marco a pierotti, claudio lombardo and camillo rosano- nanotechnology: going small for a giant leap...

8/3/2019 Marco A Pierotti, Claudio Lombardo and Camillo Rosano- Nanotechnology: going small for a giant leap in cancer dia

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OECI 2008

Scientific Conference

Nanotechnology applications in cancer

prevention and treatment


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Key words: nanotechnology, nano-

medicine, drug delivery, nanoparticles

Correspondence to: Camillo Rosano,

National Institute for Cancer Re-

search, L.go Rosanna Benzi 10, 16132

Genova, Italy.

Tel +39-010-5737 337

[email protected]

Nanotechnology: going small for a giant leap incancer diagnostics and therapeutics

Marco A Pierotti1, Claudio Lombardo2, and Camillo Rosano2

1Fondazione IRCCS Istituto Nazionale dei Tumori, Milan; 2Istituto Nazionale per la Ricerca sul Cancro,

Genoa, Italy

ABSTRACT

There is Plenty of Room at the Bottom - not just There is Room at the

Bottom.What I have demonstrated is that there is room - that you can

decrease the size of things in a practical way. I now want to show that

there is plenty of room.

Richard Feynman, December 29, 1959

More than 30 years ago Richard Feynman pointed out that physicists knew no limitsto prevent us from doing engineering at the level of atoms. Until recently, though,while the lack of physical limits was accepted as commonplace, molecular engineer-ing was thought of as impractical, unnecessary, or requiring breakthroughs in knowl-edge and technique that placed it somewhere in the distant future. Many visionariesintimately familiar with the development of silicon technology still forecast it wouldtake between 20 and 50 years before molecular engineering became a reality. This iswell beyond the planning horizon of most companies. But recently, everything hasbegun to change. After the industrial revolution and the computer age, are we real-ly facing a newera?

Introduction

Recent years have witnessed an unprecedented rapid growth in the area of biolog-ical sciences. In this context, the explosion of nanosciences could not have happenedin a better period. Nanotechnologies represent a paradigm change in the study of andinteraction with normal and cancer cells in real time and at a molecular scale. Al-though there is increasing optimism that nanotechnologies applied to medicine willbring significant advances in cancer diagnostics and therapy, many challenges havestill to be overcome. Nanotechnology is a concept that refers to the research and tech-nological development of different objects in a scale range of 1 to 100 nanometers. Inthis range, matter shows properties that are quite different from those seen in thebulk scale. Interestingly to biomedical scientists, nanotechnologies are opening newresearch avenues: the novel properties of nanomaterials are offering new possibilitiesto interact with complex biological functions operating at the very same scale of bio-molecules. In these first years of the 21st century, scientists have begun to understandthe unique atomic and molecular properties at this nanometer scale. Manipulation ofthe chemico-physical properties on this scale gives researchers the ability to buildand use nanoparticles for different purposes, as drug delivery vectors, image contrastagents, and diagnostic tools.

The OECI 2008 Scientific Conference focuses on the emerging field of nanobiotech-nology and involves leading European experts. The discussions and recommenda-tions that will be presented in Genoa should ensure continuing European cutting-edge research and development in the field of nanobiotechnology and nanomedicinewhilst reducing healthcare costs.

Tumori, 94: 191-196,2008


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icine. A number of possible carriers are currently being

studied (Figure 3).Colloidal drug carrier systems are encapsulated or

dispersed systems which typically have a diameter of10-400 nm. Their properties make these micellar sys-tems very promising13. High-molecular-weight micellarsystems are formed by self-assembly of short am-phiphilic block copolymers (5-50 nm) in aqueous solu-tions. Due to their structure, they are able to accumulatein solid tumors, using the enhanced permeability andretention (EPR) effect. The active molecules can be en-trapped in the core of micelles and transported even atconcentrations that can exceed their intrinsic water sol-

ubility14. Moreover, micelles can effectively protectdrugs (proteins, nucleic acids or polysaccharides) fromexternal hydrolysis and enzymatic degradation15.

Liposomes are a form of phospholipid vesicles con-sisting of different or just one phospholipid bilayer.Their diameter size ranges between 50 and 250 nm, thusenabling polar drug molecules to be encapsulated intheir core. These carriers display interesting propertiessuch as the ability to use the EPR effect to accumulatedrugs in solid tumors. Amphiphilic and lipophilic mole-cules could be solubilized within the phospholipid bi-layer. In this way channel proteins could be incorporat-

ed into a liposome shell without losing their activity.Acting as a size-selective filter, these proteins could al-low passive diffusion of small solutes such as ions, pro-tecting the drugs from degradation by proteolytic en-zymes. At the same time, the drug molecule is able todiffuse through the channel, driven by the differentconcentration between the core and the exterior of theliposome. While at first the liposomes showed high in-stability under physiological conditions resulting in to-tal release of the internal content (burst effect), the sta-bility improved thanks to the introduction of multi-vesicular liposomes.

