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Surface Modification of Nanoparticles for

Biomedical Applications

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• Consequently a multi-component nanomedical system can be constructed in reverse order of controlling events, namely from the inside out. The outer components are the first to be used. The inner components are the last.

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

The drug can be inserted into the core

Ligands for targeting

Cell Targeting

A. antibodiesB. PeptidesC. AptamersD. Other ligands

Antibodies• Antibodies directed against tissue-specific

antigens.

Examples

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

αvβ3 IntegrinMatrix metalloproteinases

APTAMERS

Aptamers are oligonucleic acid molecules 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 as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.

Aptamer target protein or molecule ApplicationPSMA Prostate cancer diagnosis and therapyWT1 Understanding Wilm's tumor pathogenesis4,4 -methylenedianiline′ Detecting DNA-damaging compoundsVEGF 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 cancersNF-κB Targeting a transcription factor implicated in many diseasesPhosphatidylcholine:cholesterol liposomes Triggering liposome degradationRaf-1 Inhibiting pro-growth signalingαvβ3 integrin Targeting tumor-associated vasculatureHuman keratinocyte growth factor Inhibiting pro-growth signali

Properties of aptamers versus antibodies

Aptamers

Binding affinity nanomolar to picomolar

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

Can select for ligands under a variety of conditions for in vitro diagnostics

Uniform activity regardless of batch synthesis

PK parameters can be changed on demand

Investigator determines target site of protein

Wide variety of chemical modifications to molecule for diverse functions of molecule

Return to original conformation after temperature insult

Unlimited shelf-life

No evidence of immunogenicity

AntibodiesBinding affinity nanomolar to picomolarSelection requires a biological system, thus it is difficult to raise antibodies to toxins (not tolerated by animal) or non-immunogenic targets.Limited to physiologic conditions for diagnosticsScreening monoclonal antibodies time consuming and expensiveActivity of antibodies vary from batch to batchDifficult to modify PK parametersImmune system determines target site of proteinTemperature sensitive and undergo irreversible denaturationLimited shelf-lifeSignificant immunogenicity

PEPTIDES

• Peptide sequences recognized by receptors responsible 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 constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range).The variable loop 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-A is the most used scaffold protein, the variable loop being inserted within the reducing active site, which is a -Cys-Gly-Pro-Cys- loop in the wild protein, the two Cysteines lateral chains being able to form a disulfide bridge.Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system.

OTHER LIGANDS

• Natural ligands for receptors can be employed.Examples:Folate ApoETrasferrin

+

Via succinimide

biotin

biotin

streptavidin

nanoparticlenanoparticle

streptavidin

antibody

nanoparticle

biotin

antibody

LNA• A locked nucleic acid (LNA), often

referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. Such oligomers are commercially available. The locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the hybridization properties (melting temperature) of oligonucleotides.[1]

cystein