Health Sciences Degree Level: PhD Abstract ID# 752
Graduate Category: Health Sciences Degree Level: PhD Abstract ID# 752
Targeted Delivery of Polymer Pro-Drug Conjugate to Treat Drug Resistance in Ovarian Cancer Prashant Raj Bhattarai, Ankita Pandey, Weiting Chang, Ban-An Khaw Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115 Methods (contd.)
Introduc#on Clinical use of the anBcancer drugs is limited by its poor water solubility and dose-‐dependent toxicity. We have conjugated paclitaxel and doxorubicin to Poly-‐Glutamic acid polymers and have been able to improve the hydrophilicity of drugs and overcome cardio-‐toxicity associated to free drugs. We propose that using bispecific anBbody comprising Affibody (specific to Her2/neu receptors over expressed in various cancers) and AnB-‐DTPA anBbody we will be able to target our polymer pro-‐drugs to cancer cells and overcome mulBdrug resistance.
Methods
Free Paclitaxel
PGA-‐ Paclitaxel
Free PGA
120
2.000
100
1.500
80
1.000 Sample_DPGA_P1 BSA-DTPA
0.500
PGA-‐only 0 0.46875
-0.500 0.0001
0.001
0.01
0.1
1
10
Figure 2. TLC to determine the conjugation of Paclitaxel to PGA
Polymer Drug Conjugate
-‐20
DTPA-‐DOX-‐PGA
-‐11.475
DTPA-‐Paclitaxel-‐PGA
-‐15.754
80
Figure 4. Zeta potential Values for the various Polymer drug conjugates compared to the PGA alone
Characteriza#on of Her2/neu receptors in SKOV3 (sensi#ve) and SKOV3 TR (resistant) ovarian cancer cell lines Hoechst
0
1
2
3
4
5
6
7
8
9
10
Paclitaxel Concentra#on (μM)
Figure 9. Dose vs. response curve comparison for the free paclitaxel ( blue diamond) vs. DPGAPaclitaxel vs. DPGA-Paclitaxel pre-targeted with 10µg/ml of BispAb
C
E
DPGA-‐PTXL pre-‐targeted with 10ug/ml BispAb
0
FITC
B
DPGA-‐Paclitaxel alone
20
Bright field
Free Paclitaxel
60
40
F
! Figure 5. Epi- fluorescent microscopy of SKOV3 TR (resistant) ( Panel A – C) and SKOV3 (sensitive) (Panel D – F) cells with 5µg/ml of Affibody - FITC
0.500
0.000
!
Figure 10. Transformed dose vs. response curve for free paclitaxel vs. DPGA-‐paclitaxel treatment aVer pre-‐targe#ng with 10μg/ml of BispAb
Summary and Conclusion
• ConjugaBon of Paclitaxel to PGA was achieved. • TLC confirms conjugaBon of paclitaxel to PGA. • Using fluorescein labeled anB-‐Her2/neu Affibody expression of Her2 receptors in SKOV3 and SKOV3 TR (resistant) cell lines were confirmed. Her2 receptors were expressed at a significantly higher level in SKOV3 TR than in SKOV3 Paclitaxel sensiBve ovarian cancer cells. • DTPA-‐Paclitaxel-‐PGA pre-‐targeted with bispecific anB-‐Her2 affibody-‐anB-‐DTPA resulted in cell killing of SKOV3 TR (resistant) cancer cells with an IC50 of 0.8622μM compared to IC50 of 2.94μM with unconjugated free paclitaxel. • The mode of targeted delivery of paclitaxel-‐polymer-‐drug-‐conjugate to drug resistant ovarian cancer cells may lead to a novel treatment that can overcome drug resistance.
