Oral delivery of nanomedicine for genetic kidney disease.

Chronic and genetic kidney diseases such as autosomal dominant polycystic kidney disease (ADPKD) have few therapeutic options, and clinical trials testing small molecule drugs have been unfavorable due to low kidney bioavailability and adverse side effects. Although nanoparticles can be designed to deliver drugs directly to the diseased site, there are no kidney-targeted nanomedicines clinically available, and most FDA-approved nanoparticles are administered intravenously which is not ideal for chronic diseases. To meet these challenges of chronic diseases, we developed a biomaterials-based strategy using chitosan particles (CP) for oral delivery of therapeutic, kidney-targeting peptide amphiphile micelles (KMs). We hypothesized that encapsuling KMs into CP would enhance the bioavailability of KMs upon oral administration given the high stability of chitosan in acidic conditions and mucoadhesive properties enabling absorption within the intestines. To test this, we evaluated the mechanism of KM access to the kidneys via intravital imaging and investigated the KM biodistribution in a porcine model. Next, we loaded KMs carrying the ADPKD drug metformin into CP (KM-CP-met) and measured in vitro therapeutic effect. Upon oral administration in vivo, KM-CP-met showed significantly greater bioavailability and accumulation in the kidneys as compared to KM only or free drug. As such, KM-CP-met treatment in ADPKD mice (Pkd1fl/fl;Pax8-rtTA;Tet-O-Cre which develops the disease over 120 days and mimics the slow development of ADPKD) showed enhanced therapeutic efficacy without affecting safety despite repeated treatment. Herein, we demonstrate the potential of KM-CP as a nanomedicine strategy for oral delivery for the long-term treatment of chronic kidney diseases.

Single cell transcriptional changes across the blood stages of artemisinin resistant K13C580Y mutant Plasmodium falciparum upon dihydroartemisinin exposure.

Artemisinins have been a cornerstone of malaria control, but resistance in Plasmodium falciparum, due to mutations in the Kelch 13 gene, threaten these advances. Artemisinin exposure results in a dynamic transcriptional response across multiple pathways, but most work has focused on ring stages and ex vivo transcriptional analysis, limiting evaluation of all life cycle stages. We applied single cell RNAseq to two unsynchronized isogenic parasite lines (K13C580 and K13580Y) over 6 hrs after a pulse exposure to dihydroartemisinin (DHA). Transcription was altered across all stages, with the greatest occurring at the early trophozoite and mid ring stage in both lines. This response involved the arrest of metabolic processes and the enhancement of protein trafficking and the unfolded protein response. While similar, the response was enhanced in the K13580Y mutant, which may lead to the dormancy phenomenon upon treatment. Increased surface protein expression was seen in mutant parasites at baseline and upon drug exposure, highlighted by the increased expression of PfEMP1 and GARP, a potential therapeutic target. Antibody targeting GARP maintained anti-parasitic efficacy in mutant parasites. This work provides single cell insight of gene transcription across all life cycle stages revealing transcriptional changes that could initiate dormancy state and mediate survival.

Long-term, in vivo therapeutic effects of a single dose of miR-145 micelles for atherosclerosis.

Atherosclerosis is a chronic inflammatory disease that is characterized by the build-up of lipid-rich plaques in the arterial walls. The standard treatment for patients with atherosclerosis is statin therapy aimed to lower serum lipid levels. Despite its widespread use, many patients taking statins continue to experience acute events. Thus, to develop improved and alternative therapies, we previously reported on microRNA-145 (miR-145 micelles) and its ability to inhibit atherosclerosis by targeting vascular smooth muscle cells (VSMCs). Importantly, one dose of miR-145 micelles significantly abrogated disease progression when evaluated two weeks post-administration. Thus, in this study, to evaluate how long the sustained effects of miR-145 micelles can be maintained and towards identifying a dosing regimen that is practical for patients with chronic disease, the therapeutic effects of a single dose of miR-145 micelles were evaluated for up to two months in vivo. After one and two months post-treatment, miR-145 micelles were found to reduce plaque size and overall lesion area compared to all other controls including statins without causing adverse effects. Furthermore, a single dose of miR-145 micelle treatment inhibited VSMC transdifferentiation into pathogenic macrophage-like and osteogenic cells in plaques. Together, our data shows the long-term efficacy and sustained effects of miR-145 micelles that is amenable using a dosing frequency relevant to chronic disease patients.

Expression Microdissection for the Analysis of miRNA in a Single-Cell Type.

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries.

Therapeutic Response of miR-145 Micelles on Patient-Derived Vascular Smooth Muscle Cells.

