In Vitro Prostate Cancer Treatment via CRISPR-Cas9 Gene Editing Facilitated by Polyethyleneimine-Derived Graphene Quantum Dots.

CRISPR-Cas9 is a programmable gene editing tool with a promising potential for cancer gene therapy. This therapeutic function is enabled in the present work via the non-covalent delivery of CRISPR ribonucleic protein (RNP) by cationic glucosamine/PEI-derived graphene quantum dots (PEI-GQD) that aid in overcoming physiological barriers and tracking genes of interest. PEI-GQD/RNP complex targeting the TP53 mutation overexpressed in ~50% of cancers successfully produces its double-stranded breaks in solution and in PC3 prostate cancer cells. Restoring this cancer "suicide" gene can promote cellular repair pathways and lead to cancer cell apoptosis. Its repair to the healthy form performed by simultaneous PEI-GQD delivery of CRISPR RNP and a gene repair template leads to a successful therapeutic outcome: 40% apoptotic cancer cell death, while having no effect on non-cancerous HeK293 cells. The translocation of PEI-GQD/RNP complex into PC3 cell cytoplasm is tracked via GQD intrinsic fluorescence, while EGFP-tagged RNP is detected in the cell nucleus, showing the successful detachment of the gene editing tool upon internalization. Using GQDs as non-viral delivery and imaging agents for CRISPR-Cas9 RNP sets the stage for image-guided cancer-specific gene therapy.

Dual-Mode Fluorescence/Ultrasound Imaging with Biocompatible Metal-Doped Graphene Quantum Dots.

Sonography offers many advantages over standard methods of diagnostic imaging due to its non-invasiveness, substantial tissue penetration depth, and low cost. The benefits of ultrasound imaging call for the development of ultrasound-trackable drug delivery vehicles that can address a variety of therapeutic targets. One disadvantage of the technique is the lack of high-precision imaging, which can be circumvented by complementing ultrasound contrast agents with visible and, especially, near-infrared (NIR) fluorophores. In this work, we, for the first time, develop a variety of lightly metal-doped (iron oxide, silver, thulium, neodymium, cerium oxide, cerium chloride, and molybdenum disulfide) nitrogen-containing graphene quantum dots (NGQDs) that demonstrate high-contrast properties in the ultrasound brightness mode and exhibit visible and/or near-infrared fluorescence imaging capabilities. NGQDs synthesized from glucosamine precursors with only a few percent metal doping do not introduce additional toxicity in vitro, yielding over 80% cell viability up to 2 mg/mL doses. Their small (<50 nm) sizes warrant effective cell internalization, while oxygen-containing surface functional groups decorating their surfaces render NGQDs water soluble and allow for the attachment of therapeutics and targeting agents. Utilizing visible and/or NIR fluorescence, we demonstrate that metal-doped NGQDs experience maximum accumulation within the HEK-293 cells 6-12 h after treatment. The successful 10-fold ultrasound signal enhancement is observed at 0.5-1.6 mg/mL for most metal-doped NGQDs in the vascular phantom, agarose gel, and animal tissue. A combination of non-invasive ultrasound imaging with capabilities of high-precision fluorescence tracking makes these metal-doped NGQDs a viable agent for a variety of theragnostic applications.

Detection of Pancreatic Cancer miRNA with Biocompatible Nitrogen-Doped Graphene Quantum Dots.

Early-stage pancreatic cancer remains challenging to detect, leading to a poor five-year patient survival rate. This obstacle necessitates the development of early detection approaches based on novel technologies and materials. In this work, the presence of a specific pancreatic cancer-derived miRNA (pre-miR-132) is detected using the fluorescence properties of biocompatible nitrogen-doped graphene quantum dots (NGQDs) synthesized using a bottom-up approach from a single glucosamine precursor. The sensor platform is comprised of slightly positively charged (1.14 ± 0.36 mV) NGQDs bound via π-π stacking and/or electrostatic interactions to the negatively charged (-22.4 ± 6.00 mV) bait ssDNA; together, they form a complex with a 20 nm average size. The NGQDs' fluorescence distinguishes specific single-stranded DNA sequences due to bait-target complementarity, discriminating them from random control sequences with sensitivity in the micromolar range. Furthermore, this targetability can also detect the stem and loop portions of pre-miR-132, adding to the practicality of the biosensor. This non-invasive approach allows cancer-specific miRNA detection to facilitate early diagnosis of various forms of cancer.

