) offers reduced scattering, lower autofluorescence, and deeper tissue penetration than NIR-I imaging, but its adoption is limited by the need for custom-synthesized fluorophores. Here, we identify commercially available dyes that exhibit usable NIR-II emission. Eleven visible, far-red, and NIR-I fluorophores were screened under twelve acquisition configurations combining 670, 760, and 808 nm excitation with band-pass (950 nm, 1400 nm) or long-pass (1000 nm, 1250 nm) emission filters. Output varied markedly with fluorophore identity and excitation/emission configuration. Among hydrophobic dyes, DiR exhibited strong emission across almost all excitation and emission filters. Among hydrophilic dyes, strong NIR-II fluorescence was observed for IRDye 680RD (excitation at 670 nm), sulfo-cyanine 7 (excitation at 670 nm and 760 nm), and indocyanine green (excitation at 808 nm). DiR showed a linear concentration-response under 760 nm excitation with BP1400 detection. Upon encapsulation in PEGylated liposomes, strong NIR-II fluorescence was retained. In an in vivo study in mice, NIR-II resolved vasculature that NIR-I could not consistently delineate, and enabled pharmacokinetic analysis. Both windows returned similar ex vivo organ distributions. NIR-II imaging is therefore accessible using commercial off-the-shelf fluorophores, provided the dye is matched to the intended excitation/emission configuration.

Existing pooled lipid nanoparticle (LNP) screens lack spatial information on formulation localization, cellular uptake, and associated multicellular tissue responses. Here we introduce NanoSTAMP, a spatially resolved, pooled in vivo screening platform for barcoded LNP libraries that uses fluorescence in situ hybridization (FISH)-based barcode readout and is compatible with spatial omics. NanoSTAMP links LNP formulation to cell-type specific uptake, cargo expression, and nearby multicellular neighborhoods within intact tissue, which enables spatially-informed design of RNA delivery, establishing tissue architecture as a dimension of LNP performance.

mRNA-based therapeutics have emerged as a transformative class of medicines, yet their translation beyond infectious disease vaccines remains challenged by the absence of an integrated pharmacological framework accounting for the tri-component nature of these therapies - the lipid nanoparticle, the mRNA, and the expressed protein. Here, we present a modular, multiscale computational platform integrating two complementary mechanistic models covering the full pharmacological cascade of mRNA-based immunotherapies. The first is a Quantitative Systems Pharmacology (QSP) model describing the immunological response to mRNA vaccines, from antigen expression in antigen-presenting cells through B cell activation and circulating antibody production. The second is a Physiologically Based Pharmacokinetic (PBPK) model tracking whole-body disposition of mRNA-encoded therapeutic antibodies, incorporating a molecular layer resolving LNP uptake, endosomal mRNA escape, and intracellular translation. Both models are informed by a machine learning pipeline that maps IVT-mRNA nucleotide sequences directly onto kinetic parameters, enabling product-specific model simulations. We propose this platform as a step toward the quantitative pharmacological framework that mRNA therapeutics currently lack, and as a practical tool for model-informed design and development of this therapeutic class.

Hyperglycaemia disrupts mitochondrial homeostasis, leading to oxidative stress, ATP depletion, lysosomal dysfunction, impaired stress signalling, and proteostasis collapse. Although static magnetic fields (SMFs) have shown therapeutic potential in diabetic models, the optimal field strength for restoring subcellular organelle integrity post high glucose associated dysfunction remains unknown. Here, using Caenorhabditis elegans exposed to 40 mM glucose, we systematically evaluated SMFs ranging from 20 to 100 mT. Hyperglycaemia induced mitochondrial fragmentation, elevated reactive oxygen species, lysosomal abnormalities, reduced ATP levels, caused developmental delay, suppression of cytoprotective stress reporters, and increased polyglutamine aggregation. Among all field strengths tested, 70 mT produced the most robust recovery, restoring mitochondrial network architecture, reducing oxidative stress, normalizing lysosomal morphology, recovering ATP homeostasis, improving developmental progression, enhancing stress-responsive signalling, reducing proteotoxic aggregates, and increasing mitochondrial resilience to secondary hypoxic stress. These findings identify an optimal therapeutic SMF window and provide the first whole-organism demonstration that appropriately tuned static magnetic fields restore mitochondrial homeostasis and reverse multiple downstream consequences of hyperglycaemic stress.

