Cinzia Stigliano

Soft Discoidal Polymeric Nanoconstructs Resist Macrophage Uptake and Enhance Vascular Targeting in Tumors

Most nanoparticles for biomedical applications originate from the self-assembling of individual constituents through molecular interactions and possess limited geometry control and stability. Here, 1000 × 400 nm discoidal polymeric nanoconstructs (DPNs) are demonstrated by mixing hydrophobic and hydrophilic polymers with lipid chains and curing the resulting paste directly within silicon templates. By changing the paste composition, soft- and rigid-DPNs (s- and r-DPNs) are synthesized exhibiting the same geometry, a moderately negative surface electrostatic charge (-14 mV), and different mechanical stiffness (∼1.3 and 15 kPa, respectively). Upon injection in mice bearing nonorthotopic brain or skin cancers, s-DPNs exhibit ∼24 h circulation half-life and accumulate up to ∼20% of the injected dose per gram tumor, detecting malignant masses as small as ∼0.1% the animal weight via PET imaging. This unprecedented behavior is ascribed to the unique combination of geometry, surface properties, and mechanical stiffness which minimizes s-DPN sequestration by the mononuclear phagocyte system. Our results could boost the interest in using less conventional delivery systems for cancer theranosis.

Engineered magnetic hybrid nanoparticles with enhanced relaxivity for tumor imaging

Clinically used contrast agents for magnetic resonance imaging (MRI) suffer by the lack of specificity; short circulation time; and insufficient relaxivity. Here, a one-step combinatorial approach is described for the synthesis of magnetic lipid-polymer (hybrid) nanoparticles (MHNPs) encapsulating 5 nm ultra-small super-paramagnetic iron oxide particles (USPIOs) and decorated with Gd(3+) ions. The MHNPs comprise a hydrophobic poly(lactic acid-co-glycolic acid) (PLGA) core, containing up to ~5% USPIOs (w/w), stabilized by lipid and polyethylene glycol (PEG). Gd(3+) ions are directly chelated to the external lipid monolayer. Three different nanoparticle configurations are presented including Gd(3+) chelates only (Gd-MHNPs); USPIOs only (Fe-MHNPs); and the combination thereof (MHNPs). All three MHNPs exhibit a hydrodynamic diameter of about 150 nm. The Gd-MHNPs present a longitudinal relaxivity (r1 = 12.95 ± 0.53 (mM s)(-1)) about four times larger than conventional Gd-based contrast agents (r1 = 3.4 (mM s)(-1)); MHNPs have a transversal relaxivity of r2 = 164.07 ± 7.0 (mM s)(-1), which is three to four times larger than most conventional systems (r2 ~ 50 (mM s)(-1)). In melanoma bearing mice, elemental analysis for Gd shows about 3% of the injected MHNPs accumulating in the tumor and 2% still circulating in the blood, at 24 h post-injection. In a clinical 3T MRI scanner, MHNPs provide significant contrast confirming the observed tumor deposition. This approach can also accommodate the co-loading of hydrophobic therapeutic compounds in the MHNP core, paving the way for theranostic systems.

Positron emitting magnetic nanoconstructs for PET/MR imaging.

Hybrid PET/MRI scanners have the potential to provide fundamental molecular, cellular, and anatomic information essential for optimizing therapeutic and surgical interventions. However, their full utilization is currently limited by the lack of truly multi-modal contrast agents capable of exploiting the strengths of each modality. Here, we report on the development of long-circulating positron-emitting magnetic nanoconstructs (PEM) designed to image solid tumors for combined PET/MRI. PEMs are synthesized by a modified nano-precipitation method mixing poly(lactic-co-glycolic acid) (PLGA), lipids, and polyethylene glycol (PEG) chains with 5 nm iron oxide nanoparticles (USPIOs). PEM lipids are coupled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and subsequently chelated to (64)Cu. PEMs show a diameter of 140 ± 7 nm and a transversal relaxivity r2 of 265.0 ± 10.0 (mM × s)(-1), with a r2/r1 ratio of 123. Using a murine xenograft model bearing human breast cancer cell line (MDA-MB-231), intravenously administered PEMs progressively accumulate in tumors reaching a maximum of 3.5 ± 0.25% ID/g tumor at 20 h post-injection. Correlation of PET and MRI signals revealed non-uniform intratumoral distribution of PEMs with focal areas of accumulation at the tumor periphery. These long-circulating PEMs with high transversal relaxivity and tumor accumulation may allow for detailed interrogation over multiple scales in a clinically relevant setting.

