Therapeutic Gene Silencing Using Targeted Lipid Nanoparticles in Metastatic Ovarian Cancer.

Ovarian cancer is an aggressive tumor owing to its ability to metastasize from stage II onward. Herein, lipid nanoparticles (LNPs) that encapsulate combination of small interfering RNAs (siRNAs), polo-like kinase-1 (PLK1), and eukaryotic translation-initiation factor 3c (eIF3c), to target different cellular pathways essential for ovarian cancer progression are generated. The LNPs are further modified with hyaluronan (tNPs) to target cluster of differentiation 44 (CD44) expressing cells. Interestingly, hyaluronan-coated LNPs (tNPs) prolong functional activity and reduce growth kinetics of spheroids in in vitro assay as compared to uncoated LNPs (uNPs) due to ≈1500-fold higher expression of CD44. Treatment of 2D and 3D cultured ovarian cancer cells with LNPs encapsulating both siRNAs result in 85% cell death and robust target gene silencing. In advanced orthotopic ovarian cancer model, intraperitoneal administration of LNPs demonstrates CD44 specific tumor targeting of tNPs compared to uNPs and robust gene silencing in tissues involved in ovarian cancer pathophysiology. At very low siRNA dose, enhanced overall survival of 60% for tNPs treated mice is observed compared to 10% and 20% for single siRNA-, eIF3c-tNP, and PLK1-tNP treatment groups, respectively. Overall, LNPs represent promising platform in the treatment of advanced ovarian cancer by improving median- and overall-survival.

An ovarian spheroid based tumor model that represents vascularized tumors and enables the investigation of nanomedicine therapeutics.

The failure of cancer therapies in clinical settings is often attributed to the lack of a relevant tumor model and pathological heterogeneity across tumor types in the clinic. The objective of this study was to develop a robust in vivo tumor model that better represents clinical tumors for the evaluation of anti-cancer therapies. We successfully developed a simple mouse tumor model based on 3D cell culture by injecting a single spheroid and compared it to a tumor model routinely used by injecting cell suspension from 2D monolayer cell culture. We further characterized both tumors with cellular markers for the presence of myofibroblasts, pericytes, endothelial cells and extracellular matrix to understand the role of the tumor microenvironment. We further investigated the effect of chemotherapy (doxorubicin), nanomedicine (Doxil®), biological therapy (Avastin®) and their combination. Our results showed that the substantial blood vasculature in the 3D spheroid model enhances the delivery of Doxil® by 2.5-fold as compared to the 2D model. Taken together, our data suggest that the 3D tumors created by simple subcutaneous spheroid injection represents a robust and more vascular murine tumor model which is a clinically relevant platform to test anti-cancer therapy in solid tumors.

Combined reflectance spectroscopy and coherent light backscattering measurement differentiate cervical cancer from normal epithelial tissue in a xenograft mouse model.

Cervical cancer is a type of slow-growing cancer associated with high mortality rates. Early detection can enable lifesaving early intervention. Current cervical premalignant lesion detection methods suffer from both high miss rates and excessive referrals for unnecessary biopsies. Herein, coherent light backscatter and modifications in reflected white-light spectra were measured to specifically discriminate between cervical tumors and normal squamous epithelial tissues resected from a mouse xenograft model. The combined measurements resulted in 92% sensitivity and 93% specificity in discrimination between the two tissues. These methods can be used to develop a noninvasive portable optical probe for sensitive and objective detection of precancer and cancer epithelial lesions in the cervix and other accessible epithelial tissues.

Current Progress in Non-viral RNAi-Based Delivery Strategies to Lymphocytes.

RNAi-based therapy holds great promise, as it can be utilized for the treatment of multiple conditions in an accurate manner via sequence-specific manipulation of gene expression. To date, RNAi therapeutics have advanced into clinical trials for liver diseases and solid tumors; however, delivery of RNAi to leukocytes in general and to lymphocytes in particular remains a challenge. Lymphocytes are notoriously hard to transduce with RNAi payloads and are disseminated throughout the body, often located in deep tissues; therefore, developing an efficient systemic delivery system directed to lymphocytes is not a trivial task. Successful manipulation of lymphocyte function with RNAi possesses immense therapeutic potential, as it will enable researchers to resolve lymphocyte-implicated diseases such as inflammation, autoimmunity, transplant rejection, viral infections, and blood cancers. This potential has propelled the development of novel targeted delivery systems relying on the accumulating research knowledge from multiple disciplines, including materials science and engineering, immunology, and genetics. Here, we will discuss the recent progress in non-viral delivery strategies of RNAi payloads to lymphocytes. Special emphasis will be made on the challenges and potential opportunities in manipulating lymphocyte function with RNAi. These approaches might ultimately become a novel therapeutic modality to treat leukocyte-related diseases.

