Exposure to environmental airborne particulate matter caused wide-ranged transcriptional changes and accelerated Alzheimer's-related pathology: A mouse study.

Air pollution poses a significant threat to human health, though a clear understanding of its mechanism remains elusive. In this study, we sought to better understand the effects of various sized particulate matter from polluted air on Alzheimer's disease (AD) development using an AD mouse model. We exposed transgenic Alzheimer's mice in their prodromic stage to different sized particulate matter (PM), with filtered clean air as control. After 3 or 6 months of exposure, mouse brains were harvested and analyzed. RNA-seq analysis showed that various PM have differential effects on the brain transcriptome, and these effects seemed to correlate with PM size. Many genes and pathways were affected after PM exposure. Among them, we found a strong activation in mRNA Nonsense Mediated Decay pathway, an inhibition in pathways related to transcription, neurogenesis and survival signaling as well as angiogenesis, and a dramatic downregulation of collagens. Although we did not detect any extracellular Aß plaques, immunostaining revealed that both intracellular Aß1-42 and phospho-Tau levels were increased in various PM exposure conditions compared to the clean air control. NanoString GeoMx analysis demonstrated a remarkable activation of immune responses in the PM exposed mouse brain. Surprisingly, our data also indicated a strong activation of various tumor suppressors including RB1, CDKN1A/p21 and CDKN2A/p16. Collectively, our data demonstrated that exposure to airborne PM caused a profound transcriptional dysregulation and accelerated Alzheimer's-related pathology.

ß-Amyloid targeting nanodrug for neuron-specific delivery of nucleic acids in Alzheimer's disease mouse models.

Delivery of therapeutic substances into the brain poses a significant challenge in the treatment of neurological disorders. This is primarily due to the blood-brain barrier (BBB), which restricts access, alongside the limited stability and distribution of these agents within the brain tissue. Here we demonstrate an efficient delivery of microRNA (miRNA) and antisense RNA preferentially to neurons compared to astroglia in the brain of healthy and Alzheimer's disease mice, via disulfide-linked conjugation with poly(ß-L-malic acid-trileucine)-copolymer a biodegradable, amphiphilic, and multivalent platform. By conjugating a D-configured (D3)-peptide (vector) for specific targeting, highly efficient delivery across the BBB is achieved through the Low-Density Lipoprotein Receptor-Related Protein-1 (LRP-1) transcytosis pathway, amyloid beta (Aß) peptides. Nanodrug distribution was determined by fluorescent labeling and analyzed by microscopy in neurons, astroglia, and in extracellular amyloid plaques typical for Alzheimer's disease. Whereas D-configured BBB-vectors can efficiently target neurons, L-configured (e.g., AP2-peptide) guided vector can only cross BBB but not seem to bind neurons. An analysis of post-injection fluorescence distribution, and RNA-seq followed by real-time PCR validation, confirmed a successful in vivo delivery of morpholino-miRNA-186 nanoconjugates into mouse brain. The size and fluorescence intensity of the intracellular nanodrug particulates were analyzed and verified by a competition with non-fluorescent conjugates. Differentially expressed genes (DEGs) from RNA-seq were identified in the nanodrug injected mice, and the changes of selected DEGs related to Alzheimer's disease were further validated by western blot and real-time PCR. Collectively, these results demonstrated that D3-peptide-conjugated nanopolymer drug is able to achieve neuron-selective delivery of miRNA and can serve as an efficient brain delivery vehicle in Alzheimer's disease (AD) mouse models.

Signature Effects of Vector-Guided Systemic Nano Bioconjugate Delivery Across Blood-Brain Barrier of Normal, Alzheimer's, and Tumor Mouse Models.

