Protocol to engineer nanofilms embedded lipid nanoparticles for controlled and targeted drug delivery (NECTAR).

We present a protocol to engineer a substrate-mediated delivery platform comprising hyaluronic acid-coated lipid nanoparticles (HALNPs) embedded into polyelectrolyte multilayer (PEM) films. This platform allows controlled spatiotemporal release of lipid nanoparticles (LNP) by embedding them within the polyelectrolyte multilayer films matrix. HALNP conjugate with antibodies also adds the ability for targeted delivery. The use of LNP enables this platform to encapsulate both hydrophobic and hydrophilic drugs. This platform can easily be reproduced and utilized for various biomedical drug delivery applications. For complete details on the use and execution of this protocol, please refer to Hayward et al. (2015, 2016a, 2016b), Hayward and Kidambi (2018), and Kidambi and Hayward (2022).

Independent Blinded Validation of a Tissue Systems Pathology Test to Predict Progression in Patients With Barrett's Esophagus.

INTRODUCTION: A risk prediction test was previously validated to predict progression to high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus (BE). The aim of our study was to independently validate this test to predict the risk of progression to HGD/EAC in BE patients with nondysplastic (ND), indefinite for dysplasia and low-grade dysplasia (LGD). METHODS: A single-blinded, case-control study was conducted to stratify patients with BE as low, intermediate, or high risk for progression to HGD/EAC within 5 years using a previously described risk prediction test. Patients with BE who progressed to HGD/EAC after at least 1 year (n = 58) were matched to patients undergoing surveillance without progression (n = 210, median surveillance 7 years). Baseline biopsies with subspecialist diagnoses of ND, indefinite for dysplasia, or LGD were tested in a blinded manner, and the predictive performance of the test was assessed. RESULTS: This risk prediction test stratified patients with BE based on progression risk with the high-risk group at 4.7-fold increased risk for HGD/EAC compared with the low-risk group (95% confidence interval 2.5-8.8, P < 0.0001). Prevalence-adjusted positive predictive value at 5 years was 23%. The high-risk class and male sex provided predictive power that was independent of pathologic diagnosis, age, segment length, and hiatal hernia. Patients with ND BE who scored high risk progressed at a higher rate (26%) than patients with subspecialist-confirmed LGD (21.8%) at 5 years. DISCUSSION: A risk prediction test identifies patients with ND BE who are at high risk for progression to HGD/EAC and may benefit from early endoscopic therapy or increased surveillance.

Depletion of mitochondrial protease OMA1 alters proliferative properties and promotes metastatic growth of breast cancer cells.

Metastatic competence of cancer cells is influenced by many factors including metabolic alterations and changes in mitochondrial biogenesis and protein homeostasis. While it is generally accepted that mitochondria play important roles in tumorigenesis, the respective molecular events that regulate aberrant cancer cell proliferation remain to be clarified. Therefore, understanding the mechanisms underlying the role of mitochondria in cancer progression has potential implications in the development of new therapeutic strategies. We show that low expression of mitochondrial quality control protease OMA1 correlates with poor overall survival in breast cancer patients. Silencing OMA1 in vitro in patient-derived metastatic breast cancer cells isolated from the metastatic pleural effusion and atypical ductal hyperplasia mammary tumor specimens (21MT-1 and 21PT) enhances the formation of filopodia, increases cell proliferation (Ki67 expression), and induces epithelial-mesenchymal transition (EMT). Mechanistically, loss of OMA1 results in alterations in the mitochondrial protein homeostasis, as reflected by enhanced expression of canonic mitochondrial unfolded protein response genes. These changes significantly increase migratory properties in metastatic breast cancer cells, indicating that OMA1 plays a critical role in suppressing metastatic competence of breast tumors. Interestingly, these results were not observed in OMA1-depleted non-tumorigenic MCF10A mammary epithelial cells. This newly identified reduced activity/levels of OMA1 provides insights into the mechanisms leading to breast cancer development, promoting malignant progression of cancer cells and unfavorable clinical outcomes, which may represent possible prognostic markers and therapeutic targets for breast cancer treatment.

Ultraspecific and Amplification-Free Quantification of Mutant DNA by Single-Molecule Kinetic Fingerprinting.

Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by 3 orders of magnitude, with a dynamic range of up to 5 orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug-resistance-conferring cancer mutation EGFR T790M (a single C → T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.

CD44 Targeted Lipid Nanoparticles for MicroRNA Therapy.

MicroRNAs are small noncoding RNAs that function as powerful endogenous regulators of gene expression. Dysregulation of MicroRNA biogenesis has been correlated with the onset and progression of many human diseases. MicroRNA therapy involves the re-equilibration of aberrant intracellular MicroRNA expression profiles for long-term disease management. Despite the significant potential of MicroRNA therapy, the utilization of MicroRNA-based therapeutics has been drastically hindered in practice by the lack of a targeted and translatable delivery vehicle. CD44 is a cell surface glycoprotein that facilitates cellular communication and motility through cell-cell and cell-extracellular matrix interactions. CD44 has been shown to be elevated in multiple disease states including cancer making it a potential diagnostic biomarker and an ideal receptor for targeted drug delivery systems. We describe a method for targeting CD44 using a lipid nanocarrier for the cytoplasmic delivery of active MicroRNA.

