Advanced Therapeutics

Biotherapeutics are required to achieve high remission rates in patients with severe ulcerative colitis (UC); however, adverse effects, complex dosing regimens, administration routes, and low patient compliance may limit their widespread clinical use. Given the localized nature of UC, this study aimed to develop and evaluate a localized delivery strategy for infliximab (IFX), an anti-tumor necrosis factor- (TNF-) monoclonal antibody (mAb) recommended by the European Crohns and Colitis Organization (ECCO) and the American Crohns & Colitis Foundation for moderately-to-severely active UC. Exploiting the intrinsic biocompatibility, mucoadhesivity, and protein-entrapment capacity of lipid mesophases (LMPs), IFX was encapsulated within the gel matrix, providing protection against enzymatic and environmental degradation. IFX-loaded LMPs were designed for targeted delivery to inflamed colonic tissues via rectal or oral administration, with patient-centric oral dosage forms manufactured using a 3D printing approach. A comprehensive physicochemical characterization was performed to elucidate mesophase self-assembly and its relationship with IFX release profiles in biorelevant fluids. Therapeutic efficacy was evaluated in vivo using a dextran sulfate sodium (DSS)-induced colitis rat model, which demonstrated rectal gel retention for at least 8 h and colonic targeting of the oral formulation within 6 h. Under severe inflammatory conditions, LMP-based formulations reduced disease activity, inflammatory biomarkers (TNF- and fecal lactoferrin), and colon shortening to values comparable to those of healthy controls, outperforming the therapeutic efficacy of subcutaneous IFX. Overall, this study establishes a biocompatible delivery platform that enables targeted colonic IFX release and suppresses systemic absorption, representing a promising advancement in the biotherapeutic treatment of UC.

Tumor extracellular matrix (ECM) forms a net negative charged network that interacts with and hinders the transport of molecules partly based on electrostatic interactions. The focus on drug delivery in solid tumors has traditionally been on developing neutral charge coatings to minimize interactions with the ECM for improved transport. In contrast to this prior work, we recently found a cationic peptide that interacted electrostatically with the negatively charged components of the ECM, resulting in enhanced uptake and retention of nanoparticles in tumor ECM and tumor tissue. Based on this previous study, here, we hypothesize that the electrostatically driven interactions of the cationic peptide will improve the binding and retention of immune checkpoint blockade antibodies (ICBs), ultimately enhancing their antitumor immunogenic responses. We prepared peptide antibody (Ab) conjugates by conjugating the cationic peptide to ICBs, anti-cytotoxic T lymphocyte antigen 4 ({propto}-CTLA4) and anti-programmed cell death ligand-1 ({propto}-PD-L1) Abs, using copper-free click chemistry. We confirmed an average of 1 - 2 peptides per Ab. The cationic peptide electrostatically interacted with the net negatively charged tumor ECM and improved the binding of the Abs to the tumor ECM without affecting their antigen recognition capacities. Modifying the Abs due to cationic peptide conjugation reduced the systemic exposure of the Abs and did not induce treatment-related toxicities. We quantified a significantly higher population of tumor-infiltrating CD8+ T cells and a significant depletion of regulatory T cells in the tumor and tumor-draining lymph nodes upon peptide conjugation, which resulted in a better therapeutic outcome of the ICBs. ONE SENTENCE SUMMARYElectrostatic interaction-based intratumoral retention enhances antitumor responses of immune checkpoint blockade antibodies upon local administration.

