Doktorranden har varit inne på GlycoImagings område med att studera hur nanoteknik kan användas för att målsöka en specifik cancertumör och via tekniken applicera det mest effektiva läkemedlet på identifierad tumör.
Doktoranden ifråga har haft "vår" forskare Ed Luther från universitet som handledare för att förstå sig på och sen använda universitets HoloMonitor för sina studier.
Targeted Nanopreparations For Cancer Therapy
STATEMENT OF HYPOTHESIS
Considering the advantages of targeted drug delivery in cancer therapy, I hypothesize that the
development of the tumor-targeted nanomedicines such as phage protein-targeted micellar
formulation for pancreatic cancer and the antibody-targeted liposomal formulation for breast
cancer will facilitate specific drug delivery to the tumor site and produce optimal therapeutic effects
by minimizing the adverse effects associated with the drug and maximizing the therapeutic benefits.
Abstract:
Cancer therapy in the recent years has evolved with the development of novel targeted drug delivery
systems. The conventional chemotherapy approach is plagued by side effects and numerous
disadvantages such as low bioavailability, poor solubility of drugs, toxicity, non-specific drug action
and so forth. With the main goal of producing a drug delivery vehicle that is optimally designed to
address the many limitations associated with the conventional chemotherapy, the work has been
designed as follows: Different lipid-based targeted formulations were prepared, such as the tumor-
targeting micelles and liposomes, and the in vitro effects of the formulations on pancreatic and
breast cancer cells have been studied to determine their optimal therapeutic effects and their ability
to minimize adverse off-target effects. Keeping in mind the need for an ideal therapy requirement
for life threatening illnesses, hard to treat pancreatic cancer and the very commonly diagnosed
cancer among women, breast cancer, were chosen as the targets for testing designed nano-
preparations.
The first approach was to develop polymeric micelles self-assembled with a single drug, paclitaxel
and a targeting agent, a peptide sequence derived from phage coat protein. The drug is hydrophobic
and poorly soluble in aqueous solvents that limit its pharmaceutical applications despite high
efficacy. Cell viability studies on PANC-1 pancreatic cancer cells using the targeted phage micelle
preparation produced significantly higher cell death of ~45 % while the non-targeted preparation
caused only about ~30% cell death at 750ng/ml concentration of paclitaxel. Significantly higher cell
death was observed at 1.5 and 2.75 mg/ml concentrations of paclitaxel-loaded in the formulation, that was dose and time dependent. More than a 30% increase in caspase3/7 activity was observed
in vitro in a monolayer of PANC-1 cells while the in vivo data on lactate dehydrogenase release
indicated a significantly enhanced apoptosis in the PANC-1 spheroid model upon treatment with the
targeted micelle preparation.
The second approach utilized liposomes modified with monoclonal antibody 2C5 (mAb 2C5) as
targeting ligand for breast cancer therapy. The study is important in that a combination of 2 drugs,
paclitaxel, a microtubule inhibitor, and salinomycin, an anti-cancer stem cell targeting agent, has
been used. The main goal with the use of combination chemotherapeutics was to eliminate not just
the bulk cancer cells as with conventional chemotherapy, but also the cancer stem cells, or the
tumor-initiating cells, in the core of the tumor to eliminate possibilities of cancer metastasis and
recurrence. The study produced promising results for cellular interaction, uptake and cytotoxicity in
vitro as was observed both quantitively and qualitatively. Interaction of the mAb 2C5-targeted
liposomes was greater than ~3.25-fold with SK-BR-3 breast cancer cells, while the same formulation
showed over a ~1.3-fold increase in interaction with the MDA-MB-231 breast cancer cells in
comparison to the other controls used. A significantly enhanced fluorescence signal upon treatment
of both the breast cancer cells with rhodamine-labeled mAb 2C5-targeted liposomes was observed
in comparison to the unmodified liposomes that supported cellular specificity and enhanced cellular
uptake of the antibody-targeted formulation. The average of the corrected total cell fluorescence
(CTCF) plotted for the mAb 2C5-modified liposome-treated cells from three different locations on the fluorescence image was statistically significant (p 0.05) in comparison to that of the unmodified liposome-treated cells in both the cell lines. There was a ~4.5-fold and a ~3.0-fold increase in average CTCF with the mAb 2C5-modified liposomes in comparison to the unmodified liposomes in SK-BR-3 and MDA-MB-231 cells respectively. The quantitative data was a further confirmation for the enhanced cellular uptake of the formulations in both the cell lines.
