Presentazione di PowerPoint

Nanomedicine

PremisesSince the human body is basically an extremely complex system of interacting molecules (i.e., a molecular machine), the technology required to truly understand and repair the body is the molecular machine technology : nanotechnology A natural consequence of this level of technology will be the ability to analyze and repair the human body as completely and effectively as we can repair any conventional machine today: nanomedicine

NANO 1 - 100 nm

MAJOR BIOLOGICAL STRUCTURES IN SCALE

The numbers of nanomedicineThe Nanomedicine Market Global Industry Analysis, Size, Share, Growth, Trends and Forecast, 2013 - 2019," predicts that the total nanomedicine market globally will be worth USD 177.60 billion by 2019.

Number of publications related to nanomedicine in Medline1995 1997 1999 2001 2003 2005 2007 2009 2011 2013

6

Topics in nanomedicineTherapy: Drug Delivery: Use nanodevices specifically targeted to cells, to guide delivery of drugs, proteins and genesDiagnosis n vivo and in vitro : Prevention and Early Detection of diseases:Use nanodevices to detect specific changes in diseased cells and organism.Reparative/Regenerative Medicine

7

Nanoparticles (NP): Smart Nanostructures for diagnosis and therapy

Why Nanoparticles1) Drugs, contrast agents, paramagnetic or radiolabeled probes can be vehiculated by NPs2) NPs can be multi-functionalized to confer differents features on them

An ideal Multi-functional nanoparticle vectorAntibody

Targeting

Polietilenglycol(PEG)

StealthPrevents lysosomal digestion

Tat peptide

Cell intrusionProbe

Imaging MRI / PETDrug

Examples of nanoparticulate carriersLIPID-BASEDPOLYMERICMETALLIC

11

Liposomes

50 500 nm Composed by natural non-toxic phospholipids and cholesterol.

ExtrusionUnilamellar liposomes are formed by pushing MLV through polycarbonate microfilters in extruders, which results in the narrow distribution in size of the liposomal population.

Liposofast Extruder

13

Metal-core nanoparticles (Gold / Iron oxide

gold nanoparticles (1-20 nm) are produced by reduction of chloroauric acid (H[AuCl4]),

To the rapidly-stirred boiling HAuCl4 solution, quickly add 2 mL of a 1% solution of trisodium citrate dihydrate, Na3C6H5O7.2H2O. The gold sol gradually forms as the citrate reduces the gold(III). Remove from heat when the solution has turned deep red or 10 minutes has elapsed.

Polymeric(e.g.PLGA, PAA, PACA)spherical polymers of uniformmolecular weight madefrom branched monomers are provingparticularly adapt at providingmultifunctional modularity.

Mesoporous silica (SiO2)

Nanobodies: antibodies derived from camelidae (camels and llamas), much smaller than traditional antibodies. Antibodies are a conglomerate of two heavy protein chains and two light chains. Nanobodies are about a tenth the size of human antibodies and just a few nanometers in length. Nanobodies are being researched for multiple pharmaceutical applications These shortened proteins are more chemically agile, able to engage targets--including the active sites of enzymes and clefts in cell membranes--too small to admit an antibody.Because nanobodies are more resistant to heat and pH, may retain their activity as they pass through the gastrointestinal tract, raising the prospect of oral nanobody pills

The leading application segment in the past years within the nanomedicine market was that of oncology, holding a 38% share of the overall market in 2012, as a vast number of commercially available products prevail in this sector.

The fastest growing segment is the cardiovascular market. Growth in this segment has been fuelled by the presence of a sizeable patient population, and a simultaneous growth in the demand for device and drugs that are based on nanomedicine. These factors are collectively anticipated to further fuel the growth of the cardiovascular segment within the nanomedicine market.MEDICAL APPLICATIONS

2014:67 commercialized nanodevices 43 marketed nanotherapeutics

A total of 25 devices and 122 therapeutics currently in development accounted for 789 ongoing clinical trials

Intravenous/intraperitoneal injectionis the most common administration

NP kinetics depends on size charge and functional coating.Tissues with a leaky endothelial wall contribute to a significant uptake of NP. In liver, spleen and bone marrow, NP uptake is also due to the macrophages residing in the tissues (RES) .

What happens when NP enter the blood?

