Meir Goldsmith

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.

Localized RNAi Therapeutics of Chemoresistant Grade IV Glioma Using Hyaluronan-Grafted Lipid-Based Nanoparticles

Glioblastoma multiforme (GBM) is one of the most infiltrating, aggressive, and poorly treated brain tumors. Progress in genomics and proteomics has paved the way for identifying potential therapeutic targets for treating GBM, yet the vast majority of these leading drug candidates for the treatment of GBM are ineffective, mainly due to restricted passages across the blood-brain barrier. Nanoparticles have been emerged as a promising platform to treat different types of tumors due to their ability to transport drugs to target sites while minimizing adverse effects. Herein, we devised a localized strategy to deliver RNA interference (RNAi) directly to the GBM site using hyaluronan (HA)-grafted lipid-based nanoparticles (LNPs). These LNPs having an ionized lipid were previously shown to be highly effective in delivering small interfering RNAs (siRNAs) into various cell types. LNPs surface was functionalized with hyaluronan (HA), a naturally occurring glycosaminoglycan that specifically binds the CD44 receptor expressed on GBM cells. We found that HA-LNPs can successfully bind to GBM cell lines and primary neurosphers of GBM patients. HA-LNPs loaded with Polo-Like Kinase 1 (PLK1) siRNAs (siPLK1) dramatically reduced the expression of PLK1 mRNA and cumulated in cell death even under shear flow that simulate the flow of the cerebrospinal fluid compared with control groups. Next, a human GBM U87MG orthotopic xenograft model was established by intracranial injection of U87MG cells into nude mice. Convection of Cy3-siRNA entrapped in HA-LNPs was performed, and specific Cy3 uptake was observed in U87MG cells. Moreover, convection of siPLK1 entrapped in HA-LNPs reduced mRNA levels by more than 80% and significantly prolonged survival of treated mice in the orthotopic model. Taken together, our results suggest that RNAi therapeutics could effectively be delivered in a localized manner with HA-coated LNPs and ultimately may become a therapeutic modality for GBM.

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.

A ceramide-1-phosphate analogue, PCERA-1, simultaneously suppresses tumour necrosis factor-α and induces interleukin-10 production in activated macrophages

Tight regulation of the production of the key pro‐inflammatory cytokine tumour necrosis factor‐α (TNF‐α) is essential for the prevention of chronic inflammatory diseases. In vivo administration of a synthetic phospholipid, named hereafter phospho‐ceramide analogue‐1 (PCERA‐1), was previously found to suppress lipopolysaccharide (LPS)‐induced TNF‐α blood levels. We therefore investigated the in vitro anti‐inflammatory effects of PCERA‐1. Here, we show that extracellular PCERA‐1 potently suppresses production of the pro‐inflammatory cytokine TNF‐α in RAW264.7 macrophages, and in addition, independently and reciprocally regulates the production of the anti‐inflammatory cytokine interleukin‐10 (IL‐10). Specificity is demonstrated by the inability of the phospholipids ceramide‐1‐phosphate (C1P), sphingosine‐1‐phosphate (S1P) and lysophosphatidic acid (LPA) to perform these activities. Similar TNF‐α suppression and IL‐10 induction by PCERA‐1 were observed in macrophages when activated by Toll‐like receptor 4 (TLR4), TLR2 and TLR7 agonists. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that, while PCERA‐1 does not block activation of nuclear factor (NF)‐κB and mitogen‐activated protein kinases by LPS, it elevates the intracellular cAMP level. In conclusion, the anti‐inflammatory activity of PCERA‐1 seems to be mediated by a cell membrane receptor, upstream of cAMP production, and eventually TNF‐α suppression and IL‐10 induction. Thus, identification of the PCERA‐1 receptor may provide new pharmacological means to block inflammation.