Dysbiosis is linked to autoimmune diseases such as rheumatoid arthritis (RA), where microbial metabolites, such as short chain fatty acids (SCFAs), mediate the so-called gut-joint axis. The therapeutic potential of SCFAs is limited due to the frequent and high oral dosage requirements. RA is characterized by aberrant activation of peripheral T cells and myeloid cells. We aim to deliver butyrate, an SCFA, directly to the lymphatics using a polymeric micelle as a butyrate prodrug, creating a depot for inducing long-lasting immunomodulatory effects. Notably, negatively charged micelles (Neg-ButM) demonstrate superior efficacy in targeting the lymphatics post-subcutaneous administration, and were retained in the draining lymph nodes, spleen, and liver for over a month. In a mouse RA model, we found that Neg-ButM substantially mitigated arthritis symptoms and promoted tolerogenic phenotypes in T cells and myeloid cells, both locally and systemically. These findings suggest potential applications of this approach in treating inflammatory autoimmune diseases.

Accurate and precise delineation of gross tumour volumes remains a barrier to radiotherapy dose escalation and boost dosing in the treatment of solid tumours, such as prostate cancer. Magnetic resonance imaging of tumour molecular targets has the power to enable focal dose boosting, particularly when combined with technological advances such as MRI-LINAC. Fibroblast activation protein (FAP) is a transmembrane protein overexpressed in stromal components of >90% of epithelial carcinomas. Herein we compare targeted MRI of gold standard PSMA with FAP in the delineation of orthotopic tumours in a mouse model of prostate cancer. Control (no ligand), FAP and PSMA-targeting iron oxide nanoparticles were prepared with modification of an MRI agent (FerroTrace). Mice with orthotopic LNCaP tumours underwent T2-weighted 3D MRI 24 hours after intravenous injection of contrast agents. FAP and PSMA nanoparticles produced contrast enhancement on MRI when compared to control nanoparticles, which was most pronounced on the tumour periphery. FAP-targeted MRI increased the proportion of tumour contrast enhancing black pixels by 13.37% when compared to PSMA. Furthermore, analysis of changes in R2 values between healthy prostates and LNCaP tumours indicated an increase in contrast enhancing pixels in the tumour border of 15%, when targeting FAP, in contrast to PSMA This study demonstrates preclinical feasibility of PSMA and FAP-targeted MRI which can enable targeted image-guided focal therapy of localized prostate cancer.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone and neurotransmitter secreted from intestinal L-cells in response to nutrients to stimulate insulin and block glucagon secretion in a glucose-dependent manner. GLP-1 in itself is rapidly degraded, but long-acting GLP-1 receptor agonists (GLP-1 RAs) have become central in the treatment of T2D because of the beneficial effects extending also beyond glucose control. Currently, these therapeutics must be injected either daily or weekly or taken daily orally, leaving room for technological innovations that enable less frequent administrations, which will reduce patient burden and increase patient compliance. An ideal GLP-1 RA drug product would provide continuous therapy for upwards of four months from a single administration to match the cadence with which T2D patients typically visit their physician. In this work, we leveraged an injectable hydrogel depot technology to develop a long-acting GLP-1 RA drug product. By modulating the hydrogel properties to tune GLP-1 RA retention within the hydrogel depot, we engineered formulations capable of months-long GLP-1 RA delivery. Using a rat model of T2D, we confirmed that a single injection of hydrogel-based therapies exhibits sustained exposure of GLP-1 RA over 42 days, corresponding to a once-every four month therapy in humans. Moreover, these hydrogel therapies maintained optimal management of blood glucose and weight comparable to daily injections of a leading GLP-1 RA drug molecule. The pharmacokinetics and pharmacodynamics of these hydrogel-based long-acting GLP-1 RA treatments are promising for development of novel therapies reducing treatment burden for more effective management of T2D. Progress and PotentialWhile insufficient access to quality healthcare is problematic for consistent management of Type II diabetes (T2D), poor adherence to burdensome treatment regimens is one of the greatest challenges for disease management. Glucagon-like peptide 1 (GLP1) drugs have become central to the treatment of T2D due to their many beneficial effects beyond improving glucose control. Unfortunately, while optimization of GLP1 drugs has reduced treatment frequency from daily to weekly, significant patient burden still leads to poor patience compliance. In this work we developed an injectable hydrogel technology to enable GLP1 drugs only requiring administration once every four months. We showed in a rat model of T2D that one injection of a hydrogel-based therapy improves management of blood glucose and weight when compared with daily injections of the leading drug used clinically. These hydrogel-based GLP1 treatments are promising for reducing treatment burden and more effectively managing T2D. Future ImpactA GLP-1-based drug product providing four months of continuous therapy per administration could be transformational for the management of Type II diabetes (T2D). One of the most challenging aspects of diabetes management with GLP-1 mimics is maintenance of consistent levels of the drugs in the body, which is complicated by poor patient compliance on account of the high frequency of dosing required for current treatments. By leveraging a unique sustained release hydrogel depot technology we develop a months-long GLP-1 drug product candidate that has the potential to reduce patient burden and improving diabetes management. Overall, the hydrogel technology we describe here can dramatically reduce the frequency of therapeutic interventions, significantly increasing patient quality of life and reducing complications of diabetes management. Our next steps will focus on optimization of the drug formulations in a swine model of T2D, which is the most advanced and translationally-relevant animal model for these types of therapeutics. The long-term vision for this work is to translate lead candidate drug products towards clinical evaluation, which will also require comprehensive safety evaluation in multiple species and manufacturing our these materials according to Good Manufacturing Practices. The months-long-acting GLP-1 drug product that will come from this work has the potential to afford thus far unrealized therapeutic impact for the hundreds of millions of people with diabetes worldwide.

