Overcoming biological barriers Peptide nanotubes …...•Biological barriers inhibit drug delivery...
Transcript of Overcoming biological barriers Peptide nanotubes …...•Biological barriers inhibit drug delivery...
Self-assembled Peptides as Drug Delivery Molecules for transportacross the Biological Barriers
Porter, S., McCarthy, H., & Laverty, G. (2017). Self-assembled Peptides as Drug Delivery Molecules fortransport across the Biological Barriers. Paper presented at 39th All Ireland Schools of Pharmacy ResearchConference, Cork, Ireland.
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Download date:01. Dec. 2020
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].
NH2
O
NHOH
O
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
Peptide structure
Self assembled peptide nanotubes
B: Adsorptive endocytosis (Previously demonstrated for nanotube structures)
Astrocyte
Characterising the assembled secondary structure
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Circular dichroism spectra for assembled peptide nantotube structures:
• Large positive peak ~ 217 nm• Characteristic of β-sheet assembly in peptides• Useful to confirm presence of peptide nanotubes in
absence of environmental microscope
Measuring zeta potential
pH (n=3) Mean Zeta Potential (mV)
7.4 -21.10(±0.49)
5.5 -13.57(±2.37)
• 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
Water World. 2016. ONLINE ZETA POTENTIAL MEASUREMENT PROVIDES WATER TREATMENT CONTROL, COST REDUCTION. [ONLINE] Available at: http://www.waterworld.com/articles/print/volume-30/issue-10/features/automation-technology/online-zeta-potential-measurement-provides-water-treatment-control-cost-reduction.html. [Accessed 16 April 2017].
Encapsulation of Rhodamine B
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P e p t i d e c o n c e n t r a t i o n ( m g / m l )
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• 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• 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 TEERBrunswick 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].
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Permeation across model
• 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 de-assemble 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
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Biocompatibility studies
Haemolysis of equine erythrocytes:
• 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
Percentage of remaining viable NCTC 929 cells following 6-hour
treatment with a range of peptide nanotube suspension concentrations
using a MTS assay. Key: black column:. NS: no significant difference
(P≥0.05), *: P<0.01 significant difference between percentage viability of
peptide nanotube treated cells and negative control.
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LIVE/DEAD® stain of NCTC 929 cells following 6-hour treatment
with peptide nanotube suspension. Live cells are stained green and
dead cells are stained red. Scale bar represents 200 µm.
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