Advanced Therapeutics

Post-traumatic osteoarthritis (PTOA) is a progressive joint disease for which no disease-modifying osteoarthritis drugs (DMOADs) have been approved. Although injectable drug delivery systems can prolong therapeutic retention within the joint, it remains unclear whether local drug release kinetics influence disease-modifying efficacy. Here, we developed a modular platform of injectable supramolecular hydrogels using biocompatible, generally recognized as safe (GRAS) amphiphilic molecules and systematically engineered a range of degradation and drug release profiles. Using the cathepsin-K inhibitor L-006235 as a model DMOAD, we generated hydrogels with distinct release kinetics and evaluated their therapeutic performance in PTOA. Hydrogels exhibiting slower degradation and more sustained drug release like Sucrose Stearate (SS hydrogel) showed prolonged intra-articular retention and improved therapeutic outcomes. In a destabilization of the medial meniscus (DMM) mouse model, sustained-release formulations significantly reduced cartilage degeneration, preserved aggrecan expression, improved joint histopathology, and enabled effective monthly dosing. In contrast, formulations with faster degradation and release kinetics required more frequent administration to achieve comparable benefits. To our knowledge, this is the first study to establish local drug release kinetics as a critical determinant of disease-modifying efficacy in PTOA. This work provides one of the clearest demonstrations to date that engineering intra-articular release kinetics, rather than merely prolonging residence time, can improve disease-modifying outcomes. Our findings establish local release kinetics as a key design parameter for osteoarthritis therapeutics and highlight the potential of tunable supramolecular hydrogels for long-acting drug delivery.

Glioblastoma represents a highly aggressive brain tumor with low survival and no response to chemotherapy and radiation therapy. Temozolomide, the current standard of care chemotherapy, improves patient survival by only about 6 months because of several resistance mechanisms, including unmethylated MGMT, which enables repair of chemotherapy-induced DNA damage. Thus, additional treatments strategies are necessary to investigate efficient responses towards glioblastoma. Doxorubicin (DOX) is a chemotherapeutic agent that is independent of MGMT methylation and instead works through inhibition of topoisomerase (TOPO) II, an enzyme necessary for DNA replication of the tumor. The inability of doxorubicin to cross the blood-brain barrier (BBB) precludes its use in glioblastoma. Polymersome nanoparticles have the potential to transport agents across the BBB. Here, we develop hyaluronic acid-b-polylactic acid (HA-PLA) polymeric nanoparticles called polymersomes, encapsulate them with DOX and investigate the ability of our system to induce apoptosis in a human glioblastoma cell line. The HA-PLA polymersomes show specificity and receptor-mediated endocytosis towards CD44-positive U87 glioblastoma cells due to the natural affinity of HA (hyaluronic acid) to CD44. Our HLA-PLA-DOX system promotes apoptosis of glioblastoma through inhibition of topoisomerase (TOPO) II. Thus, our system could allow tumor specificity through HA-CD44 affinity and slow drug release through pH sensitivity of PLA in the acidic tumor microenvironment.

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

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

Alzheimers disease is characterized by progressive cognitive decline, amyloid-{beta} deposition, neuroinflammation, and neurodegeneration, yet effective and well-tolerated therapies remain limited. Because dysregulated myeloid responses are increasingly recognized as important drivers of disease progression, we investigated the therapeutic potential of poly(lactic-co-glycolic acid) immunomodulatory nanoparticles in the 5xFAD mouse model of amyloid-driven neurodegeneration. Poly(lactic-co-glycolic acid) immunomodulatory nanoparticles and fluorescently labeled particles displayed the expected size range and negative surface charge. After intraperitoneal administration, fluorescent particles were preferentially associated with myeloid cells in the blood, spleen, and brain, with greater uptake by brain myeloid populations in 5xFAD mice than in wild-type controls. Therapeutic treatment of 6.5-month-old 5xFAD mice, a stage at which behavioral abnormalities are already established, resulted in significant improvement in elevated plus maze behavior and a more modest improvement in Barnes maze performance. Flow cytometric analysis performed 9 weeks after the final treatment demonstrated persistent changes in brain immune composition, with the most prominent effects observed in P2RY12+ microglial populations, particularly the CD11c+ subset, and comparatively limited sustained effects in CD11b+P2RY12- myeloid cells. These changes were accompanied by reduced expression of activation- and disease-associated markers and lower pro-inflammatory cytokine production within microglial populations. Histological analysis further showed reduced cortical amyloid plaque burden, decreased CD68 immunoreactivity, and reduced neurodegeneration in treated 5xFAD mice. Together, these findings show that systemically administered poly(lactic-co-glycolic acid) immunomodulatory nanoparticles produce durable behavioral, immunological, and pathological benefits in 5xFAD mice and support further investigation of this biodegradable myeloid-targeted platform as a therapeutic strategy for Alzheimers disease.

