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#1. Hello, this is Kyuha Chong. My major field of interests is how to cure the malignant
brain tumors, especially glioblastoma multiforme which is known as incurable disease. Recently,
I and my advisor, professor Choi, have focused about the application of nano-scale drug delivery
system for the diagnosis and treatment of malignant brain tumors. The topic I will going
to discuss and talk about today is about the result that acquired lately, enhancement of
the photocytotoxic efficiency of sub-12 nm size therapeutic polymeric micelles at malignant
brain tumor cells. From now on, I will use malignant brain tumor as abbreviation, MBT.
#2. Prior to the beginning, I just want to instruct you about the current problems and
causes that limit the treatment efficacy of MBTs. Brief in summary, current treatment
modalities for MBTs are surgical resection, chemotherapy, and radiation therapy. Even
though surgical resection is known as most effective treatment modality, it is nearly
impossible to resect tumor totally. Hence, a great deal of clinical trials are ongoing,
and theranosis with photosensitizer is suggested as an alternative. But, to enhance the efficiencies,
drug delivery to MBT is main problem to overcome. Either with chemotherapy or radiation therapy,
the drug delivery to MBT is a problem of the greatest importance, also.
#3. The main factor that limits drug delivery to MBT is presence of a barrier which is called
blood-brain tumor barrier, the BBTB. Function as a double-edged sword, the organ specific
structure for the protection of brain from toxins and inflammatory cells, blocks drug
delivery to MBT. #4. From the 1960s and 70s, there were lots
of studies to investigate the BBTB. Comparison between MBT and other cancers, and between
the environmental differences, non-cranial vs. cranial, revealed that under the 100 nm
size might be the relative size for the penetration that over-cross the BBTB.
#5. And recent studies identified that, because of the high interstitial fluid pressure, the
physiologic size is much smaller, so below 12 nm size of nanoparticles are only effectively
penetrate the BBTB. To the best of our knowledge, there were no other reports that evaluated
sub-12 nm sized polymeric micelles as phototherapeutics for MBTs.
#6. So, to validate the hypothesis of sub-12 nm size polymeric micelles as an efficient
phototherapeutics for the MBTs, the study was design with the aims of 1) successful
engineering of polymeric micelles and 2) evaluation of its phototherapeutic efficiency with in
vitro experiments. #7. To evaluate their phototherapeutic effect,
a photosensitizer hypericin was chosen because it is a hydrophobic compound which is expected
to be feasibly incorporated into the micelles, and also because of that has fluorescence
with photocytotoxic effects, simultaneously. And to optimize the size, three different
kinds of micelles were prepared by varying the length of DSPE-mPEG. The synthesis of
the micelles was performed with major 3 steps. After drying the lipid components under vacuum,
the lipid film was hydrated with water or PBS under continuous sonication. And micelles
of the desired size were obtained by filtration. #8. To verify the size of engineered micelles,
dynamic light scattering and transmission electron microscopy imaging were performed.
With the analysis of the results, the size of engineered micelles were below the 12 nm
in size, which is satisfied with the aims of this study.
#9. As a proof-of-concept experiment, two other groups were prepared, the parental aggregated
form and PEGylated form, which are previously used forms with in vitro, in vivo experiments,
and clinical trials. These three formulations of a drug was applied to U251MG human malignant
brain tumor cells, for the evaluation of phototherapeutic efficiency.
#10. Compared with other anticancer chemotherapeutic agents, photosensitizers exhibit negligible
cytotoxicity in the absence of irradiation of light. Therefore, by using a photosensitizer
as a drug, nonspecific uptake of the nanoparticles will not pose a high risk to the cells because
the toxicity is negligible under normal circumstances. To ensure this, dark cytotoxicity, the toxicity
of the agents without a light irradiation was evaluated, firstly. Within phototherapeutic
dosage, below 10 μM of hypericin, the dark cytotoxicity was negligible, and either with
various formulations of the drug, including engineered polymeric micelles, cytotoxicity
were negligible also, as expected. #11. Subsequently, evaluation of the phototherapeutic
effect between drug delivery systems was performed. For the investigation, LED based light irradiation
system was established, with the analysis of dosimetry. Under the light irradiation
system, photocytotoxic efficiency was evaluated with the following of 2 hours of incubation
of 5 μM of hypericin, and irradiation of light at a dosage of 0.1 J/cm2. Interestingly,
the engineered polymeric micelles were exhibited more than 2.5 times higher photocytotoxic
efficiency, compare with two other formulations. #12. Because 1) main mechanism that brings
photocytotoxicity to the cells with photodynamic therapy is related with the actions of photosensitizers
in mitochondria or in lysosome, and 2) hypericin is local-distributed mainly in the mitochondria,
ER, and lysosome, evaluation of co-localization of hypericin in the mitochondria, ER, and
lysosome for the all formulations was performed. For the evaluation, fluorescence based analysis
was taken. #13. The fluorescence images and co-localization
coefficients indicated that all of the hypericin formulations were similarly co-localized in
the lysosomal compartment. Notably, the co-localization value of hypericin in the mitochondria and
in the ER was not statistically different for parental aggregated form and PEGylated
form. However, the calculated values for the engineered micelles in the mitochondria and
in the ER were significantly higher and lower, respectively, than those for two other formulations.
#14. By encapsulating a photosensitizer with our polymeric micelle, the photocytotoxic
efficiency induced by the delivered hypericin was significantly higher in U251MG human malignant
brain tumor cells. This finding was explained in part by the increased co-localization of
hypericin in the mitochondria. We hypothesized that a difference might exist in the intracellular
organelle-specific drug delivery between micelles and the other formulations. Specifically,
we assumed that co-localization or uptake of the HY in the mitochondria might be enhanced
with engineered micelles. #15. The differential uptake in the mitochondria
might be due to increased permeability of the mitochondrial membrane by local action
of disassembled polymeric chains from the internalized micelles. But still this is a
hypothesis, so to make certain of it, I think further experiments and evaluations should
be followed by. #16. To date, various nanoparticles have been
reported and are anticipated to overcome some limitations of conventional drug delivery
to MBTs. Our engineered micelle is the first example of a sub-12 nm sized micelle, which
has been proved to be more efficient in photodynamic therapy than its parental drug form or PEGylated
form. Although this engineered micelle was neither designed for active targeting nor
lipid modification for enhancing cellular uptake, the agent was highly accumulated in
the mitochondria, consistent with phenotypic analysis of photocytotoxicity. So, we expect
that the conjugation of cancer cells or mitochondria specific targeting ligands on the surface
of the micelle would significantly increase either the cellular uptake of the agent or
the photocytotoxic efficacy. And we anticipate, this engineered micelle may act as a superb
therapeutic agent for MBTs and spur the application of sub-12 nm micelles for MBT therapeutics.
The contents are published online at ‘Chemical Communications’ at September 11th of this
year, and will be published at December 21st as a front cover story. I hope you enjoyed
the talk. Thank you. Have a nice day.