Tumor targeting profiling of hyaluronan-coated lipid based-nanoparticles

Hyaluronan (HA), a naturally occurring high Mw (HMw) glycosaminoglycan, has been shown to play crucial roles in cell growth, embryonic development, healing processes, inflammation, and tumor development and progression. Low Mw (LMw, <10 kDa) HA has been reported to provoke inflammatory responses, such as induction of cytokines, chemokines, reactive nitrogen species and growth factors. Herein, we prepared and characterized two types of HA coated (LMw and HMw) lipid-based targeted and stabilized nanoparticles (tsNPs) and tested their binding to tumor cells expressing the HA receptor (CD44), systemic immunotoxicity, and biodistribution in tumor bearing mice. In vitro, the Mw of the surface anchored HA had a significant influence on the affinity towards CD44 on B16F10 murine melanoma cells. LMw HA-tsNPs exhibited weak binding, while binding of tsNPs coated with HMw HA was characterized by high binding. Both types of tsNPs had no measured effect on cytokine induction in vivo following intravenous administration to healthy C57BL/6 mice suggesting no immune activation. HMw HA-tsNPs showed enhanced circulation time and tumor targeting specificity, mainly by accumulating in the tumor and its vicinity compared with LMw HA-tsNPs. Finally, we show that methotrexate (MTX), a drug commonly used in cancer chemotherapy, entrapped in HMw HA-tsNPs slowly diffused from the particles with a half-life of 13.75 days, and improved the therapeutic outcome in a murine B16F10 melanoma model compared with NPs suggesting an active cellular targeting beyond the Enhanced Permeability and Retention (EPR) effect. Taken together, these findings have major implications for the use of high molecular weight HA in nanomedicine as a selective and safe active cellular targeting moiety.

Precision nanomedicine in neurodegenerative diseases

The treatment of neurodegenerative diseases remains a tremendous challenge due to the limited access of molecules across the blood-brain barrier, especially large molecules such as peptides and proteins. As a result, at most, a small percentage of a drug that is administered systemically will reach the central nervous system in its active form. Currently, research in the field focuses on developing safer and more effective approaches to deliver peptides and proteins into the central nervous system. Multiple strategies have been developed for this purpose. However, noninvasive approaches, such as nanostructured protein delivery carriers and intranasal administration, seem to be the most promising strategies for the treatment of chronic diseases, which require long-term interventions. These approaches are both target-specific and able to rapidly bypass the blood-brain barrier. In this Perspective, we detail some of these strategies and discuss some of the potential pitfalls and opportunities in this field. The next generation strategies will most likely be more cell-type-specific. Devising these strategies to target the brain may ultimately become a novel therapeutic modality to treat neurodegenerative diseases.

Grand challenges in modulating the immune response with RNAi nanomedicines

RNAi is a ubiquitous and highly specific, endogenous, evolutionarily conserved mechanism of gene silencing. RNAi holds great promise as a novel therapeutic modality. Despite the rapid progress in the understanding and utilization of RNAi in vitro, the application of RNAi in vivo has been met with great difficulties, mainly in the delivery of these molecules into specific cell types. Here, we describe the major systemic nanomedicine platforms that have been developed. Focus is given to the development of new strategies to target subsets of leukocytes, which are among the most difficult cells to transduce with RNAi. Finally, we discuss the hurdles and potential opportunities for in vivo manipulation of the immune response utilizing RNAi nanomedicines.

Synergistic IL-10 induction by LPS and the ceramide-1-phosphate analog PCERA-1 is mediated by the cAMP and p38 MAP kinase pathways

