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.

Ultrasound-activatable drug-loaded nanocarriers enable noninvasive and spatiotemporally-precise on-demand drug delivery throughout the body. However, most systems for ultrasonic drug uncaging utilize cavitation or heating as the drug release mechanism and often incorporate relatively exotic excipients into the formulation that together limit the drug-loading potential, stability, and clinical translatability and applicability of these systems. Here we describe an alternate strategy for the design of such systems in which the acoustic impedance and osmolarity of the internal liquid phase of a drug-loaded particle is tuned to maximize ultrasound-induced drug release. No gas phase, cavitation, or medium heating is necessary for the drug release mechanism. Instead, a non-cavitation-based mechanical response to ultrasound mediates the drug release. Importantly, this strategy can be implemented with relatively common pharmaceutical excipients, as we demonstrate here by implementing this mechanism with the inclusion of a few percent sucrose into the internal buffer of a liposome. Further, the ultrasound protocols sufficient for in vivo drug uncaging with this system are achievable with current clinical therapeutic ultrasound systems and with intensities that are within FDA and society guidelines for safe transcranial ultrasound application. Finally, this current implementation of this mechanism should be versatile and effective for the loading and uncaging of any therapeutic that may be loaded into a liposome, as we demonstrate for four different drugs in vitro, and two in vivo. These acoustomechanically activatable liposomes formulated with common pharmaceutical excipients promise a system with high clinical translational potential for ultrasonic drug uncaging of myriad drugs of clinical interest. One Sentence SummaryIncorporating a few percent sucrose into a liposome transforms it into an immediately translatable vehicle for noninvasive, on-demand ultrasound-targeted drug delivery.

Niemann-Pick Disease Type C (NPC) is a severe neurovisceral disorder that is pathophysiologically characterized by intracellular transport abnormalities leading to cytoplasmic accumulation of lipids such as cholesterol and multiple sphingolipids, including sphingosine. The compound 2-hydroxypropyl-{beta}-cyclodextrin (HP{beta}CD) is a compound with high cholesterol complexation capacity and is currently under clinical investigation for the treatment of NPC. However, due to its short blood half-life, high doses are required to produce a therapeutic effect. It has been reported in mice that HP{beta}CDs circulation time and efficacy can be improved by increasing its size via polymerization, but the biodegradable nature of these systems did not allow the contribution of the macromolecule to the activity to be determined. In this work, stable forms of polymerized HP{beta}CD were generated (via epichlorohydrin crosslinking) to investigate their in vitro mechanisms of action and in vivo effects. Crosslinked CDs (8-312 kDa) displayed a 10-fold greater complexation capacity towards cholesterol than monomeric HP{beta}CD but were taken up by cells to a lower extent (in a size-dependent fashion), resulting in an overall comparable in vitro effect on intracellular cholesterol accumulation that was dependent on cholesterol complexation. When tested in vivo, the crosslinked 19.3 kDa HP{beta}CD exhibited a longer terminal half-life than the monomeric HP{beta}CD. However, it did not increase the life span of Npc1 mice, possibly due to reduced organ penetration and brain diffusion consequence of its large molecular weight. This could be circumvented by the application of magnetic resonance imaging-guided low intensity-pulsed focused ultrasound (MRIg-FUS), which increased the brain penetration of the CD. In conclusion, stable forms of polymerized HP{beta}CD constitute valuable tools to elucidate CDs mechanism of action. Moreover, the use of MRIg-FUS to maximize CDs tissue penetration warrants further investigation, as it may be key to harnessing CDs full therapeutic potential in the treatment of NPC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/230136v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@3dbaf1org.highwire.dtl.DTLVardef@bc255dorg.highwire.dtl.DTLVardef@390e7borg.highwire.dtl.DTLVardef@1e5e5c5_HPS_FORMAT_FIGEXP M_FIG C_FIG The 2-hydroxypropyl-{beta}-cyclodextrin (HP{beta}CD) is a well-established pharmaceutical excipient that can complex cholesterol and is currently under clinical investigation to treat Niemann-Pick Disease Type C (NPC). However, high doses of the drug are needed to achieve a therapeutic effect. Using stable and long circulating crosslinked HP{beta}CDs, this study attempts to further understand the mechanisms behind CDs activity.

