Trafficking through the blood-brain barrier is directed by core and outer surface components of layer-by-layer nanoparticles.

Drug-carrying nanoparticles are a promising strategy to deliver therapeutics into the brain, but their translation requires better characterization of interactions between nanomaterials and endothelial cells of the blood-brain barrier (BBB). Here, we use a library of 18 layer-by-layer electrostatically assembled nanoparticles (NPs) to independently assess the impact of NP core and surface materials on in vitro uptake, transport, and intracellular trafficking in brain endothelial cells. We demonstrate that NP core stiffness determines the magnitude of transport, while surface chemistry directs intracellular trafficking. Finally, we demonstrate that these factors similarly dictate in vivo BBB transport using intravital imaging through cranial windows in mice. We identify that hyaluronic acid surface chemistry increases transport across the BBB in vivo, and flow conditions are necessary to replicate this finding in vitro. Taken together, these findings highlight the importance of assay geometry, cell biology, and fluid flow in developing nanocarriers for delivery to the brain.

Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Layer-by-Layer Polymer Functionalization Improves Nanoparticle Penetration and Glioblastoma Targeting in the Brain.

Glioblastoma is characterized by diffuse infiltration into surrounding healthy brain tissues, which makes it challenging to treat. Complete surgical resection is often impossible, and systemically delivered drugs cannot achieve adequate tumor exposure to prevent local recurrence. Convection-enhanced delivery (CED) offers a method for administering therapeutics directly into brain tumor tissue, but its impact has been limited by rapid clearance and off-target cellular uptake. Nanoparticle (NP) encapsulation presents a promising strategy for extending the retention time of locally delivered therapies while specifically targeting glioblastoma cells. However, the brain's extracellular structure poses challenges for NP distribution due to its narrow, tortuous pores and a harsh ionic environment. In this study, we investigated the impact of NP surface chemistry using layer-by-layer (LbL) assembly to design drug carriers for broad spatial distribution in brain tissue and specific glioblastoma cell targeting. We found that poly-l-glutamate and hyaluronate were effective surface chemistries for targeting glioblastoma cells in vitro. Coadsorbing either polymer with a small fraction of PEGylated polyelectrolytes improved the colloidal stability without sacrificing cancer cell selectivity. Following CED in vivo, gadolinium-functionalized LbL NPs enabled MRI visualization and exhibited a distribution volume up to three times larger than liposomes and doubled the retention half-time up to 13.5 days. Flow cytometric analysis of CED-treated murine orthotopic brain tumors indicated greater cancer cell uptake and reduced healthy cell uptake for LbL NPs compared to nonfunctionalized liposomes. The distinct cellular outcomes for different colayered LbL NPs provide opportunities to tailor this modular delivery system for various therapeutic applications.

The Blood-Brain Barrier: Implications for Experimental Cancer Therapeutics.

The blood-brain barrier is critically important for the treatment of both primary and metastatic cancers of the central nervous system (CNS). Clinical outcomes for patients with primary CNS tumors are poor and have not significantly improved in decades. As treatments for patients with extracranial solid tumors improve, the incidence of CNS metastases is on the rise due to suboptimal CNS exposure of otherwise systemically active agents. Despite state-of-the art surgical care and increasingly precise radiation therapy, clinical progress is limited by the ability to deliver an effective dose of a therapeutic agent to all cancerous cells. Given the tremendous heterogeneity of CNS cancers, both across cancer subtypes and within a single tumor, and the range of diverse therapies under investigation, a nuanced examination of CNS drug exposure is needed. With a shared goal, common vocabulary, and interdisciplinary collaboration, the field is poised for renewed progress in the treatment of CNS cancers.

Massively parallel pooled screening reveals genomic determinants of nanoparticle delivery.

To accelerate the translation of cancer nanomedicine, we used an integrated genomic approach to improve our understanding of the cellular processes that govern nanoparticle trafficking. We developed a massively parallel screen that leverages barcoded, pooled cancer cell lines annotated with multiomic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. We identified both materials properties and cell-intrinsic features that mediate nanoparticle-cell association. Using machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers. We validated one such biomarker: gene expression of SLC46A3, which inversely predicts lipid-based nanoparticle uptake in vitro and in vivo. Our work establishes the power of integrated screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations.

A predictive microfluidic model of human glioblastoma to assess trafficking of blood-brain barrier-penetrant nanoparticles.

The blood­brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood­brain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood­brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood­brain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.

Glioblastoma and the search for non-hypothesis driven combination therapeutics in academia.

Glioblastoma (GBM) remains a cancer of high unmet clinical need. Current standard of care for GBM, consisting of maximal surgical resection, followed by ionisation radiation (IR) plus concomitant and adjuvant temozolomide (TMZ), provides less than 15-month survival benefit. Efforts by conventional drug discovery to improve overall survival have failed to overcome challenges presented by inherent tumor heterogeneity, therapeutic resistance attributed to GBM stem cells, and tumor niches supporting self-renewal. In this review we describe the steps academic researchers are taking to address these limitations in high throughput screening programs to identify novel GBM combinatorial targets. We detail how they are implementing more physiologically relevant phenotypic assays which better recapitulate key areas of disease biology coupled with more focussed libraries of small compounds, such as drug repurposing, target discovery, pharmacologically active and novel, more comprehensive anti-cancer target-annotated compound libraries. Herein, we discuss the rationale for current GBM combination trials and the need for more systematic and transparent strategies for identification, validation and prioritisation of combinations that lead to clinical trials. Finally, we make specific recommendations to the preclinical, small compound screening paradigm that could increase the likelihood of identifying tractable, combinatorial, small molecule inhibitors and better drug targets specific to GBM.