Targeted Delivery of MicroRNA125a-5p by Engineered Lipid Nanoparticles for the Treatment of HER2 Positive Metastatic Breast Cancer.

MicroRNAs (miRNAs) are endogenous regulators of gene expression that play a pivotal role in biological processes spanning from global homeostasis to disease onset and progression. The ability to manipulate and induce cellular reequilibrium of deregulated miRNA expression profiles by inhibition of oncogenic miRNA or overexpression of tumor suppressor miRNA is a promising cancer strategy, but is currently hindered in application by the lack of nonviral delivery systems. Here we present a lipid nanoparticle (LNP) platform surface coated with Hyaluronic Acid (HA) for the delivery of mature tumor suppressor MicroRNA125a-5p to treat HER2 positive metastatic breast cancer. The delivery platform actively targets patient-derived metastatic breast cancer cells (21MT-1) isolated from the metastatic pleural effusion over normal breast tissue via an intrinsic HA-CD44 mediated endocytosis event, and has the ability to escape from the intracellular endolysosomal pathway for potent gene silencing. Knockdown of the HER2 proto-oncogene at the level of transcription and translation was achieved following HA-LNP mediated transfection with MicroRNA125a-5p. In addition, the PI3K/AKT and MAPK hyperactivated signaling pathways, cellular proliferation, and migration potential were also potently suppressed. Furthermore, the therapeutic efficacy of MicroRNA125a-5p by the HA-LNP platform was demonstrated to be significantly improved as compared to a commercial transfection reagent. This study highlights the therapeutic potential of MicroRNA125a-5p as a standalone treatment of HER2+ metastatic breast cancer via a translational nonviral delivery platform. These findings have major implications on future gene therapy regimens for breast cancer.

Hyaluronic acid-conjugated liposome nanoparticles for targeted delivery to CD44 overexpressing glioblastoma cells.

Glioblastoma Multiforme (GBM) is a highly prevalent and deadly brain malignancy characterized by poor prognosis and restricted disease management potential. Despite the success of nanocarrier systems to improve drug/gene therapy for cancer, active targeting specificity remains a major hurdle for GBM. Additionally, since the brain is a multi-cell type organ, there is a critical need to develop an approach to distinguish between GBM cells and healthy brain cells for safe and successful treatment. In this report, we have incorporated hyaluronic acid (HA) as an active targeting ligand for GBM. To do so, we employed HA conjugated liposomes (HALNPs) to study the uptake pathway in key cells in the brain including primary astrocytes, microglia, and human GBM cells. We observed that the HALNPs specifically target GBM cells over other brain cells due to higher expression of CD44 in tumor cells. Furthermore, CD44 driven HALNP uptake into GBM cells resulted in lysosomal evasion and increased efficacy of Doxorubicin, a model anti-neoplastic agent, while the astrocytes and microglia cells exhibited extensive HALNP-lysosome co-localization and decreased antineoplastic potency. In summary, novel CD44 targeted lipid based nanocarriers appear to be proficient in mediating site-specific delivery of drugs via CD44 receptors in GBM cells, with an improved therapeutic margin and safety.

Astrogliosis in a dish: substrate stiffness induces astrogliosis in primary rat astrocytes.

Astrogliosis due to brain injury or disease can lead to varying molecular and morphological changes in astrocytes. Magnetic resonance elastography and ultrasound have demonstrated that brain stiffness varies with age and disease state. However, there is a lack in understanding the role of varied stiffness on the progression of astrogliosis highlighting a critical need to engineer in vitro models that mimic disease stages. Such models need to incorporate the dynamic changes in the brain microenvironment including the stiffness changes. In this study we developed a polydimethyl siloxane (PDMS) based platform that modeled the physiologically relevant stiffness of brain in both a healthy (200 Pa) and diseased (8000 Pa) state to investigate the effect of stiffness on astrocyte function. We observed that astrocytes grown on soft substrates displayed a consistently more quiescent phenotype while those on stiff substrates displayed an astrogliosis-like morphology. In addition to morphological changes, astrocytes cultured on stiff substrates demonstrated significant increase in other astrogliosis hallmarks - cellular proliferation and glial fibrillary acidic protein (GFAP) protein expression. Furthermore, culturing astrocytes on a stiff surface resulted in increased reactive oxygen species (ROS) production, increased super oxide dismutase activity and decreased glutamate uptake. Our platform lends itself for study of potential therapeutic strategies for brain injury focusing on the intricate brain microenvironment-astrocytes signaling pathways.

Ionic Driven Embedment of Hyaluronic Acid Coated Liposomes in Polyelectrolyte Multilayer Films for Local Therapeutic Delivery.

