RES
Graduate Category: Health Sciences Degree Level: Ph.D. Abstract ID# 1249
Transferrin-Targeted, Resveratrol-Loaded Liposomes for the Treatment of Glioblastoma Aditi Jhaveri and Vladimir Torchilin Ph.D. , D.Sc. Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston MA 02115
Results
Abstract Glioblastomas (GBMs) are the most lethal brain tumors with a dismal prognosis. They are known to harbor subsets of cells known as tumor-initiating cells (TICs), that are resistant to therapy and are responsible for the formation, maintenance, invasiveness and recurrence of GBMs. Most conventional chemotherapeutics act on the rapidly dividing cells sparing the TICs, and result in tumor relapse. Resveratrol, (RES) a natural polyphenol, has been shown to exhibit chemopreventive effects in all the major stages of cancer including initiation, promotion and progression, but poor water solubility, chemical instability, low bioavailability and poor pharmacokinetics limit its use as a free drug. To overcome these limitations, we developed a liposomal formulation of RES (RES-L) as a means to eradicate both the bulk tumor cells and TICs in GBMs. Since both these sub-populations of cells are known to over express transferrin receptors (TfRs), we utilized Tf as a targeting ligand and developed Tf-targeted RES-L (Tf-RESL) to enhance their tumor-specific delivery. In vitro, RES was found to inhibit the anchorage-independent growth of neurospheres in two GBM TIC models. Cytotoxicity studies showed time and dose-dependent toxicity of RES on GBM cells. Tf-RES-L induced significantly greater apoptosis and cytotoxicity in GBM cells compared to RESL. RES acted as a pro-oxidant in cancer cells at higher concentrations and arrested cells in the S-phase of cell-cycle at low concentrations. Tf-L also exhibited a greater association with and internalization into cancer cells versus non-targeted liposomes. TfRES-L thus seem like a promising platform for the treatment of GBMs.
Development and characterization of TIC cultures (Neurospheres)
Cell-cycle effects
Expression of CD133 on NS
Neurosphere (NS) cultures U-87 MG NS P-1
General mechanisms of action of RES
LN-18 NS P-1
Figure 1. Images of U-87 MG NS (day 5) and LN-18 NS (day 3) after seeding in 10 cm dishes in NS media (DMEM/F-12 with N2 and B27 supplements+EGF+FGF)
In-vitro limiting dilution assays
Cell-cycle distribution
Figure 8. U-87 MG cells were grown in 6-well plates and treated with free RES or RES-L (0-300 µM RES) for 24 h. Following the incubation, cells were stained with FxCycle™ solution and the DNA content was analyzed using flow cytometry.
U-87 MG NS,P-4
Tf-targeted liposomes Mechanism of uptake of Tf-L
Figure 3. Single cells dissociated from U-87 MG and LN-18 NS were serially diluted and seeded at a density of 1 cell/well in 96-well ultra-low attachment (ULA) plates. The wells containing a single NS were followed over the next few days. The NS were imaged at day 13 using a Nikon Eclipse E400 microscope.
•GBMs are difficult to treat, poor prognosis (5 year survival: < 5%) • Sub-population of cells known as tumor-initiating cells (TICs) • Conventional therapies: Toxicity to normal tissues, act on bulk tumor cells, leave behind TICs , cause tumor relapse
Table 1. Composition, size , charge and drug-loading characteristics of liposomes Lipid composition (molar ratios)
• Drugs which act on both the cell populations (bulk and TICs) • Resveratrol (RES): Natural polyphenol found in red wine • Exhibits various health benefits • Demonstrated effectiveness in inhibiting both populations of cells in GBMs • Issues with RES as a free drug: Compromised • Poor water solubility pharmacological effects • Chemical instability • Low bioavailability • Poor pharmacokinetic (PK) properties
EggPC, Chol, DSPE-PEG2000, DOPE, CHEMS (61:24:3:6:6)
Our approach: • Encapsulating RES in liposomes • Improved stability, solubility and PK parameters • PEGylation of liposomes to improve in vivo circulation • Exploiting tumor-specific receptors to target liposomes specifically to cancer cells: • Transferrin receptors (TfRs): Involved in irontransport, over-expressed in GBMs and other cancers, also expressed in TICs in GBMs • Tf attached to the distal ends of PEG to make Tftargeted RES-loaded liposomes (Tf-RES-L)
Figure 4. Cell-cycle profiles of the parent GBM cell lines (monolayers) were compared with NS. Cells growing as monolayers and as neurospheres were collected post-trypsinization and stained with PI/RNAse solution to analyze their DNA content using flow cytometry.
