Nanomedicine

Premises

• Since 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.

MAJOR BIOLOGICAL STRUCTURES

IN SCALE

NANOTECHNOLOGYFeynman: "There is plenty of room at the bottom"

• Seminal speech on December1959 at CalTech

• " Why can’t be compressed 24 volumes of EncyclopediaBritannica on a pin head ?“

• " The biological example of writinginformation on a small scale has inspired me to think of somethingthat should be possible "

• In 1990, IBM scientists wrote the logo IBM using 35 xenon atoms on nickel.

NANO ≈ < 100 nm

Nanomedicine:

EC European Technology Platform

(ETP)

E.C.-ETP

“Nanomedicine, is defined as the application ofnanotechnology to achieve breakthroughs inhealthcare. It exploits the improved and often novelphysical, chemical and biological properties ofmaterials at the nanometer scale. Nanomedicinehas the potential to enable early detection andprevention, and to essentially improve diagnosis,treatment and follow-up of diseases.……………………….

Nanomedicine:

European Science Foundation (ESF)

“The field of Nanomedicine is the science and technology ofdiagnosing, treating and preventing disease and traumaticinjury, of relieving pain, and ofpreserving and improving humanhealth, using molecular tools and molecular knowledge of the human body. It embraces sub-disciplines which are in many waysoverlapping and are underpinnedby common technical issues.”

The numbers of nanomedicine

The global nanomedicine market

reached $63.8 billion in 2010 and

$72.8 billion in 2011. The market

is expected to grow to $130.9

billion by 2016 at a compound

annual growth rate (CAGR) of

12.5% between years 2011 and

2016.

The “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.

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 development of nanomedicine-based treatments and products

that are able to directly target tumors in the brain and other bodily

sites is poised to be a significant factor affecting growth in this

market.

Though the largest market segment within the nanomedicine

market is that of oncology, 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.

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Number of publications related to “nanomedicine” in Medline

1995 1997 1999 2001 2003 2005 2007 2009 2011 2013

1966

Topics in nanomedicine

• Therapy:

Drug Delivery: Use nanodevices specifically

targeted to cells, to guide delivery of drugs,

proteins and genes

Drug targeting : Whole body, cellular ,

subcellular delivery

Drug discovery : Novel bioactives and

delivery systems

Topics in nanomedicine

• Diagnosis:

Prevention and Early Detection of diseases: Usenanodevices to detect specific changes in diseased cells and organism.

Nanoparticles (NP):

Smart Nanostructures for diagnosis

and therapy

Why Nanoparticles

1) Drugs, contrast agents,

paramagnetic or radiolabeled

probes can be vehiculated by NPs

2) NPs can be multi-functionalized

to confer differents features on

them

• Targeting: nanoparticles control the drug delivery.

The drug follows the NP

• Drugs are concentrated to target. Less systemic

toxicity.

• Less drug is necessary

• Drugs are protected inside NPs and are not degraded.

Longer drug halflife (if NP have long halflife).

• Biologicals (proteins, genes, Antibodies) are most

favourable candidates for NP

1) Drugs, contrast agents, paramagnetic or

radiolabeled probes can be vehiculated by NPs

• Multi-functionalization: Controls drug targeting,

drug dosage, and drug release characteristics

2) NPs can be multi-functionalized to confer

differents features on them

An ideal Multi-functional nanoparticle vector

Anticorpo

Indirizza la NP verso un antigene specifico sulla cellula da colpire

Polietilenglicol(PEG)

Evita che la NP venga rimossa dal circolo

Evita che NP venga digerita nei lisosomi

Tat peptide

Determina Fusione e ingresso della NP nella cellula

Probe magnetico

Permette imagingtramite MRI

Farmaco

LIPOSOMES

DENDRIMERS

MICELLES

NANOTUBES

GOLD NP MAGNETIC NP

QUANTUM DOTS SILICA NP

SOLID‐LIPID NP

POLYMERIC NP

POLYMERIC MICELLE

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LIPOPLEX

Examples of nanoparticulate carriers

LIPID-BASED

POLYMERIC

METALLIC

Carbon-based: Buckyballs and

Nanotubes

C60 1nm

What are Carbon Nanotubes?

Carbon nanotubes are

hexagonally shaped

arrangements of carbon

atoms that have been

rolled into tubes.

Human hair fragment

(the purplish thing) on

top of a network of

single-walled carbon

nanotubes

Nanotubes are members of the

fullerene structural family, which

also includes the spherical

buckyballs. Their name is derived

from their size, since the diameter

of a nanotube is on the order of a

few nanometers, while they can be

up to tenths of centimeters in

length

Nanotubes are categorized as

single-walled nanotubes (SWNTs)

and multi-walled nanotubes

(MWNTs)

Single-walled

• Most single-walled

nanotubes (SWNT) have

a diameter of close to 1

nanometer, with a tube

length that can be many

millions of times longer..

