targeting

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

8. The drug can be inserted into the core

9. Ligands for targeting

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

15. 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)

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

17. 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
Matrix metalloproteinases

20. APTAMERS

21. 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.

22. 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.

24. Aptamer target protein or molecule Application
PSMA Prostate cancer diagnosis and therapy
WT1 Understanding Wilm's tumor
pathogenesis
4,4′-methylenedianiline Detecting DNA-damaging compounds
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; targeting
demonstrated
PDGF/PDGFR Improving transport to tumors and targeting brain cancers
NF-κB Targeting a transcription factor implicated in many
diseases
Raf-1 Inhibiting pro-growth signaling
αvβ3 integrin Targeting tumor-associated vasculature
Human keratinocyte growth factor Inhibiting pro-growth signali

25. 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
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

26. 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

27. 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.

28. 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

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

30. Peptides vs. antibodies
• 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)

31. 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

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

33. Antibodies vs
physiological
ligands

34. 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

41. +
Via succinimide

43. biotin
nanoparticle nanoparticle
biotin
streptavidin

44. streptavidin
antibody
nanoparticle
biotin
antibody
100 nm
10 nm

47. 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]

53. cystein