NANOMEDICINE A.A

An expanding field, Nanomedicine represents an active field of pharmacological research. However, only a small part of nanodrugs and nanosized devices potentially usefull for the exploitation in humans have reached the clinical experimental phase; even fewer are approved for use in humans.

Nanosized drugs: main properties. 1.Enhanced Permeabilization and Retention (EPR): accumulation in newly forming endothelia 2.Compartimentalization 3.Accessibility to districts with blood barriers (e.g. brain, posterior pole of the eye) Main results : changes of the bioavailability and of other pharmacological paramenters in comparison with traditional formulations.

The STHEALTH technology. The EPR effect, while useful for targeting newly vascularized tissues, can be indesirable if it reduces the half life of the nanodrug. En efficient way to reduce this effect is to cover the nanoparticle with a layer of PEG. This procedure is a technology customized under the name STHEALTH®.

STHEALTH nAu Uncoated nAu (on the left ) enters the phagocyte in very larger amount than PEG-coated nAu (on the right) of similar size and shape.

Surface functionalization In addition to the STHEALTH, nanodrugs can be functionalized through the addition of layers of antibodies directed to protein specifically expressed by the targed tissue, or of the substrate for specificaly bounding receptors. Modular molecules are designed, able to disassemble gradually when approaching their target.

Clinical advantages The main clinical advantages of therapy with nanosized and functionalized drugs are: Higher concentrations of the active drug at the site of action Possible targeting to desired cellular type to be targeted, or even to selected cellular districts Lower general toxicity of the active principle

Major toxicity hazard The toxicity of the nanomaterial itself is mostly unknown, it is not possible to infer it from the properties of the equivalent bulk material Adequate models for toxicity studies “in vivo” and in humans are mostly lacking The dissolvation of elements from complex nanomaterials is at the present unpredictable, especially in complex environment like the fluids of the body.

Nanoparticles can enter the cell. Co 3 O 4 nanoparticles form small aggregates inside the cell. Courtesy Lab. Cell Biol. University of Insubria

Nanoparticles can be toxic for the cells Courtesy Lab. Cell Biol. University of Insubria

Main fields of exploitation in human clinics Cancer, especially if advanced, refractory or affecting poorly accessible tissues Drug-resistant, life-threatening bacterial and parasite infections Diseases affecting the posterior pole of the eye and the Central Nervous System (CNS)

Carriers for nanodrugs: lipid-based. From the left: liposomes and STHEALTH liposomes (embedded with PEG), liquid and solid lipid nanoparticles (LLN, SLN). Cattaneo et al J. Appl. Toxicol. 30: 730–744. DOI /jat.1609

A multilamellar liposome in equilibrium with planar membrane. This technology was conceived to get a system similar to the cell membrane bilayer, possibly integrated with it when used to carry chemicals inside the cell. Updated, customized technologies use both multi- and monolayered liposomes. (Pidgeon & McNeely, 1987, Biochemistry 26:17-29, modified) Liposomes Sucrose

TEM image of doxorubicine, an antineoplastic agent, embedded in bilayered liposomes (Doxil). (Gabizion et al., Eur. J. Pharm. Sci, 45: 388–398)

Pharmacokinetics Liposomal doxorubicin is partially protected from rapid renal clearance after three cycles (B), therefore the plasma levels increase (A). Data are taken in cancer experimentally induced in mice. (Gabizion et al., Eur. J. Pharm. Sci, 45: 388–398)

Effect on experimental cancer Doxorubicin in tissues appears red-orange. Soluble doxorubicin (A) does not accumulate in tumoral nodules. The nanoformulation of the drug, embedded in bilayered liposomes (B), clearly accumulates. (Gabizon et al., Eur. J. Pharm. Sci, 45: 388–398)

1.Nanogold, nAu 2.Nanosilver, nAg 3.UltraSmall Paramagnetic Iron Oxides (USPIO) For diagnostics and therapeutics. USPIO greatly enhance the signal of (Magnetic Resonance Imaging (MRI) Some metallic nanodrugs.

nAu coated with TNF (Aurimune) in oncology Au nanoparticles aggregates inside the tumoral cells

The Combidex: an USPIO for cancer diagnostics. Combidex is a customized formulation of dextran- coated USPIO. The pictures show its 3D structure (the iron oxide cluster at the center of dextran molecules is in violet- blue) and the aspect of particles at TEM. The particles mean diameter is 21 nm.