Surface modification is necessary to avoid the removal by RES

Stealth Nanoparticles INVISIBLE TO THE RES

Monuclear phagocyte system (MPS) is the major contributor for the clearance of nanoparticles. Reducing the rate of MPS uptake by minimizing the opsonization is the best strategy for prolonging the circulation of nanoparticles..Surface modifications

PEGylated NP in Brush configuration attract less Opsonins from plasma

MetabolismInert NP are not metabolized (gold and silver, fullerenes, carbon nanotubes).Functionalized or biocompatible NP can be metabolized effectively by enzymes in the body, especially present in liver and kidney.The intracellularly released drug is metabolized according to the usual pathways.

Functionalization for targeting

Targeting moleculesA. antibodiesB. PeptidesC. AptamersD. Other ligands

Ligand should be selected to target cell membrane surface molecules that 1)are physiologically overexpressed on healthy target organs or cells ( e.g. Transferrin receptor on blood brain barrier)or2) are oversexpressed as a consequence of a pathology (e.g. tumour markers)

Examples of antibodies against:

Receptors: Vascular endothelial growth factor (VEGF); folate (highly expressed in tumours); Transferrin, opiod peptides (Brain), Apolipoproteins ( Brain) , Human epidermal growth factor (EGF)

v3 IntegrinMatrix metalloproteinases

Aptamer target protein or moleculeApplicationPSMAProstate cancer diagnosis and therapyWT1Wilm's tumor 4,4-methylenedianiline Detecting DNA-damaging compoundsVEGFInhibiting angiogenesisRET Inhibition of pro-growth signalingHER-3Reducing drug resistance in HER+ cancersTCF-1Colon cancer growth inhibitionTenascin-CGlioblastoma (brain cancer) detectionMUC1 Breast, pancreatic, ovarian cancersPDGF/PDGFRImproving transport to tumors and targeting brain cancersNF-B Targeting a transcription factor implicated in many diseasesRaf-1Inhibiting pro-growth signalingv3 integrinTargeting tumor-associated vasculatureHuman keratinocyte growth factorInhibiting pro-growth signali

Drug release from Nanoparticles

Stimuli-responsive NPs

pHLightTemperature Ultrasound Magnetic fieldEnzymes

pH in living systemsCompartment pHGastric acid1Lysosomes4.5Granules of chromaffin cells5.5Human skin5.5Urine6.0Cytosol7.2Cerebrospinal fluid (CSF)7.5Blood7.347.45Mitochondrial matrix7.5Pancreas secretions8.1Solid tumours 6.5

pH Sensitive

Microwaves

39C41C43C39C39C

CANCER THERAPY/DIAGNOSIS

Tumors generally cant grow beyond 2 mm in size without becoming angiogenic (attracting new capillaries) because difficulty in obtaining oxygen and nutrients. Tumors produce angiogenic factors to form new capillary structures.Permeability-enhancing factors such as VEGF (vascular endothelial growth factor) are secreted to increase the permeability of the tumor blood vessels.In solid tumors the uptake of NP depends on the so-called enhanced permeability and retention effect (EPR). EPR, in principle, is based on passive targeting

38

How EPR works

1- nanovehicles passively target to vasculature and extravasate through fenestrated tumor vasculature.

2- the extended circulation time (stealth features) allows accumulation in tumor tissue

3- lack of lymphatic drainage prevents removal of nanoparticles after extravasation

The use of nanoparticulate pharmaceutical drug delivery systems (NDDSs)to enhance the effectiveness of drugs is now well established. The developmentof multifunctional and stimulus-sensitive NDDSs is an active area of current research.Such NDDSs can have long circulation times, target the site of the disease and enhancethe intracellular delivery of a drug. This type of NDDS can also respond to local stimulithat are characteristic of the pathological site by, for example, releasing an entrappeddrug or shedding a protective coating, thus facilitating the interaction betweendrug-loaded nanocarriers and target cells or tissues.

Research in Cardio Vascular area

Nitric Oxide (NO)-releasing nanoparticles

NO produced by endothelial cells (eNOS) plays a key role in the cardiovascular system by controlling blood flow and blood pressure and inhibiting thrombus formation. eNOS produces pico-to nanomolar concentrations of NO for short periods of time to promote vasodilation, thereby increasing blood flow.

The combination of NO and nanocarriers has emerged as an efficient approach to overcome the difficulties associated with the biomedical administration of NO.NO acts as a key modulator in cardiovascular, immunological, neurological, and respiratory systems, and deficiencies in the production of NO or its inactivation has been associated with several pathologic conditions, ranging from hypertension to sexual dysfunction.