The blood-brain barrier (BBB) limits drug delivery to the brain and the movement of neurological biomarkers between the brain and blood. Focused ultrasound-mediated blood-brain barrier opening (FUS-BBBO) noninvasively opens the BBB, allowing increased molecular transport to and from the brain parenchyma. Despite being initially developed as a drug delivery method, FUS-BBBO has shown promise both as a neuroimmunotherapeutic modality, and as a way of improving neurological disease diagnosis via amplification of disease biomarker circulation. Recently, the role of extracellular vesicles (EVs) in modulating the neuroimmune system and in improving biomarker detection has sparked research interest. However, despite their potential role in modulating FUS-BBBO-induced neuroimmunotherapy and their ability to improve biomarker specificity after treatment, the EV response to FUS-BBBO had not been extensively characterized prior to this study. In this study, we investigated the effect of FUS-BBBO on EV concentration and content in the serum of mice and Alzheimers Disease (AD) patients. We observed a 164% increase in murine EV concentration one hour after treatment, as well as an increase in EV RNA associated with FUS-BBBO neuroimmunotherapy. Patient EV concentration also increased one hour after treatment and was dependent on the volume of BBB opening three days post-treatment. Furthermore, EV isolation was found to significantly enhance the amplification of AD biomarker detection by FUS-BBBO. Overall, we present the first evidence of altered murine and AD patient EV concentration and content in response to FUS-BBBO, providing evidence of EVs role within FUS-BBBO neuroimmunotherapy as well as their utility in improving FUS-BBBO biomarker amplification.

The U.S. opioid epidemic is an extraordinary public health crisis that started in 1990 and significantly accelerated in the last decade. Since 2020, over 100,000 fatal drug overdoses have been reported annually, and 75% of those involved fentanyl and its analogs (F/FA). Accelerating the translation of innovative, effective, and safe treatments is needed to augment existing measures to counteract such a crisis. Active immunization against F/FA and other opioids represents a promising therapeutic and prophylactic strategy for opioid use disorder (OUD) and opioid-induced overdose toxicity. Previously we demonstrated that the anti-fentanyl vaccine comprising a fentanyl-based hapten (F) conjugated to the diphtheria cross-reactive material (CRM), admixed with the novel lipidated toll-like receptor 7/8 (TLR7/8) agonist INI-4001 adsorbed on Alhydrogel(R) (alum) induced high-affinity fentanyl-specific polyclonal antibodies that protected against fentanyl-induced pharmacological effects in mice, rats, and mini-pigs. Here, INI-4001 was formulated into liposomes with different surface charges, and their impact on F-CRM adsorption, INI-4001 adjuvanticity, and vaccine efficacy were explored. Additionally, as the role of innate immunity in mediating the efficacy of addiction vaccines is largely unknown, we tested these formulations on the activation of innate immunity in vitro. Cationic INI-4001 liposomes surpassed other liposomal and aluminum-based formulations of INI-4001 by enhancing the efficacy of fentanyl vaccines and protecting rats against bradycardia and respiratory depression by blocking the distribution of fentanyl to the brain. Fentanyl vaccines adjuvanted with either cationic INI-4001 liposomes or the aqueous INI-4001 adsorbed to alum induced significant surface expression of co-stimulatory molecules and maturation markers in a murine dendritic cell line (JAWS II), while the former was superior in enhancing the macrophages surface expression of CD40, CD86 and inducible nitric oxide synthase (iNOS), indicative of maturation and activation. These results warrant further investigation of liposome-based formulations of TLR7/8 agonists for improving the efficacy of vaccines targeting F/FA and other opioids of public health interest. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/631964v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@178c8fdorg.highwire.dtl.DTLVardef@d38c09org.highwire.dtl.DTLVardef@8ec41forg.highwire.dtl.DTLVardef@edcf82_HPS_FORMAT_FIGEXP M_FIG C_FIG