Armchair (n,n)

• Single-walled nanotubes

are an important variety

of carbon nanotube

because they exhibit

electric properties that

are not shared by the

multi-walled carbon

nanotube (MWNT)

variants.One useful

application of SWNTs is

in the development of the

first intramolecular field

effect transistors (FET).

• (Used for

nanobiosensors).

Multi-walled• Multi-walled nanotubes (MWNT)

consist of multiple rolled layers

(concentric tubes) of graphite.

• In the Russian Doll model, sheets

of graphite are arranged in

concentric cylinders, e.g. a (0,8)

single-walled nanotube (SWNT)

within a larger (0,10) single-walled

nanotube.

• In the Parchment model, a single

sheet of graphite is rolled in

around itself, resembling a scroll

of parchment or a rolled

newspaper. The interlayer

distance in multi-walled

nanotubes is close to the distance

between graphene layers in

graphite, approximately 3.4 Å.

Properties of Carbon

Nanotubes

Nanotubes have a very broad range of electronic,

thermal, and structural properties that change

depending on diameter, length. They exhibit

extraordinary strength and unique electrical

properties, and are efficient conductors of heat.

Strength

• Carbon nanotubes are the strongest

and stiffest materials yet discovered in

terms of tensile strength and elastic

modulus respectively. This strength

results from the covalent sp2 bonds

formed between the individual carbon

atoms. In 2000, a multi-walled carbon

nanotube was tested to have a tensile

strength of 63 gigapascals (GPa).

(This, for illustration, translates into the

ability to endure tension of a weight

equivalent to 6422 kg on a cable with

cross-section of 1 mm2.) Since carbon

nanotubes have a low density for a

solid of 1.3 to 1.4 g·cm−3, its specific

strength of up to 48,000 kN·m·kg−1 is

the best of known materials, compared

to high-carbon steel's 154 kN·m·kg−1.

Electrical properties

• Depending how the graphene sheet

is rolled up, the nanotube can be

metallic; semiconducting or moderate

semiconductor.

Thermal property• All nanotubes are expected to be very good

thermal conductors along the tube,

exhibiting a property known as "ballistic

conduction," but good insulators laterally to

the tube axis.

Defects

• As with any material, the existence of a

crystallographic defect affects the material

properties. Defects can occur in the form of

atomic vacancies. High levels of such defects can

lower the tensile strength by up to 85%.

Crystallographic defects also affect the tube's

electrical properties. A common result is lowered

conductivity through the defective region of the

tube.

Natural, incidental, and

controlled flame environments• Fullerenes and carbon nanotubes are not

necessarily products of high-tech laboratories;

they are commonly formed in such places as

ordinary flames,produced by burning

methane,ethylene,and benzene,and they have

been found in soot from both indoor and outdoor

air. However, these naturally occurring varieties

can be highly irregular in size and quality

because the environment in which they are

produced is often highly uncontrolled.

Potential and current

applications of CNT

In electrical circuits

• Nanotube based transistors

have been made that operate

at room temperature and that

are capable of digital switching

using a single electron.The first

nanotube integrated memory

circuit was made in 2004.

Nanotube Transistor

Proposed as a vessel for transporting drugsinto the body. The ends of a nanotube might be capped with a

hemisphere of the buckyball structureThe drug can be attached to

the side or trailed behind, or the drug can actually be placed inside

the nanotube.

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NanotubeNanocap

Covalent Functionalization

Non-Covalent Functionalization

Non-covalent funzionalization (DNA)

ToxicityTheir final usage, however, may be limited by

their potential toxicity.

Results 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, a cancer of the lungs, or

peritoneal mesothelioma (both caused by exposure

to asbestos). Available data suggest that under

certain conditions, especially those involving

chronic exposure, carbon nanotubes can pose a

serious risk to human health.

Lipid-based NPs :Liposomes and

solid lipid nanoparticles (SLN)

50 – 500 nm 40-1000nm

•LIPOSOMES are the smallest spherical structure technically produced by natural non-toxic

phospholipids and cholesterol.

Metal-core nanoparticles

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.

In cancer research, colloidal gold can be used to target

tumors and provide detection using SERS (Surface

Enhanced Raman Spectroscopy) in vivo.

