Evaluation of MUC1-Aptamer Functionalized Hybrid Nanoparticles for Targeted Delivery of miRNA-29b to Nonsmall Cell Lung Cancer.

The objective of this study was to evaluate the therapeutic efficacy and pharmacokinetic study of mucin1-aptamer functionalized miRNA-29b-loaded hybrid nanoparticles (MAFMILHNs) in lung tumor-bearing SCID mice. MAFMILHNs were manufactured using an isoelectric point based nanotechnology. They were then fully characterized for particle size, loading capacity, zeta potential, and encapsulation efficiency. The ability of MAFMILHNs to downregulate oncoprotein DNMT3B both at the cellular level and in vivo was monitored using Western blot, while the effect of the downregulation of DNMT3B on tumor growth was assessed using bioluminescence. Results indicate that the presence of MUC1-aptamer conjugated to the surface of the nanoparticles enhanced the selective delivery of miRNA-29b to tumor cells and tissues. Further, the downregulation of DNMT3B by MAFMILHNs resulted in the inhibition of tumor growth in mouse models.

Improvement in Therapeutic Efficacy and Reduction in Cellular Toxicity: Introduction of a Novel Anti-PSMA-Conjugated Hybrid Antiandrogen Nanoparticle.

Second generation antiandrogens have improved overall survival for men with metastatic castrate resistant prostate cancer; however, the antiandrogens result in suppression of androgen receptor (AR) activity in all tissues resulting in dose limiting toxicity. We sought to overcome this limitation through encapsulation in a prostate specific membrane antigen (PSMA)-conjugated nanoparticle. We designed and characterized a novel nanoparticle containing an antiandrogen, enzalutamide. Selectivity and enhanced efficacy was achieved through coating the particle with PSMA. The PSMA-conjugated nanoparticle was internalized selectively in AR expressing prostate cancer cells. It did not elicit an inflammatory effect. The efficacy of enzalutamide was not compromised through insertion into the nanoparticle; in fact, lower systemic drug concentrations of enzalutamide resulted in comparable clinical activity. Normal muscle cells were not impacted by the PSMA-conjugated containing antiandrogen. This approach represents a novel strategy to increase the specificity and effectiveness of antiandrogen treatment for men with castrate resistant prostate cancer. The ability to deliver higher drug concentrations in prostate cancer cells may translate into improved clinical end points including overall survival.

Novel targeted siRNA-loaded hybrid nanoparticles: preparation, characterization and in vitro evaluation.

BACKGROUND: siRNAs have a high potential for silencing critical molecular pathways that are pathogenic. Nevertheless, their clinical application has been limited by a lack of effective and safe nanotechnology-based delivery system that allows a controlled and safe transfection to cytosol of targeted cells without the associated adverse effects. Our group recently reported a very effective and safe hybrid nanoparticle delivery system composing human IgG and poloxamer-188 for siRNA delivery to cancer cells. However, these nanoparticles need to be optimized in terms of particle size, loading capacity and encapsulation efficiency. In the present study, we explored the effects of certain production parameters on particle size, loading capacity and encapsulation efficiency. Further, to make these nanoparticles more specific in their delivery of siRNA, we conjugated anti-NTSR1-mAb to the surface of these nanoparticles to target NTSR1-overexpressing cancer cells. The mechanism of siRNA release from these antiNTSR1-mAb functionalized nanoparticles was also elucidated. RESULTS: It was demonstrated that the concentration of human IgG in the starting nanoprecipitation medium and the rotation speed of the magnetic stirrer influenced the encapsulation efficiency, loading capacity and the size of the nanoparticles produced. We also successfully transformed these nanoparticles into actively targeted nanoparticles by functionalizing with anti-NTSR1-mAb to specifically target NTSR1-overexpressing cancer cells, hence able to avoid undesired accumulation in normal cells. The mechanism of siRNA release from these nanoparticles was elucidated to be by Fickian diffusion. Using flow cytometry and fluorescence microscopy, we were able to confirm the active involvement of NTSR1 in the uptake of these anti-NTSR1-mAb functionalized hybrid nanoparticles by lung adenocarcinoma cells. CONCLUSIONS: This hybrid nanoparticle delivery system can be used as a platform technology for intracellular delivery of siRNAs to NTSR1-overexpressing tumor cells.

