Advanced Therapeutics

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.

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.

Leptomeningeal disease (LD), involving the metastasis of cells to the leptomeningeal membranes in the central nervous system (CNS), can be a deadly complication of several different types of cancer originating in the periphery or CNS, including breast cancer (BC) and pediatric medulloblastoma (MB). Targeted therapy represents a promising new approach to improve overall survival for LD patients. To this date, angiopep-2 (Ang2) and transactivating transcriptional activator (TAT), two well-known peptides for their brain delivery capability, have been reported to transport therapeutic cargos into the CNS for treatment of disease. Current administration strategies, however, still rely on oral delivery or intravenous injection (IV), where the substances need to travel through complex biological barriers to reach the subarachnoid space (SAS), which is the primary location of LD. Our research group has focused on the intrathecal (IT) route of administration as an alternative approach that can potentially enable high exposure of drug to CSF exposed tissues. However, there is a major field gap in understanding how targeting peptides can access (or not access) LD as a function of their route of administration. Therefore, our work was focused on comparing the targeting capability of Ang2 vs TAT by IT vs IV routes of administration. We first generated two xenograft models of LD by directly infusing breast cancer cells (MDA-MB231) or medulloblastoma cells (HDMB03) into the SAS via intracisternal magna injection (ICM) to form BC-LD and MB-LD models, respectively. These tumor models were characterized for overall survival, tumor growth patterns, and presence of hydrocephalus. Second, we further administered fluorescently labeled Ang2 or TAT peptides either IV or ICM into tumor bearing mice. Neuraxial fluorescence images were examined to evaluate the targeting ability of these two peptides based on colocalization between peptide signal and tumor tissues ex vivo. We discovered that the median survival of both models was negatively related to the number of the cells infused. While HDMB03 cells tended to metastasize preferentially to the brain region, MDA-MB231 cells tended to metastasize preferentially to the spinal cord. Both models present hydrocephalus as one of the common clinical symptoms in LD patients. Compared to the healthy control, MB-LD yielded a 7.3-fold increase and BC-LD a 26.5-fold increase in ventricular volume. Furthermore, targeting achieved by TAT was significantly higher than targeting achieved by Ang2 in thoracic spine for the MB-LD model. For BC-LD model, TAT signal was found to be significantly higher than Ang2 signal in the olfactory bulbs, brain stem, thoracic spine, and lumbar spine regions. While both peptides showed a strong signal at 2 hours post ICM injection, signal was not detectable 24 hours after administration, reflecting washout or degradation. Significantly, these data provide evidence that ICM will be a preferable route of administration over IV for the purpose of maximally targeting LD.

Low response rates to immune checkpoint inhibitors (ICIs) in solid tumors are often driven by insufficient tumor-infiltrating CD8 T cells and immunosuppressive tumor microenvironment (TME). Although interleukin-2 (IL-2) potently expands and activates CD8 T cells, its clinical use is limited by rapid clearance, dose-limiting toxicity, and regulatory T cell (Treg) stimulation. Engineered IL-2 variants have not yet achieved meaningful clinical efficacy. Here, polymer-modified mesoporous silica nanoparticles displaying dense, unmodified wild-type IL-2 on their surface (IL2-NP) are developed, conferring proteolytic stability and tumor retention. IL2-NP enables avidity-mediated CD8 T cell binding and enhances proliferation and effector function without increased Treg binding or proliferation. Intratumoral IL2-NP expands CD8 T cells, increases CD8/Treg ratios, and reprograms TME through dendritic cell activation and M1-like macrophage polarization. IL2-NP induces regression of both treated and untreated distant colorectal tumors in a CD8 T cell-dependent manner. IL2-NP synergizes with ICIs and leads to complete tumor regression and immunological memory that protect against rechallenge. Treatment is well tolerated, with strong efficacy also observed in triple-negative breast and metastatic ovarian cancer models. Overall, intratumoral IL2-NP elicits robust systemic antitumor immunity, offering a promising strategy to enhance ICIs, cancer vaccines, and adoptive T-cell therapies. Graphical abstractThis work introduces a nanoparticle platform that overcomes major shortcomings of IL-2 immunotherapy by presenting wild-type IL-2 at high density on the nanoparticle surface, thereby increasing binding avidity to effector T cells. The resulting IL-2 nanoparticles enhance cytotoxic T cell expansion, reprogram the tumor microenvironment, and augment responses to immune checkpoint blockade to achieve robust ant-tumor immune response in mouse tumor models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/738558v1_ufig1.gif" ALT="Figure 1"> View larger version (82K): org.highwire.dtl.DTLVardef@12f8c8corg.highwire.dtl.DTLVardef@b46b1forg.highwire.dtl.DTLVardef@e4efc5org.highwire.dtl.DTLVardef@3993e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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

