Periodic Reporting for period 1 - CLIMB (Development of a Cavity Supported Lipid Membranes Biomimetic drug permeability models (CLIMB))
Periodo di rendicontazione: 2018-01-01 al 2019-12-31
Majority of therapeutic molecules must overcome membrane barrier before reaching their site of action. However, optimal delivery of drugs has always remained elusive in the pharmaceutical industry. This cannot be more true for diseases such as neurological disorders, cancers, and viral infections, which have affected millions globally, causing billion-dollar worth of productivity loss worldwide. Yet, delivery issues remain largely unresolved. In principle, one could increase the chance of Identifying a drug with both good delivery efficiency and the desired therapeutic efficacy by screening hundreds of thousands or more drug candidates. Unfortunately, such an endeavor is formidably uneconomical. The current project is to address this challenge – to devise a platform for the accurate prediction of drug’s membrane permeability in a high-throughput manner.
To meet this vision, the main objective of the current project is to provide a proof-of-principle demonstration of how prudently designed nano-photonic structures can be made to mimic an actual cell in terms of drug uptake. While cell-based assay is conventionally used, it is laborious and expensive as biological cells needed to be cultured and kept in a sterilized condition. To this end, this project seeks to develop a novel platform bearing photonics-based ‘artificial cells’, for the study of drug plasma membrane interactions. The cells provide cell membrane analogues across which molecule binding, entry and diffusion dynamics can be monitored using multiple distinct analytical modalities including Electrochemsitry, Raman and Fluorescence Spectroscopy. The modalities can be combined and the methods selected depend on the characteristics of the drug.
In conclusion, it is showed that the ‘artificial cells’ can display uptake behavior characteristic of real biological cells for representative libraries of drugs from different drug categories. More importantly, using a multi-modal approach, the current method is also able to reveal the underlying mechanism of drug-transport across the membrane, that was normally obscured in traditional cell-based assays.
For more details on this project and related publications, please visit https://sites.google.com/dcu.ie/keyes-research-group/research(si apre in una nuova finestra).
To meet this vision, the main objective of the current project is to provide a proof-of-principle demonstration of how prudently designed nano-photonic structures can be made to mimic an actual cell in terms of drug uptake. While cell-based assay is conventionally used, it is laborious and expensive as biological cells needed to be cultured and kept in a sterilized condition. To this end, this project seeks to develop a novel platform bearing photonics-based ‘artificial cells’, for the study of drug plasma membrane interactions. The cells provide cell membrane analogues across which molecule binding, entry and diffusion dynamics can be monitored using multiple distinct analytical modalities including Electrochemsitry, Raman and Fluorescence Spectroscopy. The modalities can be combined and the methods selected depend on the characteristics of the drug.
In conclusion, it is showed that the ‘artificial cells’ can display uptake behavior characteristic of real biological cells for representative libraries of drugs from different drug categories. More importantly, using a multi-modal approach, the current method is also able to reveal the underlying mechanism of drug-transport across the membrane, that was normally obscured in traditional cell-based assays.
For more details on this project and related publications, please visit https://sites.google.com/dcu.ie/keyes-research-group/research(si apre in una nuova finestra).
The project commenced by investigating the various techniques for fabricating nano-patterned substrates. Here, three fabrication techniques were developed, nano-sphere self-assembly, in-cavity hierarchical nano-patterning, and Au over 3D-nanoprinted nano-patterns. The former approach is a multi-stage technique in which Au cavity array was formed by depositing Au electrochemically through a nano-sphere template. Large uniform array (up to 6 × 6 mm2) can be successfully prepared. The second approach aim to produce sub-micron structures within the cavities, which lead to 3 times improvement in detection sensitivity compared to Au cavities without in-cavity sub-structures. The third approach involves duplication of nano-patterns from a 3D-nanoprinted mould. Au was then sputter-coated over the replicated pattern. To this end, FE-SEM was then used to verify the nano-gemeotries of these substrates. All arrays are generally uniform over large area, with less than 15% inter- as well as intra-variations in cavity diameters. FDTD simulations were carried out using the dimensional information obtained via FE-SEM. Simulated electromagnetic fields verified the plasmonic hot-spots at the bottom of the cavities as intended. Experimentally, substrates with in-cavity substructures were shown to exhibit the largest SERS enhancement factors in consistent with simulation prediction.
For the current proof-of-principle study, Au cavities without in-cavity sub-structures were used for drug-membrane interaction studies. Prior to membrane-deposition, the substrate was rendered hydrophilic via surface functionalization with 6-mercapto-1-hexanol. Lipid bilayers were then deposited using a Langmuir Blodgett/vesicle fusion method developed in the Keyes group. Both the EIS and SERS techniques were used to assess the integrity of the suspended membranes, which was shown to last up to 21hrs. Such an excellent stability was attributed to the ‘cushioning effect’ brought about by the rounded-edge around the mouth of the cavity supports. Permeability test using SERS with a membrane-impermeable probe confirmed proper sealing of the cavities, suggesting the absence of any leaky defects in the membrane.