Hepatic stellate cells and portal fibroblasts are the major cellular sources of collagens and lysyl oxidases in normal liver and early after injury.

Liver fibrosis is characterized by excessive deposition of extracellular matrix proteins by myofibroblasts derived from hepatic stellate cells and portal fibroblasts. Activation of these precursors to myofibroblasts requires matrix stiffness, which results in part from increased collagen cross-linking mediated by lysyl oxidase (LOX) family proteins. The aims of this study were to characterize the mechanical changes of early fibrosis, to identify the cells responsible for LOX production in early injury, and to determine which cells in normal liver produce collagens and elastins, which serve as substrates for LOXs early after injury. Hepatocytes and liver nonparenchymal cells were isolated from normal and early-injured liver and examined immediately for expression of LOXs and matrix proteins. We found that stellate cells and portal fibroblasts were the major cellular sources of fibrillar collagens and LOXs in normal liver and early after injury (1 day after bile duct ligation and 2 and 7 days after CCl(4) injury). Activity assays using stellate cells and portal fibroblasts in culture demonstrated significant increases in LOX family enzymatic activity as cells became myofibroblastic. LOX family-mediated deoxypyridinoline and pyridinoline cross-links increased after CCl(4)-mediated injury. There was a significant association between liver stiffness (as quantified by the shear storage modulus G') and deoxypyridinoline levels; increased deoxypyridinoline levels were also coincident with significantly increased elastic resistance to large strain deformations, consistent with increased cross-linking of the extracellular matrix. These data suggest a model in which the liver is primed to respond quickly to injury, activating potential mechanical feed-forward mechanisms.

Therapeutic Challenge with a CDK 4/6 Inhibitor Induces an RB-Dependent SMAC-Mediated Apoptotic Response in Non-Small Cell Lung Cancer.

Purpose: The retinoblastoma tumor suppressor (RB), a key regulator of cell-cycle progression and proliferation, is functionally suppressed in up to 50% of non-small cell lung cancer (NSCLC). RB function is exquisitely controlled by a series of proteins, including the CyclinD-CDK4/6 complex. In this study, we interrogated the capacity of a CDK4/6 inhibitor, palbociclib, to activate RB function.Experimental Design and Results: We employed multiple isogenic RB-proficient and -deficient NSCLC lines to interrogate the cytostatic and cytotoxic capacity of CDK 4/6 inhibition in vitro and in vivo We demonstrate that while short-term exposure to palbociclib induces cellular senescence, prolonged exposure results in inhibition of tumor growth. Mechanistically, CDK 4/6 inhibition induces a proapoptotic transcriptional program through suppression of IAPs FOXM1 and Survivin, while simultaneously augmenting expression of SMAC and caspase-3 in an RB-dependent manner.Conclusions: This study uncovers a novel function of RB activation to induce cellular apoptosis through therapeutic administration of a palbociclib and provides a rationale for the clinical evaluation of CDK 4/6 inhibitors in the treatment of patients with NSCLC. Clin Cancer Res; 24(6); 1402-14. ©2018 AACR.

siRNA-Encapsulated Hybrid Nanoparticles Target Mutant K-ras and Inhibit Metastatic Tumor Burden in a Mouse Model of Lung Cancer.

There is an unmet need in the development of an effective therapy for mutant K-ras-expressing non-small-cell lung cancer (NSCLC). Although various small molecules have been evaluated, an effective therapy remains a dream. siRNAs have the potential to downregulate mutant K-ras both at the protein and mRNA levels. However, a safe and effective delivery of siRNAs to tumors remains a limitation to their translational application in the treatment of this highly debilitating disease. Here we developed a novel hybrid nanoparticle carrier for effective delivery of anti-mutant K-ras to NSCLC (AKSLHN). The ability of this treatment modality to regress lung tumors in mouse models was evaluated as a monotherapy or as a combination treatment with erlotinib. Further, the toxicity of this treatment modality to healthy tissues was evaluated, along with its ability to elicit immune/inflammatory reactions. The results suggest that this treatment modality is a promising prospect for the treatment of mutant K-ras-expressing NSCLC without any accompanying toxicity. However, further understanding of the cellular-level interaction between AHSLHN and erlotinib needs to be attained before this promising treatment modality can be brought to the bedside.