Characterization of liposomal formulations
Potential Solution:
Resveratrol
Liposomes
Particle diameter (nm± S.D)
PDI
Zeta potential (mv± S.D)
PL
180.5±5.8
0.11
-39.4±1.3
RES-L (3%w/w RES)
182.3±12.1
0.11
-41.7±1.6
Tf-L (1 mol% Tf)
203.8±7.8
0.10
-35.0±1.5
Tf-RES-L (3%w/w RES, 1 mol%Tf)
217.4±0.8
0.09
-34.1±0.9
Figure 10. U-87 MG cells were grown in 12 well plates for 24 h. 30 min before incubation with rhodamine labeled Tf-L, free Tf (2.5mg/ml) was added to the medium. After 4 h incubation, cells were processed for flow cytometry and geometric means of fluorescence were recorded..
PL-1h
Tf-L 1h
% Drug loading
PL
70-75%
Tf-L
RES-L
PL-4h
Tf-L 4h
Cytotoxicity of Tf-RES-formulations
Figure 11. U-87 MG cells were grown on glass coverslips in 6-well plates for 24 h, following which they were incubated with rhodamine labeled PL or Tf-L for 1 h and 4 h. 20 min before the end of incubation, the cells were stained with Hoechst (5µg/mL) and Tf-Alexa fluor 680 (5µg/mL), fixed with 4% PFA and imaged using Zeiss confocal microscope.
Apoptosis induction
Tf-RES-L
70-75% Figure 5. TEM images taken at a direct magnification of 30000X
Effects of RES formulations on NS and monolayers Growth inhibitionof NS by RES
Aqueous core
Cytotoxicity on monolayers Figure 12. Cells were grown for 24 h in 96 well plates and incubated with RES formulations (0-200 µM RES). CellTiter-Blue® assay was used to determine the percent of viable cells at 24 h following the exposure of cells.
Lipid bilayer
Figure 13. Cells were grown in 12 well plates for 24h, followed by incubation with various RES-formulations for 24 h. Cells were then trypsinized and stained with Annexin-V/propidium iodide using a standard apoptosis assay kit. Percentages of apoptotic and necrotic cells were analyzed using flow cytometry
PEG layer
Conclusions
Transferrin
Two TIC models of GBM were developed and characterized using various functional assays Liposomal formulation of RES was successfully developed, characterized and tested on GBM cells in vitro Free RES and RES-L exhibited cytotoxicity on GBM cell lines Free RES inhibited the growth of NS in vitro, suggesting its potential to be used as a drug that inhibits both Control
DMSO
20 uM
40 uM
60 uM
80 uM
1. Kaiser, J. (2015) Science 347:6219, 226-229 2. Jang, M. et al. (1997) Science 275(5297) 218-20
4. Schonberg, D.L. et al. (2015) Cancer Cell 28(4):441-55
Confocal imaging: Internalization of Tf-L into cells
TEM images of formulations
References
3. Neves, A.R. et al. (2012) Curr. Med. Chem. 19:1663-81
Figure 9. U-87 MG cells were grown in 12-well plates. They were treated with CMH2DCFDA dye (1 µM) for 30 min, followed by treatments for 1 h with various RES formulations (20-400 µM RES) Following the incubation, cells were processed for flow cytometry.
Figure 2. Determining the expression of CD133 on NS. NS were dissociated and incubated with CD133-PE or the isotype IgG2b-PE antibodies respectively, for 20 min at 4°C in the dark. The expression of CD133 was determined using flow cytometry.
Background The Problem:
Reactive-oxygen species generation
Figure 6. NS were dissociated into single cells and 1 x103 were seeded in each well of an ULA 6-well plate. RES was added to the wells from 20-80 µM and the NS were imaged at day 8 using a fluorescence microscope. LN-18 (left), U-87 MG (right)
Figure 7. Cells were seeded at 5 X 103 and 3x103cells/well respectively in 96 well plates and incubated with RES formulations (0-200 µM RES). CellTiterBlue® assay was used to determine the percent of viable cells at 24 h and 48 h following exposure of cells.
