Self-assembled Peptides as Drug Delivery Molecules for transport
across the Biological Barriers
Porter, S., McCarthy, H., & Laverty, G. (2017). Self-assembled Peptides as Drug Delivery Molecules for
transport across the Biological Barriers. Paper presented at 39th All Ireland Schools of Pharmacy Research
Conference, Cork, Ireland.
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Download date:31. Jul. 2017
OVERCOMING
BIOLOGICAL BARRIERS:
PEPTIDE NANOTUBES
FOR DRUG DELIVERY
Biological barriers
•
Biological barriers inhibit drug delivery
throughout the body
•
For efficient drug delivery, drugs must be able
to reach their target site of action
•
The blood brain barrier is very problematic for
drug delivery
•
Large gap in adequate care for patients for
particular diseases: brain tumors, Parkinson's,
Alzheimer's
CRS. 2015. Controlled release society. [ONLINE] Available at: http://www.crsaustralia.org/?page_id=971. [Accessed 16 April 2017].
The blood brain barrier
For Transmembrane diffusion of small
molecules: <400 Daltons and high lipid
solubility
Approaches to bypassing this barrier:
• Disruption of the blood brain barrier tight
junctions (detergents or ultrasound)
• Receptor mediated transcytosis – engage
cell-surface receptors over expressed by
brain cells.
• Adsorptive endocytosis of nanoparticles
Jonathan C.H. Choi . 2016. Jonathan C.H. Choi Research Group. [ONLINE] Available at: http://www.bme.cuhk.edu.hk/jchchoi/r_SHIAE14-16.php. [Accessed 16 April 2017].
Peptide structure
NH2-(D)Phe-(D)Phe-COOH
•
Ultra short peptide structure
consisting of two amino acids
•
Phenylalanine-Phenylalanine
•
Molecular weight of 312.36 Daltons
•
Relatively easy manufacture process
due to the short length – peptides
become increasingly difficult to
manufacture at high purities with
increasing chain length
O
NH
H2N
OH
O
Self assembled peptide nanotubes
B: Adsorptive endocytosis (Previously
demonstrated for nanotube structures)
Astrocyte
Characterising the assembled
secondary structure
Circular dichroism spectra for assembled peptide
nantotube structures:
1 0 0
• Large positive peak ~ 217 nm
• Characteristic of β-sheet assembly in peptides
• Useful to confirm presence of peptide nanotubes in
absence of environmental microscope
m d e g
5 0
0
2 2 0
W a v e le n g th
-5 0
2 4 0
(n m )
Measuring zeta potential
• Particle charge is a key consideration when
considering formulation of any nanoparticle
• Unfavourable charge pairing will lead to the
repelling of the drug cargo from the nanoparticle
• Extremes of either positive or negative charge
unfavourable for delivering across cell layers
because it can lead to entrapment
pH (n=3)
Mean Zeta Potential (mV)
7.4
-21.10(±0.49)
5.5
-13.57(±2.37)
Water World. 2016. ONLINE ZETA POTENTIAL MEASUREMENT PROVIDES WATER TREATMENT CONTROL, COST REDUCTION. [ONLINE] Available at: http://www.waterworld.com/articles/print/volume30/issue-10/features/automation-technology/online-zeta-potential-measurement-provides-water-treatment-control-cost-reduction.html. [Accessed 16 April 2017].
Encapsulation of Rhodamine B
4 0
2 0
P e p tid e
c o n c e n tr a tio n
5
.2
1
2
.5
.0
0
0
0
5
R h o d a m in e
B
e n c a p s u la tio n
(%
)
6 0
( m g /m l)
• Instead of beginning encapsulation and
permeation studies with a drug, a
fluorescent molecule is used for ease of
screening
• Rhodamine B is chosen as a tracer
molecule due to the previous zeta
potential data, rhodamine should be
positively charged at pH during the
assembly process
• Rhodamine B is added while the peptide
nanotubes are self assembling and are
spontaneously incorporated into the
tubes
• A maximum of 56 % encapsulation
efficiency was observed
In-vitro blood brain
barrier model
Brunswick laboratories. 2016. Caco-2 Permeability Screening Assay (unidirectional/bidirectional). [ONLINE] Available at: https://brunswicklabs.com/capabilities/clinical-study/adme-tox-studies/caco-2-permeability-screening/.
[Accessed 16 April 2017].
•
Transwell inserts used to grow a brain
endothelial cell layer on
•
Semi-porous membrane containing
defined pore size (0.4 micron)
•
Cell layer grown on insert membrane –
creates two separate chambers
separated by a brain endothelial cell
layer (hCMEC/D3 Cell)
•
Nanotube suspensions loaded with a
fluorescent marker can be loaded into
top chamber
•
Validate using TEER
Permeation across model
•
•
(% )
4
to ta l d o s e
•
At time zero peptide nanotube suspension
loaded with rhodamine b is added to the top
chamber of the transwell insert
The bottom chamber is sampled over time
and the fluorescence intensity of rhodamine b
at 562/583 nm excitation/emission
wavelengths is recorded
Peptide nanotubes transport rhodamine over
the cell monolayer and then begin to deassemble once they have reached the bottom
compartment and are no longer in a peptide
monomer saturated solution
After 4 hours 2.4% of the total dose had
crossed, which is reasonable considering the
short time point
P e rc e n ta g e
•
3
2
1
0
0
6 0
1 2 0
T im e
( m in )
1 8 0
2 4 0
Biocompatibility
studies
4
2
1
P e p tid e
c o n c e n tr a tio n
( m c g /m l)
3
.9
0
1
7
.8
3
1
5
.6
5
1
3
2
6
.2
0
.5
0
1
2
5
.0
0
.0
0
2
5
0
0
5
0
0
0
.0
.0
0
0
0
1
• Peptide nanotubes are incubated with
horse red blood cells
• The percentage of red blood cells lysed
can be calculated by measuring
absorbance
• Gives an indicator of how compatible the
treatments would be as an injectable
H a e m o ly s is
Haemolysis of equine erythrocytes:
(% )
3
N S
N S
N S
N S
P e rc e n ta g e
v ia b ility
(% )
N S
1 0 0
5 0
*
*
*
*
3
0
.6
5
1
.5
2
.2
0
0
.0
5
1
0
.0
0
0
P e p tid e
c o n c e n tr a tio n
( m g /m l)
Percentage of remaining viable NCTC 929 cells following 6-hour
LIVE/DEAD® stain of NCTC 929 cells following 6-hour treatment
treatment with a range of peptide nanotube suspension concentrations
with peptide nanotube suspension. Live cells are stained green and
using a MTS assay. Key: black column:. NS: no significant difference
dead cells are stained red. Scale bar represents 200 µm.
(P≥0.05), *: P<0.01 significant difference between percentage viability of
peptide nanotube treated cells and negative control.
Conclusions
• Phenylalanine-phenylalanine peptides can self assemble into nanotube structures which can
encapsulate fluorescent markers
• Preliminary studies have shown they have high biocompatibility and can permeate an in
vitro blood brain barrier model
Thank you