Avi Schroeder

Treating metastatic cancer with nanotechnology

Metastasis accounts for the vast majority of cancer deaths. The unique challenges for treating metastases include their small size, high multiplicity and dispersion to diverse organ environments. Nanoparticles have many potential benefits for diagnosing and treating metastatic cancer, including the ability to transport complex molecular cargoes to the major sites of metastasis, such as the lungs, liver and lymph nodes, as well as targeting to specific cell populations within these organs. This Review highlights the research, opportunities and challenges for integrating engineering sciences with cancer biology and medicine to develop nanotechnology-based tools for treating metastatic disease.

Lipid-based nanotherapeutics for siRNA delivery

RNA interference (RNAi) is a specific gene‐silencing mechanism triggered by small interfering RNA (siRNA). The application of RNAi in the clinic requires the development of safe and effective delivery systems. Inspired by progress with lipid‐based systems in drug delivery, efforts have been dedicated to the development of liposomal siRNA delivery systems. Many of the lipid‐based delivery vehicles self‐assemble with siRNA through electrostatic interactions with charged amines, generating multi‐lamellar lipoplexes with positively charged lipid bilayers separated from one another by sheets of negatively charged siRNA strands. Internalization of lipid‐based siRNA delivery systems into cells typically occurs through endocytosis; accordingly, delivery requires materials that can facilitate endosomal escape. The size of the carrier is important as carriers <100 nm in diameter have been reported to have higher accumulation levels in tumours, hepatocytes and inflamed tissue, whereas larger particles tend to be taken up by Kupffer cells or other components of the reticuloendothelial system (RES). To reduce RES uptake and increase circulation time, carriers have been modified on the surface with hydrophilic materials, such as polyethyleneglycol. Herein, we review the molecular and structural parameters of lipid‐based siRNA delivery systems.

Ultrasound, liposomes, and drug delivery: principles for using ultrasound to control the release of drugs from liposomes.

Ultrasound is used in many medical applications, such as imaging, blood flow analysis, dentistry, liposuction, tumor and fibroid ablation, and kidney stone disruption. In the past, low frequency ultrasound (LFUS) was the main method to downsize multilamellar (micron range) vesicles into small (nano scale) unilamellar vesicles. Recently, the ability of ultrasound to induce localized and controlled drug release from liposomes, utilizing thermal and/or mechanical effects, has been shown. This review, deals with the interaction of ultrasound with liposomes, focusing mainly on the mechanical mechanism of drug release from liposomes using LFUS. The effects of liposome lipid composition and physicochemical properties, on one hand, and of LFUS parameters, on the other, on liposomal drug release, are addressed. Acoustic cavitation, in which gas bubbles oscillate and collapse in the medium, thereby introducing intense mechanical strains, increases release substantially. We suggest that the mechanism of release may involve formation and collapse of small gas nuclei in the hydrophobic region of the lipid bilayer during exposure to LFUS, thereby inducing the formation of transient pores through which drugs are released. Introducing PEG-lipopolymers to the liposome bilayer enhances responsivity to LFUS, most likely due to absorption of ultrasonic energy by the highly hydrated PEG headgroups. The presence of amphiphiles, such as phospholipids with unsaturated acyl chains, which destabilize the lipid bilayer, also increases liposome susceptibility to LFUS. Application of these principles to design highly LFUS-responsive liposomes is discussed.

In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

Nanoparticles targeting the infarcted heart.

We report a nanoparticulate system capable of targeting the heart after myocardial infarction (MI). Targeting is based on overexpression of angiotensin II type 1 (AT1) receptor in the infarcted heart. Liposomes 142 nm in diameter were conjugated with a ligand specific to AT1. The nanoparticles were able to specifically target cardiac cells in vitro, and in the infarcted heart after intravenous injection in vivo. This system may be useful for delivering therapeutic agents specifically to the infarcted heart.

Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates

Significance The safe, selective, and efficient delivery of siRNA is a key challenge to the broad application of siRNA therapeutics in humans. Motivated by the structure of lipoproteins, we developed lipopeptide nanomaterials for siRNA delivery. In vivo in mice, siRNA–lipopeptide particles provide the most potent delivery to hepatocytes (ED50 ∼ 0.002 mg/kg for FVII silencing), with the highest selectivity of delivery to hepatocytes over nontarget cell types (orders of magnitude), yet reported. These materials also show efficacy in nonhuman primates. siRNA therapeutics have promise for the treatment of a wide range of genetic disorders. Motivated by lipoproteins, we report lipopeptide nanoparticles as potent and selective siRNA carriers with a wide therapeutic index. Lead material cKK-E12 showed potent silencing effects in mice (ED50 ∼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg). Apolipoprotein E plays a significant role in the potency of cKK-E12 both in vitro and in vivo. cKK-E12 was highly selective toward liver parenchymal cell in vivo, with orders of magnitude lower doses needed to silence in hepatocytes compared with endothelial cells and immune cells in different organs. Toxicity studies showed that cKK-E12 was well tolerated in rats at a dose of 1 mg/kg (over 100-fold higher than the ED50). To our knowledge, this is the most efficacious and selective nonviral siRNA delivery system for gene silencing in hepatocytes reported to date.