Neuroinflammation mediated by reactive microgliosis is a central driver of Parkinsons disease (PD) pathogenesis. This inflammatory process unfolds years before clinical symptoms, creating an opportunity for early intervention. In vivo imaging technologies that could detect and quantify microglial reactivity are therefore essential for early diagnosis, patient stratification, and evaluating emerging immunomodulatory therapies that target this fundamental driver of PD progression. Yet no standardized, sensitive, and specific technology currently achieves this goal. Molecular magnetic resonance imaging (mMRI) is uniquely suitable to address this problem because it integrates inherent high spatial resolution and soft tissue contrast of conventional MRI with molecularly targeted contrast agents, enabling simultaneous acquisition of anatomical detail and functional/biological information at submillimeter isotropic resolution. Here we present a novel mMRI probe designed to specifically target colony stimulating factor-1 receptor, expressed primarily on microglia in the brain. In silico data show that the targeting ligand binds the extracellular Ig domain of the receptor. In vitro cell uptake studies with both murine and human microglia cell lines show that the probe binds the receptor triggering active cell uptake and in vivo MRI enabled effective separation of the A53T mouse model of PD from control mice using radiomics-assisted MR image analysis. Ex-vivo immunohistochemical analysis showed signal from the probe largely in the cytosolic compartment of IBA-1 reactive cells, confirming that the observed in vivo MRI signal is due primarily to retention of the agent by microglia. This novel technology has the potential to interrogate the regional presentation of microglial activation in PD.

Vodobatinib (VBN) is a weakly basic (pKa {approx} 2.3), anticancer treatment with poor enteric solubility and low oral bioavailability. This study demonstrates how an emerging polymeric amorphization technique, slurry conversion, can yield amorphous drug-polymer salts with enhanced dissolution rates. The technique had not previously been applied to a weakly basic drug, so design rules for this class of active were unknown. Two acidic polymers, poly(styrene sulfonic acid) (PSSA) and poly(acrylic acid) (PAA), were individually evaluated for salt formation with VBN. Formulation involved blending the drug and polymer in a 1:2 (v/v) ratio of a protic liquid to solvent and a 1:9 (w/w) ratio of solid to solvent. Design rules for effective combinations of solvents and protic liquids were developed and optimized to thread the needle between dissolution of all species and acid-base interactions, both of which were required to form amorphous salts. Drug loadings of 10%, 20%, and 40% by mass were tested. X-ray photoelectron spectroscopy was employed to evaluate protonation of the quinoline nitrogen atoms on VBN, a key indicator of successful salt formation. Powder X-ray diffraction was used to confirm that the resulting slurry contained amorphous VBN, and 1H NMR spectroscopy indicated residual solvent remained after drying, which remains an area for improvement. In dissolution kinetics tests in FeSSIF, the lead drug-polymer salt formulation achieved a concentration of dissolved VBN up to 140 {micro}g/mL, an improvement of >35-fold compared to <4 {micro}g/mL (LLD) for crystalline VBN. These results demonstrate that slurry conversion is a viable polymeric amorphization technique even for weakly basic drugs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/734800v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1812ceforg.highwire.dtl.DTLVardef@1ad06dcorg.highwire.dtl.DTLVardef@9d8bb7org.highwire.dtl.DTLVardef@13fcbe8_HPS_FORMAT_FIGEXP M_FIG C_FIG