During atherosclerosis, vascular smooth muscle cells (VSMCs) undergo a phenotypic transition from a healthy contractile state into pathological phenotypes including a proliferative and migratory, synthetic phenotype and osteochondrogenic-like phenotype that exacerbate plaques. Thus, inhibiting the transition of healthy, quiescent VSMCs to atherogenic cell types has the potential to mitigate atherosclerosis. To that end, previously, we reported that delivery of microRNA-145 (miR-145, a potent gatekeeper of the contractile VSMC phenotype) using nanoparticle micelles limited atherosclerotic plaque growth in murine models of atherosclerosis. Building on this preclinical data and toward clinical application, in this study, we tested the therapeutic viability of miR-145 micelles on patient-derived VSMCs and evaluated their effects based on disease severity. We collected vascular tissues from 11 patients with healthy, moderate, or severe stages of atherosclerosis that were discarded following vascular surgery or organ transplant, and isolated VSMCs from these tissues. We found that with increasing disease severity, patient-derived VSMCs had decreasing levels of contractile markers (miR-145, ACTA2, MYH11) and increasing levels of synthetic markers (KLF4, KLF5, and ELK1). Treatment with miR-145 micelles showed that an increase in disease severity correlated with a more robust response to therapy in VSMCs. Notably, miR-145 micelle therapy rescued contractile marker expression to baseline contractile levels in VSMCs derived from the most severely diseased tissues. As such, we demonstrate the use of miR-145 micelles across different stages of atherosclerosis disease and present further evidence of the translatability of miR-145 micelle treatment for atherosclerosis.

Exosomes in Atherosclerosis, a Double-Edged Sword: Their Role in Disease Pathogenesis and Their Potential as Novel Therapeutics.

Cardiovascular disease (CAD) due to atherosclerosis is a major cause of death worldwide. The development of atherosclerosis involves intercellular communication facilitated by exosomes secreted from vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs), immune cells, and platelets. In this review, we summarize the current understanding of exosome biogenesis and uptake, and discuss atherogenic and atheroprotective functions of exosomes secreted from these cell types. In addition, we examine the potential of enhancing the therapeutic and targeting ability of exosomes exhibiting atheroprotective function by drug loading and surface modification with targeting ligands. We conclude with current challenges associated with exosome engineering for therapeutic use.

miR-145 micelles mitigate atherosclerosis by modulating vascular smooth muscle cell phenotype.

In atherosclerosis, resident vascular smooth muscle cells (VSMCs) in the blood vessels become highly plastic and undergo phenotypic switching from the quiescent, contractile phenotype to the migratory and proliferative, synthetic phenotype. Additionally, recent VSMC lineage-tracing mouse models of atherosclerosis have found that VSMCs transdifferentiate into macrophage-like and osteochondrogenic cells and make up to 70% of cells found in atherosclerotic plaques. Given VSMC phenotypic switching is regulated by microRNA-145 (miR-145), we hypothesized that nanoparticle-mediated delivery of miR-145 to VSMCs has the potential to mitigate atherosclerosis development by inhibiting plaque-propagating cell types derived from VSMCs. To test our hypothesis, we synthesized miR-145 micelles targeting the C-C chemokine receptor-2 (CCR2), which is highly expressed on synthetic VSMCs. When miR-145 micelles were incubated with human aortic VSMCs in vitro, >90% miR-145 micelles escaped the lysosomal pathway in 4 hours and released the miR cargo under cytosolic levels of glutathione, an endogenous reducing agent. As such, miR-145 micelles rescued atheroprotective contractile markers, myocardin, α-SMA, and calponin, in synthetic VSMCs in vitro. In early-stage atherosclerotic ApoE-/- mice, one dose of miR-145 micelles prevented lesion growth by 49% and sustained an increased level of miR-145 expression after 2 weeks post-treatment. Additionally, miR-145 micelles inhibited 35% and 43% plaque growth compared to free miR-145 and PBS, respectively, in mid-stage atherosclerotic ApoE-/- mice. Collectively, we present a novel therapeutic strategy and cell target for atherosclerosis, and present miR-145 micelles as a viable nanotherapeutic that can intervene atherosclerosis progression at both early and later stages of disease.

Oral delivery of metformin by chitosan nanoparticles for polycystic kidney disease.

Nanoparticle drug delivery has many advantages over small molecule therapeutics, including reducing off-target side effects and increasing drug potency. However, many nanoparticles are administered parenterally, which is challenging for chronic diseases such as polycystic kidney disease (PKD), the most common hereditary disease worldwide in which patients need continuous treatment over decades. To address this clinical need, we present the development of nanoparticles synthesized from chitosan, a widely available polymer chosen for its ability to improve oral bioavailability. Specifically, we optimized the synthesis parameters of chitosan nanoparticles and demonstrate mucoadhesion and permeation across an intestinal barrier model in vitro. Furthermore, when administered orally to mice, ex vivo imaging of rhodamine-loaded chitosan nanoparticles showed significantly higher accumulation in the intestines compared to the free model drug, as well as 1.3 times higher serum area under the curve (AUC), demonstrating controlled release and improved serum delivery over 24 h. To test its utility for chronic diseases such as PKD, we loaded the candidate PKD drug, metformin, into chitosan nanoparticles, and upon oral administration to a PKD murine model (Pkd1fl/fl;Pax8-rtTA;Tet-O cre), a lower cyst burden was observed compared to free metformin, and was well tolerated upon repeated dosages. Blood urea nitrogen (BUN) and creatinine levels were similar to untreated mice, demonstrating kidney and biocompatibility health. Our study builds upon previous chitosan-based drug delivery approaches, and demonstrates a novel, oral nanoformulation for PKD.