References
-0.500 100
Anti body Di l uti ons
Figure 1. AnB-‐DTPA anBbody Elisa for PGA-‐DTPA and BSA-‐DTPA
Drug Concentra#on (μg/ml)
100
Curve_DPGA1 DT PA-BSA
10
30
120
Zeta Potential at 25 0C (mV)
1.000
1
15
1.500
0.1
7.5
2.000
0.01
3.75
Figure 7. Cardio-toxicity associated to free drug was overcome using the various PGA drug conjugates
-‐21.425
D
1.875
Figure 3. Anti-DTPA antibody Elisa for DTPA-Paclitaxel-PGA (yellow diamonds) and BSA-DTPA (orange diamonds)
PGA
A
0.9375
100
2.500
0.001
PGA-‐Dox 40
20
0.000
Characteriza#on of Polymer Drug Conjugates
0.0001
Dox
60
PGA-‐PTXL
Results
Absorbance 450nm
Conjuga#on of Doxorubicin to DTPA-‐PGA: 1. 10mg/ml of DTPA-‐PGA in 0.1M PBS (pH=7.4) is mixed with 9 mg of doxorubicin in minimal amount of DMSO (200μl). 2. 17.2 mg of 1-‐ethyl-‐3-‐(3-‐dimethylaminopropyl) carbodiimide (EDC) is dissolved in the minimal amount of DMSO (250μl) and added to the mixture of PGA-‐DTPA and doxorubicin 3. Final soluBon is incubated in dark for 2hrs at 4 0C followed by overnight at room temperature. 4. Gel filtraBon using Sephadex G-‐25 column (1x10cm column) is carried out to separate free doxorubicin from the DTPA-‐Dox-‐PGA complex.
Cardi-‐otoxicity studies of PGA-‐drug conjugates in H9c2 rat cardiomyocytes 2.500
% Cell Death
Prepara#on of DTPA-‐PGA-‐Paclitaxel complex: 1. To an aliquot of PGA in dry N,N dimethylformamide Paclitaxel, Dicyclohexylcarbodiimide, and a trace amount of dimethylaminopyridine were added and reacBon allowed to proceed overnight at room temperature. TLC was undertaken to assess successful conjugaBon of Paclitaxel to PGA. 2. The reacBon was then stopped by addiBon of chloroform. The polymer-‐ drug-‐conjugate was subjected to RotavaporaBon . 3. ResulBng power dissolved in 0.5 M NaHCO3, pH 9.6. The polymer drug conjugate was then dialyzed overnight in 0.1 M NaHCO3, pH 9.6. 4. Then several Molar excess of DTPA dissolved in DMSO was added drop wise to the polymer-‐drug-‐conjugate soluBon. The reacBon was allowed to proceed for 2 hrs at room temperature and then was dialyzed overnight in 0.1 M PBS pH 7.4. 5. AnB-‐DTPA ELISA was uBlized to determine conjugaBon of DTPA. Prepara#on of Affibody-‐FITC: 1. 0.5 mg/ml of Affibody was incubated with 20mmol/L of dithiothreitol (DTT) at pH 7.4 for 2 hrs at room temperature. 2. A]er the reducBon of oxidized cysteine, affibody soluBon was dialyzed extensively against 0.1M PBS buffer containing 10mM EDTA for 24 hrs at 370C. 3. Fluorescein-‐malemide dye were then dissolved in DMSO and then added to the reduced affibody and reacBon was allowed to proceed overnight at 40C. 4. Unreacted dye was removed by Sephadex G-‐10 desalBng column chromatography. Epi-‐fluorescent microscopy studies for characteriza#on of Her2/neu receptors in SKOV3 and SKOV3 TR cell lines: 1. Approximately 500μl of culture media containing 80,000 SKOV3 or SKOV3 TR cells were added to the 12 well culture plates coverslip and incubated overnight. 2. Wells were washed with 0.1M PBS, a]er which the cells were fixed and permeabilized by adding Acetone to the wells for 10 mins at room temperature, followed by blocking with 3% BSA for 2 hours and washing. 4. Aliquots of 5 μg/ml of Affibody-‐FITC was added to the cells aLached to the coverslips and incubated in dark for 1 hr in a humidifier. 5. Coverslips were washed 5X Bmes with PBS-‐T followed by counterstaining with Hoechst, and then, mounted on slide. In Vitro tumorotoxicity assay on SKOV3 and SKOV3 TR cell lines: 1. 5000 cells/well were seeded in 96 well plates and incubated overnight for cell aLachment. 2. Medium of each well was removed and was replaced with medium containing 10 μg/ml of BispAb or culture medium alone and incubated for 1 hr at 370. Following which medium containing serial diluBon of 7.5, 3.75, 1.875, 0.9375, 0.46875, 0.234375, 0.1171875 μg/ml of free paclitaxel or equivalent concentraBon of DTPA-‐paclitaxel-‐PGA. 3. A]er 24hr incubaBon at 370C, viability was assessed using Cell Titer Blue assay.
