What are the SSTRs? ⢠SSTRs are members of the GPCRs super family. ⢠There are five different subtypes of SSTRs (SSTR1-5),. ⢠SSTR2 are classified into two ...
Fluorescent Nanoparticles for Targeting of Somatostatin Receptors
Dr. Ahmed Abdellatif Faculty of Pharmacy, Al-Azhar University at Assiut
u Overview Ø How to deliver these particles effectively to specific target cells? Ø To answer this question; Delivery of nanoparticles to receptor. Ø SSTRs are one class of receptors that could serve this purpose.
What are the SSTRs? • • • •
SSTRs are members of the GPCRs super family. There are five different subtypes of SSTRs (SSTR1-5), SSTR2 are classified into two subtypes, SSTR2A and SSTR2B. The five receptor subtypes bind with the natural SST and its analogues with low nanomolar affinity, and produce defined biological effects in many normal and diseased cells. • The blocking of SSTRs with antagonist suppresses the interaction of the peptide agonist with SSTRs.
QDsOCT
² OCT is a SST analogue and a shorter peptide compared to SST. It has 8 amino acids. ² It selectively binds to SSTR2, SSTR5, especially more selective to SSTR2, and less selective to SSTR3. ² OCT is more stable, highly potent SST analogues with prolonged duration of action. Octreotide
H2N O H N
NH
O
HO NH
NH
NH O
H2N
O S S
NH O
NH
N H
O OH
O
OH
• The aim of this research was to formulate nanopar3cles that are capable of selec3vely reaching specific sites in the human body via receptor targe3ng for diagnos3c as well as therapeu3c purposes. • Qdots that carry PEG-amine have very low non-specific binding to the cell membranes. OCT can be conjugated to Qdots at low pH selec3vely to its N-terminus. Qdots
Ø The main advantages Qdots; are detectable by fluorescence microscopy and FACS.
Outlines u Formulation and characterization of OCT decorated Qdots for targeting of SSTR2 u Cellular uptake of Qdots-OCT nanoparticles u I n v i v o a s s e s s m e n t o f t a r g e t i n g Q d o t s - O C T nanoparticles
Ø The main advantage of Qdots; are detectable by fluorescence Microscopy and FACS. Qdots
Octreotide H2N O H N
NH
O
HO NH
NH
NH O
H2N
O S S
NH O
NH
N H
O
OH
O OH
² OCT is more stable, highly potent SST analogues with prolonged duration of action.
Conjugation of octreotide to quantum dot nanoparticles
Characterization of OCT-decorated Qdots
Signal intensity (Volts)
SEC of OCT and Qdots using UV detection at 280 nm.
3
3
2
2 Qdot-OCT
1
1
Qdots OCT
0 0
2
0 4
6
8
10
Time (Minutes)
12
14
16
Determination of the surface charge 15 10 Zeta Potential (mV)
5 0 -5 -10 -15 -20 -25 -30
Qdots
Q.dots-OCT
The zeta potential of Qdots-OCT showed reversal to positive values.
Fourier transform spectroscopy analysis of Qdots
Cellular uptake of OCT-decorated Qdots CLSM images binding of Qdots to BON-1 cells. Cells: BON-1 cells that express SSTR2 Conc.: 10 nM Control (No Qdots)
Unmodified Qdots
Qdots-OCT
Qdots-OCT in presence of 100 µM free OCT
500 450 400 350 300 250
SSTR2 expression ratio relative to GAPDH
Fluorescence intensity (661 nm)/a.u.
FACS analysis for Qdots-OCT with different cell lines. BON 1 cells
330 300 270 240 210 180 150 120 90 60 30 0
HELA cells
HeLa cells
200
BON-1
RT-PCR for receptor expression in different cell line.
150 100 50 0 Control cells
10 nM Native Q.dots
10 nM Q.dots-OCT
10 nM Q.dots-OCT + 100 µM OCT
In vivo assessment of targeting OCT-decorated Qdots 60
Qdots-OCT
Qdots
% ID/g tissue
50 40
•
Male mice NMRI (nu/nu),
•
Incubation time; 1hr,
•
Dose injected; 100 pmol
30 20 10 0 Liver
Blood
spleen
kidney
Pancreas
Lung
Heart
Eyes
High blood circulation of Qdots-OCT could be due to decoration with OCT, which could be bound to SSTRs in blood cells (3-5). 3- Petrak, K., Essential properties of drug-targeting delivery systems. Drug Discov Today, 2005. 10(23-24): p. 1667-73. 4- Lichtenauer-Kaligis, E.G., et al., Differential expression of somatostatin receptor subtypes in human peripheral blood mononuclear cell subsets. European Journal of Endocrinology, 2004. 150(4): p. 565-77. 5- Reynaert, H., et al., Expression of somatostatin receptors in splanchnic blood vessels of normal and cirrhotic rats. Liver Int, 2007. 27(6): p. 825-31.
CLSM images of liver after animal experiment CLSM (EX: 500/ Em: 700), Male mice NMRI (nu/nu), Incubation time; 1hr, Dose; 100 pmol. Green Color: Liver cells Pink color: Qdots
Unmodified Qdots
Qdots-OCT
How to deliver these particles effectively to specific target cells? OCT can deliver NPs to the targeted sites in vitro & in vivo.
Conclusions; Ø The nanoparticles conjugated SSTRs agonist were successfully characterized. Ø Cellular uptake showed highly specific interactions with cells. Ø The Nanoparticles decorated SSTRs agonist can be displaced from their receptor, which relates to the specificity of the interaction with cells. Ø In vivo study showed a massive accumulation of Qdots-OCT in the blood as compared to the other organs.
Acknowledgements Prof. Dr. Achim Goepferich. Dr. Joerg Tessmar Dr. Miriam Breunig Dr. Ferdinand Brandl
Dr. Shaaban Osman, Dr. Mohammed Darwish, Dr. Alaa Zaky, Dr. Asmaa El-Bakry, Dr. Gamal Zayed and Prof. Dr. Hatem Sarhan.
Dr. Klaus Pollinger Renate Liebl Robert Hennig Angelika Berié Andrea Blaimer and Viktoria Messmann Stefan Kolb
To my family (my mother and my brothers) My wife, Laila Ahmed,
Eva-Christina Wurster Susanne Kirchhof Dr. Andreas Sellmer Dr. Abdellatif Bouazzaoui Fouad Darras
Egyptian Ministry of Higher
Dr. Mohammad Tajuldin
Education due to the financial support to study
Dr. Thilo Spruss Dr. Wendy Patterson, Jean Marie Reiter and Dinette Brink
my PhD in Germany.