Dendrimers are repeated, highly branched molecules

characterized by their symmetry and monodispersion.They consist of a central core, different symmetricalbranching units and functional groups on the molecu-lar surface. The most interesting aspect of dendrimers isthat their size and composition can be easily controlledby pH, temperature and concentration. The internalcore can be so designed as to determine the environ-ment of the cage occupied by the drug, whereas the ex-ternal groups determine the solubility and chemical be-havior of these polymers16. Dendrimers may also bebuilt around a template that can act as the core. It isthen possible to cross-link the structure and remove the

NANOTECHNOLOGY IN CANCER DIAGNOSIS AND TREATMENT 193

Figure 2 - A) The enhanced permeability and retention (EPR) effect.B) Active targeting of functionalized nanocapsules.

Figure 3 - Different examples of pharmaceutical carriers.

Tumour

Drug

Blood vessel

A

B

Micelles

Gold nanoshellRNA aptamer Carbon nanotube

Liposome Dendrimer


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template, creating a structure at the core of the den-drimer that has a specific binding site with the ability toseparate enantiomers. In addition, one can easily en-trap metal ions within dendrimers, thus creating fluo-rescent nanoparticles. By using a low concentration ofmetal-dendrimer composite in order to minimize toxic-

ity, it would then be possible to perform intracellularfluorescent assays17.

Nanoparticles are in the solid state and can be eitheramorphous or crystalline. They are able to encapsulatea drug, thus protecting it against enzymatic degrada-tion. Nanocapsules are vesicular systems consisting of ashell and an empty core in which the drug is confined, while nanospheres are matrix systems, presenting auniform cross-section in which the drug is physicallyand uniformly dispersed. The most studied nanoparti-cles for drug delivery are metallic nanoshells made of adielectric core and covered by a thin layer of metal. The

particular composition of metallic nanoshells (e.g.,gold-silica nanoshells) gives them unique physicalproperties such as light absorption in the near infraredregion, where the absorption of biological matter is low.Once the particle has reached the tumor site, externalradiation can exit the nanoshell and destroy the tumorby local thermal heating.

Hydrogels are 3-dimensional, hydrophilic polymernetworks (i.e., collagen, gelatin, dextrans etc.). They canswell and thus change their diffusion properties whenexposed to water or biological fluids. The networks canbe composed of homopolymers or copolymers. Thepresence of chemical cross-links (tie-points, junctions)

makes them insoluble. Hydrogels are used to regulatedrug release in the presence of a reservoir. It is possibleto build hydrogels that are sensitive to an appliedcharge, to particular enzymes or antigens, or to pHchanges, so that drug release can be programmed to oc-cur within specific areas of the body or via specific sites.

Covalent conjugation of biological (peptides/pro-teins) and synthetic polymers is another efficient way ofdrug delivery. The modification of proteins relies on theparticularity of the functional groups of some aminoacid residues such as lysines, cysteines, glutamic andaspartic acid, and tyrosines. The use of a polymer bio-

conjugated to an active peptide or protein can reducetoxicity, also preventing immunogenic or antigenic sideeffects. It is possible to use cavities to host other ele-ments like covalently bound small molecules. Recentlythe heat-shock protein has been modified by introduc-ing cysteine residues on the protein surface and thenselectively attaching an antitumoral drug, doxorubicin,to the thiol groups of these residues using a pH-sensi-tive linker. The modified protein assembled into a 24-subunit structure with a diameter of about 12 nm allowsthe exchange of small molecules between the interiorand the bulk solvent solution18,19.

194 MA PIEROTTI , C LOMBARDO, C ROSANO

Applications in imaging and early detection

Nanoparticle applications are not limited to the body:indeed, one of the most promising fields of application ofnanotechnology is the development of tools at thenanometer scale for in vitro diagnostics. Nanocantilevers

coated with antigens are studied for use as detectors ofantibodies and functionalized CNTs could be used also ashighly specific biosensors20. Another area with near-termapplicability is the detection of mutations and genomeinstability in situ. Nanopores are being investigated aspotential real-time DNA sequencers, making it possibleto distinguish among different types of tumors quicklyand accurately. Nanosensors could also be used to detectenvironmental and/or lifestyle cancer risk factors; thesetools will be useful not only to identify subjects at risk ofdeveloping cancer, but also to start new studies on gene-environment interactions and on the relationship be-tween these interactions and the development of (or the