The blood-brain barrier (BBB) significantly hinders the treatment of central nervous system (CNS) disorders and brain tumors with intact BBB by restricting the entry of most therapeutic agents, including small-molecule drugs and particularly larger macromolecules. Liposomal formulations, such as PEGylated liposomes with long blood half-lives, high drug-carrying capacity, and reduced off-site toxicity, can be useful for brain drug delivery, but their large size often limits BBB penetration. A novel liposomal doxorubicin formulation, Talidox(R), with a smaller size ([~]36 nm), increased blood circulation half-life, and better stability than previous clinical formulations, can be a suitable choice for brain delivery. This study investigated Talidox(R) delivery to the brain through focused ultrasound (FUS)-mediated BBB transient opening. Radiolabelling of Talidox(R) via intraliposomal 89Zr enabled Positron Emission Tomography (PET) imaging for whole-body non-invasive, real-time monitoring of biodistribution and pharmacokinetics. Following FUS-mediated BBB opening in mice, PET imaging revealed a significant increase in brain uptake compared to non-FUS controls, achieving a 14-fold higher accumulation. Additional validation using passive acoustic detection, microscopy, autoradiography, and cryo-fluorescence tomography demonstrated successful brain distribution that correlated with PET imaging results. These findings underscore the potential of combining Talidox(R) with FUS for effective, non-invasive drug delivery to the brain and highlight the advantages of PET imaging as a modality for non-invasive, longitudinal quantification of drug delivery to the brain.

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 Multiforme (GBM) represents a significant clinical challenge amongst central nervous system (CNS) tumors, with a dismal mean survival rate of less than 8 months, a statistic that has remained largely unchanged for decades (National Brain Society, 2022). The specialized intricate anatomical features of the brain, notably the blood-brain barrier (BBB), pose significant challenges to effective therapeutic interventions, limiting the potential reach of modern advancements in immunotherapy to impact these types of tumors. This study introduces an innovative, actively targeted immunotherapeutic nanoconjugate (P12/AP-2/NCs) designed to serve as an immunotherapeutic agent capable of traversing the BBB via LRP-1 receptor-mediated transcytosis. P12/AP-2/NCs exert its immune-modulating effects by inhibiting the PD-1/PD-L1 axis through a small-size PD-L1/ PD-L2 antagonist peptide Aurigene NP-12 (P12). P12/AP-2/NCs are synthesized from completely biodegradable, functionalized high molecular weight {beta}-poly(L-malic acid) (PMLA) polymer, conjugated with P12 and Angiopep-2 (AP2) to yield P12/AP-2/NCs. Evaluating nanoconjugates for BBB permeability and 3-D tumor model efficacy using an in vitro BBB-Transwell spheroid based model demonstrating successful crossing of the BBB and internalization in brain 3D tumor environments. In addition, the nanoconjugate mediated T cells cytotoxicity on 3D tumor region death in a U87 GBM 3-D spheroid model. AP2/P12/NCs is selectively inhibited in PD1/PDL1 interaction on T cells and tumor site, increasing inflammatory cytokine secretion and T cell proliferation. In an in-vivo murine brain environment, rhodamine fluorophore-labeled AP2/P12/NCs displayed significantly increased accumulation in the brain during 2-6 h time intervals post-injection with a prolonged bioavailability over unconjugated peptides. AP2/P12/NCs demonstrated a safety profile at both low and high doses based on major organ histopathology evaluations. Our findings introduce a novel, programmable nanoconjugate platform capable of penetrating the BBB for directed delivery of small peptides and significant immune environment modulation without utilizing antibodies, offering promise for treating challenging brain diseases like glioblastoma multiforme and beyond.