Holographic monitoring confirmed improved cellular killing by showing visible differences in cellular morphology, cell division and proliferation over time (48 hours) that validated the efficacy of the mAb-targeted liposomal formulation in MDA-MB-231 cancer cells. The study also confirmed the specificity of mAb 2C5 for two different breast cancer cell types viz, the triple negative MDA-MB-231 cells and Her2-positive SK-BR-3 cells. Also, preliminary data on hemolysis (%) in female athymic nude mice confirmed lower toxicity for the antibody-targeted combination drug-loaded liposomal formulation in vivo in comparison to the single drug-loaded targeted formulations.
Transitioning from the studies using targeted polymeric micelles for pancreatic cancer therapy to
mAb-targeted liposomes for breast cancer combination drug therapy, additional studies to
demonstrate the stem cell-like properties of the cancer cells were performed. In order to also
confirm the potential of the combination therapeutics for breast cancer, in vitro studies were
performed using free drugs on a monolayer of breast cancer cells. A cell viability study using free
drugs and the study of effects of free drugs (both single drugs and in combination) on cellular
morphology and metastasis were performed to demonstrate efficacy of the combination
therapeutics on breast cancer cells. Lipodox in combination with salinomycin and paclitaxel in
combination with salinomycin were used to treat breast cancer cells in vitro.
In comparison to Lipodox alone, the combination therapy showed significantly improved cellular killing over time and with different doses and produced about 50% more cellular killing at 72 hours in MDA-MB-231 cells.
In comparison to salinomycin alone, the combination killed about 70% cells in 72 hours. MCF-7 cells were more sensitive to the therapy with lower dose of Lipodox giving enhanced cell killing in combination with salinomycin and achieving significantly higher cell killing percentages at 48 hours and 72 hours after treatment.
Significantly improved cell killing percentages were observed with both the cell lines in presence of the combination of drugs as opposed to single agents in a dose and time dependent manner. Characterization of the breast cancer cell lines for presence of cancer stem cell specific markers CD44+/CD24- showed enriched populations of cells expressing these biomarkers over several generations in both the cell lines. Also, unique properties of stem cells such as their differentiation potential and self-renewal properties were demonstrated using the breast cancer cells to successfully confirm the presence of stem cells and to validate the mammospheres generated for their stem cell-like properties. Although the regular expression of CD44+/CD24- marker is variable in different types of breast cancer, the mammospheres generated in MCF-7 cells showed high levels of CD44+/CD24- expression (that is different from the usual expression profile of CD44, CD24 in such basal/ epithelial breast cancer cells) validating the enrichment of cells with stem cell-like properties over generations using the 3D mammosphere culture system. The MDA-MB-231 cancer cells with intrinsically higher expression of CD44+/CD24- marker (as expected with the basal/mesenchymal cancer cells) showed consistent high expression of the marker in the various generations of the cancer cells in the mammospheres. The results also highlighted the biological heterogeneity of different types of breast cancers.
Additional holographic studies on wound healing of a monolayer of breast cancer cells revealed that the combination of free drugs (salinomycin and paclitaxel) treatment of wounds on a monolayer of MDA-MB-231 cells showed a reduction in gap width at the wound site from ~355um at the beginning of holographic monitoring to only ~219um at the end of the wound healing assay in comparison to a reduction to ~7um with salinomycin-only and to ~104um with paclitaxel-only treated wounds. The ability of the combination chemotherapy to prevent cellular migration at the wound suggested its potential to inhibit cellular proliferation and metastasis in vivo.