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.

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

Controlled release systems for subcutaneous peptide delivery often exhibit a pronounced initial burst release followed by inadequate maintenance of therapeutic exposure, limiting depot lifetime and increasing pharmacokinetic variability. Here, we engineer a dynamic, injectable hydrogel depot technology for months-long delivery of lipidated peptides. Using semaglutide as a model, we establish a modular formulation framework integrating: (i) formulation-driven tuning of depot mechanics to control release kinetics, (ii) cargo complexation strategies leveraging hydrophobic and multivalent ion-mediated interactions, and (iii) oxidative stabilization through sacrificial antioxidant excipients. We evaluated depot performance by rheology, in vitro cargo release, and in vivo pharmacokinetic and pharmacodynamic studies in rodents. Optimized formulations sustained semaglutide exposure for over six weeks from a single administration with two-fold reduction in peak-to-trough exposure and comparable total bioavailability relative to daily dosing, resulting in improved glucose control, weight regulation, and preservation of pancreatic islet content. These results suggest potential for quarterly dosing in humans. Together, this work establishes integrated and generalizable structure-property-performance relationships that account for cargo-matrix and cargo-excipient interactions across burst, diffusion, and erosion regimes to inform a practical formulation framework for engineering long-acting depots for sustained peptide delivery.

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

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.

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.

Calcineurin (Cn) is a protein phosphatase that initiates T-cell activation by dephosphorylating the transcription factor NFAT, driving its nuclear translocation and the transcription of immune-related genes. While clinical immunosuppressants like Cyclosporine A (CsA) potently inhibit Cn, they completely block its catalytic site, leading to non-specific inhibition and severe off-target toxicity. Selectively targeting the specific protein-protein interaction (PPI) between Cn and NFAT presents a safer therapeutic strategy. We previously identified the C16orf74 (C16) peptide as a high-affinity Cn-NFAT PPI inhibitor; however, its utility in cellular systems is restricted by poor membrane permeability. In this study, we evaluated cell-penetrating peptide (CPP) conjugates of C16 with an N-terminus transactivator of transcription (TAT) and polyarginine (R11) to enable efficient intracellular delivery. Structural modeling, fluorescence polarization displacement, and pull-down assays confirmed that the CPP-C16 conjugates retain the ability to compete with an NFAT-derived peptide and bind Cn. Fluorescence microscopy demonstrated efficient intracellular entry of TAT-C16 and R11-C16 in mammalian cells, and effective inhibition of NFAT nuclear translocation and attenuation of downstream NFAT-dependent transcriptional activity of the IL-2 gene in human T cells at concentrations of 10 {micro}M or lower. Crucially, unlike CsA, the CPP-C16 peptides exhibited minimal cytotoxicity even at high concentrations of up to 50 {micro}M, establishing a potential safe therapeutic window. These findings establish CPP-C16 conjugates as effective, cell-permeable, and non-toxic inhibitors of the Cn-NFAT signaling axis, providing the basis for the development of PPI-directed immunosuppressants.

Controlled drug delivery systems have important implications in many therapeutic applications to improve patient health, but it remains challenging to deliver therapeutics to specific target sites while being released at controlled rates to minimize the off-target accumulation. Here, we report a drug (e.g., vancomycin)-modulating other drug release strategies in human serum albumin (HSA)-based nanoparticles (NPs) to achieve multiple drug encapsulation and controlled release of drugs. Without external stimuli but only with water, the release kinetics of drugs such as sulfasalazine and epidermal growth factor can be modulated by adding vancomycin. The mechanistic study suggests that the release kinetics are related to the secondary structure of albumin through the interactions between drugs and HSA. Our strategy achieved controlled drug delivery through modulating the secondary structure of albumin with a water-soluble drug as a modulator, which provides a promising way to simultaneously deliver multiple drugs while releasing in a predicted and sustained manner.

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.

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.