Mechanistic Investigation of Site-specific DNA Methylating Agents Targeting Breast Cancer Cells.

We previously described the development of a DNA-alkylating compound that showed selective toxicity in breast cancer cells. This compound contained an estrogen receptor α (ERα)-binding ligand and a DNA-binding/methylating component that could selectively methylate the N3-position of adenines at adenine-thymine rich regions of DNA. Herein, we describe mechanistic investigations that demonstrate that this class of compounds facilitate the translocation of the ERα-compound complex to the nucleus and induce the expression of ERα target genes. We confirm that the compounds show selective toxicity in ERα-expressing cells, induce ERα localization in the nucleus, and verify the essential role of ERα in modulating the toxicity. Minor alterations in the compound structure significantly affects the DNA binding ability, which correlates to the DNA-methylating ability. These studies demonstrate the utility of DNA-alkylating compounds to accomplish targeted inhibition of the growth of specific cancer cells; an approach that may overcome shortcomings of currently used chemotherapy agents.

Effects of Doxorubicin Delivery by Nitrogen-Doped Graphene Quantum Dots on Cancer Cell Growth: Experimental Study and Mathematical Modeling.

With 18 million new cases diagnosed each year worldwide, cancer strongly impacts both science and society. Current models of cancer cell growth and therapeutic efficacy in vitro are time-dependent and often do not consider the Emax value (the maximum reduction in the growth rate), leading to inconsistencies in the obtained IC50 (concentration of the drug at half maximum effect). In this work, we introduce a new dual experimental/modeling approach to model HeLa and MCF-7 cancer cell growth and assess the efficacy of doxorubicin chemotherapeutics, whether alone or delivered by novel nitrogen-doped graphene quantum dots (N-GQDs). These biocompatible/biodegradable nanoparticles were used for the first time in this work for the delivery and fluorescence tracking of doxorubicin, ultimately decreasing its IC50 by over 1.5 and allowing for the use of up to 10 times lower doses of the drug to achieve the same therapeutic effect. Based on the experimental in vitro studies with nanomaterial-delivered chemotherapy, we also developed a method of cancer cell growth modeling that (1) includes an Emax value, which is often not characterized, and (2), most importantly, is measurement time-independent. This will allow for the more consistent assessment of the efficiency of anti-cancer drugs and nanomaterial-delivered formulations, as well as efficacy improvements of nanomaterial delivery.

Formation of Platinum Nanocrystals on Silicon Nanotubes and Corresponding Anti-Cancer Activity in Vitro.

Biodegradable porous silicon nanotubes (pSiNTs), functionalized with primary amine moieties via the use of 3-aminopropyltriethoxysilane (APTES), is demonstrated as a template for formation of platinum nanocrystals (Pt NCs) (1-3 nm). Transmission electron microscopy-energy dispersive X-ray analysis (TEM-EDX) indicates a relatively high and tunable concentration of Pt uniformly immobilized on a given nanotube (wt % Pt: 20-60%). In vitro viability and cellular uptake studies are consistent with a time-dependent toxicity of Pt NCs-pSiNTs against HeLa cells that is influenced by the degradation kinetics of the pSiNTs; internalization of the composites inside the cells exerts cellular damage in an apoptotic manner, therefore suggesting promising future applications in anticancer treatments.

Plant-Derived Tandem Drug/Mesoporous Silicon Microcarrier Structures for Anti-Inflammatory Therapy.