Overall, the study below is valuable for the development of targeted drug delivery systems for
pancreatic and breast cancer therapy. We hope that the work translates in the clinic and provides an
optimal outcome in people with this life-threatening malady.
Urval av studiens innehåll.
Additional information on the behavior of breast cancer cells in the presence of the targeted
formulation using holographic imaging was obtained to assess the effectiveness of the targeted
combination chemotherapeutics and their mode of action on the cancer cells. The specific
morphological changes induced by the action of the combination chemotherapeutics in the targeted
liposomal formulation were studied in the triple negative breast cancer cell line, MDA-MB-231, using holographic imaging. The cancer cell line is triple negative since it lacks estrogen receptor (ER) and progesterone receptor (PR) expression, as well as HER2 (human epidermal growth factor receptor 2) amplification. Specific information on cell count, proliferation, motility, and killing mechanism over time was assessed using this advanced imaging technique.
The wound was also imaged holographically over 48 hours (at 10-minute intervals) to secure quantitative data on gap widths and the percent cell-free areas following the drug treatments.
12.3.15. Holographic monitoring of MDA-MB-231 cellular morphology and proliferation
Cells, when ~90% confluent, were split and ~350,000 cells/well were seeded. Cells were incubated
for 24 hours and treated with the targeted formulation, the non-targeted formulation or the free
drugs (at starting concentrations of 2.5ug/ml for the single drug formulations and 1.25ug/ml of each
drug in combination preparations) before imaging using a holomonitor. The final concentrations of
drugs per well were diluted ~4x with the anti-condensation strips dipped thoroughly inside each well
before the start of imaging. The wells were holographically monitored for ~ 48 hours by recording
images every 10 minutes. After 48 hours, independent frames were collected from the beginning,
middle and the end of the monitoring to assay cellular proliferation, division and any other
morphological changes. Videos recorded changes over time.
13.10. Free drug effects on cellular migration and proliferation at the wound site imaged using
holographic monitoring
The wound generated was also imaged holographically over 48 hours (at intervals of 10 minutes) to
determine the rate of change of gap widths following the drug treatments. From about 355um gap
width at the start of the monitoring, the width reduced steadily to less than half over 24 hours and was ~104um wide at the end of 48 hours in the paclitaxel treated group. Likewise, from a 63% cell-
free area at the wound from start of the monitoring, the area decreased to ~18% in the paclitaxel-
treated wound. The other groups, salinomycin-only and untreated, showed an opposite trend, with
increasing cell numbers at the wound over time (reducing wound gaps and cell-free area
percentages). For the salinomycin, combination treated and the untreated wounds, the gap widths
were reduced (from ~355um to) ~7um, ~219um and ~47um respectively. The combination drug
treated wound had a significantly wider gap and a higher percentage of cell- free area at the wound
in comparison to any of the single drug treatments or the untreated wounds. This observation
supported the potential for anti-metastatic and anti-proliferative effects of combination therapy
over either of the independent drug treatments in vivo.
13.15. Holographic imaging of morphology, proliferation and division of breast cancer cells after treatment with drug-loaded mAb 2C5 modified liposomes
Holographic imaging of breast cancer cells treated with the mAb 2C5-modified drug containing
paclitaxel, salinomycin and combination-loaded liposomes, unmodified liposomes loaded with the
drugs, free drugs, and the untreated cells was performed for 48 hours. The monitoring helped
visualize live changes in morphology, proliferation and division of cells after the various treatments (Figure 43 a-g). The images gave a clear idea on how the different treatments (free drugs, vs. targeted
or non-targeted formulation loaded with drugs (as single agents and in combination)) affected the
viability of breast cancer cells.