The ability to control the spatial distribution and temporal release of a therapeutic remains a central challenge for biomedical research. Here, we report the development and optimization of a novel substrate mediated therapeutic delivery system comprising of hyaluronic acid covalently functionalized liposomes (HALNPs) embedded into polyelectrolyte multilayer (PEM) platform via ionic stabilization. The PEM platform was constructed from sequential deposition of Poly-L-Lysine (PLL) and Poly(Sodium styrene sulfonate) (SPS) "(PLL/SPS)4.5" followed by adsorption of anionic HALNPs. An adsorption affinity assay and saturation curve illustrated the preferential HALNP deposition density for precise therapeutic loading. (PLL/SPS)2.5 capping layer on top of the deposited HALNP monolayer further facilitated complete nanoparticle immobilization, cell adhesion, and provided nanoparticle confinement for controlled linear release profiles of the nanocarrier and encapsulated cargo. To our knowledge, this is the first study to demonstrate the successful embedment of a translatable lipid based nanocarrier into a substrate that allows for temporal and spatial release of both hydrophobic and hydrophilic drugs. Specifically, we have utilized our platform to deliver chemotherapeutic drug Doxorubicin from PEM confined HALNPs. Overall, we believe the development of our HALNP embedded PEM system is significant and will catalyze the usage of substrate mediated delivery platforms in biomedical applications.

Mitochondrial dysfunction and loss of glutamate uptake in primary astrocytes exposed to titanium dioxide nanoparticles.

Titanium dioxide (TiO2) nanoparticles are currently the second most produced engineered nanomaterial in the world with vast usage in consumer products leading to recurrent human exposure. Animal studies indicate significant nanoparticle accumulation in the brain while cellular toxicity studies demonstrate negative effects on neuronal cell viability and function. However, the toxicological effects of nanoparticles on astrocytes, the most abundant cells in the brain, have not been extensively investigated. Therefore, we determined the sub-toxic effect of three different TiO2 nanoparticles (rutile, anatase and commercially available P25 TiO2 nanoparticles) on primary rat cortical astrocytes. We evaluated some events related to astrocyte functions and mitochondrial dysregulation: (1) glutamate uptake; (2) redox signaling mechanisms by measuring ROS production; (3) the expression patterns of dynamin-related proteins (DRPs) and mitofusins 1 and 2, whose expression is central to mitochondrial dynamics; and (4) mitochondrial morphology by MitoTracker® Red CMXRos staining. Anatase, rutile and P25 were found to have LC50 values of 88.22 ± 10.56 ppm, 136.0 ± 31.73 ppm and 62.37 ± 9.06 ppm respectively indicating nanoparticle specific toxicity. All three TiO2 nanoparticles induced a significant loss in glutamate uptake indicative of a loss in vital astrocyte function. TiO2 nanoparticles also induced an increase in reactive oxygen species generation, and a decrease in mitochondrial membrane potential, suggesting mitochondrial damage. TiO2 nanoparticle exposure altered expression patterns of DRPs at low concentrations (25 ppm) and apoptotic fission at high concentrations (100 ppm). TiO2 nanoparticle exposure also resulted in changes to mitochondrial morphology confirmed by mitochondrial staining. Collectively, our data provide compelling evidence that TiO2 nanoparticle exposure has potential implications in astrocyte-mediated neurological dysfunction.

Titanium Dioxide Nanoparticles Trigger Loss of Function and Perturbation of Mitochondrial Dynamics in Primary Hepatocytes.

Titanium dioxide (TiO2) nanoparticles are one of the most highly manufactured and employed nanomaterials in the world with applications in copious industrial and consumer products. The liver is a major accumulation site for many nanoparticles, including TiO2, directly through intentional exposure or indirectly through unintentional ingestion via water, food or animals and increased environmental contamination. Growing concerns over the current usage of TiO2 coupled with the lack of mechanistic understanding of its potential health risk is the motivation for this study. Here we determined the toxic effect of three different TiO2 nanoparticles (commercially available rutile, anatase and P25) on primary rat hepatocytes. Specifically, we evaluated events related to hepatocyte functions and mitochondrial dynamics: (1) urea and albumin synthesis using colorimetric and ELISA assays, respectively; (2) redox signaling mechanisms by measuring reactive oxygen species (ROS) production, manganese superoxide dismutase (MnSOD) activity and mitochondrial membrane potential (MMP); (3) OPA1 and Mfn-1 expression that mediates the mitochondrial dynamics by PCR; and (4) mitochondrial morphology by MitoTracker Green FM staining. All three TiO2 nanoparticles induced a significant loss (p < 0.05) in hepatocyte functions even at concentrations as low as 50 ppm with commercially used P25 causing maximum damage. TiO2 nanoparticles induced a strong oxidative stress in primary hepatocytes. TiO2 nanoparticles exposure also resulted in morphological changes in mitochondria and substantial loss in the fusion process, thus impairing the mitochondrial dynamics. Although this study demonstrated that TiO2 nanoparticles exposure resulted in substantial damage to primary hepatocytes, more in vitro and in vivo studies are required to determine the complete toxicological mechanism in primary hepatocytes and subsequently liver function.