Nanoparticles can be functionalized for biomedical applications by modifying their surface with multiple components in a defined order from the innermost to outermost. Common steps include adding a drug to the core, then a targeting ligand on the outer layer. Possible ligands include antibodies, peptides, aptamers and other molecules that bind receptors overexpressed on target cells. Aptamers have advantages over antibodies like controlled synthesis and lack of immunogenicity. Peptides identified from in vivo phage display libraries can also serve as targeting ligands. The choice of ligand depends on the drug and intended application.

1. 1
Surface Modification of
Nanoparticles for
Biomedical Applications
1

2. Multifunctionalization
• A nanomedical device should perform several
functions alltogether

4. • 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.

5. Possible steps
It is possible but rare for a single molecule
to perform two or more functions

7. Choice depending on the
drug and utilization of NP

8. Step 2 – Add a drug

9. Step 3- Add a ligand for targeting

11. Sometimes an additional ligand for “AND” or “ NOT” instruction
may be added

15. Targeting molecules
A. antibodies
B. Peptides
C. Aptamers
D. Other ligands

16. 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)
or
2) are oversexpressed as a consequence of a
pathology (e.g. tumour markers)

17. Antibodies
• Antibodies directed against tissue-specific
antigens.

18. 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 Integrin (tumours)
Matrix metalloproteinases (tumours)
CD 66 (atherosclerotic plaque)

20. HLA-DR MHC class II cell surface receptor
DCSIGNCluster of Differentiation 209

22. Problems in functionalization with Ab

23. APTAMERS

24. Aptamers are oligonucleotides that bind a specific
target molecule. Aptamers are usually created by
selecting them from a large random sequence pool.
Aptamers can be used for both basic research and
clinical purposes as macromolecular drugs.

25. 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.

27. APTAMER TARGET PROTEIN OR MOLECULE APPLICATION
PSMA Prostate cancer diagnosis and therapy
WT1 Wilm's tumor pathogenesis
4,4′-methylenedianiline Detecting DNA-damaging compound
VEGF Inhibiting angiogenesis
RET Inhibition of pro-growth signaling
HER-3 Reducing drug resistance in HER-2+ cancers
TCF-1 Colon cancer growth inhibition
Tenascin-C Glioblastoma (brain cancer) detection
MUC1 Breast, pancreatic, ovarian cancers;
PDGF/PDGFR Improving transport to tumors
NF-κB Targeting a transcription factor
Raf-1 Inhibiting pro-growth signaling
αvβ3 integrin Targeting tumor-associated vasculature
Human keratinocyte growth factor Inhibiting pro-growth signali

30. 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
Wide variety of chemical modifications to
molecule for diverse functions of molecule
Uniform activity regardless of batch
synthesis
PK parameters can be changed on demand
Investigator determines target site of
protein
Return to original conformation after
temperature insult
Unlimited shelf-life
No evidence of immunogenicity
Antibodies
Binding affinity nanomolar to picomolar
Selection 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
diagnostics
Screening monoclonal antibodies time
consuming and expensive
Activity of antibodies vary from batch to
batch
Difficult to modify PK parameters
Immune system determines target site of
protein
Temperature sensitive and undergo
irreversible denaturation
Limited shelf-life
Significant immunogenicity

31. PEPTIDES
• Peptide sequences recognized by receptors
responsible of binding can be identified and
synthesized.

34. 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.

35. In vivo phage display
Bacteriophage is a virus that infects and replicates
within a bacterium
Phage display technology is based on the ability to
express foreign (poly)peptides as fusions to capsid
proteins on the surface of bacteriophage
A phage random peptide library displays as many as
1011 different peptides

36. Phage library
• E. coli can be infected to
multiply the number of
bacteriophages.
• Can quickly create large
libraries of phage clones
displaying different
peptides.

37. In vivo phage display
Tissue or vascular targeting ligand•Specific
organs or tumors•Tumor blood vessels
•Ischemic or inflammatory lesions

38. • 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 mimetics available
• Fast blood-pool clearance; less background
• Weaker affinity to antigen (epitope)
Peptides vs. antibodies

39. OTHER LIGANDS
• Natural ligands for receptors can be employed
to functionalize NP surface .
Examples:
Folate …..binds to folate receptor
ApoE ……. binds to LDL receptor
Trasferrin…. binds to Tf receptor

40. • Problems : competition from circulating
Folate, ApoE(lipoproteins), Transferrin

41. Folate receptors
Antibodies

42. Antibodies are mores specific than natural
ligands
Much more expensive
Approach:
1- to eliminate physiological competitor in
blood,
2- to inject NP functionalized with the
ligand

43. Multi ligands increase binding
After the binding of one ligand to its target,
the interaction between the second
ligand(green) to its target (yellow) isfavoured
by their proximity.

50. +
Via succinimide

52. biotin
biotin
streptavidin
nanoparticlenanoparticle

53. streptavidin
antibody
nanoparticle
biotin
antibody
100 nm
10 nm

56. 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]

61. Ellman’s reagent

62. Click Chemistry

63. cystein