Pharmacokinetic Principles and Their Application to Central Nervous System Tumors.

Despite increasing knowledge of the biologic drivers of central nervous system tumors, most targeted agents trialed to date have not shown activity against these tumors in clinical trials. To effectively treat central nervous system tumors, an active drug must achieve and maintain an effective exposure at the tumor site for a long enough period of time to exert its intended effect. However, this is difficult to assess and achieve due to the constraints of drug delivery to the central nervous system. To address this complex problem, an understanding of pharmacokinetic principles is necessary. Pharmacokinetics is classically described as the quantitative study of drug absorption, distribution, metabolism, and elimination. The innate chemical properties of a drug, its administration (dose, route and schedule), and host factors all influence these four key pharmacokinetic phases. The central nervous system adds a level of complexity to standard plasma pharmacokinetics as it is a coupled drug compartment. This review will discuss special considerations of pharmacokinetics in the context of therapeutic development for central nervous system tumors.

Theranostic Layer-by-Layer Nanoparticles for Simultaneous Tumor Detection and Gene Silencing.

Layer-by-layer nanoparticles (NPs) are modular drug delivery vehicles that incorporate multiple functional materials through sequential deposition of polyelectrolytes onto charged nanoparticle cores. Herein, we combined the multicomponent features and tumor targeting capabilities of layer-by-layer assembly with functional biosensing peptides to create a new class of nanotheranostics. These NPs encapsulate a high weight percentage of siRNA while also carrying a synthetic biosensing peptide on the surface that is cleaved into a urinary reporter upon exposure to specific proteases overexpressed in the tumor microenvironment. Importantly, this biosensor reports back on a molecular signature characteristic to metastatic tumors and associated with poor prognosis, MMP9 protease overexpression. This nanotheranostic mediates noninvasive urinary-based diagnostics in mouse models of three different cancers with simultaneous gene silencing in flank and metastatic mouse models of ovarian cancer.

Embryonic stem cell secreted factors decrease invasiveness of triple-negative breast cancer cells through regulome modulation.

Stem cell microenvironments decrease the invasiveness of cancer cells, and elucidating the mechanisms associated with disease regression could further the development of targeted therapies for aggressive cancer subtypes. To this end, we applied an emerging technology, TRanscriptional Activity CEll aRray (TRACER), to investigate the reprogramming of triple-negative breast cancer (TNBC) cells in conditions that promoted a less aggressive phenotype. The repressive environment was established through exposure to mouse embryonic stem cell conditioned media (mESC CM). Assessment of carcinogenic phenotypes indicated that mESC CM exposure decreased proliferation, invasion, migration, and stemness in TNBC cells. Protein expression analysis revealed that mESC CM exposure increased expression of the epithelial protein E-cadherin and decreased the mesenchymal protein MMP9. Gene expression analysis showed that mESC CM decreased epithelial to mesenchymal transition (EMT) markers fibronectin, vimentin, and Snail. Over a period of 6 d, TRACER quantified changes in activity of 11 transcription factors (TFs) associated with oncogenic progression. The EMT profile was decreased in association with the activity of 7 TFs (Smad3, NF-κΒ, MEF2, GATA, Hif1, Sp1, and RXR). Further examination of Smad3 and GATA expression and phosphorylation revealed that mESC CM exposure decreased noncanonical Smad3 phosphorylation and Smad3-mediated gene expression, increased GATA3 expression and phosphorylation, and resulted in a synergistic decrease in migration of GATA3 overexpressing MDA-MB-231 cells. Collectively, the application of TRACER to examine TF activity associated with the transition of cancer cells to a less aggressive phenotype, as directed by mESC CM, identified novel mechanistic events linking the embryonic microenvironment to both favorable changes and cellular plasticity in TNBC cell phenotypes.

The Benefits and Burdens of Cancer: A Prospective Longitudinal Cohort Study of Adolescents and Young Adults.

BACKGROUND: Adolescents and early young adults (AYAs) with cancer are at high risk for poor outcomes. Positive psychological responses such as benefit-finding may buffer the negative impacts of cancer but are poorly understood in this population. OBJECTIVE: We aimed to prospectively describe the content and trajectory of benefit- and burden-finding among AYAs to develop potential targets for future intervention. PATIENTS AND METHODS: One-on-one semistructured interviews were conducted with English-speaking AYA patients (aged 14-25 years) within 60 days of diagnosis of a noncentral nervous system malignancy requiring chemotherapy, 6-12 and 12-18 months later. Interviews were coded using directed content analyses with a priori schema defined by existing theoretical frameworks, including changed sense of self, relationships, philosophy of life, and physical well-being. We compared the content, raw counts, and ratios of benefit-to-burden by patient and by time point. SETTING/SUBJECTS: Seventeen participants at one tertiary academic medical center (mean age 17.1 years, SD = 2.7) with sarcoma (n = 8), acute leukemia (n = 6), and lymphoma (n = 3) completed 44 interviews with >100 hours of transcript-data. RESULTS: Average benefit counts were higher than average burden counts at each time point; 68% of interviews had a benefit-to-burden ratio >1. Positive changed sense-of-self was the most common benefit across all time points (44% of all reported benefits); reports of physical distress were the most common burden (32%). Longitudinal analyses suggested perceptions evolved; participants tended to focus less on physical manifestations and more on personal strengths and life purpose. CONCLUSIONS: AYAs with cancer identify more benefits than burdens throughout cancer treatment and demonstrate rapid maturation of perspectives. These findings not only inform communication practices with AYAs but also suggest opportunities for interventions to potentially improve outcomes.

CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin.

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFß superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.