Results (contd.)
% Cell Viability
Ra#onale: MulBdrug resistance of malignant cancer cells is a common mechanism leading to failure of chemotherapy. Drug resistance occurs in both hematological and solid cancers. We propose that targeted delivery of polymer pro-‐drug conjugates (PPDCs) to drug resistant ovarian cancer cells could lead to beLer therapeuBc outcome by uptake of PPDCs and subsequent release of acBve drug internally in cancer cells. Methods: Bispecific anBbody consisBng of Affibody, specific to HER2 receptors and conjugated to capture anB-‐DTPA anBbody was made. Poly-‐L-‐glutamic Acid (PGA) polymer is conjugated with doxorubicin and paclitaxel. SKOV3 and SKOV3TR (paclitaxel resistant) ovarian cancer cells that express HER 2 receptors were selected to demonstrate enhanced cytotoxicity. Results: In vitro studies using H9c2 cardio-‐myocytes demonstrated significant reducBon of cardio-‐toxicity with PGA-‐doxorubicin compared to doxorubicin alone. However, Paclitaxel and PGA-‐ paclitaxel were minimally cardio-‐toxic. A]er pre-‐targeBng with 10μg/ ml of bispecific anBbody for 1hr serial diluBons of doxorubicin and paclitaxel, and equivalent concentraBon of DTPA-‐Dox-‐PGA, or DTPA-‐ paclitaxel-‐PGA were added to both SKOV3 and SKOV3 TR. Higher concentraBon of cell death was observed in targeted delivery of DTPA-‐paclitaxel-‐PGA (IC50 0.8622μM) as compared to the free paclitaxel (IC50 2.94μM). Also DTPA-‐Dox-‐PGA and free dox had similar IC50 of 0.4μM. Conclusion: Targeted delivery of DTPA-‐Paclitaxel-‐PGA led to the higher cell killing in SKOV3 TR ovarian cancer cell line. OpBmizaBon of targeted delivery of DTPA-‐paclitaxel-‐PGA, pre-‐targeted with bispecific anBbodies and co-‐delivery of DTPA-‐Dox-‐PGA may lead to enhanced therapeuBc efficacy and overcome the drug resistance in ovarian cancer.
Results (contd.)
Absorbance 450nm
Abstract
Figure 6. Fluorescent intensity of FITC on SKOV3 and SKOV3 TR (resistant) cells targeted with Affibody - FITC
1.R. Duncan, L.W. Seymour, K.B. O'Hare, P.A. Flanagan, S. Wedge, I.C. Hume, M. Faro and A. Surato: Preclinical evaluaBon of polymer-‐bound doxorubicin. Journal of controlled release, 19 (1992) 331-‐346 2.Li, C., D. F. Yu, R. A. Newman, F. Cabral, L. C. Stephens, N. Hunter, L. Milas, and S. Wallace. 1998. Complete regression of well-‐established tumors using a novel water-‐ soluble poly (L-‐glutamic acid)-‐paclitaxel conjugate. Cancer Res 58:2404-‐2409.