Advanced Strategies in Immune Modulation of Cancer Using Lipid-Based Nanoparticles.

Immunotherapy has a great potential in advancing cancer treatment, especially in light of recent discoveries and therapeutic interventions that lead to complete response in specific subgroups of melanoma patients. By using the body's own immune system, it is possible not only to specifically target and eliminate cancer cells while leaving healthy cells unharmed but also to elicit long-term protective response. Despite the promise, current immunotherapy is limited and fails in addressing all tumor types. This is probably due to the fact that a single treatment strategy is not sufficient in overcoming the complex antitumor immunity. The use of nanoparticle-based system for immunotherapy is a promising strategy that can simultaneously target multiple pathways with the same kinetics to enhance antitumor response. Here, we will highlight the recent advances in the field of cancer immunotherapy that utilize lipid-based nanoparticles as delivery vehicles and address the ongoing challenges and potential opportunities.

Immunomodulation of hematological malignancies using oligonucleotides based-nanomedicines.

Hematological malignancies are a group of diseases characterized by clonal proliferation of blood-forming cells. Malignant blood cells are classified as myeloid or lymphoid cells depending on their stem cell origin. Lymphoid malignancies are characterized by lymphocyte accumulation in the blood stream, in the bone marrow, or in lymphatic nodes and organs. Several of these diseases are associated with chromosomal translocations, which cause gene fusion and amplification of expression, while others are characterized with aberrant expression of oncogenes. Overall, these genes play a major role in development and maintenance of malignant clones. The discovery of antisense oligonucleotides and RNA interference (RNAi) mechanisms offer new tools to specifically manipulate gene expression. Systemic delivery of inhibitory oligonucleotides molecules for manipulation of gene expression in lymphocytes holds a great potential for facilitating the development of an oligonucleotides -based therapy platform for lymphoid blood cancer. However, lymphocytes are among the most difficult targets for oligonucleotides delivery, as they are resistant to conventional transfection reagents and are dispersed throughout the body, making it difficult to successfully localize or deliver oligonucleotides payloads via systemic administration. In this review, we will survey the latest progress in the field of oligonucleotides based nanomedicine in the heterogeneous group of hematological malignancies with special emphasis on RNA based strategies. We will describe the most advanced non-viral nanocarriers for RNA delivery to malignant blood cells. We will also discuss targeted strategies for cell specific delivery of RNA molecules using nanoparticles and the therapeutic benefit of manipulating gene function in hematological malignancies. Finally, we will focus on the ex vivo, in vivo, and clinical trial strategies, that are currently under development in hematological malignancies - strategies that might increase the arsenal of drugs available to hematologists in the upcoming years.

Advances in RNAi therapeutic delivery to leukocytes using lipid nanoparticles.

Small interfering RNAs (siRNAs) therapeutics has advanced into clinical trials for liver diseases and solid tumors, but remain a challenge for manipulating leukocytes fate due to lack of specificity and safety issues. Leukocytes ingest pathogens and defend the body through a complex network. They are also involved in the pathogeneses of inflammation, viral infection, autoimmunity and cancers. Modulating gene expression in leukocytes using siRNAs holds great promise to treat leukocyte-mediated diseases. Leukocytes are notoriously hard to transduce with siRNAs and are spread throughout the body often located deep in tissues, therefore developing an efficient systemic delivery strategy is still a challenge. Here, we discuss recent advances in siRNA delivery to leukocyte subsets such as macrophages, monocytes, dendritic cells and lymphocytes. We focus mainly on lipid-based nanoparticles (LNPs) comprised of new generation of ionizable lipids and their ability to deliver siRNA to primary or malignant leukocytes in a targeted manner. Special emphasis is made on LNPs targeted to subsets of leukocytes and we detail a novel microfluidic mixing technology that could aid in changing the landscape of process development of LNPs from a lab tool to a potential novel therapeutic modality.