They are being investigated as photothermal converters

of near infrared light for in-vivo applications, as ablation

components for cancer, and other targets since near

infrared light transmits readily through human skin and

tissue

Polymeric/Dendrimers

(e.g.PLGA, PAA, PACA)

spherical polymers of uniform

molecular weight made

from branched monomers are proving

particularly adapt at providing

multifunctional modularity.

Polymeric

PLGA Poly-Lactic-Glycolic Acid

PLGA POLY-LACTIC-GLYCOLIC ACID

Polyacrylamide (PACA)

In solvente organico In acqua

Dendrimers are repetitively

branched molecules.

PLGA

PAA

PAA= POLI

AMMINO AMMIDE

Polyamidoamines (PAA or PAMAM)

HYDROGELSCo-polymers (e.g. acrylamide and acrylic acid) create water-

impregnated nanoparticles with pores of well-defined size.

Water flows freely into these particles, carrying proteins and other small

molecules into the polymer matrix.

By controlling the pore size, huge proteins such as albumin and

immunoglobulin are excluded while smaller peptides and other

molecules are allowed.

The polymeric component acts as a negatively

charged "bait" that attracts positively

charged proteins, improving the particles'

performance.

Mesoporous

silica (SiO2)

Mesoporous silica particles: nano-sized spheres filled with a regular

arrangement of pores with controllable pore size from 3 to 15nm and outer

diameter from 20nm to 1000 nm .

The large surface area of the pores allows the particles to be filled with a

drug or with a fluorescent dye that would normally be unable to pass

through cell walls. The MSN material is then capped off with a molecule that

is compatible with the target cells. When are added to a cell culture, they

carry the drug across the cell membrane.

These particles are optically transparent,

so a dye can be seen through the silica walls.

The types of molecules that

are grafted to the outside will control what

kinds of biomolecules are allowed inside

the particles to interact with the dye.

EM

Quantum dots

3 nm

Dots are crystalline fluorophores made of binary compounds such as

lead sulfide, lead selenide, cadmium selenide, cadmium sulfide,

indium arsenide, and indium phosphide.

Dots may also be made from ternary compounds such as cadmium

selenide sulfide. These quantum dots can contain as few as 100 to

100,000 atoms within the quantum dot volume, with a diameter of 10

to 50 atoms. This corresponds to about 2 to 10 nanometers.

A quantum dot is a semiconductor whose excitons are

confined in all three spatial dimensions.

An immediate optical feature of colloidal quantum

dots is their coloration

First attempts have been made to use quantum dots

for tumor targeting under in vivo conditions.

Generically toxic

61

High quantum yield compared to common fluorescent dyes

Broadband absorption: light that has a shorter wavelength than

the emission maximum wavelength can be absorbed, peak

emission wavelength is independent of excitation source

Tunable and narrow emission, dependent on composition and

size

High resistance to photo bleaching: inorganic particles are more

photostable than organic molecules and can survive longer

irradiation times

Long fluorescence lifetime: fluorescent of quantum dots are 15

to 20 ns, which is higher than typical organic dye lifetimes.

Improved detection sensitivity: inorganic semiconductor

nanoparticles can be characterized with electron microscopes

Quantum Dot Properties

Quantum Dots

• Raw quantum dots are toxic

• But they fluoresce brilliantly, better than dyes

(imaging agents)

• Only way of clearance of protected QDs from the body

is by slow filtration and excretion through the kidney

Quantum Dots

• QD technology helps cancer researchers to observe fundamental

molecular events occurring in the tumor cells by tracking the

QDs of different sizes and thus different colors, tagged to

multiple different biomoleules, in vitro by fluorescent

microscopy.

• QD technology holds a great potential for applications in

nanobiotechnology and medical diagnostics where QDs could be

used as labels.

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Figure 2. Phase contrast images (top row) and

fluorescence image NIH-3T3 cells incubated with QDs2;

(c) SKOV3 cells were incubated with QDs2

FPP-QDs specifically bind to tumor cells via the membrane expression of

FA receptors on cell surface

Quantum Dots for Imaging of Tumor Cells

Y. Zhao et al. Journal of Colloid and Interface Science 350 (2010) 44–50.

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Quantum dots conjugated with folate–PEG–PMAM

for targeting tumor cells

Folate–poly(ethylene glycol)–polyamidoamine ligands encapsulate and solubilize

CdSe/ZnS quantum dots and target folate receptors in tumor cells.