The ability to cross the blood-brain barrier (BBB) is critical for targeted therapy of the central nerve system (CNS). Six peptide vectors were covalently attached to a 50 kDa poly(ß-l-malic acid)-trileucine polymer forming P/LLL(40%)/vector conjugates. The vectors were Angiopep-2 (AP2), B6, Miniap-4 (M4), and d-configurated peptides D1, D3, and ACI-89, with specificity for transcytosis receptors low-density lipoprotein receptor-related protein-1 (LRP-1), transferrin receptor (TfR), bee venom-derived ion channel, and Aß/LRP-1 related transcytosis complex, respectively. The BBB-permeation efficacies were substantially increased ("boosted") in vector conjugates of P/LLL(40%). We have found that the copolymer group binds at the endothelial membrane and, by an allosterically membrane rearrangement, exposes the sites for vector-receptor complex formation. The specificity of vectors is indicated by competition experiments with nonconjugated vectors. P/LLL(40%) does not function as an inhibitor, suggesting that the copolymer binding site is eliminated after binding of the vector-nanoconjugate. The two-step mechanism, binding to endothelial membrane and allosteric exposure of transcytosis receptors, is supposed to be an integral feature of nanoconjugate-transcytosis pathways. In vivo brain delivery signatures of the nanoconjugates were recapitulated in mouse brains of normal, tumor (glioblastoma), and Alzheimer's disease (AD) models. BBB permeation of the tumor was most efficient, followed by normal and then AD-like brain. In tumor-bearing and normal brains, AP2 was the top performing vector; however, in AD models, D3 and D1 peptides were superior ones. The TfR vector B6 was equally efficient in normal and AD-model brains. Cross-permeation efficacies are manifested through modulated vector coligation and dosage escalation such as supra-linear dose dependence and crossover transcytosis activities.

Author Correction: Blood-brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20129-9.

Single- and Multi-Arm Gadolinium MRI Contrast Agents for Targeted Imaging of Glioblastoma.

BACKGROUND: Position of gadolinium atom(s) plays a key role in contrast enhancement of gadolinium-based contrast agents. To gain a better understanding of effects of distance of gadolinium in relation to the nanoconjugate platform, we designed and synthesized single- and multi-arm ("star") gadolinium conjugates equipped with antibody and peptides for targeting. The contrast agents were studied for their tumor imaging performance in a glioma mouse model. MATERIALS AND METHODS: Antibody- and peptide-targeted nano contrast agents (NCAs) were synthesized using polymalic acid platforms of different sizes. Gadolinium-DOTA and intermediates were attached as amides and targeting agents such as antibodies and peptides as thioethers. For in vivo experiments, we used human U87MG xenografts as glioma models. Magnetic resonance imaging (MRI) was performed on a Bruker BioSpec 94/20USR 9.4 T small-animal scanner. Delivery of contrast agents across the blood-brain barrier was studied by fluorescent microscopy. RESULTS: All contrast agents accumulated into tumor and showed composition-dependent imaging performance. Peptide-targeted mini-NCAs had hydrodynamic diameters in the range 5.2-9.4 nm and antibody-targeted NCAs had diameters in the range 15.8-20.5 nm. Zeta potentials were in the range of -5.4--8.2 mV and -4.6--8.8 mV, respectively. NCAs showed superior relaxivities compared to MultiHance at 9.4 T. The signal enhancement indicated maximum accumulation in tumor 30-60 minutes after intravenous injection of the mouse tail vein. Only targeted NCAs were retained in tumor for up to 3 hours and displayed contrast enhancement. CONCLUSION: The novel targeted NCAs with star-PEG features displayed improved relaxivity and greater contrast compared with commercial MultiHance contrast agent. The enhancement by mini-NCAs showed clearance of tumor contrast after 3 hours providing a suitable time window for tumor diagnosis in clinics. The technology provides a great tool with the promise of differential MRI diagnosis of brain tumors.

Author Correction: Blood-brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cranial and ventricular size following shunting or endoscopic third ventriculostomy (ETV) in infants with aqueductal stenosis: further insights from the International Infant Hydrocephalus Study (IIHS).

PURPOSE: The craniometrics of head circumference (HC) and ventricular size are part of the clinical assessment of infants with hydrocephalus and are often utilized in conjunction with other clinical and radiological parameters to determine the success of treatment. We aimed to assess the effect of endoscopic third ventriculostomy (ETV) and shunting on craniometric measurements during the follow-up of a cohort of infants with symptomatic triventricular hydrocephalus secondary to aqueductal stenosis. METHODS: We performed a post hoc analysis of data from the International Infant Hydrocephalus Study (IIHS)-a prospective, multicenter study of infants (< 24 months old) with hydrocephalus from aqueductal stenosis who were treated with either an ETV or shunt. During various stages of a 5-year follow-up period, the following craniometrics were measured: HC, HC centile, HC z-score, and frontal-occipital horn ratio (FOR). Data were compared in an analysis of covariance, adjusting for baseline variables including age at surgery and sex. RESULTS: Of 158 enrolled patients, 115 underwent an ETV, while 43 received a shunt. Both procedures led to improvements in the mean HC centile position and z-score, a trend which continued until the 5-year assessment point. A similar trend was noted for FOR which was measured at 12 months and 3 years following initial treatment. Although the values were consistently higher for ETV compared with shunt, the differences in HC value, centile, and z-score were not significant. ETV was associated with a significantly higher FOR compared with shunting at 12 months (0.52 vs 0.44; p = 0.002) and 3 years (0.46 vs 0.38; p = 0.03) of follow-up. CONCLUSION: ETV and shunting led to improvements in HC centile, z-score, and FOR measurements during long-term follow-up of infants with hydrocephalus secondary to aqueductal stenosis. Head size did not significantly differ between the treatment groups during follow-up, however ventricle size was greater in those undergoing ETV when measured at 1 and 3 years following treatment.