Geometrical confinement of Gd(DOTA) molecules within mesoporous silicon nanoconstructs for MR imaging of cancer

Porous silicon has been used for the delivery of therapeutic and imaging agents in several biomedical applications. Here, mesoporous silicon nanoconstructs (SiMPs) with a discoidal shape and a sub-micrometer size (1000×400nm) have been conjugated with gadolinium-tetraazacyclododecane tetraacetic acid Gd(DOTA) molecules and proposed as contrast agents for Magnetic Resonance Imaging. The surface of the SiMPs with different porosities - small pore (SP: ∼5nm) and huge pore (HP: ∼40nm) - and of bulk, non-porous silica beads (1000nm in diameter) have been modified with covalently attached (3-aminopropyl)triethoxysilane (APTES) groups, conjugated with DOTA molecules, and reacted with an aqueous solution of GdCl3. The resulting Gd(DOTA) molecules confined within the small pores of the Gd-SiMPs achieve longitudinal relaxivities r1 of ∼17 (mMs)(-)(1), which is 4 times greater than for free Gd(DOTA). This enhancement is ascribed to the confinement and stable chelation of Gd(DOTA) molecules within the SiMP mesoporous matrix. The resulting nanoconstructs possess no cytotoxicity and accumulate in ovarian tumors up to 2% of the injected dose per gram tissue, upon tail vein injection. All together this data suggests that Gd-SiMPs could be efficiently used for MR vascular imaging in cancer and other diseases.

Synthesis of multifunctional magnetic nanoflakes for magnetic resonance imaging, hyperthermia, and targeting.

Iron oxide nanoparticles (IOs) are intrinsically theranostic agents that could be used for magnetic resonance imaging (MRI) and local hyperthermia or tissue thermal ablation. Yet, effective hyperthermia and high MR contrast have not been demonstrated within the same nanoparticle configuration. Here, magnetic nanoconstructs are obtained by confining multiple, ∼ 20 nm nanocubes (NCs) within a deoxy-chitosan core. The resulting nanoconstructs—magnetic nanoflakes (MNFs)—exhibit a hydrodynamic diameter of 156 ± 3.6 nm, with a polydispersity index of ∼0.2, and are stable in PBS up to 7 days. Upon exposure to an alternating magnetic field of 512 kHz and 10 kA m–1, MNFs provide a specific absorption rate (SAR) of ∼75 W gFe–1, which is 4–15 times larger than that measured for conventional IOs. Moreover, the same nanoconstructs provide a remarkably high transverse relaxivity of ∼500 (mM s)−1, at 1.41T. MNFs represent a first step toward the realization of nanoconstructs with superior relaxometric and ablation properties for more effective theranostics.

Predicting the growth of glioblastoma multiforme spheroids using a multiphase porous media model

Tumor spheroids constitute an effective in vitro tool to investigate the avascular stage of tumor growth. These three-dimensional cell aggregates reproduce the nutrient and proliferation gradients found in the early stages of cancer and can be grown with a strict control of their environmental conditions. In the last years, new experimental techniques have been developed to determine the effect of mechanical stress on the growth of tumor spheroids. These studies report a reduction in cell proliferation as a function of increasingly applied stress on the surface of the spheroids. This work presents a specialization for tumor spheroid growth of a previous more general multiphase model. The equations of the model are derived in the framework of porous media theory, and constitutive relations for the mass transfer terms and the stress are formulated on the basis of experimental observations. A set of experiments is performed, investigating the growth of U-87MG spheroids both freely growing in the culture medium and subjected to an external mechanical pressure induced by a Dextran solution. The growth curves of the model are compared to the experimental data, with good agreement for both the experimental settings. A new mathematical law regulating the inhibitory effect of mechanical compression on cancer cell proliferation is presented at the end of the paper. This new law is validated against experimental data and provides better results compared to other expressions in the literature.