A microfluidic cell-migration assay for the prediction of progression-free survival and recurrence time of patients with glioblastoma.

Clinical scores, molecular markers and cellular phenotypes have been used to predict the clinical outcomes of patients with glioblastoma. However, their clinical use has been hampered by confounders such as patient co-morbidities, by the tumoral heterogeneity of molecular and cellular markers, and by the complexity and cost of high-throughput single-cell analysis. Here, we show that a microfluidic assay for the quantification of cell migration and proliferation can categorize patients with glioblastoma according to progression-free survival. We quantified with a composite score the ability of primary glioblastoma cells to proliferate (via the protein biomarker Ki-67) and to squeeze through microfluidic channels, mimicking aspects of the tight perivascular conduits and white-matter tracts in brain parenchyma. The assay retrospectively categorized 28 patients according to progression-free survival (short-term or long-term) with an accuracy of 86%, predicted time to recurrence and correctly categorized five additional patients on the basis of survival prospectively. RNA sequencing of the highly motile cells revealed differentially expressed genes that correlated with poor prognosis. Our findings suggest that cell-migration and proliferation levels can predict patient-specific clinical outcomes.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. METHODS: Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. RESULTS: Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). CONCLUSIONS: We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Collagenase-Cleavable Peptide Amphiphile Micelles as a Novel Theranostic Strategy in Atherosclerosis.

Atherosclerosis is an inflammatory disease characterized by plaques that can cause sudden myocardial infarction upon rupture. Such rupture-prone plaques have thin fibrous caps due to collagenase degradation, and a noninvasive diagnostic tool and targeted therapy that can identify and treat vulnerable plaques and may inhibit the onset of acute cardiac events. Toward this goal, monocyte-binding, collagenase-inhibiting, and gadolinium-modified peptide amphiphile micelles (MCG PAMs) are developed. Monocyte chemoattractant protein-1 (MCP-1) binds to C-C chemokine receptor-2 expressed on pathological cell types present within plaques. Through the peptide binding motif of MCP-1, MCG PAMs bind to monocytes and vascular smooth muscle cells in vitro. Moreover, using magnetic resonance imaging, MCG PAMs show enhanced targeting and successful detection of plaques in diseased mice in vivo and act as contrast agents for molecular imaging. Through the collagenase-cleaving peptide sequence of collagen [VPMS-MRGG], MCG PAMs can compete for collagenases that degrade the fibrous cap of plaques, providing therapy. MCG PAM-treated mice show increased fibrous cap thickness by 61% and 113% histologically compared to nontargeting micelle- or PBS-treated mice (p = 0.0075 and 0.001, respectively). Overall, this novel multimodal nanoparticle offers new theranostic opportunities for noninvasive diagnosis and treatment of atherosclerotic plaques.

Hydroxyapatite-binding micelles for the detection of vascular calcification in atherosclerosis.

Atherosclerosis is a chronic disease characterized by the formation of calcified, arterial plaques. Microcalcifications (5 µm to 100 µm), mainly composed of hydroxyapatite (HA, Ca5(PO4)3(OH)), develop in the fibrous caps of atherosclerotic plaques and can trigger plaque rupture due to the loss of compliance and elasticity. Ultimately, plaque rupture can cause arterial occlusion and embolization and result in ischemic events such as strokes and myocardial infarctions. Unfortunately, current imaging technologies used to detect calcifications are limited by low signal-to-noise ratio or use invasive procedures that pose risk of arterial dissection. To mitigate these drawbacks, in our study, we developed a novel, fluorescently-labeled peptide amphiphile micelle (PAM) that uses a 12 amino acid HA-binding peptide (HABP) [SVSVGMKPSPRP] to target and detect atherosclerotic calcification (HA PAM). Our results show HA PAMs can successfully target HA microcrystals with a strong binding affinity (KD = 6.26 ± 1.2 µM) in vitro. In addition, HA PAMs detected HA mineralization (HA PAM vs. non-targeting micelle, p≤ 0.001; HA PAM vs. scrambled HABP PAM, p≤ 0.01) formed by calcifying mouse aortic vascular smooth muscle cells (MOVAS). Moreover, HA PAMs successfully detected calcifications in atherosclerotic mouse models as well as in patient-derived arteries. Our studies show that HA PAMs show promise as calcium-targeting nanoparticles for the detection of calcifications in atherosclerosis.

Calcium-binding nanoparticles for vascular disease.

Cardiovascular disease (CVD) including atherosclerosis is the leading cause of death worldwide. As CVDs and atherosclerosis develop, plaques begin to form in the blood vessels and become calcified. Calcification within the vasculature and atherosclerotic plaques have been correlated with rupture and consequently, acute myocardial infarction. However, current imaging methods to identify vascular calcification have limitations in determining plaque composition and structure. Nanoparticles can overcome these limitations due to their versatility and ability to incorporate a wide range of targeting and contrast agents. In this review, we summarize the current understanding of calcification in atherosclerosis, their role in instigating plaque instability, and clinical methodologies to detect and analyze vascular calcification. In addition, we highlight the potential of calcium-targeting ligands and nanoparticles to create novel calcium-detecting tools.