Magnetic iron oxide nanoparticles for imaging, targeting and treatment of primary and metastatic tumors of the brain.

Magnetic nanoparticles in general, and iron oxide nanoparticles in particular, have been studied extensively during the past 20 years for numerous biomedical applications. The main applications of these nanoparticles are in magnetic resonance imaging (MRI), magnetic targeting, gene and drug delivery, magnetic hyperthermia for tumor treatment, and manipulation of the immune system by macrophage polarization for cancer treatment. Recently, considerable attention has been paid to magnetic particle imaging (MPI) because of its better sensitivity compared to MRI. In recent years, MRI and MPI have been combined as a dual or multimodal imaging method to enhance the signal in the brain for the early detection and treatment of brain pathologies. Because magnetic and iron oxide nanoparticles are so diverse and can be used in multiple applications such as imaging or therapy, they have attractive features for brain delivery. However, the greatest limitations for the use of MRI/MPI for imaging and treatment are in brain delivery, with one of these limitations being the brain-blood barrier (BBB). This review addresses the current status, chemical compositions, advantages and disadvantages, toxicity and most importantly the future directions for the delivery of iron oxide based substances across the blood-brain barrier for targeting, imaging and therapy of primary and metastatic tumors of the brain.

Blood-brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy.

Brain glioma treatment with checkpoint inhibitor antibodies to cytotoxic T-lymphocyte-associated antigen 4 (a-CTLA-4) and programmed cell death-1 (a-PD-1) was largely unsuccessful due to their inability to cross blood-brain barrier (BBB). Here we describe targeted nanoscale immunoconjugates (NICs) on natural biopolymer scaffold, poly(ß-L-malic acid), with covalently attached a-CTLA-4 or a-PD-1 for systemic delivery across the BBB and activation of local brain anti-tumor immune response. NIC treatment of mice bearing intracranial GL261 glioblastoma (GBM) results in an increase of CD8+ T cells, NK cells and macrophages with a decrease of regulatory T cells (Tregs) in the brain tumor area. Survival of GBM-bearing mice treated with NIC combination is significantly longer compared to animals treated with single checkpoint inhibitor-bearing NICs or free a-CTLA-4 and a-PD-1. Our study demonstrates trans-BBB delivery of tumor-targeted polymer-conjugated checkpoint inhibitors as an effective GBM treatment via activation of both systemic and local privileged brain tumor immune response.

Polymalic acid chlorotoxin nanoconjugate for near-infrared fluorescence guided resection of glioblastoma multiforme.

Maximal surgical resection of glioma remains the single most effective treatment. Tools to guide the resection while avoiding removal of normal brain tissues can aid surgeons in achieving optimal results. One strategy to achieve this goal is to rely upon interoperative fluorescence staining of tumor cells in vivo, that can be visualized by the surgeon during resection. Towards this goal we have designed a biodegradable fluorescent mini nano imaging agent (NIA) with high specificity for U87MG glioma cells and previously unmet high light emission. The NIA is the conjugate of polymalic acid (PMLA) with chlorotoxin for tumor targeting, indocyanine green (ICG) for NIR fluorescence and the tri-leucin peptide as fluorescence enhancer. PMLA as a multivalent platform carries several molecules of ICG and the other ligands. The NIA recognizes multiple sites on glioma cell surface, demonstrated by the effects of single and combined competitors. Systemic IV injection into xenogeneic mouse model carrying human U87MG glioblastoma indicated vivid tumor cell binding and internalization of NIA resulting in intensive and long-lasting tumor fluorescence. The NIA is shown to greatly improve tumor removal supporting its utility in clinical applications.

Blockade of a Laminin-411-Notch Axis with CRISPR/Cas9 or a Nanobioconjugate Inhibits Glioblastoma Growth through Tumor-Microenvironment Cross-talk.

