טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKajal Ashima
SubjectDeveloping Barcoded Nanotherapeutics for Treating Cancer
DepartmentDepartment of Chemical Engineering
Supervisor Professor Avi Schroeder


Abstract

Cancer is a leading cause of death worldwide. Nanotechnologies are becoming impactful medical tools, granting therapeutic accuracy and potency that cannot be attained using systems of a larger scale. However, to realize their full potential, a better understanding of the mechanism by which nanotechnologies interface with cancer cells is needed. To date, in most studies, an extremely high number of drug-loaded nanoparticles are injected to treat the diseased tissue. Furthermore, even though targeting approaches have greatly improved, the cumulative administered drug dose using nanoparticles has not decreased.

In this Ph.D. research program, I studied the mechanism at which a single drug-loaded nanoparticle interacts with a corresponding single cancer cell. We used synthetic DNA barcodes, introduced into 100-nm drug-loaded liposomes as mediators to correlate the number of liposomes taken up by each cancer cell, in order to calculate the dose of an anticancer drug required to kill a single cancer cell. Specifically, 4T1 triple-negative breast cancer (TNBC) cells were treated with 100-nm liposomes, loaded with anticancer drug doxorubicin and corresponding synthetic DNA barcode, sorted to live and dead cells using FACS and extracted dead cells barcodes were amplified by real-time (RT)-PCR to quantify the cellular uptake of the liposome and drug. We sought to use this approach to explore and optimize the therapeutic payload a single drug-loaded nanoparticle must carry in order to be therapeutically active.


Characterization of the liposome properties indicated that each liposome was encapsulated with 10,000±50 doxorubicin molecules and 1± 0.3 DNA barcode. We extracted six barcodes from a single dead cancer cell, meaning each dead cell took approximately six liposomes. Correspondingly, the total dose of doxorubicin found inside a dead cancer cell was approximately 9.6*10-20±0.3 moles of doxorubicin, meaning that 60,000±25 doxorubicin molecules are required to kill a single TNBC cell. 

Further exploring the liposomal uptake dynamics, we determined that when the same concentration of liposomal and free form of doxorubicin is used the free doxorubicin was more effective in killing cancer cells. At the same concentration, after 4 hours of treatment with cancer cells, the amount of extracted doxorubicin from dead cancer cells was 20 times greater in case of free doxorubicin compared to liposomal doxorubicin. This suggests that to increase drug activity the liposomal uptake should be improved, for example, using targeting ligands.

To summarize, it is proposed herein, that as targeting modalities are improving, future nanomedicines must carry the effective dose to treat only the targeted diseased cell. Although there are many benefits of loading anticancer drugs into nanoparticles, such as targeting the disease site and reducing toxicity to healthy organs, this study demonstrated that it takes a higher dose of liposomal drug to achieve the same efficacy when compared to free drug. This requires elucidating the therapeutic payload each nanomedicine must carry to the target cancer cell and liposome-based nano-drugs utilize and benefit from tumor's unique features. The mode of delivery and the payload of the nanoparticle should be coupled with its activity to achieve a biologically and therapeutically relevant dose.