The immunoregulatory cation channel TMEM176B plays a dual role in tumor immunity. On one hand, TMEM176B promotes antigen cross-presentation to CD8+ T cells by regulating phagosomal pH in dendritic cells (DCs). On the other hand, TMEM176B inhibits NLRP3 inflammasome activation through ionic mechanisms in DCs, monocytes and macrophages. Moreover, the TMEM176B blocker BayK8644 controls tumor progression through mechanisms involving inflammasome activation in prophylactic but not in therapeutic protocols. We speculated that the limited therapeutic efficacy of the compound may be linked to its potential capacity to inhibit antigen cross-presentation. Here we show that free BayK8644 inhibits antigen cross-presentation by splenic DCs. To prevent such inhibition, we reasoned that formulating BayK8644 in nanoparticles may delay the release of the compound in endosomes. Avoiding TMEM176B inhibition during the first 30 minutes of nanoparticle internalization by DCs may allow efficient cross-presentation to occur during this critical time frame. Indeed, we observed that NP-PEG-BayK8644 did not inhibit antigen cross-presentation, in contrast to the free compound. Moreover, NP-PEG-BayK8644 triggered inflammasome activation in a Tmem176b-dependent manner. We then injected eNP-PEG or NP-PEG-BayK8644 to mice bearing established tumors. NP-PEG-BayK8644 significantly controlled tumor growth and mice survival, as compared to eNP-PEG and free BayK8644, in a Tmem176b-dependent manner in mouse melanoma and lymphoma tumors. Responding animals treated with NP-PEG-BayK8644 showed reinforced tumor infiltration by total and tumor-specific CD8+ T cells. Overall, we rationally developed a formulating method of BayK8644 that improves its anti-tumoral therapeutic efficacy by uncoupling the dual role of TMEM176B on innate and adaptive immunity.

The widespread use of Covid-19 mRNA vaccines has highlighted the need to address rare but concerning side effects. Systemic off-target gene expression has been identified as a primary cause of acute adverse reactions and side effects associated with nucleoside-modified mRNA vaccines. In this study, we incorporated the permanent cationic lipid Dotap component into the mRNA-LNP formula associated with the FDA-approved mRNA vaccine Comirnaty to create a novel positively charged LNP carrier for mRNA vaccine delivery. Using the optimized LNP formula to prepare SARS-Cov-2 Spike mRNA vaccines for immunogenicity testing, Balb/c mice exhibited improved immunogenicity kinetics with initial antibody titers being lower but showing a continuous upward trend, ultimately reaching levels comparable to those of control mRNA vaccines 8 weeks after boost immunization. The mRNA vaccines encapsulated in the modified LNPs have demonstrated a superior safety profile in respect to systemic delivery of LNP constituents, off-target gene expression, and the systemic pro-inflammatory stimulation. Consequently, it may represent a safer alternative of conventional mRNA-LNP vaccines.

Many neurological and psychiatric diseases are characterized by pathological neuronal activity. Current treatments involve drugs, surgeries, and implantable devices to modulate or remove the affected region. However, none of these methods can be simultaneously non-invasive and possess site- and cell type specificity. Here, we apply a non-invasive neuromodulation approach called Acoustically-Targeted Chemogenetics, or ATAC, to increase the seizure threshold. Here, the ATAC approach used a multi-point focused ultrasound to transiently open the blood-brain barrier of the whole hippocampus and transduce pyramidal neurons with engineered G-protein-coupled receptors to inhibit their activity. To express the engineered receptors in the mouse hippo-campus, we used a recently engineered viral vector optimized for ultrasound-based gene delivery to the brain, AAV.FUS.3. In a mouse flurothyl seizure model, we showed successful gene delivery throughout the hippocampus, a significant neuronal activity inhibition as evidence by an increase in seizure threshold. Finally, we benchmarked these effects against a clinically prescribed drug that acts without spatial precision.