Background & Aims: Adeno-associated virus (AAV) vectors are attractive delivery vehicles for therapeutic gene delivery, and a notable feature of most AAVs is their natural tropism for the liver, which leads to significant hepatic uptake following systemic administration. In previous work, we identified 266G as a conserved motif on a variable region on the capsid of many commonly used AAV variants that controls liver uptake in both mice and non-human primates. This single amino acid could be functionally leveraged to engineer AAVs to either de-target from or enhance tropism to the liver. Here, we explored whether these observations extended to the human context. Methods: Two human hepatocyte models were tested: Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice with humanized livers and a bioengineered human microliver platform in vitro. A barcoded AAV capsid library including standard control serotypes were used to assess the role of the 266G motif on gene transfer and transgene expression in both liver systems. Results: In vivo, 266G containing AAVs indeed targeted human hepatocytes superiorly, with some noted dependency on the degree of human-hepatocyte replacement in the chimeric mouse model. Initial studies in the micropatterned primary human hepatocyte co-culture model however demonstrated enrichment of heparin-binding AAVs, and not 266G variants. Notably, incorporation of polyethylene glycol (PEG) into the system modified the AAV transduction potential of those capsids including the liver-targeting motif, recapitulating the hepatocyte transduction pattern observed in vivo. Importantly, when PEG was used, the two human models, both at the DNA and RNA level, did correlate significantly. Conclusions: Our results showed the potential of a combinatorial AAV library for model validation and revealed the human microliver platform-PEG as a reliable system for the development of AAV therapeutics.

IntroductionTherapeutic ultrasound has been extensively studied in ablative and sonodynamic contexts, leaving the intrinsic bioactivity of continuous non-thermal low-intensity ultrasound (LIU) largely uncharacterized. ObjectivesTo characterize the tumor biological and immunomodulatory effects of non-thermal continuous LIU in complementary in vitro and in vivo breast cancer models, underpinned by a standardized exposure platform characterized through finite element simulations and experimental validation. MethodsAcoustic and thermal fields were characterized and optimized using in silico simulations and validated against hydrophone and temperature measurements to ensure homogeneous, non-thermal exposure (1MHz, 1W/cm2, 100% duty cycle). 4T07 murine mammary carcinoma spheroids received 20min LIU treatment, and metabolic activity, apoptosis, and intracellular stress-associated markers were assessed. In a syngeneic orthotopic 4T07 mammary carcinoma model in BALB/c mice, up to six LIU treatment cycles were administered; tumor growth, survival, histopathology, immunohistochemistry, bulk tumor RNA sequencing, spleen volume and plasma cytokine profiles were assessed. ResultsIn vitro and intratumoral temperatures remained within the physiological range ([&le;]39{degrees}C) throughout exposure. In spheroids, LIU reduced ATP content by more than 40% and significantly increased apoptotic, Hsp70 and Hsp90 cell fractions. In vivo, cyclic LIU slowed tumor growth, increased intratumoral necrosis, and significantly prolonged time to humane endpoint compared to untreated controls. LIU promoted early intratumoral myeloid cell infiltration and shifted the tumor transcriptome (2,573 differentially expressed genes), with enrichment in gene sets associated with immunogenic cell death, pattern-recognition, inflammatory, and innate and adaptive immune programs and downregulation of pro-tumorigenic pathways. LIU enriched the transcriptional signatures of M1 macrophage polarization and, notably, B-cell compartment engagement, which has not previously been reported for standalone continuous mechanical ultrasound. LIU significantly attenuated tumor-associated splenomegaly and elevated plasma IL-1, TNF-, and IL-10. ConclusionThese results establish a reproducible preclinical platform and provide a hypothesis-generating mechanistic basis for evaluating LIU as an adjunct to immune checkpoint blockade. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/743931v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@31d366org.highwire.dtl.DTLVardef@12df6aborg.highwire.dtl.DTLVardef@9d91adorg.highwire.dtl.DTLVardef@c72b8a_HPS_FORMAT_FIGEXP M_FIG C_FIG