The properties of nanostructured plant-derived porous silicon (pSi) microparticles as potential candidates to increase the bioavailability of plant extracts possessing anti-inflammatory activity are described in this work. pSi drug carriers were fabricated using an eco-friendly route from the silicon accumulator plant bamboo (tabasheer) powder by magnesiothermic reduction of plant-derived silica and loaded with ethanolic extracts of Equisetum arvense, another silicon accumulator plant rich in polyphenolic compounds. The anti-inflammatory properties of the active therapeutics present in this extract were measured by sensitive luciferase reporter assays; this active extract was subsequently loaded and released from the pSi matrix, with a clear inhibition of the activity of the inflammatory signaling protein NF-κB over a period of hours in a sustained manner. Our results showed that after loading the extracts of E. arvense into pSi microparticles derived from tabasheer, enhanced anti-inflammatory activity was observed owing to enhanced solubility of the extract.

Doped Graphene Quantum Dots for Intracellular Multicolor Imaging and Cancer Detection.

Despite significant advances of nanomedicine, the issues of biocompatibility, accumulation-derived toxicity, and the lack of sensing and in vivo imaging capabilities hamper the translation of most nanocarriers into clinic. To address this, we utilize nitrogen, boron/nitrogen, and sulfur-doped graphene quantum dots (GQDs) as fully biocompatible multifunctional platforms allowing for multicolor visible/near-IR imaging and cancer-sensing. These GQDs are scalably produced in one-step synthesis from a single biocompatible glucosamine precursor, are water-soluble, show no cytotoxicity at high concentrations of 1 mg/mL, and demonstrate substantial degradation at 36 h in biological environments as verified by TEM imaging. Because of their small sizes, GQDs exhibit efficient internalization maximized at 12 h followed by further degradation/excretion. Their high-yield intrinsic fluorescence in blue/green and near-infrared allows for multicolor in vitro imaging on its own or in combination with other fluorophores, and offers the capabilities for in vivo near-IR fluorescence tracking. Additionally, nitrogen- and sulfur-doped GQDs exhibit pH-dependent fluorescence response that is successfully utilized as a sensing mechanism for acidic extracellular environments of cancer cells. It allows for the deterministic, ratiometric spectral discrimination between cancerous (HeLa and MCF-7 cell) versus healthy (HEK-293 cell) environments with substantial intensity ratios of 1.6 to 8. These results suggest fully biocompatible GQDs developed in this work as multifunctional candidates for in vitro delivery of active agents, multicolor visible/near-IR fluorescence imaging, and pH-sensing of cancerous environments.

In Vitro Gene Delivery with Large Porous Silicon Nanoparticles Fabricated Using Cost-Effective, Metal-Assisted Chemical Etching.

The cytocompatibility, cell membrane affinity, and plasmid DNA delivery from surface oxidized, metal-assisted stain-etched mesoporous silicon nanoscale particles (pSiNPs) to human embryonic kidney (HEK293) cells is demonstrated, suggesting the possibility of using such material for targeted transfection and drug delivery.

DNA site-specific N3-adenine methylation targeted to estrogen receptor-positive cells.

A compound that can target cells expressing the estrogen receptor (ER), and produce predominantly 3-MeA adducts in those cells has been designed and synthesized. This compound produces mainly the 3-MeA adduct upon reaction with calf thymus DNA, and binds to the ER with a relative binding affinity of 51% (estradiol = 100%). The compound is toxic to ER-expressing MCF-7 breast cancer cells, and pre-treatment with the ER antagonist fulvestrant abrogates the toxicity. Pre-treatment of MCF-7 cells with netropsin, which inhibits N3-adenine methylation by the compound, resulted in a threefold decrease in the toxicity. These results demonstrate the feasibility of this strategy for producing 3-MeA adducts in targeted cells.

The role of nanostructured mesoporous silicon in discriminating in vitro calcification for electrospun composite tissue engineering scaffolds.

The impact of mesoporous silicon (PSi) particles-embedded either on the surface, or totally encapsulated within electrospun poly (ε-caprolactone) (PCL) fibers-on its properties as a tissue engineering scaffold is assessed. Our findings suggest that the resorbable porous silicon component can sensitively accelerate the necessary calcification process in such composites. Calcium phosphate deposition on the scaffolds was measured via in vitro calcification assays both at acellular and cellular levels. Extensive attachment of fibroblasts, human adult mesenchymal stem cells, and mouse stromal cells to the scaffold were observed. Complementary cell differentiation assays and ultrastructural measurements were also carried out; the levels of alkaline phosphatase expression, a specific biomarker for mesenchymal stem cell differentiation, show that the scaffolds have the ability to mediate such processes, and that the location of the Si plays a key role in levels of expression.