Transforming Nanomedicines From Lab Scale Production to Novel Clinical Modality.

The use of nanoparticles as anticancer drug carriers has been studied for over 50 years. These nanoparticles that can carry drugs are now termed "nanomedicines". Since the approval of the first FDA "nanodrug", DOXIL in 1995, tremendous efforts have been made to develop hundreds of nanomedicines based on different materials. The development of drug nanocarriers (NCs) for cancer therapy is especially challenging and requires multidisciplinary approach. Not only is the translation from a lab scale production of the NCs to clinical scale a challenge, but tumor biology and its unique physiology also possess challenges that need to be overcome with cleverer approaches. Yet, with all the efforts made to develop new strategies to deliver drugs (including small molecules and biologics) for cancer therapy, the number of new NCs that are reaching clinical trials is extremely low. Here we discuss the reasons most of the NCs loaded with anticancer drugs are not likely to reach the clinic and emphasize the importance of understanding tumor physiology and heterogeneity, the use of predictive animal models, and the importance of sharing data as key denominators for potential successful translation of NCs from a bench scale into clinical modality for cancer care.

Nanomedicine as an emerging platform for metastatic lung cancer therapy.

Metastatic lung cancer is one of the most common cancers leading to mortality worldwide. Current treatment includes chemo- and pathway-dependent therapy aiming at blocking the spread and proliferation of these metastatic lesions. Nanomedicine is an emerging multidisciplinary field that offers unprecedented access to living cells and promises the state of the art in cancer detection and treatment. Development of nanomedicines as drug carriers (nanocarriers) that target cancer for therapy draws upon principles in the fields of chemistry, medicine, physics, biology, and engineering. Given the zealous activity in the field as demonstrated by more than 30 nanocarriers already approved for clinical use and given the promise of recent clinical results in various studies, nanocarrier-based strategies are anticipated to soon have a profound impact on cancer medicine and human health. Herein, we will detail the latest innovations in therapeutic nanomedicine with examples from lipid-based nanoparticles and polymer-based approaches, which are engineered to deliver anticancer drugs to metastatic lung cells. Emphasis will be placed on the latest and most attractive delivery platforms, which are developed specifically to target lung metastatic tumors. These novel nanomedicines may open new avenues for therapeutic intervention carrying new class of drugs such as RNAi and mRNA and the ability to edit the genome using the CRISPER/Cas9 system. Ultimately, these strategies might become a new therapeutic modality for advanced-stage lung cancer.

Quaternized starch-based carrier for siRNA delivery: from cellular uptake to gene silencing.

RNAi therapeutics is a powerful tool for treating diseases by sequence-specific targeting of genes using siRNA. Since its discovery, the need for a safe and efficient delivery system for siRNA has increased. Here, we have developed and characterized a delivery platform for siRNA based on the natural polysaccharide starch in an attempt to address unresolved delivery challenges of RNAi. Modified potato starch (Q-starch) was successfully obtained by substitution with quaternary reagent, providing Q-starch with cationic properties. The results indicate that Q-starch was able to bind siRNA by self-assembly formation of complexes. For efficient and potent gene silencing we monitored the physical characteristics of the formed nanoparticles at increasing N/P molar ratios. The minimum ratio for complete entrapment of siRNA was 2. The resulting complexes, which were characterized by a small diameter (~30 nm) and positive surface charge, were able to protect siRNA from enzymatic degradation. Q-starch/siRNA complexes efficiently induced P-glycoprotein (P-gp) gene silencing in the human ovarian adenocarcinoma cell line, NCI-ADR/Res (NAR), over expressing the targeted gene and presenting low toxicity. Additionally, Q-starch-based complexes showed high cellular uptake during a 24-hour study, which also suggested that intracellular siRNA delivery barriers governed the kinetics of siRNA transfection. In this study, we have devised a promising siRNA delivery vector based on a starch derivative for efficient and safe RNAi application.

Toxicity profiling of several common RNAi-based nanomedicines: a comparative study.