The immunoactivation effects of senescent tumor cells are a potential avenue for cancer therapy. They can act as antigen reservoirs for cancer vaccination, but how to maintain strong immunogenicity to induce a robust immunity is underexplored. In this study, we developed an engineered live vaccine composed of hydrogel-encapsulated senescent tumor cells and liposomal celecoxib (STCs+CLX-Lipo@Gel). This vaccine prolongs the in vivo persistence of senescent tumor cells and utilizes liposomal celecoxib (COX2 inhibitor) to promote the recruitment and maturation of dendritic cells (DC). Notably, a single dose can significantly delay melanoma growth by eliciting robust immunity. The vaccine extended the survival of mice with melanoma brain metastases. Moreover, this strategy also demonstrated high efficacy against orthotopic pancreatic tumors. This study presents a comprehensive strategy to boost the immunogenicity of whole-tumor-cell vaccines by leveraging senescent tumor cells and COX2 inhibition, with treatment efficacy in various tumor models. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/694320v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@f08fforg.highwire.dtl.DTLVardef@117d0aborg.highwire.dtl.DTLVardef@849665org.highwire.dtl.DTLVardef@adcf83_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic summary.C_FLOATNO Schematic illustration of the preparation of live-cell vaccines and the tumor-specific immune responses elicited by the vaccine. Created with BioRender.com. C_FIG