Targeted Delivery of Polymer Pro-Drug Conjugate to Treat Drug Resistance in Ovarian Cancer Prashant Raj Bhattarai, Ankita Pandey, Weiting Chang, Ban-An Khaw Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115 Methods (contd.)
Introduc#on Clinical use of the anBcancer drugs is limited by its poor water solubility and dose-‐dependent toxicity. We have conjugated paclitaxel and doxorubicin to Poly-‐Glutamic acid polymers and have been able to improve the hydrophilicity of drugs and overcome cardio-‐toxicity associated to free drugs. We propose that using bispecific anBbody comprising Affibody (specific to Her2/neu receptors over expressed in various cancers) and AnB-‐DTPA anBbody we will be able to target our polymer pro-‐drugs to cancer cells and overcome mulBdrug resistance.
Methods
Free Paclitaxel
PGA-‐ Paclitaxel
Free PGA
120
2.000
100
1.500
80
1.000 Sample_DPGA_P1 BSA-DTPA
0.500
PGA-‐only 0 0.46875
-0.500 0.0001
0.001
0.01
0.1
1
10
Figure 2. TLC to determine the conjugation of Paclitaxel to PGA
Polymer Drug Conjugate
-‐20
DTPA-‐DOX-‐PGA
-‐11.475
DTPA-‐Paclitaxel-‐PGA
-‐15.754
80
Figure 4. Zeta potential Values for the various Polymer drug conjugates compared to the PGA alone
Characteriza#on of Her2/neu receptors in SKOV3 (sensi#ve) and SKOV3 TR (resistant) ovarian cancer cell lines Hoechst
0
1
2
3
4
5
6
7
8
9
10
Paclitaxel Concentra#on (μM)
Figure 9. Dose vs. response curve comparison for the free paclitaxel ( blue diamond) vs. DPGAPaclitaxel vs. DPGA-Paclitaxel pre-targeted with 10µg/ml of BispAb
C
E
DPGA-‐PTXL pre-‐targeted with 10ug/ml BispAb
0
FITC
B
DPGA-‐Paclitaxel alone
20
Bright field
Free Paclitaxel
60
40
F
! Figure 5. Epi- fluorescent microscopy of SKOV3 TR (resistant) ( Panel A – C) and SKOV3 (sensitive) (Panel D – F) cells with 5µg/ml of Affibody - FITC
0.500
0.000
!
Figure 10. Transformed dose vs. response curve for free paclitaxel vs. DPGA-‐paclitaxel treatment aVer pre-‐targe#ng with 10μg/ml of BispAb
Summary and Conclusion
• ConjugaBon of Paclitaxel to PGA was achieved. • TLC confirms conjugaBon of paclitaxel to PGA. • Using fluorescein labeled anB-‐Her2/neu Affibody expression of Her2 receptors in SKOV3 and SKOV3 TR (resistant) cell lines were confirmed. Her2 receptors were expressed at a significantly higher level in SKOV3 TR than in SKOV3 Paclitaxel sensiBve ovarian cancer cells. • DTPA-‐Paclitaxel-‐PGA pre-‐targeted with bispecific anB-‐Her2 affibody-‐anB-‐DTPA resulted in cell killing of SKOV3 TR (resistant) cancer cells with an IC50 of 0.8622μM compared to IC50 of 2.94μM with unconjugated free paclitaxel. • The mode of targeted delivery of paclitaxel-‐polymer-‐drug-‐conjugate to drug resistant ovarian cancer cells may lead to a novel treatment that can overcome drug resistance.