RNAi-based nanomedicine platforms (RNPs) have progressed from tools to study gene expression in vitro into clinical trials. Numerous RNPs strategies have been documented with an efficient ability to condense RNAi payloads and induce potent gene silencing. Moreover, some of these RNPs have been explored in various animal models, and some have even made it to the clinic. Still, there is lack of a clinically approved RNAi-based delivery strategy most probably due to unpredicted clinical toxicity. In this study, we prepared common RNPs such as cationic liposomes, polyamines, and hyaluronan-coated lipid-based nanoparticles and tested these strategies for global toxicity parameters such as changes in bodyweight, liver enzyme release, and hematological profiling. We found that polyamines such as polyethyleneimine and Poly-L-lysine released high levels of liver enzymes into the serum and reduced C57BL/6 mice bodyweight upon three intravenous injections. In addition, these polyamines dramatically reduced the total number of leukocytes, suggesting an immune suppression mechanism, while cationic liposomes, which also increased liver enzymes levels in the serum, elevated the total number of leukocytes probably by activation of Toll-like receptors 2 and 4. Coating the liposomes with hyaluronan, a hydrophilic glycosaminoglycan, provided a protective layer and did not induce adverse effects upon multiple intravenous injections. These findings suggest that there is an urgent need to develop gold standards for nanotoxicity in the field of RNAi that will be embraced by the RNAi community.

eIF3c: a potential therapeutic target for cancer.

Cancer cells are rapidly evolving due to their unstable genome, which contributes to the development of new cancer clones with different gene expression profile (GEP). Manipulating the expression of the genes vital for the progression of the disease is essential to overcome its heterogeneity. However, targeting overexpressed genes, retrieved from GEP analysis, would be efficient for a specific kind of a malignancy. Alternatively, manipulating the expression of genes that are part of a fundamental mechanism in the cell would be effective against a wide range of malignancies. To test this hypothesis we characterized, using RNAi approaches, the therapeutic potential of the housekeeping eIF3c gene in five different cancer cell lines NCI-ADR/RES (NAR), HeLa, MCF7, HCT116 and B16F10. eIF3c is one of the core subunit of the eukaryote translation initiation factor (eIF) 3 complex, which has a crucial role in the translation initiation process. In this study, we demonstrated that eIF3c is vital to translation initiation in vivo, as its downregulation decreases the global protein synthesis and causes a polysome run-off. In addition, reducing the expression of eIF3c mediates G0/G1 or G2/M arrest in a tissue dependent manner, which leads to a reduction in cell proliferation and eventually to cell death. Moreover, we demonstrated the efficiency of the hyaluronan (HA)-coated lipid-based nanoparticles (LNPs) platform to deliver eIF3c-siRNAs to mouse melanoma cells. Taking together, our results emphasize the importance of seeking ubiquitously expressed housekeeping genes such as eIF3c rather than tumor associated overexpressed genes as therapeutic targets for the heterogeneous malignancies.

Hyaluronan grafted lipid-based nanoparticles as RNAi carriers for cancer cells.

RNA interference (RNAi), a natural cellular mechanism for RNA-guided regulation of gene expression could in fact become new therapeutic modality if an appropriate efficient delivery strategy that is also reproducible and safe will be developed. Numerous efforts have been made for the past eight years to address this challenge with only mild success. The majority of these strategies are based on cationic formulations that condense the RNAi payload and deliver it into the cell cytoplasm. However, most of these formulations also evoke adverse effects such as mitochondrial damage, interfering with blood coagulation cascade, induce interferon response, promote cytokine induction and activate the complement. Herein, we present a strategy that is devised from neutral phospholipids and cholesterol that self-assembled into lipid-based nanoparticles (LNPs). These LNPs were then coated with the glycosaminoglycan, hyaluronan (HA). HA-LNPs bound and internalized specifically into cancer cells compared with control, non-coated particles. Next, loaded with siRNAs against the multidrug resistance extrusion pump, p-glycoprotein (P-gp), HA-LNPs efficiently and specifically reduced mRNA and P-gp protein levels compared with control particles and with HA-LNPs loaded with control, non-targeted siRNAs. In addition, no cellular toxicity or cytokine induction was observed when these particles were cultured with human Peripheral Blood Mononuclear Cells (PBMCs). The HA-LNPs may offer an alternative approach to cationic lipid-based formulations for RNAi delivery into cancer cells in an efficient and safe manner.

Nanoparticle hydrophobicity dictates immune response.