Dendrimer ligands with multivalent amino groups can react with Zn2+ on the surface

of CdSe/ZnS QDs based on direct ligand-exchange reactions with ODA ligands

Y. Zhao et al. Journal of Colloid and Interface Science 350 (2010) 44–50.

QD nanocrystals are highly toxic to cultured cells under UV

illumination. The energy of UV irradiation is close to that of the

covalent chemical bond energy of CdSe nanocrystals. As a result,

semiconductor particles can be dissolved, in a process known as

photolysis, to release toxic Metal ions into the culture medium. In

the absence of UV irradiation, however, quantum dots with a stable

polymer coating have been found to be essentially nontoxic. NP

encapsulation of quantum dots allows for quantum dots to be

introduced into a stable aqueous solution, reducing the possibility of

Metal leakage.Then again, only little is known about the excretion

process of quantum dots from living organisms.. ] These and other

questions must be carefully examined before quantum dot

applications can be approved for human clinical use.

Brand name Description

Emend

(Merck & Co. Inc.)

Nanocrystal (antiemetic) in a capsule

Rapamune

(Wyeth-Ayerst Laboratories)

Nanocrystallized Rapamycin (immunosuppressant) in a

tablet

Abraxane

(American Biosciences, Inc.)

Paclitaxel (anticancer drug)- bound albumin particles

Rexin-G

(Epeius Biotechnology

corporation)

A retroviral vector carrying cytotoxic gene

Olay Moisturizers

(Procter and Gamble)

Contains added transparent, better protecting nano zinc

oxide particles

Trimetaspheres (Luna Nanoworks) MRI images

Silcryst

(Nucryst Pharmaceuticals)

Enhance the solubility and sustained release of silver

nanocrystals

Nano-balls

(Univ. of South Florida)

Nano-sized plastic spheres with drugs (active against

methicillin-resistant staph (MRSA) bacteria) chemically

bonded to their surface that allow the drug to be dissolved

in water.

Nano-particulate pharmaceuticals

Company Product

• CytImmune Gold nanoparticles for targeted delivery of drugs to tumors

• Nucryst Antimicrobial wound dressings using silver nanocrystals

• NanobiotixNanoparticles that target tumor cells, when irradiated by xrays the

nanoparticles generate electrons which cause localized destruction of the tumor

cells.

• Oxonica Disease identification using gold nanoparticles (biomarkers)

• Nanotherapeutics Nanoparticles for improving the performance of drug delivery

by oral, inhaled or nasal methods

• NanoBio Nanoemulsions for nasal delivery to fight viruses (such as the flu

and colds) and bacteria

• BioDelivery Sciences Oral drug delivery of drugs encapuslated in a

nanocrystalline structure called a cochleate

• NanoBioMagnetics Magnetically responsive nanoparticles for targeted drug

delivery and other applications

• Z-Medica Medical gauze containing aluminosilicate nanoparticles which help bood

clot faster in open wounds

Some liposome -based pharmaceuticals

Open Problems

Manufacturing NPs for medical use:

Putting the drug on the particleAssessment of NPs:

Dynamic structural

features in vivo

Kinetics of drug

release

Triggered drug release

Maintaining the drug in the particle

Making the drug come off the

particle once application is done

Purity and homogeneity of

nanoparticles

Open Problems

Toxicity:

short term - no toxicity in animals

long term- not known

Toxicity for both the host and the environment should be addressed

Open Problems

Delivery:

Ensuring Delivery to target

organ/cell

SOLUTION:

detection of NPs

at target, organs ,

cells , subcellular

location et al.

Tissue

distribution

Removal of nanoparticles from the body

Open Problems:

Targeting the brain

• Brain micro-vessel endothelial cells build

up the blood brain barrier (BBB)

• The BBB hinders water soluble molecules

and those with MW > 500 from getting into

the brain

NPs

The blood-brain

barrier (BBB)

Open Problems

GMP Challenges

• No standards for:

Purity and homogeneity of nanoparticles

Manufacturing Methods

Testing and Validation

Good manufacturing practices (GMP) are the practices required in order to

conform to guidelines recommended by agencies that control authorization

and licensing for manufacture and sale of food, drug products, and active

pharmaceutical products. These guidelines provide minimum requirements

that a pharmaceutical or a food product manufacturer must meet to assure that

the products are of high quality and do not pose any risk to the consumer or

public.

Good manufacturing practices, along with good laboratory practices and

good clinical practices, are overseen by regulatory agencies in the United

States, Canada, Europe, China, and other countries.

• Toxicities of nanomaterials are unknown

• to best target the nanomaterials so that systemic administration can be used

• to uncage the drug so it gets out at the desired location

• to “re-cage” the drug when it is no longer desired

• Removal of nanoparticles from the body

• Mathematical modeling of nanostructures

• Barrier crossing (BBB, G.I., et al.)

• GMP production

Summary