Aptamer-hybrid nanoparticle bioconjugate efficiently delivers miRNA-29b to non-small-cell lung cancer cells and inhibits growth by downregulating essential oncoproteins.

MicroRNAs (miRNAs) are potentially attractive candidates for cancer therapy. However, their therapeutic application is limited by lack of availability of an efficient delivery system to stably deliver these potent molecules intracellularly to cancer cells while avoiding healthy cells. We developed a novel aptamer-hybrid nanoparticle bioconjugate delivery system to selectively deliver miRNA-29b to MUC1-expressing cancer cells. Significant downregulation of oncoproteins DNMT3b and MCL1 was demonstrated by these MUC1 aptamer-functionalized hybrid nanoparticles in A549 cells. Furthermore, downregulation of these oncoproteins led to antiproliferative effect and induction of apoptosis in a superior version when compared with Lipofectamine 2000. This novel aptamer-hybrid nanoparticle bioconjugate delivery system could potentially serve as a platform for intracellular delivery of miRNAs to cancer cells, hence improving the therapeutic outcome of lung cancer.

Biodistribution and Pharmacokinetics Study of siRNA-loaded Anti-NTSR1-mAb-functionalized Novel Hybrid Nanoparticles in a Metastatic Orthotopic Murine Lung Cancer Model.

Small interfering RNA (siRNA) is effective in silencing critical molecular pathways in cancer. The use of this tool as a treatment modality is limited by lack of an intelligent carrier system to enhance the preferential delivery of this molecule to specific targets in vivo. In the present study, the in vivo behavior of novel anti-NTSR1-mAb-functionalized antimutant K-ras siRNA-loaded hybrid nanoparticles, delivered by i.p. injection to non-small-cell lung cancer in mice models, was investigated and compared to that of a naked siRNA formulation. The siRNA in anti-NTSR1-mAb-functionalized hybrid nanoparticles was preferentially accumulated in tumor-bearing lungs and metastasized tumor for at least 48 hours while the naked siRNA formulation showed lack of preferential accumulation in all of the organs monitored. The plasma terminal half-life of nanoparticle-delivered siRNA was 11 times higher (17-1.5 hours) than that of the naked siRNA formulation. The mean residence time and AUClast were 3.4 and 33 times higher than the corresponding naked siRNA formulation, respectively. High-performance liquid chromatography analysis showed that the hybrid nanoparticle carrier system protected the encapsulated siRNA against degradation in vivo. Our novel anti-NTSR1-mAb-functionalized hybrid nanoparticles provide a useful platform for in vivo targeting of siRNA for both experimental and clinical purposes.

Gradually softening hydrogels for modeling hepatic stellate cell behavior during fibrosis regression.

The extracellular matrix (ECM) presents an evolving set of mechanical cues to resident cells. We developed methacrylated hyaluronic acid (MeHA) hydrogels containing both stable and hydrolytically degradable crosslinks to provide cells with a gradually softening (but not fully degradable) milieu, mimicking physiological events such as fibrosis regression. To demonstrate the utility of this cell culture system, we studied the phenotype of rat hepatic stellate cells, the major liver precursors of fibrogenic myofibroblasts, within this softening environment. Stellate cells that were mechanically primed on tissue culture plastic attained a myofibroblast phenotype, which persisted when seeded onto stiff (∼20 kPa) hydrogels. However, mechanically primed stellate cells on stiff-to-soft (∼20 to ∼3 kPa) hydrogels showed reversion of the myofibroblast phenotype over 14 days, with reductions in cell area, expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA), and Yes-associated protein/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) nuclear localization when compared to stellate cells on stiff hydrogels. Cells on stiff-to-soft hydrogels did not fully revert, however. They displayed reduced expression of glial fibrillary acidic protein (GFAP), and underwent abnormally rapid re-activation to myofibroblasts in response to re-stiffening of the hydrogels through introduction of additional crosslinks. These features are typical of stellate cells with an intermediate phenotype, reported to occur in vivo with fibrosis regression and re-injury. Together, these data suggest that mechanics play an important role in fibrosis regression and that integrating dynamic mechanical cues into model systems helps capture cell behaviors observed in vivo.