Systemic delivery of immunotherapy is dose-limited and often causes serious immune-related adverse events. Intratumoral injections can reduce systemic immunotoxicities and increase immunotherapy concentrations within a tumor. However, high pressures associated with direct tumor injection limits injectate retention, as low viscosity, saline-based solutions rapidly leak out of tumors. Viscoelastic solids, such as hydrogels, can improve local retention of co-formulated immunotherapies and provide sustained delivery. Prior work demonstrated that a chitosan-based hydrogel, XCSgel, was shear-thinning, self-healing, injectable, biocompatible, and clinically imageable. Here, we investigated XCSgel as a localized intratumoral delivery platform in the context of murine models of orthotopic triple-negative breast cancer. The intratumoral retention of immunotherapeutics co-formulated in XCSgel was characterized both ex vivo and in vivo via fluorescence imaging. Histopathological responses to intratumoral injections of XCSgel alone were scored by a veterinary pathologist. Initial antitumor studies evaluated a range of antitumor cytokines co-formulated with XCSgel. Subsequent antitumor and rechallenge studies evaluated the efficacy of a single intratumoral injection of interleukin-12 (IL-12) co-formulated in XCSgel (XCSgel-IL12) to control the growth of primary and abscopal tumors while inducing protective immunity. Pharmacokinetics studies quantified the systemic dissemination of IL-12 and consequent production of interferon-gamma following intratumoral injection with XCSgel co-formulation. Spectral flow cytometry was used to document changes in the tumor-immune microenvironment (TIME). XCSgel resisted tumor leakage and slowly released three model cytokines. XCSgel could be tuned for faster or slower release of embedded therapeutics. XCSgel-IL12 outperformed XCSgel formulations with other commonly used antitumor cytokines. A single injection of XCSgel-IL12 eliminated 86% E0771 and 20% mWnt orthotopic primary TNBC tumors. Mice rendered tumor-free resisted a live tumor challenge. XCSgel-IL12 also eliminated 67% untreated abscopal E0771 tumors. XCSgel-IL12 induced profound changes to the TIME, including a 3-fold reduction in the frequency of exhausted CD8+ T cells and a 3.2-fold increase in activated, proliferating CD8+ T cells. XCSgel is a promising localized delivery platform well-suited to enhance the retention and antitumor activity of potent immunotherapeutics. A single injection of XCSgel-IL12 can eliminate both primary and abscopal solid tumors, indicating that systemic immunotherapy may not be required for systemic control of cancer. SynopsisA novel injectable hydrogel, XCSgel, can localize and slowly release immunotherapies to eliminate primary and abscopal murine triple negative breast cancer tumors with a single injection.

Interleukin-15 (IL-15) emerges as a promising immunotherapeutic candidate in oncology because of its pivotal role in modulating both innate and adaptive immunity. However, the therapeutic utility remains concern due to the unexpected toxicity. We propose here that the mRNA lipid nanoparticle (mRNA-LNP) system can balance the issue through targeted delivery to increase IL-15 concentration in the tumor area and reduce leakage into the circulation. Utilizing the Structure-driven TARgeting (STAR) platform, we acquired intellectual property LNP vectors for effective and selective mRNA delivery to local (LNPLocal) and to pulmonary (LNPLung). Then the promising IL-15 superagonists mRNAs were obtained through structural optimization and sequence screening, showing better activity compared with benchmarker N-803. Subsequently, the anti-tumor efficacy of IL-15 superagonists mRNAs were evaluated by intratumoural (i.t.) injection and intravenous (i.v.) injection via LNPLocal and LNPLung, respectively. As a result, such superagonists exhibited better anti-tumor activity, less systematic exposure, and less cytokine related risks than N-803. We finally verified the selective delivery and well tolerability of LNPLung in non-human primates (NHPs), confirming the potential for clinical application. This finding may open up new possibilities for the treatment of lung cancers and lung metastasis cancers.