Control Contrast: Gd Contrast: Combidex USPIO in cancer diagnostics. Enhanced MRI of metastatic cancer in the brain. From the left: without contrasting agents, with Gd as contrast, with USPIO (Combidex).

Functionalized Iron oxide as a targeted carrier for drugs Boyer et al., NPG Asia Materials, 23–30 (2010) | doi: /asiamat

Size-dependent Magnetic properties of IONPs doi: /asiamat ; doi: /ja A)TEM of differently sized Iron Oxide NanoParticles (IONPs) B)Size-dependent T2-weighted MR images of IONPs in aqueous solution at 1.5 T C)As before, color-coded D)Graph of T 2 value versus size of water soluble IONPs. E)Magnetization of water soluble IONPs measured by a SQUID magnetometer.

Mesoporous silicon, with nanopores, shows properties useful for: 1.Sustained, localized and prolonged release of drugs 2.Enhanced reconstruction of tissues through cell growth stimulation or promoting accelerated mineralization of bones. Carriers for nanodrugs: bioactive silicon.

Carriers for nanodrugs: organic compounds. Those exploited for the use in humans are in the following categories: 1.Polymers (polylactide, polyglycolide) 2.Dendrimers (polyamidoamides) 3.Albumin nanotubes 2 3 J. Appl. Toxicol. 2010, 30: ; wileyonlinelibrary.com/journal/pat

Theranostics and “modular” nanoparticles. Theranostics: combining diagnosis with therapy. NPs with high imaging properties and able to kill the cell when activated (es. light sensitive molecules) are coated to prolonge their half-life, conjugated at the surface to be targeted to specific cells (e.g. tumoral) and with molecules improving the uptake into the target cell. Or: NPs able to kill the cells (e.g. radioactive isotopes) are coated and functionalized for targeting, and injected locally in the bloody supply of the tumor. Or: NPs with high imaging properties (e.g. USPIO) are coated and functionalized for targeting and killing the cell, than directed to the target by functionalization or by a directional magnetic field.

NP Protective coating Cell permeabilization agent Sensor (e.g. Ab to recognize tumor cell) An hypothetical modular nanocarrier. And a scheme of how it works….

Degradation Signal for imaging DIAGNOSIS Activation Cell damage: THERAPY Binding to the target

A modular nanoparticle for “in situ” cancer treatment Cai & Chen, 2007, Small, 3: 1840 (modified) Monoclonal antibodies (ChL6) PEG Dextran Isotope: In111 (with chelator, DOTA) 20 nm Iron oxide

Binding to the target Ionizing radiation: Cell death Therapy Degradation Internalization of IONPs Internalization of In111 Enhanced signal for NMI Diagnosis

1.Tissues and coating releasing nanometals (e.g. nAg, with antibacteric properties) 2.Creams eluting active, nanosized compounds (nAg for antibacteric gels, nTiO 2 for solar creams) 3.Drugs eluting devices (silicon scaffolds for wluting drugs to the the posterior pole of the eye, central venous catheter with nAg). Other nanosized materials

How to test toxicity? “in silico”: nano-QSAR and PSAR (pseudo-structure-activity-relationships) “in vitro”: toxicity test on monocellular organisms, tissues and cells “in vivo”: toxicity tests on model organisms Metabolomics: newer methodology to get contemporary informations on a complete panel of biological parameters