References
-0.500 100
Anti body Di l uti ons
Figure 1. AnB-‐DTPA anBbody Elisa for PGA-‐DTPA and BSA-‐DTPA
Drug Concentra#on (μg/ml)
100
Curve_DPGA1 DT PA-BSA
10
30
120
Zeta Potential at 25 0C (mV)
1.000
1
15
1.500
0.1
7.5
2.000
0.01
3.75
Figure 7. Cardio-toxicity associated to free drug was overcome using the various PGA drug conjugates
-‐21.425
D
1.875
Figure 3. Anti-DTPA antibody Elisa for DTPA-Paclitaxel-PGA (yellow diamonds) and BSA-DTPA (orange diamonds)
PGA
A
0.9375
100
2.500
0.001
PGA-‐Dox 40
20
0.000
Characteriza#on of Polymer Drug Conjugates
0.0001
Dox
60
PGA-‐PTXL
Results
Absorbance 450nm
Conjuga#on of Doxorubicin to DTPA-‐PGA: 1. 10mg/ml of DTPA-‐PGA in 0.1M PBS (pH=7.4) is mixed with 9 mg of doxorubicin in minimal amount of DMSO (200μl). 2. 17.2 mg of 1-‐ethyl-‐3-‐(3-‐dimethylaminopropyl) carbodiimide (EDC) is dissolved in the minimal amount of DMSO (250μl) and added to the mixture of PGA-‐DTPA and doxorubicin 3. Final soluBon is incubated in dark for 2hrs at 4 0C followed by overnight at room temperature. 4. Gel filtraBon using Sephadex G-‐25 column (1x10cm column) is carried out to separate free doxorubicin from the DTPA-‐Dox-‐PGA complex.
Cardi-‐otoxicity studies of PGA-‐drug conjugates in H9c2 rat cardiomyocytes 2.500
% Cell Death
Prepara#on of DTPA-‐PGA-‐Paclitaxel complex: 1. To an aliquot of PGA in dry N,N dimethylformamide Paclitaxel, Dicyclohexylcarbodiimide, and a trace amount of dimethylaminopyridine were added and reacBon allowed to proceed overnight at room temperature. TLC was undertaken to assess successful conjugaBon of Paclitaxel to PGA. 2. The reacBon was then stopped by addiBon of chloroform. The polymer-‐ drug-‐conjugate was subjected to RotavaporaBon . 3. ResulBng power dissolved in 0.5 M NaHCO3, pH 9.6. The polymer drug conjugate was then dialyzed overnight in 0.1 M NaHCO3, pH 9.6. 4. Then several Molar excess of DTPA dissolved in DMSO was added drop wise to the polymer-‐drug-‐conjugate soluBon. The reacBon was allowed to proceed for 2 hrs at room temperature and then was dialyzed overnight in 0.1 M PBS pH 7.4. 5. AnB-‐DTPA ELISA was uBlized to determine conjugaBon of DTPA. Prepara#on of Affibody-‐FITC: 1. 0.5 mg/ml of Affibody was incubated with 20mmol/L of dithiothreitol (DTT) at pH 7.4 for 2 hrs at room temperature. 2. A]er the reducBon of oxidized cysteine, affibody soluBon was dialyzed extensively against 0.1M PBS buffer containing 10mM EDTA for 24 hrs at 370C. 3. Fluorescein-‐malemide dye were then dissolved in DMSO and then added to the reduced affibody and reacBon was allowed to proceed overnight at 40C. 4. Unreacted dye was removed by Sephadex G-‐10 desalBng column chromatography. Epi-‐fluorescent microscopy studies for characteriza#on of Her2/neu receptors in SKOV3 and SKOV3 TR cell lines: 1. Approximately 500μl of culture media containing 80,000 SKOV3 or SKOV3 TR cells were added to the 12 well culture plates coverslip and incubated overnight. 2. Wells were washed with 0.1M PBS, a]er which the cells were fixed and permeabilized by adding Acetone to the wells for 10 mins at room temperature, followed by blocking with 3% BSA for 2 hours and washing. 4. Aliquots of 5 μg/ml of Affibody-‐FITC was added to the cells aLached to the coverslips and incubated in dark for 1 hr in a humidifier. 5. Coverslips were washed 5X Bmes with PBS-‐T followed by counterstaining with Hoechst, and then, mounted on slide. In Vitro tumorotoxicity assay on SKOV3 and SKOV3 TR cell lines: 1. 5000 cells/well were seeded in 96 well plates and incubated overnight for cell aLachment. 2. Medium of each well was removed and was replaced with medium containing 10 μg/ml of BispAb or culture medium alone and incubated for 1 hr at 370. Following which medium containing serial diluBon of 7.5, 3.75, 1.875, 0.9375, 0.46875, 0.234375, 0.1171875 μg/ml of free paclitaxel or equivalent concentraBon of DTPA-‐paclitaxel-‐PGA. 3. A]er 24hr incubaBon at 370C, viability was assessed using Cell Titer Blue assay.
