Graduate Category: Health Sciences Degree Level: Doctoral ...
Graduate Category: Health Sciences Degree Level: Doctoral candidate Abstract ID# 1436
The use of 3-dimensional cancer spheroids to study the lipid-based delivery systems for anti-cancer drug, NCL-240 B. 1Center
1 Pattni ,
S.
1 Nagelli ,
I.
1 Dutta ,
1 B. Aryasomayajula ,
A.
2 Degterev ,
V.
1 Torchilin
for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA. 2Department of Biochemistry, Tufts University, Boston, MA
ABSTRACT: Current in vitro cell culture studies use monolayer (2-dimensional) cell culture techniques to study the efficacy of various anti-cancer drugs and drug delivery systems (DDS). However, the monolayer cell culture fails to replicate the in vivo conditions of solid tumors at cellular and tumor microenvironment levels. Spheroid (3-dimensional) cell culture, which encourages multiple cell-cell contacts similar to physiological conditions, exhibits solid tumor features like a distribution gradient of O2, nutrients and proliferation, similar ratios of gene expression, accumulation of metabolites and development of a necrotic core. Resistant ovarian cancer cells, NCI/ADR-RES, were used to develop spheroids. A novel anti-cancer drug, NCL-240, in combination with TRAIL (tumor necrosis factor α (TNF-α)-related apoptosis-inducing ligand) was encapsulated in micelles and liposomes to study the efficacy in spheroids. Although NCI/ADR-RES cells, grown as monolayers, were sensitive to TRAIL, the ovarian cancer cells spheroids were resistant to TRAIL. Combination of TRAIL with NCL-240 reduced the drug resistance and improved therapeutic efficacy. Targeting the NCL-240–loaded DDS with transferrin (Tf) improved the cytotoxic efficacy of NCL-240. This effect correlated with improved penetration and association of targeted DDS in the spheroids. Thus, the application of spheroids to study anti-cancer drugs and DDS can be useful in oncology research.
INTRODUCTION
RESULTS Spheroid formation
Spheroids
Spheroid 2
Spheroid 1
The tumor microenvironment (TME) has an important role in the progression of cancer. To treat cancers effectively, it is very important to fully understand the changes associated with the TME. The figure below demonstrates the regions in the spheroid and solid tumors consisting of gradient of cell proliferation, oxygen distribution, nutrient distribution and pH. This kind of heterogeneous phenomenon are not represented by monolayer cell culture.
Association of micelles and liposomes with NCI/ADR-RES spheroids Spheroid 3
DAY 1
DAY 3
DAY 4
DAY 7
DAY 10
Measuring size of the spheroids
Incubation
Lipid based delivery systems
Mean Diameter (in µm)
1200 1000
MEAN DIAMETER OF NCI ADR SPHEROIDS 940.17
800 594.20 600
542.50
547.42
568.83
Day 4
Day 7
Day 10
400 200 0
Day 1
NCL-240
Day 3
Cytotoxicity studies
ePC/CHOL/CHEMS/DOPE 1x PBS, pH 7.4
0.2 → 0.1 µm
Nanoparticle characterization Formulation
Size (in nm)
TEM images Zeta potential (mV)
PEG2000-PE micelles
17.2±0.4
-6.8±1.7
0.5 μg/ml Tf-micelles
36.8±3.6
-30.6±2.8
1 μg/ml Tf-micelles
38.2±5.2
-31.4±0.9
2 μg/ml Tf-micelles
45.8±5.6
-37.5±1.3
Plain liposomes
144.2±3.7
-42.3±6.6
0.5 mol% Tf-liposomes 1 mol% Tf-liposomes 2 mol% Tf-liposomes
178.8±1.5 195.9±3.0 217.9±4.5
-36.1±3.3 -30.4±1.4 -32.2±2.1
Uniformly distributed liposomes
CONCLUSIONS Presence of transferrin on the surface of liposomes
3D NCI/ADR-RES spheroids were formed with sufficient reproducibility and consistency. As compared to the monolayer model of in vitro testing, the spheroids represented the solid tumor much better with visual presentation of a necrotic core as well as tight junction bindings and expression of diffusion profiles. As a result, the dose of drug required for the spheroids was higher than the monolayers The Tf-conjugated micelles and liposomes loaded with NCL-240 are capable drug delivery systems to target specific cancers A significant improvement in the toxicity profile of the NCL-240 drug was observed with targeting Penetration and uptake studies demonstrate how the targeted micelles and liposomes penetrate the deeper layers of the tumor.
ACKNOWLEDGEMENTS This work was supported NCI grant 5U54CA151881 to V. Torchilin
REFERENCES 1. 2. 3. 4.
Swartz, M.A., et al., Tumor microenvironment complexity: emerging roles in cancer therapy. Cancer Res, 2012. 72(10): p. 2473-80. Patel, N.R., et al., Nanopreparations to overcome multidrug resistance in cancer. Adv Drug Deliv Rev, 2013. 65(13-14): p. 1748-62 Hirschhaeuser, F., et al., Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol, 2010. 148(1): p. 3-15. Riehle, R.D., et al., Micellar formulations of pro-apoptotic DM-PIT-1 analogs and TRAIL in vitro and in vivo. Drug Deliv. 2013 Feb;20(2):78-85.