Stiffening hydrogels for investigating the dynamics of hepatic stellate cell mechanotransduction during myofibroblast activation.

Tissue fibrosis contributes to nearly half of all deaths in the developed world and is characterized by progressive matrix stiffening. Despite this, nearly all in vitro disease models are mechanically static. Here, we used visible light-mediated stiffening hydrogels to investigate cell mechanotransduction in a disease-relevant system. Primary hepatic stellate cell-seeded hydrogels stiffened in situ at later time points (following a recovery phase post-isolation) displayed accelerated signaling kinetics of both early (Yes-associated protein/Transcriptional coactivator with PDZ-binding motif, YAP/TAZ) and late (alpha-smooth muscle actin, α-SMA) markers of myofibroblast differentiation, resulting in a time course similar to observed in vivo activation dynamics. We further validated this system by showing that α-SMA inhibition following substrate stiffening resulted in attenuated stellate cell activation, with reduced YAP/TAZ nuclear shuttling and traction force generation. Together, these data suggest that stiffening hydrogels may be more faithful models for studying myofibroblast activation than static substrates and could inform the development of disease therapeutics.

Normal and Fibrotic Rat Livers Demonstrate Shear Strain Softening and Compression Stiffening: A Model for Soft Tissue Mechanics.

Tissues including liver stiffen and acquire more extracellular matrix with fibrosis. The relationship between matrix content and stiffness, however, is non-linear, and stiffness is only one component of tissue mechanics. The mechanical response of tissues such as liver to physiological stresses is not well described, and models of tissue mechanics are limited. To better understand the mechanics of the normal and fibrotic rat liver, we carried out a series of studies using parallel plate rheometry, measuring the response to compressive, extensional, and shear strains. We found that the shear storage and loss moduli G' and G" and the apparent Young's moduli measured by uniaxial strain orthogonal to the shear direction increased markedly with both progressive fibrosis and increasing compression, that livers shear strain softened, and that significant increases in shear modulus with compressional stress occurred within a range consistent with increased sinusoidal pressures in liver disease. Proteoglycan content and integrin-matrix interactions were significant determinants of liver mechanics, particularly in compression. We propose a new non-linear constitutive model of the liver. A key feature of this model is that, while it assumes overall liver incompressibility, it takes into account water flow and solid phase compressibility. In sum, we report a detailed study of non-linear liver mechanics under physiological strains in the normal state, early fibrosis, and late fibrosis. We propose a constitutive model that captures compression stiffening, tension softening, and shear softening, and can be understood in terms of the cellular and matrix components of the liver.

Hydrogels with differential and patterned mechanics to study stiffness-mediated myofibroblastic differentiation of hepatic stellate cells.

The differentiation of hepatic stellate cells (HSCs) into myofibroblasts is a key event in liver fibrosis. Due to the local stiffening of the extracellular matrix (ECM) during fibrosis, it is of great interest to develop mimics that can be used to investigate the cellular response to changes in mechanics. Here, we used a step-wise hydrogel crosslinking technique, where macromolecules are crosslinked using a sequence of addition then UV light-mediated radical crosslinking, to generate hydrogels with tunable stiffness. Freshly isolated HSCs remained rounded with lipid droplets and high levels of PPARγ expression on soft substrates (E~2kPa); however, HSCs spread, lost their lipid droplets, and expressed high levels of α-smooth muscle actin (α-SMA) and type I collagen on stiff substrates (E~24kPa). Similarly, fully differentiated cells reverted to a quiescent state when plated on soft substrates. Stiffness-induced differentiation of HSCs was enhanced in the presence of exogenous TGF-ß1, a dominant signal in fibrosis. When the UV-induced secondary crosslinking was restricted with a photomask to spatially control mechanics, HSCs responded based on the local hydrogel stiffness, although they remained quiescent on stiff substrates if the stiff feature size was not sufficient to allow cell spreading. This hydrogel system permits the investigation of HSC response to materials with diverse levels and spatially heterogeneous mechanical properties.

Isolation of rat portal fibroblasts by in situ liver perfusion.

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis. Portal fibroblasts have been isolated using various techniques including outgrowth and liver perfusion with enzymatic digestion followed by size selection. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.