The blood-brain barrier (BBB) is a highly selective, semi-permeable border of endothelial cells that prevents solutes and therapeutic agents in systemic circulation from passively crossing into the central nervous system (CNS) parenchyma. The Transferrin receptor 1 (TfR1) endocytosis pathway for iron homeostasis is one of the most well-characterized strategies for therapeutic delivery across the BBB. The work presented here showcases the discovery of novel anti-TfR1 NANOBODY(R) shuttles. The identified anti-TfR1 NANOBODY(R) molecules display cross-reactivity and pH-dependent binding to human, cynomolgus (cyno), and mouse TfR1. Structural data further explain and support the underlying mechanism of this pH-dependent binding. These anti-TfR1 NANOBODY(R) molecules were successfully conjugated to both short-interfering RNA (siRNA) and antisense oligonucleotide (ASO) tool payloads. anti-TfR1 NANOBODY(R)-siRNA conjugates can induce up to 60% knockdown of the target mRNA transcript in skeletal muscle up to two weeks post a single IV dose in mice and up to 35-40% at four weeks post dose. Furthermore, extending the half-life of the anti-TfR1 NANOBODY(R)-ASO shuttles enhances heart, sciatic nerve, and brain exposure and enables up to 30-60% target knockdown in different CNS cell types. Altogether, these results highlight important features for the development of anti-TfR1 shuttles for the purpose of downregulating target mRNA transcripts in muscle and CNS for a variety of neurologic and neuromuscular indications.

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

Innate immune agonists are promising therapeutic agents to induce immune responses against cancer. However, these agents have been limited by toxicity associated with systemic accumulation and activity in off-target cells. In this work, a targeted nanoparticle (NP) platform to encapsulate and protect the Toll-like receptor 3 (TLR3) agonist polyinosinic-polycytidylic acid (poly(I:C)) and promote its specific delivery to antigen presenting cells (APCs), macrophages and dendritic cells, for activation of this cell population was designed. To determine NP physiochemical properties that promote APC delivery, we developed a library of NP surface chemistries formed by electrostatic adsorption of polyanion coatings onto liposomes using layer-by-layer (LbL) assembly and screened the particles on APCs and off-target cells. Dextran sulfate was identified as a promising coating to enhance specific APC delivery. We applied these design parameters to develop a poly(I:C)-loaded NP for an APC-targeted immunotherapy. In a model of metastatic ovarian cancer, the LbL NP prolonged poly(I:C) retention in the peritoneal space--with 2-fold remaining 24-48hr after administration compared to free poly(I:C)--ultimately reducing systemic accumulation and associated toxicities. Compared to free drug, the NP reduced the increase in serum levels of TNF, IL-6, and CXCL10 by 9-, 4-, and 31-fold respectively. NP-treated mice experienced lower weight loss and recovered more quickly at a higher poly(I:C) dose, indicating a widening of the therapeutic window. The NP formulation enhanced accumulation of poly(I:C) in the tumor 2-fold and activation of the target APC population compared to free drug, and ultimately slowed tumor growth and extended survival in combination with doxorubicin chemotherapy. Overall, this work demonstrates a modular NP delivery strategy to improve the delivery, safety, and therapeutic window of a TLR3 agonist.

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.

Liquid crystalline mesophases exhibit structurally programmable internal architectures that enable co-loading of chemically orthogonal molecules within a single composite material. Realizing the potential of these materials for drug delivery requires a quantitative understanding of how tuning the composition affects internal mesophase architecture and consequently performance metrics such as payload release. Here, Flash NanoPrecipitation with hydrophobic ion pairing is used to prepare nanocarriers containing liquid crystalline mesophases co-encapsulating two compounds from widely different chemical classes: hydrophilic polymyxin B (logP -6) with one of four hydrophobic co-core materials (logP 7-11), achieving >75% encapsulation efficiency and up to 32% and 50% mass loadings for polymyxin and co-core. Synchrotron SAXS is used to quantify characteristic mesophase repeat spacing, which is found to be tunable as a function of composition. A strong correlation between d-spacing and polymyxin release rate is presented. Co-core chemistry and weight fraction jointly govern mesophase architecture, and repeat distance emerges as a structural metric linking these to the hydrophilic payload release kinetics. Mucus diffusivity and antibacterial efficacy are assessed as independent performance metrics, and results corroborate the release behavior. These findings establish a quantitative framework connecting material composition, mesophase architecture, and functional performance that can be applied toward rational co-formulation design. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/734853v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1d722eborg.highwire.dtl.DTLVardef@135adb7org.highwire.dtl.DTLVardef@11fe29eorg.highwire.dtl.DTLVardef@571165_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOToC Graphic TextC_FLOATNO Flash NanoPrecipitation yields liquid crystalline nanocarriers co-encapsulating with high efficiency payloads with widely distinct physicochemical properties. Synchrotron SAXS establishes characteristic repeat spacing as a quantitative structural metric directly governing hydrophilic release kinetics, providing a rational design framework linking mesophase architecture to functional performance across a range of payload structures. C_FIG