Immune checkpoint inhibitors (ICI) have revolutionized cancer therapy, yet response rates remain suboptimal across many solid tumors, and resistance mechanisms, particularly those involving glycans, are not fully understood. Recent studies have identified sialic acid-containing glycans and their interactions with Siglec receptors on tumor-associated macrophages as an important contributor to immune suppression within the tumor microenvironment (TME). Targeting this sialic acid-Siglec axis by glycan engineering with sialidases and other glycosidases has shown therapeutic potential in preclinical models. However, safe and effective delivery of sialidases to tumors remains a challenge. Here, we present a novel approach using adeno-associated virus (AAV)-mediated therapy to deliver sialidases (AAVSia) and other glycosidases, including fucosidase, directly to the TME. Intratumoral administration of AAVSia in mouse models resulted in significant tumor growth reduction, enhanced survival, and robust systemic antitumor immunity through improved cross-presentation and dendritic cell activation. Furthermore, combining local sialidase expression with fucosidase treatment and classical PD-1 blockade allowed a synergistic effect, amplifying antitumor response. Our findings highlight the therapeutic promise of glycoengineering the TME using local delivery systems and support the development of combination strategies to overcome glycan-mediated resistance in cancer immunotherapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/720097v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@dc9d72org.highwire.dtl.DTLVardef@1e4e455org.highwire.dtl.DTLVardef@4a8f93org.highwire.dtl.DTLVardef@11813a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality in men and women. Timely detection and diagnosis are key to management of CRC, which is under-diagnosed because colorectal aberrant crypt foci, hyperplastic polyps, and microadenomas are often missed with conventional colonoscopy. The enzyme cyclooxygenase-2 (COX-2) is overexpressed in early stages of colorectal carcinogenesis and plays an important regulatory role in the process, suggesting that it could be a valuable target for enhanced imaging of nascent disease. Thus, we have generated an alpaca-derived library of 73 COX-2-specific nanobody clones. Here, we describe one such nanobody, F9-K45Q-K77Q-ROX, in which two native lysine residues have been mutated followed by conjugation to a fluorophore at the N-terminus with retention of COX-2-selective binding. The site of fluorophore conjugation and COX-2 binding affinity of F9-K45Q-K77Q-ROX were determined by proteomic and microscale thermophoretic analyses, respectively. In cell culture studies using 1483 human head and neck squamous cell carcinoma cells, F9-K45Q-K77Q-ROX accumulated inside cells and bound to intracellular COX-2, as visualized by fluorescence microscopy. In vivo pharmacokinetic, and toxicological analyses revealed that F9-K45Q-K77Q-ROX is detectable in circulation with a plasma half-life of 17.9 min and there is no short-term toxicity associated with single injections of 10 mg/kg, 20 mg/kg, or 40 mg/kg doses at 24 h post-administration. Noninvasive in vivo fluorescence endoscopic imaging validated tumor-specific accumulation of F9-K45Q-K77Q-ROX in azoxymethane/dextran sodium sulfate-induced colorectal adenomas in mice. This work demonstrates the first COX-2-targeted nanobodies including a fluorescent derivative that offers significant promise for targeted endoscopic imaging of COX-2-expressing neoplasms. Significance StatementCurrent colorectal cancer screening procedures, such as white-light colonoscopy, chromoendoscopy, and narrow-band imaging aim to detect solid colon tumors and precursor lesions. However, these methods tend to detect only raised solid tumors and mature cancers, whereas precursor lesions, such as aberrant crypt foci, hyperplastic polyps, and small adenomas are frequently missed. To address the need for better visualization of early lesions, we developed a library of alpaca-derived nanobodies targeted to cyclooxygenase-2 (COX-2), an enzyme that is overexpressed in colorectal adenomas. COX-2-targeted nanobodies bearing a fluorescent tag accumulate and are retained in colonic adenomas, facilitating their endoscopic visualization. This novel COX-2-targeted nanobody platform may also be valuable for early detection of other neoplastic diseases in which COX-2 overexpression occurs. (Word counts 119, limit 120)

IntroductionIn this study, we developed a modular in vitro platform that integrates advanced polymer-drug conjugation chemistry with stepwise cytotoxicity screening in both 2D (monolayer) and 3D (spheroids) glioblastoma (GBM) models. Buparlisib was selected as the model therapeutic due to its well-characterised mechanism of action, high blood-brain barrier permeability, and relevance to PI3K-targeted therapy. MethodsTwo mechanistically distinct conjugation strategies were explored using N-(2-hydroxypropyl)methacrylamide-based copolymers. The first strategy was based on a redox-sensitive disulphide linkage designed for intracellular glutathione-triggered release, whereas the second used an azide-bearing derivative compatible with strain-promoted azide-alkyne cycloaddition. Drug release was assessed by high-performance liquid chromatography. Biological activity was systematically evaluated in U87MG, U118MG, and T98G cells under 2D conditions using a resazurin-based metabolic activity assay. Subsequently, the more promising disulphide-based formulations were assessed in 3D spheroids by metabolic activity measurements and live-cell monitoring of spheroid growth dynamics. ResultsFree Buparlisib showed the strongest inhibitory effect, while its modification and polymer conjugation reduced the apparent activity. Nevertheless, the disulphide-based derivative and polymer conjugate retained concentration-dependent activity, whereas the azide-based polymer conjugate showed minimal effects. Moreover, treatment responses differed between cell lines and between 2D and 3D models. DiscussionOverall, linker chemistry, cell-line-specific behaviour, and model dimensionality strongly influenced the biological performance of the polymeric Buparlisib formulations. The redox-sensitive polymer conjugate therefore represents the more promising strategy for further development.

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