There is an unmet need for the treatment of glioblastoma multiforme (GBM). The extracellular matrix, including laminins, in the tumor microenvironment is important for tumor invasion and progression. In a panel of 226 patient brain glioma samples, we found a clinical correlation between the expression of tumor vascular laminin-411 (α4ß1γ1) with higher tumor grade and with expression of cancer stem cell (CSC) markers, including Notch pathway members, CD133, Nestin, and c-Myc. Laminin-411 overexpression also correlated with higher recurrence rate and shorter survival of GBM patients. We also showed that depletion of laminin-411 α4 and ß1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of resultant intracranial tumors in mice and significantly increased survival of host animals compared with mice with untreated cells. Inhibition of laminin-411 suppressed Notch pathway in normal and malignant human brain cell types. A nanobioconjugate potentially suitable for clinical use and capable of crossing blood-brain barrier was designed to block laminin-411 expression. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, including the Notch axis. This study describes an efficient strategy for GBM treatment via targeting a critical component of the tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma.Significance: Laminin-411 expression in the glioma microenvironment correlates with Notch and other cancer stem cell markers and can be targeted by a novel, clinically translatable nanobioconjugate to inhibit glioma growth.

A Combination of Tri-Leucine and Angiopep-2 Drives a Polyanionic Polymalic Acid Nanodrug Platform Across the Blood-Brain Barrier.

One of the major problems facing the treatment of neurological disorders is the poor delivery of therapeutic agents into the brain. Our goal is to develop a multifunctional and biodegradable nanodrug delivery system that crosses the blood-brain barrier (BBB) to access brain tissues affected by neurological disease. In this study, we synthesized a biodegradable nontoxic ß-poly(l-malic acid) (PMLA or P) as a scaffold to chemically bind the BBB crossing peptides Angiopep-2 (AP2), MiniAp-4 (M4), and the transferrin receptor ligands cTfRL and B6. In addition, a trileucine endosome escape unit (LLL) and a fluorescent marker (rhodamine or rh) were attached to the PMLA backbone. The pharmacokinetics, BBB penetration, and biodistribution of nanoconjugates were studied in different brain regions and at multiple time points via optical imaging. The optimal nanoconjugate, P/LLL/AP2/rh, produced significant fluorescence in the parenchyma of cortical layers II/III, the midbrain colliculi, and the hippocampal CA1-3 cellular layers 30 min after a single intravenous injection; clearance was observed after 4 h. The nanoconjugate variant P/LLL/rh lacking AP2, or the variant P/AP2/rh lacking LLL, showed significantly less BBB penetration. The LLL moiety appeared to stabilize the nanoconjugate, while AP2 enhanced BBB penetration. Finally, nanoconjugates containing the peptides M4, cTfRL, and B6 displayed comparably little and/or inconsistent infiltration of brain parenchyma, likely due to reduced trans-BBB movement. P/LLL/AP2/rh can now be functionalized with intra-brain targeting and drug treatment moieties that are aimed at molecular pathways implicated in neurological disorders.

Coarse particulate matter (PM2.5-10) in Los Angeles Basin air induces expression of inflammation and cancer biomarkers in rat brains.

Air pollution is linked to brain inflammation, which accelerates tumorigenesis and neurodegeneration. The molecular mechanisms that connect air pollution with brain pathology are largely unknown but seem to depend on the chemical composition of airborne particulate matter (PM). We sourced ambient PM from Riverside, California, and selectively exposed rats to coarse (PM2.5-10: 2.5-10 µm), fine (PM<2.5: <2.5 µm), or ultrafine particles (UFPM: <0.15 µm). We characterized each PM type via atomic emission spectroscopy and detected nickel, cobalt and zinc within them. We then exposed rats separately to each PM type for short (2 weeks), intermediate (1-3 months) and long durations (1 year). All three metals accumulated in rat brains during intermediate-length PM exposures. Via RNAseq analysis we then determined that intermediate-length PM2.5-10 exposures triggered the expression of the early growth response gene 2 (EGR2), genes encoding inflammatory cytokine pathways (IL13-Rα1 and IL-16) and the oncogene RAC1. Gene upregulation occurred only in brains of rats exposed to PM2.5-10 and correlated with cerebral nickel accumulation. We hypothesize that the expression of inflammation and oncogenesis-related genes is triggered by the combinatorial exposure to certain metals and toxins in Los Angeles Basin PM2.5-10.