resistance against) cancer.Besides the other applications of different nanoparti-

cles (gold nanoshells, quantum dots, CNTs), a discus-sion about the in vitro application of nanotechnologywould not be complete without an overview of the in-struments used to build these entities. Traditional spec-troscopy (FTIR and Raman) together with electron mi-croscopy are used to characterize the functionality, pu-rity and weight of molecules; atomic force microscopy(AFM) is the technique that mostly influenced the ad-vent of the nanotechnology era. The capability and ac-curate sensitivity of AFM derive from the use of ananoscaled probe attached to a cantilever which is able

to sense and measure the interatomic interactions withthe substrate. The spatial resolution of AFM is a fewngstrm (1 = 0.1 nm) and nowadays it is widely usedin the biomedical field to elucidate biomolecular struc-tures in conditions very close to physiological, measur-ing forces in bindings (i.e., recepors and ligands, anti-body and antigens), observing the topological surface ofviruses, and imaging histological features.

Some concerns

We have illustrated how nanotechnologies offer many

benefits and will continue to do so in the future; howev-er, they may affect our lives in a way that may not be theone researchers are aiming at. A public debate is need-ed about their development. In particular, there is astrong need to improve the understanding of the toxico-logical implications of nanomedicine in relation to thespecific properties of nanomaterials. Considerationshould be given to the environmental impact and to asafety assessment of the whole manufacturing process.A risk-benefit assessment is needed in respect to bothacute and chronic effects of nanomedicine applicationfor medical purposes.


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The toxicity of nanoparticles seems to be linked totheir surface and not to the mass. Available studiesshowed different effects on animals, depending on thetype of nanoparticles. In particular nephrotoxicity, ef-fects on reproduction, granulomas, fibrosis and tumorshave been observed. However, the toxicological data

specific to nanoparticles are insufficient due to thesmall number of studies and to the short exposure peri-od. Additional studies are necessary to assess the riskassociated with inhalation and cutaneus exposure tonanoparticles.

Nanobiotechnologies: a business?

Even though the science at the basis of the physico-chemical properties of nanotechnologies is poorly un-derstood if not mysterious, the commercial potential ofthe infinitesimally small is coming sharply into focus. In

recent years, large and small companies have intro-duced more products from the lab into the market. Peo-ple are already using nanotechnologies in several goods:car manufacturers employ nanoceramics in the con-struction of stronger and lighter turbines, advancedpaints and chassis, aeronautics will extensively useCNTs in the next generation of hypersonic airplanes;even golf balls designed to go straight will be built usingnanomaterials. Computer industries are planning with-in 10 years to revolutionize the technology of comput-ing by improving the power of silicon-based machines.The nanopharma industry of the future is late to arrive

but, as shown by experience, we need to invest today if we want to be present as leading actors in thenanoworld of personalized medicine.

Europe is currently among the world leaders in nan-otechnology but nanotechnology science in the Euro-pean Union needs a sustainable environment for re-search in order to remain globally competitive and real-ize its international potential. Securing future prosperi-ty and exploiting the value of the technology requiresthe development of a specific government fundingstrategy, with a central body to coordinate research ini-tiatives. This will minimize redundancy in research andfacilitate the development of technology that addresses

the most urgent socioeconomic needs of the planet. Es-tablishment of good communication is a universal chal-lenge for research and development, particularly foremerging technologies. There is a need to promotetransdisciplinary conferences and research partner-ships between large medical centers and universities.Moreover, it is mandatory to make serious efforts to en-sure that politicians are well briefed in the topic: betterdiagnostics, treatment and prevention will bring health-care benefits. Research and development in nanomedi-cine will also offer employment and economic benefitswith a parallel reduction of healthcare budgets.

Conclusions

It is generally felt that the technological basis of nan-otechnology is already developed enough to enablephysicians and biologists to make ready use of thesematerials. This is not completely true: there are still a lot

of fundamental questions concerning these materialsthat have to be answered. Standardization assays needto be developed and risks should be carefully evaluated.The integration of nanotechnology with cancer researchand diagnostics is a rapidly advancing field and there isan urgent need to understand the concepts arising fromthis new technology.

The OECI meeting in Genoa will define the remit ofthe emerging field of nanotechnology in oncology. Thisfield is clearly multidisciplinary and builds on expertisein a wide range of scientific areas, from physics to col-loidal chemistry, from molecular biology to membranebiophysics, from medicine to cell physiology. The

strengths of the European Union have been clearlyidentified in terms of short- and long-term opportuni-ties. An open dialogue has been launched to safeguardall interested parties, including industries and the gen-eral public. It is now important to clearly distinguishwhat is science from what is science fiction.

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