Gene therapy using adeno-associated virus (AAV) vectors shows promise for cancer treatment through molecular intervention, yet achieving sufficient and targeted delivery to brain tumors via systemic administration remains limited by the biological barriers. Here, we investigate whether microbubble-enhanced focused ultrasound (MB-FUS) improves targeted delivery of systemically administered AAV9 to orthotopic gliomas, using quantitative PET imaging of 64Cu-radiolabeled AAV9 vectors and fluorescent reporter expression to assess biodistribution and functional efficacy. At 21 hours after injection, 64Cu-AAV9 accumulation was 3.2-fold higher in FUS-treated tumors compared to non-FUS-treated tumors (n=3, p=0.004). Quantitative PCR analysis of tumor tissue at the same timepoint confirmed a 6.4-fold increase in genome copies in FUS-treated tumors (p=0.0003). The enhanced vector delivery translated to a 5.3-fold increase in optical reporter protein expression in FUS-treated compared to control tumors (p=0.0002) at 17 days post-treatment. These results establish that MB-FUS enables spatially-targeted AAV delivery with quantifiable enhancement in both acute vector biodistribution and downstream transgene expression. The integration of radiolabeled AAV with PET imaging provides a non-invasive methodology for real-time assessment of vector delivery and optimization of treatment protocol for brain cancer gene therapy. HighlightsO_LIMB-FUS enables targeted systemic AAV delivery to brain tumors. C_LIO_LIMB-FUS enhanced vector delivery translates to increased transgene expression in gliomas. C_LIO_LIPET imaging of radiolabeled AAV allows non-invasive tracking of gene therapy vectors. C_LIO_LIReal-time imaging validates spatially-controlled gene delivery for brain cancer. C_LI

The therapeutic potential of mRNA is vast, and yet translating this potential into effective treatments requires overcoming significant challenges of achieving safe and efficient delivery. This process is hampered by biological barriers that limit cellular uptake, degrade exposed mRNA, and thus necessitate effective endosomal escape to reach the cytoplasm. To address these challenges, we developed a hPep peptide-based nanoparticle (PNP) system that encapsulates mRNA, forming stable and biocompatible particles, which are rapidly taken up by the cells and enable efficient mRNA delivery across various cell culture models. Following systemic administration in mice, lead hPep3/mRNA PNPs achieve broad mRNA expression across multiple tissues, including the lungs, liver, and spleen, and enable effective mRNA delivery to the central nervous system upon local administration. Furthermore, we established a high-yield ([≥]70%) microfluidics-based protocol to scale up the production of well-defined, sterile hPep3/mRNA PNP formulations (approximately 70 nm, PDI around 0.170). Most importantly, in a proof-of-concept study in nonhuman primates (NHPs), we demonstrate that hPep PNPs loaded with human erythropoietin (hEPO) mRNA induce dose-dependent expression of hEPO protein in monkey serum, reaching up to 10 ng/ml at 1.0 mg/kg dose, following both single and repeated administration, while remaining systemically well-tolerated. These findings underscore the potential of hPep peptide-based nanoparticles as a versatile platform for mRNA delivery across multiple tissues, highlighting their promise in advancing the development of mRNA therapeutics.

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

Resection surgery is the first-line therapy for glioblastoma (GBM) that is performed in >70% of patients, typically within days of suspected diagnosis. Current protocols for follow-on chemoradiotherapy have shown only modest efficacy in eliminating residual disease, leading to inevitable tumour recurrence. There remains a need for new approaches to swiftly and effectively treat post-operative residual disease to prevent the rapid early progression of recurrent GBM. Using syngeneic preclinical models of glioblastoma resection, we identified a spatially and temporally restricted window of blood brain barrier (BBB) disruption localised to the resection margin, during the immediate (15 min) and early (48-72h) postoperative periods. Intravenous administration of fluorescently labelled, clinically-used liposome nanoparticles during these periods demonstrated that selective accumulation at the postoperative resection margin, while largely being excluded from areas of the brain with an intact BBB, could be achieved. Confocal analysis confirmed the presence of extravasated nanoparticles within the margin parenchyma which largely interacted with microglial populations closely associated with residual tumour cells. Exploiting this, we performed intravenous administration of doxorubicin-loaded liposomes (DOX-Lipo) coinciding with the peak of postoperative BBB disruption and demonstrated both enhanced chemotherapy delivery and consequently complete inhibition of tumour recurrence from a single administration. Overall, this work underscores the importance of timing concomitant chemotherapy to the post-operative timeframe and demonstrates that clinically-used liposomal nanomedicines could be readily repurposed for early post-operative therapy in aggressive brain tumours.