High-porosity poly(epsilon-caprolactone)/mesoporous silicon scaffolds: calcium phosphate deposition and biological response to bone precursor cells.

In this study, the fabrication and characterization of highly porous composites composed of poly(epsilon-caprolactone) and bioactive mesoporous silicon (BioSilicon) prepared using salt-leaching and microemulsion/freeze-drying methods are described. The role of silicon, along with porosity, in the scaffolds on calcium phosphate deposition was assessed using acellular in vitro calcification analyses. The presence of bioactive silicon in these scaffolds is essential for the deposition of calcium phosphate while the samples are immersed in simulated body fluid (SBF). Silicon-containing scaffolds produced using salt-leaching methods are more likely to calcify as a consequence of SBF exposure than those produced using microemulsion methods. In vitro proliferation and cell viability assays of these porous composites using human embryonic kidney fibroblast cells indicate that no cytotoxic effects are present in the scaffolds under the conditions used. Preliminary analyses of bone sialoprotein and alkaline phosphatase expression using orthopedically relevant mesenchymal cells derived from bone marrow suggest that such scaffolds are capable of mediating osteoblast differentiation. Overall, the results show that these porous silicon-containing polymer scaffolds enhance calcification, can be considered nontoxic to cells, and support the proliferation, viability, attachment, and differentiation of bone precursor cells.

Accelerated calcification in electrically conductive polymer composites comprised of poly(epsilon-caprolactone), polyaniline, and bioactive mesoporous silicon.

In this study the fabrication and characterization of an electrically conductive composite material comprised of poly(epsilon-caprolactone) (PCL), polyaniline (PANi), and bioactive mesoporous silicon (BioSilicon) is discussed. The influence of PANi and silicon on calcium phosphate induction was assessed via ex vitro calcification analyses (by acellular simulated body fluid (SBF) exposure) both with and without electrical bias. Acceleration of calcium phosphate formation is one possible desirable feature of "smart" synthetic scaffolds for selected orthopedic-relevant applications. In addition, electrical stability assays were performed in growth medium (DMEM) to determine the stability of such structures to bias in an authentic electrolyte during a typical cell experiment. The cytocompatibility of the composites was evaluated in vitro using human kidney fibroblasts (HEK 293) cell proliferation assays, along with more orthopedically relevant mesenchymal stem cells from mouse stroma. Importantly, these composites demonstrate accelerated calcification in SBF when electrical bias is applied cathodically to the scaffold. Furthermore, these scaffolds exhibit noncytotoxic behavior in the presence of fibroblasts over an 8-day culture period, and attachment of stromal cells to the semiconducting scaffold was directly imaged via scanning electron microscopy. Overall, these results suggest that materials of this type of composition have potential merit as a biomaterial.

Activation of the interferon-beta promoter during hepatitis C virus RNA replication.

In this study we examined the impact of hepatitis C virus (HCV) RNA replication on the innate antiviral response of the host cell. Replication of an HCV subgenomic replicon stimulated the activation of the interferon (IFN)-beta promoter and the production of IFN in human hepatoma cells. Using a variety of functional assays, we found that HCV RNA replication induced the activation and DNA-binding activity of NFkappaB and interferon regulatory factor (IRF)-1. In addition, microscopy experiments revealed a higher frequency of cells containing the nuclear-localized, active form of IRF-3 in HCV replicon cultures versus control cultures. Consistent with these observations, cells harboring the HCV replicon exhibited high basal level expression of a subset of IFN-stimulated antiviral genes. Our results indicate that HCV RNA replication can stimulate cellular antiviral programs that contribute to the assembly and activation of the IFN-beta enhanceosome complex and initiation of the antiviral state. Stable HCV RNA replication in the face of the host antiviral response suggests that HCV may encode one or more proteins capable of overcoming specific antiviral processes, thereby supporting persistent infection.