Results (contd.)
% Cell Viability
Ra#onale: MulBdrug resistance of malignant cancer cells is a common mechanism leading to failure of chemotherapy. Drug resistance occurs in both hematological and solid cancers. We propose that targeted delivery of polymer pro-‐drug conjugates (PPDCs) to drug resistant ovarian cancer cells could lead to beLer therapeuBc outcome by uptake of PPDCs and subsequent release of acBve drug internally in cancer cells. Methods: Bispecific anBbody consisBng of Affibody, specific to HER2 receptors and conjugated to capture anB-‐DTPA anBbody was made. Poly-‐L-‐glutamic Acid (PGA) polymer is conjugated with doxorubicin and paclitaxel. SKOV3 and SKOV3TR (paclitaxel resistant) ovarian cancer cells that express HER 2 receptors were selected to demonstrate enhanced cytotoxicity. Results: In vitro studies using H9c2 cardio-‐myocytes demonstrated significant reducBon of cardio-‐toxicity with PGA-‐doxorubicin compared to doxorubicin alone. However, Paclitaxel and PGA-‐ paclitaxel were minimally cardio-‐toxic. A]er pre-‐targeBng with 10μg/ ml of bispecific anBbody for 1hr serial diluBons of doxorubicin and paclitaxel, and equivalent concentraBon of DTPA-‐Dox-‐PGA, or DTPA-‐ paclitaxel-‐PGA were added to both SKOV3 and SKOV3 TR. Higher concentraBon of cell death was observed in targeted delivery of DTPA-‐paclitaxel-‐PGA (IC50 0.8622μM) as compared to the free paclitaxel (IC50 2.94μM). Also DTPA-‐Dox-‐PGA and free dox had similar IC50 of 0.4μM. Conclusion: Targeted delivery of DTPA-‐Paclitaxel-‐PGA led to the higher cell killing in SKOV3 TR ovarian cancer cell line. OpBmizaBon of targeted delivery of DTPA-‐paclitaxel-‐PGA, pre-‐targeted with bispecific anBbodies and co-‐delivery of DTPA-‐Dox-‐PGA may lead to enhanced therapeuBc efficacy and overcome the drug resistance in ovarian cancer.
Results (contd.)
Absorbance 450nm
Abstract
Figure 6. Fluorescent intensity of FITC on SKOV3 and SKOV3 TR (resistant) cells targeted with Affibody - FITC
1.R. Duncan, L.W. Seymour, K.B. O'Hare, P.A. Flanagan, S. Wedge, I.C. Hume, M. Faro and A. Surato: Preclinical evaluaBon of polymer-‐bound doxorubicin. Journal of controlled release, 19 (1992) 331-‐346 2.Li, C., D. F. Yu, R. A. Newman, F. Cabral, L. C. Stephens, N. Hunter, L. Milas, and S. Wallace. 1998. Complete regression of well-‐established tumors using a novel water-‐ soluble poly (L-‐glutamic acid)-‐paclitaxel conjugate. Cancer Res 58:2404-‐2409.