the bulk tumor cells and TICs in GBM Mechanistic studies of RES reveal that its ability to cause arrest of cells in the S-phase as well as its ability to generate ROS in cells may be implicated in its cytotoxic actions. Tf-modified liposomes exhibited significantly improved cytotoxicity, showed better association with and internalization into cells and also induced increased levels of apoptosis in U-87 MG compared to the unmodified liposomes
Transferrin-Targeted, Resveratrol-Loaded Liposomes for the Treatment of Glioblastoma Aditi Jhaveri and Vladimir Torchilin Ph.D. , D.Sc. Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston MA 02115
Results
Abstract Glioblastomas (GBMs) are the most lethal brain tumors with a dismal prognosis. They are known to harbor subsets of cells known as tumor-initiating cells (TICs), that are resistant to therapy and are responsible for the formation, maintenance, invasiveness and recurrence of GBMs. Most conventional chemotherapeutics act on the rapidly dividing cells sparing the TICs, and result in tumor relapse. Resveratrol, (RES) a natural polyphenol, has been shown to exhibit chemopreventive effects in all the major stages of cancer including initiation, promotion and progression, but poor water solubility, chemical instability, low bioavailability and poor pharmacokinetics limit its use as a free drug. To overcome these limitations, we developed a liposomal formulation of RES (RES-L) as a means to eradicate both the bulk tumor cells and TICs in GBMs. Since both these sub-populations of cells are known to over express transferrin receptors (TfRs), we utilized Tf as a targeting ligand and developed Tf-targeted RES-L (Tf-RESL) to enhance their tumor-specific delivery. In vitro, RES was found to inhibit the anchorage-independent growth of neurospheres in two GBM TIC models. Cytotoxicity studies showed time and dose-dependent toxicity of RES on GBM cells. Tf-RES-L induced significantly greater apoptosis and cytotoxicity in GBM cells compared to RESL. RES acted as a pro-oxidant in cancer cells at higher concentrations and arrested cells in the S-phase of cell-cycle at low concentrations. Tf-L also exhibited a greater association with and internalization into cancer cells versus non-targeted liposomes. TfRES-L thus seem like a promising platform for the treatment of GBMs.
Development and characterization of TIC cultures (Neurospheres)
Cell-cycle effects
Expression of CD133 on NS
Neurosphere (NS) cultures U-87 MG NS P-1
General mechanisms of action of RES
LN-18 NS P-1
Figure 1. Images of U-87 MG NS (day 5) and LN-18 NS (day 3) after seeding in 10 cm dishes in NS media (DMEM/F-12 with N2 and B27 supplements+EGF+FGF)
In-vitro limiting dilution assays
Cell-cycle distribution
Figure 8. U-87 MG cells were grown in 6-well plates and treated with free RES or RES-L (0-300 µM RES) for 24 h. Following the incubation, cells were stained with FxCycle™ solution and the DNA content was analyzed using flow cytometry.
U-87 MG NS,P-4
Tf-targeted liposomes Mechanism of uptake of Tf-L
Figure 3. Single cells dissociated from U-87 MG and LN-18 NS were serially diluted and seeded at a density of 1 cell/well in 96-well ultra-low attachment (ULA) plates. The wells containing a single NS were followed over the next few days. The NS were imaged at day 13 using a Nikon Eclipse E400 microscope.
•GBMs are difficult to treat, poor prognosis (5 year survival: < 5%) • Sub-population of cells known as tumor-initiating cells (TICs) • Conventional therapies: Toxicity to normal tissues, act on bulk tumor cells, leave behind TICs , cause tumor relapse
Table 1. Composition, size , charge and drug-loading characteristics of liposomes Lipid composition (molar ratios)
• Drugs which act on both the cell populations (bulk and TICs) • Resveratrol (RES): Natural polyphenol found in red wine • Exhibits various health benefits • Demonstrated effectiveness in inhibiting both populations of cells in GBMs • Issues with RES as a free drug: Compromised • Poor water solubility pharmacological effects • Chemical instability • Low bioavailability • Poor pharmacokinetic (PK) properties
EggPC, Chol, DSPE-PEG2000, DOPE, CHEMS (61:24:3:6:6)
Our approach: • Encapsulating RES in liposomes • Improved stability, solubility and PK parameters • PEGylation of liposomes to improve in vivo circulation • Exploiting tumor-specific receptors to target liposomes specifically to cancer cells: • Transferrin receptors (TfRs): Involved in irontransport, over-expressed in GBMs and other cancers, also expressed in TICs in GBMs • Tf attached to the distal ends of PEG to make Tftargeted RES-loaded liposomes (Tf-RES-L)
Figure 4. Cell-cycle profiles of the parent GBM cell lines (monolayers) were compared with NS. Cells growing as monolayers and as neurospheres were collected post-trypsinization and stained with PI/RNAse solution to analyze their DNA content using flow cytometry.