Expression of the anti-inflammatory cytokine IL-10 can be induced either by TLR agonists such as lipopolysaccharide (LPS), or by various endogenous stimuli, in particular those acting via a cAMP-dependent signaling pathway. We have previously reported that the synthetic phospho-ceramide analogue-1 (PCERA-1) increases cAMP level and subsequently down-regulates production of TNFalpha and up-regulates production of IL-10 in LPS-stimulated macrophages. The objective of this study was to determine the mechanism of activity of PCERA-1 and the role of cAMP in LPS-induced IL-10 production. We show here that PCERA-1 induces IL-10 production in synergism with various TLR agonists in mouse RAW264.7 macrophages. Cooperativity is evident both at the mRNA and protein levels. IL-10 production by LPS and PCERA-1 is mediated by the cAMP pathway and by the p38 MAP kinase. Phosphorylation of p38 is cooperatively accomplished by LPS and PCERA-1 or other cAMP inducers. Furthermore, the activity of PCERA-1 can be partially mimicked by a cell-permeable analog of cAMP, and blocked by the protein kinase A (PKA) inhibitor H89. Finally, in the absence of PCERA-1, the residual IL-10 induction by LPS depends on the basal cAMP level as it can be largely elevated by the phosphodiesterase (PDE)-4 inhibitor rolipram. Our results thus indicate that IL-10 induction by LPS critically depends on basal cAMP level, and that a co-stimulus by a TLR agonist and a cAMP-elevating agent results in synergistic PKA-dependent and p38-dependent IL-10 production.

Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies

Significance RNA interference (RNAi) holds great promise as a novel therapeutic approach. Small interfering RNAs (siRNAs) that manipulate gene expression in leukocytes could be used to treat blood cancers. However, the lack of strategies for delivering siRNAs to leukocytes systemically has hampered the development of RNAi-based therapeutics. Here, we show that lipid-based nanoparticles coated with anti-CD38 monoclonal antibodies specifically target mantle cell lymphoma (MCL) cells and induce cell-specific therapeutic gene silencing in vivo. CD38-targeted nanoparticles that contain cyclin D1 siRNAs prolong survival of mice bearing MCL lymphomas in the bone marrow. This strategy opens a new avenue for treating MCL that might be applied to other hematological malignancies. Despite progress in systemic small interfering RNA (siRNA) delivery to the liver and to solid tumors, systemic siRNA delivery to leukocytes remains challenging. The ability to silence gene expression in leukocytes has great potential for identifying drug targets and for RNAi-based therapy for leukocyte diseases. However, both normal and malignant leukocytes are among the most difficult targets for siRNA delivery as they are resistant to conventional transfection reagents and are dispersed in the body. We used mantle cell lymphoma (MCL) as a prototypic blood cancer for validating a novel siRNA delivery strategy. MCL is an aggressive B-cell lymphoma that overexpresses cyclin D1 with relatively poor prognosis. Down-regulation of cyclin D1 using RNA interference (RNAi) is a potential therapeutic approach to this malignancy. Here, we designed lipid-based nanoparticles (LNPs) coated with anti-CD38 monoclonal antibodies that are specifically taken up by human MCL cells in the bone marrow of xenografted mice. When loaded with siRNAs against cyclin D1, CD38-targeted LNPs induced gene silencing in MCL cells and prolonged survival of tumor-bearing mice with no observed adverse effects. These results highlight the therapeutic potential of cyclin D1 therapy in MCL and present a novel RNAi delivery system that opens new therapeutic opportunities for treating MCL and other B-cell malignancies.

Assessing cellular toxicities in fibroblasts upon exposure to lipid-based nanoparticles: a high content analysis approach

Lipid-based nanoparticles (LNPs) are widely used for the delivery of drugs and nucleic acids. Although most of them are considered safe, there is confusing evidence in the literature regarding their potential cellular toxicities. Moreover, little is known about the recovery process cells undergo after a cytotoxic insult. We have previously studied the systemic effects of common LNPs with different surface charge (cationic, anionic, neutral) and revealed that positively charged LNPs ((+)LNPs) activate pro-inflammatory cytokines and induce interferon response by acting as an agonist of Toll-like receptor 4 on immune cells. In this study, we focused on the response of human fibroblasts exposed to LNPs and their cellular recovery process. To this end, we used image-based high content analysis (HCA). Using this strategy, we were able to show simultaneously, in several intracellular parameters, that fibroblasts can recover from the cytotoxic effects of (+)LNPs. The use of HCA opens new avenues in understanding cellular response and nanotoxicity and may become a valuable tool for screening safe materials for drug delivery and tissue engineering.