RATIONALEThe poor prognosis of glioblastoma is largely due to drug resistance and tumor location that, together, make it difficult to treat aggressively without affecting the rest of the brain. METHODOLOGYHigh-energy, short-range (40-80{micro}m) dendrimer-delivered -particles could address both challenges, because (1) they cause complex, highly cytotoxic double-strand DNA breaks, and (2) irradiation of the neighboring brain is minimal, since dendrimers selectively delivers them to tumors. Since cancer cells that are not directly hit by -particles will likely not be killed, the patterns of tumor irradiation affect efficacy. Systemically injected dendrimers extensively accumulate in glioblastomas, where they are taken up by tumor associated macrophages (TAMs), which tend to infiltrate tumors. We hypothesized that dendrimers labeled with -particle emitters, when being carried by TAMs, could more evenly irradiate glioblastomas, improving survival. In this study, the efficacy of dendrimers radiolabeled with the -particle emitter actinium-225 (dendrimer-radioconjugates) was evaluated when administered alone and/or after temozolomide, in a syngeneic immune-competent orthotopic GL261-C57BL/6 mouse model. RESULTSSystemically-administered dendrimer-radioconjugates, at activities that did not result in long-term toxicities, prolonged survival of mice with orthotopic GL261 tumors, compared to standard-of-care temozolomide (39 vs 31 days mean survival, p=0.0061) and non-treated animals (30 days, p=0.0009). Importantly, injection of temozolomide 24 hours before administration of dendrimer-radioconjugates further improved survival remarkably (44 days). This improvement in efficacy was attributed to: (1) the significant increase (by 33%) in tumor absorbed doses delivered by dendrimer-radioconjugates when injected after chemotherapy, without altering normal organ dosimetry, while sparing the tumor-surrounding healthy brain; (2) the potentially deeper tumor penetration of dendrimer-radioconjugates, suggested by the enhancement of dendrimer penetration within GL261-spheroids, employed as model tumor-avascular regions and/or TAM-free regions; and/or (3) the formation of a more lethal cocktail when both modalities acted on same cancer cells, that was correlated with increased levels of dendrimer-radioconjugates associating with GL261 cells in vitro and with greater incidences of karyomegaly in vivo. CONCLUSIONSThis study demonstrates the potential of a brain tumor targeted systemic actinium-225 radiopharmaceutical therapy that inhibits growth of glioblastoma cells and prolongs survival of mice with orthotopic brain tumors, further improved by standard-of-care temozolomide, without notable toxicities.

Neuroendocrine (NE) cancers include a diverse spectrum of hormone-secreting neoplasms that arise from the endocrine and nervous systems. Current chemo- and radio- therapies have marginal curative benefits. This study aimed to develop an innovative antibody-drug conjugate (ADC) to effectively treat NE tumors (NETs). We first confirmed that somatostatin receptor 2 (SSTR2) is an ideal surface target by analyzing 38 patient-derived NET tissues, 33 normal organs, and 3 NET cell lines. We then developed a new monoclonal antibody (mAb, IgG1 and kappa) to target two extracellular domains of SSTR2, which showed strong and specific surface binding to NETs. The ADC was constructed by conjugating the anti-SSTR2 mAb and antimitotic monomethyl auristatin E. In vitro evaluations indicated that the ADC can effectively bind, internalize, release payload, and kill NET cells effectively. Finally, the ADC was evaluated in vivo using a NET xenografted mouse model to determine cancer targeting, maximal tolerated dosage, pharmacokinetics, and anti-cancer efficacy. The anti-SSTR2 ADC was able to exclusively target and kill NETs with minimal toxicity and high stability in vivo. This study demonstrates that the anti-SSTR2 mAb-based ADC has high therapeutic values for NET therapy.

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.

Background and aimsCorrection of vascular DNA hypermethylation may slow atherogenesis. We tested the anti-inflammatory and anti-atherogenic activity of macrophage-targeted DNA methyltransferase (DNMT) inhibitor SGI-1027. Methods, ResultsSGI-1027 was encapsulated into human serum albumin (HSA) nanoparticles (HSANP) functionalized with the PP1 peptide, a macrophage scavenger receptor 1 ligand, fused to a FLAG epitope (S-HSANP-FLAGPP1). Nanoparticle physico-chemical characteristics predicted good marginalization towards the vascular wall, although SGI-1027 encapsulation efficiency was relatively low ([~]23%). S-HSANP-FLAGPP1 were rapidly internalized compared to non-functionalized and, surprisingly, functionalized void controls, and induced a shift towards an anti-inflammatory profile of secreted cytokines in human THP-1 macrophages. S-HSANP-FLAGPP1 colonized the atheroma and induced a significant [~]44% reduction of atherosclerosis burden in the aortic tree of ApoE-null mice compared to controls. A reduction in aortic root atherosclerosis was observed, although primarily induced by HSANP irrespective of loading or functionalization. No alteration of body weight, non-vascular tissue gross histology, plasma glucose, triglyceride or cholesterol were observed. HSA whether free or structured in nanoparticles, induced a 3-4-fold increase in HDL compared to vehicle. ConclusionsWe confirm that DNMT inhibition is anti-atherogenic and provide proof of principle that targeted HSANP are effective carriers for those molecules. SGI-1027 displayed a novel anti-inflammatory activity that is independent of cell proliferation and therefore likely unrelated to DNMT inhibition. HDL elevation may represent an additional advantage of HSA-based nanocarriers.