EXAMPLES

1- Sustained NO-releasing coated stents to reduce the risk of restenosis after implantation. An LMW-NO donor was encapsulated into polymeric NPs and used to coat the stent surface to prevent restenosis (Acharya et al., 2012). PLGA ( poly(lactic-co-glycolic acid), PEG and PCL are biocompatible, biodegradable and FDA-approved materials that have been used.NO eluting from polymer-coated stents revealed an initial burst on the first day ofmonitoring, in which 3047% of the drug was released, followed by a sustained release over a period of 11 days. As a consequence, NO-eluting polymeric stents were shown to preventplatelet activation and adhesion, indicating their potential to combat restenosis formation.

However scanning electron microscopy (SEM) images reveal an irregular and rough polymer-coated stent. It should be noted that, for clinical applications, it is of fundamental importance to obtain a homogenous surface for the coated stent. Therefore, techniques to generate NO-eluting stents are still being optimized.

2- Stents were coated with a polymeric bilayer construct able to release NO (Liu et al., 2013). The polymeric bilayer was composed of PLGA NPs containing the hydrophobic NO donor N-nitrosomelatonin (NOMela) and collagen top-coat; the stents were coated via either dip-coating or electrophoretic deposition techniques.

NO release from the coated stents reduced platelet aggregation in rabbit blood when compared with bare control stents. Moreover, the NO-releasing stent alleviated intima formation and thrombosis at the implanted sites.

However, real-time NO amperometric detection from coated stents in the descending aorta of rats revealed a release profile of a few seconds (Liu et al., 2013. Therefore, further investigation to successfully develop a temporal NO-eluting stent is required.

3- Calcium accumulation in heart valves leads to dysfunction in the regulation of blood flow at the heart's chambers.

LMW- NO donor diethylenetriamine diazeniumdiolate (DETA NONOate) containing-PLGA NPs (average size 300 nm) was embedded in a polymeric matrix. (Acharya et al., 2012b). The NO release profile showed a prolonged release (~3 weeks) after an initial burst in the first 24 h. In fact,within 3weeks, 55% of the drug was released, illustrating the prolonged kinetics of NO released from NPs. Prolonged NO release from polymeric NPs significantly decreased the accumulation of calcium in valve interstitial cells in osteoblastic medium This sustained NO release from NPs significantly increased the levels of cGMP, indicating that the observed anticalcification activity reported can be attributed to the NOcGMP signaling pathway).

4- NPs composed of polysiloxane have been utilized to influence the NO release profile from eNOS in human aortic endothelial cells (HAECs) (Nishikawa et al., 2009).Polysiloxane spontaneously forms NPs in aqueous solution due to its amphiphilic characteristic (80 nm) .The cellular uptake of these polysiloxane NPs in HAECs was characterized by endocytosis via caveolae, leading to the activation of eNOS and enhancement of NO release. This finding may be important for the design of new strategies to combat hypertension

Models toaccurately predict transvascular permeation of nanomedicinesare needed to aid in design optimization. Here we show that anendothelialized microchip with controllable permeability can beused to probe nanoparticle translocation across an endothelialcell layer.

BLOOD CLOTS/MYOCARDIAL INFARCTION

The leading drawbacks of thrombolysis and associated therapy by infusion of analogs of tissue plasminogen activator (tPA), other recombinant-based plasminogen activators (e.g., alteplase, reteplase, and tenecteplase), streptokinase, and urokinase (uPA) are represented by a significant burden of inefficacy combined with a high risk of bleeding complications.advantages of thrombus-targeted fibrinolysis, with nanoparticles:preferential localization within developing clots,enhanced safety due to substantial reduction of the dosage of fibrinolytic agents minimal extravasation and favorable clearance from the circulationdistribution of the drug within the clot, a more effective penetration, and an enhanced overall activity

Magnetically driven NanoparticlesMagnetic NP systems can be concentrated at predetermined sites under guidance of a sufficiently strong external magnetic fieldIn a rat arteriovenous shunt thrombosis model, the rate of thrombolysis obtained with uPA-loaded nanoparticles under exposure to magnetic fieldwas 2.6-fold more effective than uPA-loaded nanoparticleswith no exposure to magnetic field and 5.0-fold more effectivethan uPA alone.