The use of 3-dimensional cancer spheroids to study the lipid-based delivery systems for anti-cancer drug, NCL-240 B. 1Center
1 Pattni ,
S.
1 Nagelli ,
I.
1 Dutta ,
1 B. Aryasomayajula ,
A.
2 Degterev ,
V.
1 Torchilin
for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA. 2Department of Biochemistry, Tufts University, Boston, MA
ABSTRACT: Current in vitro cell culture studies use monolayer (2-dimensional) cell culture techniques to study the efficacy of various anti-cancer drugs and drug delivery systems (DDS). However, the monolayer cell culture fails to replicate the in vivo conditions of solid tumors at cellular and tumor microenvironment levels. Spheroid (3-dimensional) cell culture, which encourages multiple cell-cell contacts similar to physiological conditions, exhibits solid tumor features like a distribution gradient of O2, nutrients and proliferation, similar ratios of gene expression, accumulation of metabolites and development of a necrotic core. Resistant ovarian cancer cells, NCI/ADR-RES, were used to develop spheroids. A novel anti-cancer drug, NCL-240, in combination with TRAIL (tumor necrosis factor α (TNF-α)-related apoptosis-inducing ligand) was encapsulated in micelles and liposomes to study the efficacy in spheroids. Although NCI/ADR-RES cells, grown as monolayers, were sensitive to TRAIL, the ovarian cancer cells spheroids were resistant to TRAIL. Combination of TRAIL with NCL-240 reduced the drug resistance and improved therapeutic efficacy. Targeting the NCL-240–loaded DDS with transferrin (Tf) improved the cytotoxic efficacy of NCL-240. This effect correlated with improved penetration and association of targeted DDS in the spheroids. Thus, the application of spheroids to study anti-cancer drugs and DDS can be useful in oncology research.
INTRODUCTION
RESULTS Spheroid formation
Spheroids
Spheroid 2
Spheroid 1
The tumor microenvironment (TME) has an important role in the progression of cancer. To treat cancers effectively, it is very important to fully understand the changes associated with the TME. The figure below demonstrates the regions in the spheroid and solid tumors consisting of gradient of cell proliferation, oxygen distribution, nutrient distribution and pH. This kind of heterogeneous phenomenon are not represented by monolayer cell culture.
Association of micelles and liposomes with NCI/ADR-RES spheroids Spheroid 3
DAY 1
DAY 3
DAY 4
DAY 7
DAY 10
Measuring size of the spheroids
Incubation
Lipid based delivery systems
Mean Diameter (in µm)
1200 1000
MEAN DIAMETER OF NCI ADR SPHEROIDS 940.17
800 594.20 600
542.50
547.42
568.83
Day 4
Day 7
Day 10
400 200 0
Day 1
NCL-240
Day 3
Cytotoxicity studies
ePC/CHOL/CHEMS/DOPE 1x PBS, pH 7.4
0.2 → 0.1 µm
Nanoparticle characterization Formulation
Size (in nm)
TEM images Zeta potential (mV)
PEG2000-PE micelles
17.2±0.4
-6.8±1.7
0.5 μg/ml Tf-micelles
36.8±3.6
-30.6±2.8
1 μg/ml Tf-micelles
38.2±5.2
-31.4±0.9
2 μg/ml Tf-micelles
45.8±5.6
-37.5±1.3
Plain liposomes
144.2±3.7
-42.3±6.6
0.5 mol% Tf-liposomes 1 mol% Tf-liposomes 2 mol% Tf-liposomes
178.8±1.5 195.9±3.0 217.9±4.5
-36.1±3.3 -30.4±1.4 -32.2±2.1
Uniformly distributed liposomes
CONCLUSIONS Presence of transferrin on the surface of liposomes
3D NCI/ADR-RES spheroids were formed with sufficient reproducibility and consistency. As compared to the monolayer model of in vitro testing, the spheroids represented the solid tumor much better with visual presentation of a necrotic core as well as tight junction bindings and expression of diffusion profiles. As a result, the dose of drug required for the spheroids was higher than the monolayers The Tf-conjugated micelles and liposomes loaded with NCL-240 are capable drug delivery systems to target specific cancers A significant improvement in the toxicity profile of the NCL-240 drug was observed with targeting Penetration and uptake studies demonstrate how the targeted micelles and liposomes penetrate the deeper layers of the tumor.
ACKNOWLEDGEMENTS This work was supported NCI grant 5U54CA151881 to V. Torchilin
REFERENCES 1. 2. 3. 4.
Swartz, M.A., et al., Tumor microenvironment complexity: emerging roles in cancer therapy. Cancer Res, 2012. 72(10): p. 2473-80. Patel, N.R., et al., Nanopreparations to overcome multidrug resistance in cancer. Adv Drug Deliv Rev, 2013. 65(13-14): p. 1748-62 Hirschhaeuser, F., et al., Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol, 2010. 148(1): p. 3-15. Riehle, R.D., et al., Micellar formulations of pro-apoptotic DM-PIT-1 analogs and TRAIL in vitro and in vivo. Drug Deliv. 2013 Feb;20(2):78-85.