Understanding the interactions of nanomaterials with the immune system is essential for the engineering of new macromolecular systems for in vivo applications. Systematic study of immune activation is challenging due to the complex structure of most macromolecular probes. We present here the use of engineered gold nanoparticles to determine the sole effect of hydrophobicity on the immune response of splenocytes. The gene expression profile of a range of cytokines (immunological reporters) was analyzed against the calculated log P of the nanoparticle headgroups, with an essentially linear increase in immune activity with the increase in hydrophobicity observed in vitro. Consistent behavior was observed with in vivo mouse models, demonstrating the importance of hydrophobicity in immune system activation.

Altering the immune response with lipid-based nanoparticles.

Lipid-based nanoparticles (LNPs) hold great promise as delivery vectors in the treatment of cancer, inflammation, and infections and are already used in clinical practice. Numerous strategies based on LNPs are being developed to carry drugs into specific target sites. The common denominator for all of these LNPs-based platforms is to improve the payloads' pharmacokinetics, biodistribution, stability and therapeutic benefit, and to reduce to minimal adverse effects. In addition, the delivery system must be biocompatible and non-toxic and avoid undesirable interactions with the immune system. In order to achieve optimal benefits from these delivery strategies, interactions with the immune system must be thoroughly investigated. This report will center on the interactions of LNPs with different subsets of leukocytes and will detail representative examples of suppression or activation of the immune system by these carriers. By understanding the interactions of LNPs with the innate and the adaptive arms of the immune system it might be possible to attain improved therapeutic benefits and to avoid immune toxicity.

Enhanced bioavailability of polyaromatic hydrocarbons in the form of mucin complexes.

Increasing exposure of biological systems to large amounts of polycyclic aromatic hydrocarbons is of great public concern. Organisms have an array of biological defense mechanisms, and it is believed that mucosal gel (which covers the respiratory system, the gastrointestinal tract, etc.) provides an effective chemical shield against a range of toxic materials. However, in this work, we demonstrate, for the first time, that, upon complexation of polyaromatic hydrocarbons with mucins, enhanced bioavailability and, therefore, toxicity are obtained. This work was aimed to demonstrate how complexation of various highly hydrophobic polycyclic aromatic hydrocarbons with representative mucin glycoprotein could lead to the formation of previously undescribed materials, which exhibit increased toxicity versus pristine polycyclic aromatic hydrocarbons. In the present work, we show that a representative mucin glycoprotein, bovine submaxillary mucin, has impressive and unprecedented capabilities of binding and solubilizing water-insoluble materials in physiological solution. The complexes formed between the mucin and a series of polycyclic aromatic hydrocarbons were comprehensively characterized, and their toxicity was evaluated by both in vivo and in vitro assays. In addition, the bioavailability and membrane-penetration capabilities were tested using an internalization assay. Our results provide, for the first time, evidence of an unknown route by which hydrophobic materials may achieve higher bioavailability, penetrating some of the biological defense systems, in the form of water-soluble complexes with mucosal proteins.

A novel recombinant soluble splice variant of Met is a potent antagonist of the hepatocyte growth factor/scatter factor-Met pathway.

PURPOSE: The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), are involved in a wide range of biological activities, including cell proliferation, motility, invasion, and angiogenesis. The HGF/SF-Met signaling pathway is frequently activated in a variety of cancers, and uncontrolled Met activation correlates with highly invasive tumors and poor prognosis. In this study, we investigated the inhibitory effect of a novel soluble splice variant of Met on the HGF/SF-Met pathway. EXPERIMENTAL DESIGN: Using our alternative splicing modeling platform LEADS, we have identified a novel splice variant of the Met receptor, which encodes a truncated soluble form of the receptor. This variant was produced as a recombinant Fc-fused protein named Cgen-241A and was tested in various cell-based assays representing different outcomes of the HGF/SF-Met pathway. RESULTS: Cgen-241A significantly inhibited HGF/SF-induced Met phosphorylation as well as cell proliferation and survival. In addition, Cgen-241A showed a profound inhibitory effect on cell scattering, invasion, and urokinase up-regulation. The inhibitory effects of Cgen-241A were shown in multiple human and nonhuman cell types, representing different modes of Met activation. Furthermore, Cgen-241A showed direct binding to HGF/SF. CONCLUSIONS: Taken together, our results indicate that Cgen-241A is a potent antagonist of the HGF/SF-Met pathway, underlining its potential as a therapeutic agent for the treatment of a wide variety of human malignancies that are dependent on this pathway.