Three analytical approaches (Electrochemical impedance spectroscopy, Surface Enhanced Raman Spectroscopy, and Metal-enhanced Fluorescence measurements) were used to study drug transport across the membrane. Various drugs were investigated in the studies, including Ibuprofen, Diclofenac, Doxorubicin, Daunorubicin, and cell-penetrating peptides. Through the combined use of the spectroscopic techniques mentioned above, insights with regard to the transport mechanism was obtained. This includes the effect of membrane charges, drug-induced fluidity changes in the membrane and pore-formation. Lastly, the release mechanism of large molecules, such as oligonucleotides, from their delivery vector (Lipoplex in the current case) was examined in the context of gene-therapy. Various crucial intermediate stages in the release process were observed, suggesting the potential of the current technique in the rational design of gene-delivery vector.
An IDF is currently being filed for CLIMB. All experimental data will be published soon in scientific journals. Several funding programmes, such as the Wellcome career-development fellowship, are currently being pursued as a follow-on with the backing of the experimental outcomes, especially in the area of drug-delivery across blood-brain-barrier and gene-delivery. A business model has also been devised, and both Keyes and Dr. Kho are keen to seek early-stage investment.
For the current proof-of-principle study, Au cavities without in-cavity sub-structures were used for drug-membrane interaction studies. Prior to membrane-deposition, the substrate was rendered hydrophilic via surface functionalization with 6-mercapto-1-hexanol. Lipid bilayers were then deposited using a Langmuir Blodgett/vesicle fusion method developed in the Keyes group. Both the EIS and SERS techniques were used to assess the integrity of the suspended membranes, which was shown to last up to 21hrs. Such an excellent stability was attributed to the ‘cushioning effect’ brought about by the rounded-edge around the mouth of the cavity supports. Permeability test using SERS with a membrane-impermeable probe confirmed proper sealing of the cavities, suggesting the absence of any leaky defects in the membrane.
Three analytical approaches (Electrochemical impedance spectroscopy, Surface Enhanced Raman Spectroscopy, and Metal-enhanced Fluorescence measurements) were used to study drug transport across the membrane. Various drugs were investigated in the studies, including Ibuprofen, Diclofenac, Doxorubicin, Daunorubicin, and cell-penetrating peptides. Through the combined use of the spectroscopic techniques mentioned above, insights with regard to the transport mechanism was obtained. This includes the effect of membrane charges, drug-induced fluidity changes in the membrane and pore-formation. Lastly, the release mechanism of large molecules, such as oligonucleotides, from their delivery vector (Lipoplex in the current case) was examined in the context of gene-therapy. Various crucial intermediate stages in the release process were observed, suggesting the potential of the current technique in the rational design of gene-delivery vector.
An IDF is currently being filed for CLIMB. All experimental data will be published soon in scientific journals. Several funding programmes, such as the Wellcome career-development fellowship, are currently being pursued as a follow-on with the backing of the experimental outcomes, especially in the area of drug-delivery across blood-brain-barrier and gene-delivery. A business model has also been devised, and both Keyes and Dr. Kho are keen to seek early-stage investment.
This project has generated up to 5 publishable manuscripts, 4 poster presentations, 4 overseas oral presentations and 1 patent. Research collaborations with 2 research institutes (University of Connecticut and University College Cork) have been established through this project. Dublin City University (DCU) was able to leverage the research outcomes to secure funding for further development under the Enterprise-Ireland Commercialization Funding scheme. Several companies (e.g. Avectas, Nuritas) have expressed interests in the technology.
The current project also has a wider societal implication. For instance, much of the intracellular targets have yet being exploited for therapeutic purposes. Of the various challenges encountered so far in this area of research, delivery issues continue to be the bottleneck. While rational approach is a sensible route to tackle the problem, the complexity of the solution space and the costly cell-based assays have so far impeded the progress. As CLIMB is cost-effective, and amendable to a high-throughput microfluidic setup, it is thus envisioned that, by building upon the proof-of-principle studies carried out in this project, CLIMB could eventually replace cell-based assays as the go-to platform for a rational approach to the delivery challenges.
No specific web site has been developed for this project, but more information can be found via the web-link (https://sites.google.com/dcu.ie/keyes-research-group/research(si apre in una nuova finestra)) of the hosting group.
The current project also has a wider societal implication. For instance, much of the intracellular targets have yet being exploited for therapeutic purposes. Of the various challenges encountered so far in this area of research, delivery issues continue to be the bottleneck. While rational approach is a sensible route to tackle the problem, the complexity of the solution space and the costly cell-based assays have so far impeded the progress. As CLIMB is cost-effective, and amendable to a high-throughput microfluidic setup, it is thus envisioned that, by building upon the proof-of-principle studies carried out in this project, CLIMB could eventually replace cell-based assays as the go-to platform for a rational approach to the delivery challenges.
No specific web site has been developed for this project, but more information can be found via the web-link (https://sites.google.com/dcu.ie/keyes-research-group/research(si apre in una nuova finestra)) of the hosting group.