Polymalic Acid Tritryptophan Copolymer Interacts with Lipid Membrane Resulting in Membrane Solubilization.

Anionic polymers with membrane permeation functionalities are highly desirable for secure cytoplasmic drug delivery. We have developed tritryptophan containing copolymer (P/WWW) of polymalic acid (PMLA) that permeates membranes by a mechanism different from previously described PMLA copolymers of trileucine (P/LLL) and leucine ethyl ester (P/LOEt) that use the "barrel stave" and "carpet" mechanism, respectively. The novel mechanism leads to solubilization of membranes by forming copolymer "belts" around planar membrane "packages." The formation of such packages is supported by results obtained from studies including size-exclusion chromatography, confocal microscopy, and fluorescence energy transfer. According to this "belt" mechanism, it is hypothesized that P/WWW first attaches to the membrane surface. Subsequently the hydrophobic tryptophan side chains translocate into the periphery and insert into the lipid bilayer thereby cutting the membrane into packages. The reaction is driven by the high affinity between the tryptophan residues and lipid side chains resulting in a stable configuration. The formation of the membrane packages requires physical agitation suggesting that the success of the translocation depends on the fluidity of the membrane. It is emphasized that the "belt" mechanism could specifically function in the recognition of abnormal cells with high membrane fluidity and in response to hyperthermia.

HER2-positive breast cancer targeting and treatment by a peptide-conjugated mini nanodrug.

HER2+ breast cancer is one of the most aggressive forms of breast cancer. The new polymalic acid-based mini nanodrug copolymers are synthesized and specifically characterized to inhibit growth of HER2+ breast cancer. These mini nanodrugs are highly effective and in the clinic may substitute for trastuzumab (the marketed therapeutic antibody) and antibody-targeted nanobioconjugates. Novel mini nanodrugs are designed to have slender shape and small size. HER2+ cells were recognized by the polymer-attached trastuzumab-mimetic 12-mer peptide. Synthesis of the nascent cell-transmembrane HER2/neu receptors by HER2+ cells was inhibited by antisense oligonucleotides that prevented cancer cell proliferation and significantly reduced tumor size by more than 15 times vs. untreated control or PBS-treated group. We emphasize that the shape and size of mini nanodrugs can enhance penetration of multiple bio-barriers to facilitate highly effective treatment. Replacement of trastuzumab by the mimetic peptide favors reduced production costs and technical efforts, and a negligible immune response.

Simultaneous blockade of interacting CK2 and EGFR pathways by tumor-targeting nanobioconjugates increases therapeutic efficacy against glioblastoma multiforme.

Glioblastoma multiforme (GBM) remains the deadliest brain tumor in adults. GBM tumors are also notorious for drug and radiation resistance. To inhibit GBMs more effectively, polymalic acid-based blood-brain barrier crossing nanobioconjugates were synthesized that are delivered to the cytoplasm of cancer cells and specifically inhibit the master regulator serine/threonine protein kinase CK2 and the wild-type/mutated epidermal growth factor receptor (EGFR/EGFRvIII), which are overexpressed in gliomas according to The Cancer Genome Atlas (TCGA) GBM database. Two xenogeneic mouse models bearing intracranial human GBMs from cell lines LN229 and U87MG that expressed both CK2 and EGFR at different levels were used. Simultaneous knockdown of CK2α and EGFR/EGFRvIII suppressed their downstream prosurvival signaling. Treatment also markedly reduced the expression of programmed death-ligand 1 (PD-L1), a negative regulator of cytotoxic lymphocytes. Downregulation of CK2 and EGFR also caused deactivation of heat shock protein 90 (Hsp90) co-chaperone Cdc37, which may suppress the activity of key cellular kinases. Inhibition of either target was associated with downregulation of the other target as well, which may underlie the increased efficacy of the dual nanobioconjugate that is directed against both CK2 and EGFR. Importantly, the single nanodrugs, and especially the dual nanodrug, markedly suppressed the expression of the cancer stem cell markers c-Myc, CD133, and nestin, which could contribute to the efficacy of the treatments. In both tumor models, the nanobioconjugates significantly increased (up to 2-fold) animal survival compared with the PBS-treated control group. The versatile nanobioconjugates developed in this study, with the abilities of anti-cancer drug delivery across biobarriers and the inhibition of key tumor regulators, offer a promising nanotherapeutic approach to treat GBMs, and to potentially prevent drug resistance and retard the recurrence of brain tumors.