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.

We recently identified CD46 as a novel prostate cancer cell surface antigen that shows lineage independent expression in both adenocarcinoma and small cell neuroendocrine subtypes of metastatic castration resistant prostate cancer (mCRPC), discovered an internalizing human monoclonal antibody YS5 that binds to a tumor selective CD46 epitope, and developed a microtubule inhibitor-based antibody drug conjugate that is in a multi-center phase I trial for mCRPC (NCT03575819). Here we report the development of a novel CD46-targeted alpha therapy based on YS5. We conjugated 212Pb, an in vivo generator of alpha-emitting 212Bi and 212Po, to YS5 through the chelator TCMC to create the radioimmunoconjugate, 212Pb-TCMC-YS5. We characterized 212Pb-TCMC-YS5 in vitro and established a safe dose in vivo. We next studied therapeutic efficacy of a single dose of 212Pb-TCMC-YS5 using three prostate cancer small animal models: a subcutaneous mCRPC cell line-derived xenograft (CDX) model (subcu-CDX), an orthotopically grafted mCRPC CDX model (ortho-CDX), and a prostate cancer patient-derived xenograft model (PDX). In all three models, a single dose of 20 Ci 212Pb-TCMC-YS5 was well tolerated and caused potent and sustained inhibition of established tumors, with significant increases of survival in treated animals. A lower dose (10 Ci 212Pb-TCMC-YS5) was also studied on the PDX model, which also showed a significant effect on tumor growth inhibition and prolongation of animal survival. These results demonstrate that 212Pb-TCMC-YS5 has an excellent therapeutic window in preclinical models including PDXs, opening a direct path for clinical translation of this novel CD46-targeted alpha radioimmunotherapy for mCRPC treatment. SignificanceThis study reports a novel CD46 targeted 212Pb alpha particle radioimmunotherapy, 212Pb-TCMC-YS5, that is well tolerated and shows potent anti-tumor activity (tumor growth inhibition and increase of animal survival) in vivo in three prostate cancer small animal models, i.e., a subcutaneous and an intraprostate orthotopic mCRPC cell line-derived xenograft models, and a prostate cancer patient-derived xenograft model. Given that YS5 is a clinical stage human antibody, this YS5-based 212Pb alpha particle therapy has potential of translation to the clinic for treatment of mCRPC patients.

IntroductionSurgical resection and systemic chemotherapy with temozolomide remain the mainstay for treatment of glioblastoma. However, many patients are not candidates for surgical resection given inaccessible tumor location or poor health status. Furthermore, despite being first line treatment, temozolomide has only limited efficacy.\n\nMethodsThe development of injectable hydrogel-based carrier systems allows for the delivery of a wide range of chemotherapeutics that can achieve high local concentrations, thus potentially avoiding systemic side effects and wide-spread neurotoxicity. To test this modality in a realistic environment, we developed a diblock copolypeptide hydrogel (DCH) capable of carrying and releasing paclitaxel, a compound that we found to be highly potent against primary gliomasphere cells.\n\nResultsThe DCH produced minimal tissue reactivity and was well tolerated in the immune-competent mouse brain. Paclitaxel-loaded hydrogel induced less tissue damage, cellular inflammation and reactive astrocytes than cremaphor-taxol (typical taxol-carrier) or hydrogel alone. In a deep subcortical xenograft model, of glioblastoma in immunodeficient mice, injection of paclitaxel-loaded hydrogel led to a high local concentration of paclitaxel and led to local tumor control and improved survival. However, the tumor cells were highly migratory and were able to eventually escape the area of treatment.\n\nConclusionsThese findings suggest this technology may be ultimately applicable to patients with deep-seated inoperable tumors, but as currently formulated, complete tumor eradication would be highly unlikely. Future studies should focus on targeting the migratory potential of surviving cells.