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

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

Triple-negative breast cancer (TNBC) poses significant clinical challenges due to its high heterogeneity, with multiple subtypes exhibiting distinct molecular characteristics and treatment responses. The development of resistance to chemotherapy and targeted therapies remains a major obstacle, and identifying reliable biomarkers to predict therapeutic response continues to be challenging. This study aims to enhance the delivery of the immunostimulant Toll-like receptor 7/8 (TLR7/8) agonist resiquimod (R848) using a safe and highly integrated nanoemulsions (NEs) formulation, providing effective and reliable immunotherapy. A series of NEs were prepared and optimized with and without the reactive lipophilic compound ricinoleic acid. Neutral and negatively charged NE formulations encapsulating R848 were compared. The physicochemical properties and in vitro delivery of resiquimod into RAW 264.7 macrophages and 4T1 TNBC cell line models were studied. Both R848-loaded NE formulations exhibited prolonged shelf-life stability with minimal protein binding. Incorporating small portions of ricinoleic acid into the formulation (negatively charged NEs) slowed drug release and improved physical properties and overall delivery compared to ricinoleic-free formulations, likely due to its interaction with the drug. Cytotoxicity and cellular uptake studies were conducted on both NE models, showing localization in the macrophage cell membrane and 4T1 cell cytoplasm. Molecular profiling in 4T1 cells revealed R848-NEs modulated key biomarkers (TLR4/7, Cyclin D1, NF-{kappa}B) while potently inducing autophagy (evidenced by LC3II/p62/Beclin-1 alterations) and PD-L1 upregulation. These dual effects--autophagy-mediated tumor suppression and immune checkpoint modulation--suggest therapeutic synergy between R848-NEs and anti-PD-L1 antibodies, presenting a promising combinatorial strategy for TNBC treatment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/647265v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@d4daceorg.highwire.dtl.DTLVardef@692daforg.highwire.dtl.DTLVardef@13676d7org.highwire.dtl.DTLVardef@b449fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ovarian cancer immunotherapy remains a challenge based on the "cold" tumor microenvironment. Herein we present a rational design to create immunogenic nanoparticles as a multi-agent platform that promotes immune response in a mouse model of ovarian cancer. The hybrid lipid-silica nanosystem is capable of co-loading four types of cargo molecules including a model antigen, nucleic acid-based adjuvant Cytosine-p-linked to Guanine (CpG, TLR3/9 agonist), lipid-based adjuvant (MPLA, TLR4 agonist) integrated into the lipid coat, and optionally a small molecule drug, such as the chemotherapeutic agent oxaliplatin, a well-established treatment for ovarian cancer. The optimization of the nanoplatform in terms of lipid composition, functionalized silica dendritic core formation, and final charge, as well as their compatibility with the complex loading profile highlights an opportunity for enhanced survival of mice with advanced ovarian cancer compared to monotherapy. Furthermore, intraperitoneal administration led to preferential accumulation within tumor-burdened tissues with selective accumulation in myeloid cells. High myeloid cell cytotoxicity negated the benefits of oxaliplatin. The inclusion of CpG in the nanoparticle formulation enhanced the survival of mice with ovarian cancer. To interpret these outcomes and guide future design, we also developed a mathematical model of nanoparticle-driven immune activation, which quantified treatment efficacy and identified key parameters governing tumor response. The presented hybrid nanoparticle i tunable, enabling delivery of alternative molecules therefore, thereby highlighting a promising platform for the treatment of peritoneal cancers. Graphical TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/657862v1_ufig1.gif" ALT="Figure 1000"> View larger version (26K): org.highwire.dtl.DTLVardef@1434599org.highwire.dtl.DTLVardef@18e79e6org.highwire.dtl.DTLVardef@e5285corg.highwire.dtl.DTLVardef@bcc604_HPS_FORMAT_FIGEXP M_FIG C_FIG