For example, liposomes loaded with ATP or co-enzyme Q accumulated well in the infarcted areas of the myocardium and improved cardiac parameters inrat and rabbit models of myocardial infarct46,47. In a ratmodel of heart transplant, organ rejection was simultaneouslyimaged and treated using multifunctional polymericNDDSs co-loaded with superparamagnetic ironoxide nanoparticles (SPIONs) and plasmid DNA48. TheSPION acted as a magnetic resonance imaging (MRI)contrast agent, whereas theplasmid DNA suppressed the local immune response48.Blood clots NDDSs for targeted thrombolytic therapy were developed by conjugating a thrombolytic agent (recombinant tissue plasminogen activator) to dextran-coated iron oxide nanoparticles, which were additionally modified with a thrombus-targeted peptide that was sensitive to activated factor XIII49. These NDDSs had increased binding to the margins of intravascular thrombi and good thrombolytic activity in a mouse model of pulmonary embolism.

Ultrasound-activated NP Biological nanoparticles (polymeric, liposomes..) can be locally subjected to transcutaneous low-frequency ultrasound (typically set between 20 and 2,000 kHz) to release fibrinolytic agents in the site of targeted clots

rabbit model of carotid artery thrombosicoronary artery thrombosis produced in anesthetized dogs bythe injection of thrombins

streptokinase entrapped in a water-soluble polymer wasapproximately 2 and 10 times more effective in reducingclot mass and restoring arterial blood flow than liposomaland free streptokinase, respectively

shear-activated nanotherapeutics are synthesized by spray-dryingconcentrated solutions of biocompatible, biodegradable, polylactic-co-glycolic acid).SA-Particles are platelet-size aggregates (1 to 5 min diameter) that self- assemble from hydrophobic nanoparticles. Experiments with the use of a rheometer and microf luidic chambers show that Particles are stable under normal arterial shear stress but disaggregate at the high shear stresses that are typical of arterial stenosis.t-PA is retained inside SA-Particles when they pass through normal vasculature, but when they encounter pathologically high shear stress, they break into nanoparticles that expose the t-PA dose locally. High shear stress (> 100 dyn cm2) occurs at the apex of any clinically significant arterial stenosis, so the use of SA-NTs releases t-PA just where it is needed most, along the apex and just downstream of the stenosis, allowing the t-PA to bind to a growing thrombus

The results involving mouse models of acute arterial thrombosis and pulmonary embolism suggest that the total administered dose can be reduced by a factor of approximately 100, as compared with intravenously delivered t-PA. (Korin, Science 2012; Wootton , New England J Med, 2012) Shear- Activated (SA) particles

A- flow velocity and velocity gradients near the apex of the stenosis and slowerrecirculating flow behind the stenosisB-shear stress increasing to high stress (>100 dyn cm2) near the artery wall at the apex ofthe stenosisC- released nanoparticle flow pathsD- Particles are intact and expose minimal t-PA under normal arterial shear stresses but break down because of high shear stress near the stenosissurface

THERAPEUTIC-NEOVASCULARIZATIONRestoration of tissue perfusion in patients with critical limb ischemia is a major therapeutic goal.Recent clinical trials designed to induce neovascularization by administering exogenous angiogenic growth factors or cells failed to demonstrate a decisive clinical benefit. A controlled drug delivery system for a new approach to therapeutic neovascularization therefore would be more favorable.

Endothelial cell-selective delivery of pitavastatin nanoparticle increased the development of collateral arteries and improved exercise-induced ischemia in a rabbit model of chronic hind limb ischemia. Separate experiments with micedeficient for VEGF receptor tyrosine kinase demonstrated a crucial role of VEGF receptor signals in the therapeutic angiogenic effects.This nanotechnology platform is a promising strategy for the treatment of patients with severe organ ischemia and represents a significant advance in therapeutic arteriogenesis over current approaches.

Effects of pitavastatin nanoparticles (NP) on angiographicallyvisible collateral arterial development are shown 28 days aftertreatment. A, Effects of pitavastatin-NP containing 0.05, 0.15, or0.5 mg/kg pitavastatin on the angiographic score (n 3 each). B,Representative angiograms are shown of the phosphate buffered saline(PBS), pitavastatin-only, fluorescein isothiocyanate (FITC)-NP, andpitavastatin-NP groups at 28 days after treatment. Corkscrew-like collateralarteries were observed only in the pitavastatin-NP group. C,Summary of the angiographic scores obtained for the four groupsin panel B (n 6 each).