Hydroxyapatite (HA)-binding peptides are emerging as promising candidates for bone-targeted therapies due to their strong affinity for mineralized tissues and biocompatibility. However, most studies to date have focused on in vitro characterization, providing limited insight into their in vivo performance. This study bridges that gap by evaluating the in vivo behavior of HA-binding peptides D8, E8, YD8, and YE8 using fluorescence imaging to assess their biodistribution in healthy and pathological bone environments. In healthy animal models, D8 demonstrated the strongest binding across mineralized tissues, including the skull, femur, and tibia, while YD8 showed moderate binding. In contrast, E8 and YE8 exhibited limited localization influenced by peptide dosage and binding kinetics. Pathological models, including defective tibia and osteogenesis imperfecta (OIM) mice, revealed preferential accumulation of D8 and YD8 in structurally compromised regions, underscoring their potential for targeting diseased bone microenvironments. Fluorescence imaging, enhanced by spectral unmixing algorithms, proved effective for assessing peptide localization and distribution. These findings highlight the utility of HA-binding peptides for bone-targeted therapies and emphasize the importance of in vivo studies in advancing their therapeutic and diagnostic applications. This work provides a foundation for optimizing peptide designs to improve specificity and efficacy in bone repair and regeneration.

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.

Endocytosis is a critical cellular process involved in many physiological functions. Most research on endocytosis has been performed in vitro, however, understanding this process in vivo is necessary in tissues like bone that have a unique 3D extra-cellular matrix. Here, we present a live-cell study of endocytosis in osteocytes, mechanosensory cells embedded in mouse bone. We visualized real-time fluorescent nanoparticle uptake and trafficking in osteocytes by using intravital imaging combined with multiphoton microscopy within living animals. We applied pharmacologic inhibitors to distinguish between general and receptor-specific endocytosis pathways in vivo. Our findings reveal rapid nanoparticle uptake in osteocytes, with marked differences in the timescale and pattern of uptake depending on nanoparticle surface functionality. We also discovered differences in dynamin-dependent endocytosis in osteocytes between male and female animals. These results offer the first in vivo derived insights into how osteocytes take up materials and provide new evidence for chemically altering receptor-mediated endocytosis in live bone tissue.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, with limited survival outcomes due to tumor recurrence, mainly driven by GBM cell invasion and therapy resistance. Although temozolomide (TMZ) remains the standard-of-care chemotherapeutic, its long-term efficacy is often compromised by rapid emergence of acquired resistance, largely mediated by the DNA repair enzyme, methylguanine methyltransferase (MGMT). To investigate the interplay between tumor heterogeneity, drug resistance, and the extracellular matrix (ECM) microenvironment, we adapted a 3D methacrylamide-functionalized gelatin (GelMA) hydrogel model to study the behavior of mixed populations of TMZ-sensitive and TMZ-resistant GBM cells. Using both single-cell distributions and multicellular spheroids, we report the impact of heterogeneous cell populations and TMZ dosing regimens, including physiological, supraphysiological, and metronomic TMZ schedules, on drug response and migration. We show that the combination therapy of TMZ with an MGMT inhibitor, lomeguatrib, can modulate TMZ resistance in vitro. This hydrogel model enables systematic investigation of GBM heterogeneity, "go-or-grow" phenotypic plasticity, and therapeutic resistance in an ECM-rich microenvironment, offering a valuable platform for future translational research.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving response to immune checkpoint inhibitors (ICI). However, the potency and clinical efficacy of 5-triphosphate RNA (3pRNA) agonists of RIG-I is hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of [~]100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to PD-1 ICI in an orthotopic EO771 model of triple negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined PD-1 + CTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with potential to enhance response to ICIs and other immunotherapeutic modalities.