Assembly of Iron Oxide Nanocubes for Enhanced Cancer Hyperthermia and Magnetic Resonance Imaging

Multiple formulations of iron oxide nanoparticles (IONPs) have been proposed for enhancing contrast in magnetic resonance imaging (MRI) and for increasing efficacy in thermal ablation therapies. However, insufficient accumulation at the disease site and low magnetic performance hamper the clinical application of IONPs. Here, 20 nm iron oxide nanocubes were assembled into larger nanoconstructs externally stabilized by a serum albumin coating. The resulting assemblies of nanocubes (ANCs) had an average diameter of 100 nm and exhibited transverse relaxivity (r2 = 678.9 ± 29.0 mM‒1·s‒1 at 1.41 T) and heating efficiency (specific absorption rate of 109.8 ± 12.8 W·g‒1 at 512 kHz and 10 kA·m‒1). In mice bearing glioblastoma multiforme tumors, Cy5.5-labeled ANCs allowed visualization of malignant masses via both near infrared fluorescent and magnetic resonance imaging. Also, upon systemic administration of ANCs (5 mgFe·kg‒1), 30 min of daily exposure to alternating magnetic fields for three consecutive days was sufficient to halt tumor progression. This study demonstrates that intravascular administration of ANCs can effectively visualize and treat neoplastic masses.

Methotraxate-Loaded Hybrid Nanoconstructs Target Vascular Lesions and Inhibit Atherosclerosis Progression in ApoE-/- Mice

Atherosclerosis is an inflammatory disorder characterized by the progressive thickening of blood vessel walls eventually resulting in acute vascular syndromes. Here, intravenously injectable hybrid nanoconstructs are synthesized for tempering immune cell inflammation locally and systemically. Lipid and polymer chains are nanoprecipitated to form 100 nm spherical polymeric nanoconstructs (SPNs), loaded with methotrexate (MTX) and subsequently labeled with Cu64 and fluorescent probes for combined nuclear/optical imaging. Upon engulfment into macrophages, MTX SPNs intracellularly release their anti-inflammatory cargo significantly lowering the production of proinflammatory cytokine (interleukin 6 and tumor necrosis factor α) already at 0.06 mg mL-1 of MTX. In ApoE-/- mice, fed with high-fat diet up to 17 weeks, nuclear and optical imaging demonstrates specific accumulation of SPNs within lipid-rich plaques along the arterial tree. Histological analyses confirm SPN uptake into macrophages residing within atherosclerotic plaques. A 4-week treatment with biweekly administration of MTX SPNs is sufficient to reduce the plaque burden in ApoE-/- mice by 50%, kept on high-fat diet for 10 weeks. Systemic delivery of MTX to macrophages via multifunctional, hybrid nanoconstructs constitutes an effective strategy to inhibit atherosclerosis progression and induce, potentially, the resorption of vascular lesions.

Abstract 2887: Nanoconstructs for the efficient delivery of siRNA molecules against Aquaporin-1 for anti-angiogenic therapies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Aquaporins (AQPs) are water-specific, membrane-channel proteins expressed in diverse tissues. In particular, AQP1 has been reported to be specifically and strongly expressed in most tumor microvascular endothelial cells. Angiogenesis and tumor progression have been dramatically impaired, in AQP1-null mice [Saadoun et al. Nature 434:2005]. These findings support the notion that AQP1 could be considered as a specific target for the treatment of various neoplasias. The AQP1 proteins can be specifically blocked by using small interference RNA molecules (siRNAs). However, despite the considerable potential of RNA interference for the treatment of cancer, the proper systemic delivery of exogenous siRNA molecules is still a challenge. The most significant obstacle is ensuring specific and effective delivery of siRNAs to the cytoplasm of the target cell, limiting toxic effects and serum degradation by RNAses. The use of nanoparticles, as intravascular delivery vehicles, has potential in supporting the efficient, effective and safe systemic delivery of siRNAs. Herein, multiple nanotechnological platforms (nanoconstructs) are used to deliver siRNAs against AQP1. The performance of the different nanoconstructs is assessed in vitro using HeLa cells, transfected to express AQP1 molecules. Three nanoconstructs are studied: fullerenes (C60), poly(lactic-co-glycolic acid) (PLGA) nanospheres and hydrogel-template nanoparticles. Positively charged fullerenes are sub-nanometer spheres (diameter ∼ 0.71 nm) that complex with the negatively charged siRNA molecules forming a stable compound under physiological conditions (pH = 7.4). Differently, the PLGA nanospheres and hydrogel-template nanoparticles are loaded with siRNA molecules during their synthesis process. The silencing performance of the three different nanoconstructs is assessed in vitro at multiple time points, over a period of 6 days post incubation. The loading efficiency, the release rate, the cell-entry mechanisms and potential cytotoxic response are analyzed. The performance of the nanoconstructs is compared with traditional delivery systems based on the use of transfection agents (oligofectamine). AQP1 is here used as a model system, and the optimized nanoconstructs could be utilized also for other siRNA therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2887. doi:1538-7445.AM2012-2887