Cellular distribution of nanoparticles is shown in ischemicmuscles. A, Fluorescent photomicrographs show cross sections ofcontrol nonischemic muscle and ischemic muscles at 3, 7, and 28days after fluorescein isothiocyanate (FITC) nanoparticle (NP)injection. Nuclei were counterstained with 4=,6-diamidino-2-phenylindole (blue). Fluorescence microscopic settings (exposure,filter, excitation light intensity, etc.) were the same for all imagesScale bar 100 m. B, Photomicrographs of cross sections ofischemic muscle 28 days after FITC-NP injection stained immunohistochemicallywith the endothelial marker CD31 (red). MostFITC signals colocalized with the vascular endothelium (arrows).Scale bars 20 m.

DiagnosisUltrasound imaging beyond the vasculature with new generation contrast agentsMRI molecular imaging using GLUT1 antibody-Fe3O4 nanoparticles in the hemangioma animal model for differentiating infantile hemangioma from vascular malformationDiagnostics

NP have been shown to impact vascular endothelial cell (EC) integrity, which may disrupt the dynamic endothelial regulation of vascular tone, possibly altering systemic vascular resistance and impairing the appropriate distribution of blood flow throughout the circulation. Cardiac consequences of NP-induced toxicity include disruption of heart rate and electrical activity via catecholamine release, increased susceptibility to ischemia/reperfusion injury, and modified baroreceptor control of cardiac function. These and other CV outcomes likely contribute to adverse health effects promoting myocardial infarction, hypertension, cardiac arrhythmias, and thrombosis. This review will assess the current knowledge regarding the principle sites of CV toxicity following NP exposureTOXICITY ?

Carbon-based: Buckyballs and Nanotubes

C60 1nm

ToxicityResults of rodent studies show that CNTs produce inflammation, epithelioid granulomas (microscopic nodules), fibrosis, and biochemical/toxicological changes in the lungs. Comparative toxicity studies in which mice were given equal weights of test materials showed that SWCNTs were more toxic than quartz, which is considered a serious occupational health hazard when chronically inhaled. The needle-like fiber shape of CNTs is similar to asbestos fibers. This raises the idea that widespread use of carbon nanotubes may lead to pleural mesothelioma. Available data suggest that under certain conditions, especially those involving chronic exposure, carbon nanotubes can pose a serious risk to human health.

64

MECHANISMS OF TRANSPORT ACROSS THE BBBOnly molecules necessary for brain metabolism have access. + +

PassivediffusionCarrier-mediatedeffluxCarrier-mediatedinfluxReceptor-mediatedtranscytosisAdsorptive-mediatedtranscytosisOpening of the tightjunctionsLipid-solublenon-polarLipid-solubleamphiphilicdrugsGlucoseAmino acidsAminesNucleosidesSmall peptidesTransferrinInsulinHistoneAvidinCationised albumin

Polar

Cellmigration

1- CELL-PENETRATING PEPTIDE TAT-1. Exploiting the adsorptive mechanism of entry of HIV2-ANTI-Transferrin Receptor ANTIBODIES (OX-26, RI-7217). Exploiting the receptor-mediated mechanism of entry of Transferrin3-ApoE-DERIVED PEPTIDE (a.a. 141-150 monomer or dimer). Exploiting the lipoprotein-related receptor mediated and adsorptive mechanism ApoE-derived and TAT-1 PEPTIDE have been synthesized with a Cysteine terminal group. ANTI-TfR ANTIBODIES have been thiolated (Traut reaction)

LIGANDS TESTED (COVALENTLY LINKED TO PA-LIPOSOMES )

Nanorobotics is the technology of creating machines or robots at or close to the scale of nanometers. Alsoknown as nanobots or nanites, they would be constructed from molecularmachines. So far, researchers have mostly produced only parts of thesecomplex systems, such as bearings, sensors, and synthetic molecularmotors,

Possible applications include micro surgeryBy using a microrobot in the body to break up clots into smaller pieces or clean arthery walls Future: Nanorobots

A NANOROBOT

1966

LIPOSOMESDENDRIMERS MICELLESNANOTUBESGOLD NPMAGNETIC NPQUANTUM DOTSSILICA NPSOLID-LIPID NP

POLYMERIC NP

POLYMERIC MICELLE++++++++++++LIPOPLEX