Supporting data obtained via holographic monitoring on the gap-width closure and cellular morphology at the wound site over a period of 48 hours confirmed the efficacy of the combination drug therapy in preventing metastasis.
Holographic imaging was performed using the antibody-modified liposomal formulation over 48
hours to validate the efficacy of the formulation in MDA-MB-231 cell killing. Detailed imaging of
cellular division, proliferation and morphology over 48 hours showed promising results with the
targeted formulation in cell killing.
15. Conclusion:
The major issue with aggressive forms of breast cancer is that they tend to recur over time and thus
are metastatic. A combination drug therapy approach is a possible effective treatment that kills both
cancer stem cells (CSCs) and the bulk cells ofthe tumor35. We have reported a targeted combination-
drug delivery approach using a nanocarrier (liposome) with antibody (mAb 2C5) as the targeting
ligand. The approach confirmed the targeting effectiveness of mAb 2C5- modified liposomal
preparation and good cytotoxicity against two selected breast cancer cell lines, viz. triple negative
MDA-MB-231 cells and Her2-positive breast cancer SK-BR-3 cells. The cancer cell behavior observed holographically, confirmed that the liposomal paclitaxel/salinomycin formulation with the targeting ligand (mAb 2C5) facilitated cellular killing and had profound cancer cell cytotoxicity in an aggressive breast cancer cell line model in vitro. The advanced imaging technique provided a visual confirmation for the anti-cancer effect of the developed targeted liposomal formulation.
This research should be considered a step further in the development of the combination therapy for breast cancer, even in aggressive cases.
16. Concluding remarks
With the goal of developing targeted drug delivery systems for the conventional
chemotherapeutics in cancer therapy, the above study was designed. mAb 2C5-targeted
liposomes and phage coat protein-derived peptide-targeted micelles developed in the study are
expected to provide promising benefits such as improved solubility, bioavailability,
biodistribution and specificity of action in order to enhance the drug’s therapeutic benefits.
Additional advantage such as the delivery of combination chemotherapeutics to target and kill
both the cancer stem cells and the bulk cells using the liposomal preparation for aggressive
breast cancer therapy is an important highlight of the work. The developed drug delivery systems
tested on the hard-to-treat pancreatic and the very commonly diagnosed breast cancer cells, in
vitro, contribute the initial steps for the translation of such targeted formulations into clinic. We
firmly hope the designed experiments using the targeted formulations and the results recorded
thus far are helpful for additional research and prove useful to several people affected with the
life-threatening illness.
I thank Mr. Ed Luther for all help he rendered with Keyence microscopy imaging and holographic
monitoring.
Min kommentar
Denna väl utförda studie hur använda ny bättre teknik,bättre i den bemärkelsen att tekniken (nano) mer effektivt behandlar cancern som att den ger färre biverkningar (läs stycket om biverkningar) är baserad till stor del på användande av PHI`s HoloMonitor.När man läser igenom den 148-sidiga rapporten framträder hur väl man använt sig av PHI`s teknik och därigenom inser man hur viktig HoloMonitor varit för att nå dessa nya resultat.
Notera mina kursiverade markeringar hur doktoranden i sin avhandling beskriver Holotekniken.
PHI`s trogne partner sen 2014, Ed Luther/Northeastern University, producerar förhoppningsvis en riktigt vass framtida forskare i denna doktorand.Cancerforskningen behöver mer braighta snillen för att få stopp på denna best.Denna nya generation forskare kommer vara mer benägna att använda sig av ny state-of-the-art instrumentering för att nå helt nya insikter till gagn för världens alla cancerdrabbade.
Doktoranden Radhika Narayanaswamy lär vi få höra talas om framgent,förhoppningsvis med än mer ny kunskap baserad på användande av HoloMonitor.
För att ära hennes kommande utnämning till Doktor (PhD) tar jag mig friheten att klistra in nedanstående.
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