Characterization of liposomal formulations
Potential Solution:
Resveratrol
Liposomes
Particle diameter (nm± S.D)
PDI
Zeta potential (mv± S.D)
PL
180.5±5.8
0.11
-39.4±1.3
RES-L (3%w/w RES)
182.3±12.1
0.11
-41.7±1.6
Tf-L (1 mol% Tf)
203.8±7.8
0.10
-35.0±1.5
Tf-RES-L (3%w/w RES, 1 mol%Tf)
217.4±0.8
0.09
-34.1±0.9
Figure 10. U-87 MG cells were grown in 12 well plates for 24 h. 30 min before incubation with rhodamine labeled Tf-L, free Tf (2.5mg/ml) was added to the medium. After 4 h incubation, cells were processed for flow cytometry and geometric means of fluorescence were recorded..
PL-1h
Tf-L 1h
% Drug loading
PL
70-75%
Tf-L
RES-L
PL-4h
Tf-L 4h
Cytotoxicity of Tf-RES-formulations
Figure 11. U-87 MG cells were grown on glass coverslips in 6-well plates for 24 h, following which they were incubated with rhodamine labeled PL or Tf-L for 1 h and 4 h. 20 min before the end of incubation, the cells were stained with Hoechst (5µg/mL) and Tf-Alexa fluor 680 (5µg/mL), fixed with 4% PFA and imaged using Zeiss confocal microscope.
Apoptosis induction
Tf-RES-L
70-75% Figure 5. TEM images taken at a direct magnification of 30000X
Effects of RES formulations on NS and monolayers Growth inhibitionof NS by RES
Aqueous core
Cytotoxicity on monolayers Figure 12. Cells were grown for 24 h in 96 well plates and incubated with RES formulations (0-200 µM RES). CellTiter-Blue® assay was used to determine the percent of viable cells at 24 h following the exposure of cells.
Lipid bilayer
Figure 13. Cells were grown in 12 well plates for 24h, followed by incubation with various RES-formulations for 24 h. Cells were then trypsinized and stained with Annexin-V/propidium iodide using a standard apoptosis assay kit. Percentages of apoptotic and necrotic cells were analyzed using flow cytometry
PEG layer
Conclusions
Transferrin
Two TIC models of GBM were developed and characterized using various functional assays Liposomal formulation of RES was successfully developed, characterized and tested on GBM cells in vitro Free RES and RES-L exhibited cytotoxicity on GBM cell lines Free RES inhibited the growth of NS in vitro, suggesting its potential to be used as a drug that inhibits both Control
DMSO
20 uM
40 uM
60 uM
80 uM
1. Kaiser, J. (2015) Science 347:6219, 226-229 2. Jang, M. et al. (1997) Science 275(5297) 218-20
4. Schonberg, D.L. et al. (2015) Cancer Cell 28(4):441-55
Confocal imaging: Internalization of Tf-L into cells
TEM images of formulations
References
3. Neves, A.R. et al. (2012) Curr. Med. Chem. 19:1663-81
Figure 9. U-87 MG cells were grown in 12-well plates. They were treated with CMH2DCFDA dye (1 µM) for 30 min, followed by treatments for 1 h with various RES formulations (20-400 µM RES) Following the incubation, cells were processed for flow cytometry.
Figure 2. Determining the expression of CD133 on NS. NS were dissociated and incubated with CD133-PE or the isotype IgG2b-PE antibodies respectively, for 20 min at 4°C in the dark. The expression of CD133 was determined using flow cytometry.
Background The Problem:
Reactive-oxygen species generation
Figure 6. NS were dissociated into single cells and 1 x103 were seeded in each well of an ULA 6-well plate. RES was added to the wells from 20-80 µM and the NS were imaged at day 8 using a fluorescence microscope. LN-18 (left), U-87 MG (right)
Figure 7. Cells were seeded at 5 X 103 and 3x103cells/well respectively in 96 well plates and incubated with RES formulations (0-200 µM RES). CellTiterBlue® assay was used to determine the percent of viable cells at 24 h and 48 h following exposure of cells.
the bulk tumor cells and TICs in GBM Mechanistic studies of RES reveal that its ability to cause arrest of cells in the S-phase as well as its ability to generate ROS in cells may be implicated in its cytotoxic actions. Tf-modified liposomes exhibited significantly improved cytotoxicity, showed better association with and internalization into cells and also induced increased levels of apoptosis in U-87 MG compared to the unmodified liposomes
