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Surface Modification of

Nanoparticles for

Biomedical Applications

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Multifunctionalization

• A nanomedical device should perform severalfunctions alltogether

• Consequently a multi-componentnanomedical system can be constructed in reverse order of controlling events, namelyfrom the inside out. The outer componentsare the first to be used. The innercomponents are the last.

Possible steps

It is possible but rare for a single moleculeto perform two or more functions

lipids, polimers (biocompatible-biodegradablematerials)Also water is available (liposomes)

The drug can be inserted into the core

Ligands for targeting

Targeting molecules

A. antibodiesB. PeptidesC. AptamersD. Other ligands

Ligand should be selected to target cell membrane surface molecules that1)are physiologically overexpressed on healthytarget organs or cells ( e.g. Transferrin receptoron blood brain barrier)or2) are oversexpressed as a consequence of a pathology (e.g. tumour markers)

Antibodies

• Antibodies directed against tissue-specificantigens.

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)

αvβ3 IntegrinMatrix metalloproteinases

APTAMERS

Aptamers are oligonucleotides that bind a specific target molecule. Aptamers are usually created by

selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches.

Aptamers can be used for both basic research and clinical purposes as macromolecular drugs.

aptamers offer advantages over antibodies asthey can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicitlittle or no immunogenicity in therapeuticapplications.

Aptamer target protein or molecule ApplicationPSMA Prostate cancer diagnosis and therapyWT1 Understanding Wilm's tumor

pathogenesis4,4′-methylenedianiline Detecting DNA-damaging compounds

VEGF Inhibiting angiogenesisRET Inhibition of pro-growth signalingHER-3 Reducing drug resistance in HER-2+ cancersTCF-1 Colon cancer growth inhibitionTenascin-C Glioblastoma (brain cancer) detectionMUC1 Breast, pancreatic, ovarian cancers; targeting

demonstratedPDGF/PDGFR Improving transport to tumors and targeting brain cancers

NF-κB Targeting a transcription factor implicated in manydiseases

Raf-1 Inhibiting pro-growth signalingαvβ3 integrin Targeting tumor-associated vasculatureHuman keratinocyte growth factor Inhibiting pro-growth signali

Properties of aptamersversus antibodies

Aptamers

Binding affinity nanomolar to picomolar

Selection is a chemical process carried out in vitro and can therefore target anyprotein

Can select for ligands under a variety ofconditions for in vitro diagnostics

Uniform activity regardless of batchsynthesis

PK parameters can be changed on demand

Investigator determines target site ofprotein

Wide variety of chemical modifications tomolecule for diverse functions of molecule

Return to original conformation aftertemperature insult

Unlimited shelf-life

No evidence of immunogenicity

AntibodiesBinding affinity nanomolar to picomolarSelection requires a biological system, thus it is difficult to raise antibodiesto toxins (not tolerated by animal) or non-

immunogenic targets.Limited to physiologic conditions fordiagnosticsScreening monoclonal antibodies timeconsuming and expensiveActivity of antibodies vary from batch tobatchDifficult to modify PK parametersImmune system determines target site ofproteinTemperature sensitive and undergoirreversible denaturationLimited shelf-lifeSignificant immunogenicity

PEPTIDES

• Peptide sequences recognized by receptorsresponsible of binding can be identified and synthesized.

• Examples are peptide sequences derived from ApoE apolipoprotein that are recognized by LDL receptor on cell membranes

Peptides aptamers

• Peptide aptamers consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraintgreatly increases the binding affinity of the peptide aptamer tolevels comparable to an antibody's (nanomolar range).The variableloop length is typically comprised of 10 to 20 amino acids, and the scaffold may be any protein which has good solubility and compacity properties. Currently, the bacterial protein Thioredoxin-Ais the most used scaffold protein, the variable loop being insertedwithin the reducing active site, which is a -Cys-Gly-Pro-Cys- loop in the wild protein, the two Cysteines lateral chains being able to forma disulfide bridge.Peptide aptamer selection can be made usingdifferent systems, but the most used is currently the yeast two-hybrid system.

In vivo phage displayBacteriophage is a virus that infects and replicateswithin a bacteriumPhage display technology is based on the ability to express foreign (poly)peptides as fusions to capsidproteins on the surface of bacteriophageA phage random peptide library displays as many as1011 different peptides

In vivo phage display

Tissue or vascular targeting ligand•Specificorgans or tumors•Tumor blood vessels•Ischemic or inflammatory lesions

• Smaller size; better tissue penetration• Less possibility of immunogenicity• Less possibility of liver and bone marrow

toxicity• Easier processing and lower production cost• Small molecule peptide mimmeticsavailable• Fast blood-pool clearance; less background• Weaker affinity to antigen (epitope)

Peptides vs. antibodies

OTHER LIGANDS

• Natural ligands for receptors can be employedto functionalize NP surface .

Examples:

Folate …..binds to folate receptor

ApoE ……. binds to LDL receptor

Trasferrin…. binds to Tf receptor

• Problems : competition from circulatingFolate, ApoE(lipoproteins), Transferrin

Antibodies vs physiologicalligands

Antibodies are mores specific thannatural ligandsMuch more expensive

Approach: 1- to eliminate physiologicalcompetitor in blood, 2- to inject NP functionalized with the ligand

+

Via succinimide

biotin

biotin

streptavidin

nanoparticlenanoparticle

streptavidin

antibody

nanoparticle

biotin

antibody

100 nm

10 nm

LNA• A locked nucleic acid (LNA), often

referred to as inaccessible RNA, is amodified RNA nucleotide. Theribose moiety is modified with anextra bridge connecting the 2'oxygen and 4' carbon. LNAnucleotides can be mixed with DNAor RNA residues in theoligonucleotide whenever desired.Such oligomers are commerciallyavailable. The locked riboseconformation enhances basestacking and backbone pre-organization. This significantlyincreases the hybridizationproperties (melting temperature)of oligonucleotides.[1]

cystein