Lupus nephritis requires long-term immunosuppressive therapy, which is often associated with severe systemic side effects. Therefore, new therapeutic strategies that maintain high efficacy while minimizing adverse effects is essential. Although nanomedicine has advanced systemic and kidney-targeted drug delivery, a reliable method for glomerulus-specific delivery is lacking. Collagen IV (Col4)-alpha 3, located in the glomerular basement membrane (GBM) at the blood-tissue interface through fenestrated capillary endothelium, represents an ideal target for glomeruli delivery. Herein, we developed a novel liposomal nanoparticle conjugated with a Col4-alpha 3 binding peptide (Col4-3-NPs) for selective glomerular targeting. Prednisolone-loaded Col4-3-NPs were administered to lupus-prone mice twice weekly for 8 weeks. Kidney injury and function were evaluated biweekly, and renal immune cell populations were analyzed by flow cytometry at study completion. The results show that rhodamine-labeled NPs predominantly accumulate in kidney glomeruli 48 hours after intravenous injection. The Col4-NP system demonstrated stable and prolonged release of the encapsulated drug for over 48 hours. Lupus-prone mice treated with prednisolone-loaded Col4-NPs showed significantly improved renal function and histology, including a 30% increase in glomerular filtration rate (GFR), a 56% reduction in proteinuria, and decreased IgG deposition and fibrosis. Notably, treatment also enhanced renal regulatory T cell (Treg) populations. These findings suggest that glomerulus-targeted Col4-3-NPs hold significant translational promise. This platform may offer an effective, site-specific treatment for lupus nephritis while minimizing systemic side effects and could be adapted for other glomerular diseases requiring targeted therapy.

Potent antineoplastics, such as afatinib and freebase doxorubicin, are associated with systemic toxicity. To address this issue, we developed a carrier that releases drugs, including afatinib and doxorubicin, specifically at the focus of low-intensity ultrasound. This remotely triggered and focal approach enables the release of drugs specifically at the ultrasound focus, thus mitigating undesirable off-target effects, and at concentrations governed by the duration of the applied ultrasound. We produced ultrasound-sensitive microdroplets with high encapsulation efficiencies (39.6% for afatinib and 46.6% for doxorubicin). The microdroplets consist of an ultrasound-sensitive drug delivery system based on a methoxy poly(ethylene glycol)-poly(D, L-lactide) diblock copolymer (mPEG-PDLLA) and perfluorooctyl bromide (PFOB). Antineoplastic agents were encapsulated within these microdroplets via co-evaporation during particle synthesis. The microdroplets released doxorubicin and afatinib in an ultrasound-pressure-dependent manner, with fitted half-maximal release pressures (P50) of 0.61 MPa and 0.72 MPa, respectively. Together, the effective encapsulation of hydrophobic antineoplastic agents and the dose-dependent ultrasound-triggered release provide a new method for targeted drug delivery and a foundation for future targeted chemotherapies.