Small RNA combination therapy for lung cancer

Significance Small RNAs can potently and precisely regulate gene expression; as a result, they have tremendous clinical potential. However, effective delivery of small RNAs to solid tumors has remained challenging. Here we report that a lipid/polymer nanoparticle can deliver small RNAs to treat autochthonous tumors in the so-called “KP” mouse model of lung cancer. Nanoparticles formulated with mimics of the p53-regulated miRNA miR-34a downregulated target genes and delayed tumor progression, while nanoparticles formulated with siRNA targeting Kirsten rat sarcoma viral oncogene homolog (siKras) slowed tumor growth and increased apoptosis. Notably, concurrent delivery of miR-34a and siKras increased anti-tumor effects, and led to tumor regression. These results demonstrate that small RNA therapies can impact solid lung tumor growth, and that targeted RNA combination therapies may be used to improve therapeutic response. MicroRNAs (miRNAs) and siRNAs have enormous potential as cancer therapeutics, but their effective delivery to most solid tumors has been difficult. Here, we show that a new lung-targeting nanoparticle is capable of delivering miRNA mimics and siRNAs to lung adenocarcinoma cells in vitro and to tumors in a genetically engineered mouse model of lung cancer based on activation of oncogenic Kirsten rat sarcoma viral oncogene homolog (Kras) and loss of p53 function. Therapeutic delivery of miR-34a, a p53-regulated tumor suppressor miRNA, restored miR-34a levels in lung tumors, specifically down-regulated miR-34a target genes, and slowed tumor growth. The delivery of siRNAs targeting Kras reduced Kras gene expression and MAPK signaling, increased apoptosis, and inhibited tumor growth. The combination of miR-34a and siRNA targeting Kras improved therapeutic responses over those observed with either small RNA alone, leading to tumor regression. Furthermore, nanoparticle-mediated small RNA delivery plus conventional, cisplatin-based chemotherapy prolonged survival in this model compared with chemotherapy alone. These findings demonstrate that RNA combination therapy is possible in an autochthonous model of lung cancer and provide preclinical support for the use of small RNA therapies in patients who have cancer.

Liposomes act as effective biolubricants for friction reduction in human synovial joints.

Phospholipids (PL) form the matrix of biological membranes and of the lipoprotein envelope monolayer, and are responsible for many of the unique physicochemical, biochemical, and biological properties of these supermolecular bioassemblies. It was suggested that phospholipids present in the synovial fluid (SF) and on the surface of articular cartilage have major involvement in the low friction of cartilage, which is essential for proper mobility of synovial joints. In pathologies, such as impaired biolubrication (leading to common joint disorders such as osteoarthritis), the level of phospholipids in the SF is reduced. Using a human-sourced cartilage-on-cartilage setup, we studied to what extent and how phospholipids act as highly effective cartilage biolubricants. We found that large multilamellar vesicles (MLV), >800 nm in diameter, composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or of a mixture of DMPC and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) are superior lubricants in comparison to MLV composed of other phosphatidylcholines. Introducing cholesterol into liposomes resulted in less effective lubricants. DMPC-MLV was also superior to small unilamellar vesicles (SUV), <100 nm in diameter, composed of DMPC. MLV are superior to SUV due to MLV retention at and near (<200 microm below) the cartilage surface, while SUV penetrate deeper into the cartilage (450-730 microm). Superiority of specific PL compositions is explained by the thermotropic behavior (including compressibility) of the lipid bilayer. Correlating physicochemical properties of the MLV with the friction results suggests that MLV having lipid bilayers in the liquid-disordered phase and having a solid-ordered to liquid-disordered phase transition temperature slightly below physiological temperature are optimal for lubrication. High phospholipid headgroup hydration, high compressibility, and softness are the common denominators of all efficient PL compositions. The high efficiency of DMPC-MLV and DMPC/DPPC-MLV as cartilage lubricants combined with their resistance to degradation at 37 degrees C supports further evaluation of these MLV for treatment of joint impairments related to poor lubrication. This work also demonstrates the relevance of basic physicochemical properties of phospholipids to their activities in biological systems.

Boundary lubricants with exceptionally low friction coefficients based on 2D close-packed phosphatidylcholine liposomes.

Figure 1 . Cryo-SEM image of the HSPC-SUV adsorbed on freshly cleaved mica. The lower inset schematically interprets this in terms of liposomes that have fl attened (A), those with less available space for fl attening (B) and those (C) on top of the close-packed surface-attached layer that were not removed by the washing. The top right inset shows an AFM profi le of the same close-packed layer; holes in the originally close-packed layer arise through removal of liposomes by the AFM tip. Liposomes are widely used in pharmaceutical applications, primarily as drug delivery vehicles, as well as in gene therapy and for diagnostic imaging. [ 1–3 ] Here we report that certain phosphatidylcholine liposomes, when adsorbed onto sliding surfaces in a 2-dimensional close-packed array, may act as exceptionally effi cient boundary lubricants at physiologically high pressures [ 4 ] under water. We created small unilamellar vesicles (SUVs) of hydrogenated soy phosphatidylcholine (HSPC) lipids which self-assemble in close-packed layers on solid surfaces to reduce the coeffi cient μ of sliding friction between them down to values μ ≈ 10 − 4 –2 × 10 − 5 , at pressures up to at least ca. 12 MPa (ca. 120 atmospheres). Such low values of the friction at these high pressures have not been attained with any boundary lubricants. This remarkably low friction is attributed to lubrication by the highly-hydrated phosphocholine head-groups exposed at the vesicle walls, stabilized against high pressures by the closepacking and by the rigidity of the gel-phase liposomes. A dispersion of HSPC-SUV with a unimodal size distribution (diameter 65 nm) was prepared as described below (Experimental); freshly cleaved mica surfaces were incubated in the dispersion, then rinsed and mounted in a surface force balance [ 5 ] (SFB) under water.

Melanoma miRNA trafficking controls tumour primary niche formation

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.