Lipid nanoparticles (LNPs) have become a clinical standard for systemically-administered nucleic acid drugs and vaccines, but the LNP pipeline for locally-delivered LNP therapies remains much less mature. Local delivery in lung represents a particularly compelling application space for DNA-LNP therapeutics, as local gene therapies could support sustained epithelial recovery and functional restoration in various lung diseases. However, locally-delivered DNA-LNPs face multiple barriers, including the limited availability of serum components that often support conventional LNP activity, and the additional delivery barriers posed by the nucleus. We generated hybrid peptide-lipid nanoparticles (hpLNPs) for pulmonary DNA delivery by using a core-shell assembly strategy to incorporate short histone-derived peptides, selected for their DNA-transport capacity, into a clinically inspired LNP formulation. In parallel, we evaluated serum pre-coating of LNPs as a strategy to boost hpLNP activity in the serum-poor airway environment. A peptide:DNA amine/phosphate (N/P) ratio of 0.9 was identified as the highest feasible ratio to permit peptide incorporation into hpLNPs while preserving DNA encapsulation efficiency at >90%, retaining hpLNP colloidal stability, and preserving the overall pKa for LNPs. At N/P = 0.9, hpLNPs showed markedly enhanced DNA delivery in alveolar lung cells, achieving up to a 17-fold increase in transgene expression compared to peptide-free LNPs. Transgene expression levels varied depending on serum concentration, with expression peaking in the presence of 6% serum. Furthermore, serum pre-coating was necessary to enable in vivo hpLNP activity following intratracheal administration in mice, yielding robust local GFP expression. Mechanistic studies in exosome-free serum revealed that peptide incorporation in the hpLNPs enhanced transgene delivery by increasing nanoparticle interactions with serum exosome components, resulting in significant enhancements to hpLNP uptake. Together, these findings identify nanoparticle- serum interactions as a critical determinant of LNP-mediated pulmonary DNA delivery and establish peptide incorporation and serum pre-coating as promising strategies to enable robust, localized gene transfer in serum-free environments.

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.

Glioblastoma (GBM) is a highly aggressive brain tumor with a five-year survival rate of less than 5%. The current standard of care established 20 years ago includes maximal surgical resection and administration of alkylating agent temozolomide (TMZ). GBM is highly invasive, and GBM cells that evade surgical resection can become resistant to TMZ and develop new aggressive secondary tumors. Post-relapse there are few treatment options available to patients. Tissue engineering approaches suggest the opportunity to develop in vitro models of the GBM tumor microenvironment that may accelerate the discovery of novel therapies for GBM. Here, we report the adaptation of hydrogel microdroplets (microgels) to encapsulate GBM cells in a tailorable 3D matrix to assess patterns of growth and to screen TMZ drug response using patient-derived xenograft (PDX) specimens. We exploit a unique aspect of the microgel system to account for the cellular heterogeneity within the tumor microenvironment (TME). We combine cell-laden microgels generated from TMZ-resistant and TMZ responsive variants of the same PDX specimens to create heterogeneous populations with varying levels of drug sensitivity. We demonstrate a range of drug resistance phenotypes as a function of the ratio of TMZ-responsive to resistance cells and identify the population required for TMZ-resistance to overtake take the response. We then investigate the influence of tumor mimetic shifts in hyaluronic acid bioavailability and hypoxia on patterns of TMZ resistance. We show exposure to matrix-bound hyaluronan increases TMZ resistance and the glioma stem cell population in both cell variants. Lastly, we report an increase in TMZ sensitivity but divergent changes in the GSC subfraction for TMZ resistant vs responsive GBM in the presence of hypoxia. Together, we demonstrate the versatility of cell-laden microgel approach to replicate heterogenous tumor populations, model shifts in the tumor microenvironment, and rapidly screen therapeutic response.

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).

Delivering therapeutics to the brain remains one of the most persistent challenges in medicine, because the blood-brain barrier (BBB) excludes over 98% of small-molecule drugs and virtually all biologics from the central nervous system (CNS). We developed electrical BBB modulation (eBBB), an on-demand platform combining vascular-targeting poly-L-lactic acid nanoparticles with high-definition transcranial direct current stimulation to achieve spatially and temporally controlled BBB opening. eBBB produced localized, reversible increases in BBB permeability confined to the stimulated cortex, with the opening area tunable via electrode geometry. This transient window enhanced regional delivery of a small-molecule drug, full-length immunoglobulins, and adeno-associated viral vectors, which are cargo classes otherwise completely excluded by the intact BBB. Neurovascular unit architecture was preserved with no lasting histological damage. Integrating a biodegradable nanomaterial with a clinically evaluated stimulation technology, eBBB offers a programmable, minimally invasive strategy for regional CNS drug delivery across brain malignancies and neurological disorders. One sentence summaryElectrical activation of piezoelectric nanoparticles reversibly opens the blood-brain barrier for minimally invasive drug delivery to targeted cortical regions.