NIH Public Access Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
NIH-PA Author Manuscript
Published in final edited form as: PMSE Preprints. 2011 ; 105: 953–954.
Magnetothermally-triggered Drug Delivery Using Temperatureresponsive Polymeric Micelles James B. Bennett1, Amanda L. Glover2, David E. Nikles2, Jacqueline A. Nikles3, and Christopher S. Brazel1 1Department
of Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, Alabama 35487-0203 USA
2Department
of Chemistry, The University of Alabama, Tuscaloosa, Alabama 35487-0336 USA
3Department
of Chemistry, The University of Alabama at Birmingham, Birmingham, Alabama 35294-1240 USA
NIH-PA Author Manuscript
INTRODUCTION
NIH-PA Author Manuscript
The development of nanoscale systems with medicinal applications has accelerated in the last several years. Emerging developments in cancer research include polymer-based drug delivery systems, (immuno-)liposomes, and dendrimers [1,2,3]. Many proposed drug carriers incorporate a combination of targeting, imaging, and therapeutic moieties to improve diagnostics and while minimizing side effects. Magnetic nanoparticles are another developing technology gaining interest for their potential in clinical cancer therapy and MRI contrast agents [4,5]. Magnetic fluid hyperthermia has been used in Europe to improve patient response when combined with traditional localized radiation and chemotherapy [6, 7]. This project explores the design of a drug delivery system that utilizes similar superparamagnetic nanoparticles which can be localized within human tissue and heated using an AC magnetic field applied externally. The drug carrier is a targeted micelle made of a diblock-copolymer which self-assembles to encapsulate the magnetic nanoparticles and anticancer drug into temperature-responsive semi-crystalline core as depicted in Figure 1. The core material exhibits a positive thermal response so that a phase change occurs when a temperature just above physiological temperature is reached. When the system is subjected to the AC magnetic field, the incorporated magnetic nanoparticles heat the core triggering a phase change designed to allow the release of the therapeutic drug. Here we report the ability of custom-synthesized nanoparticles to heat modulate drug release of the polymeric micelles in response to temperature increase. For the application potential of our drug-delivery system to be realized, there are several important foci in our work including: •
Use of a biocompatible diblock-copolymer system which exhibits a phase-change around 40–45 °C,
•
Synthesis and characterization of magnetic nanoparticles that heat efficiently using an AC magnetic field,
Bennett et al.
Page 2
NIH-PA Author Manuscript
•
Optimizing the magnetic nanoparticles heating profile by tuning nanoparticle concentration, AC field intensity and frequency,
•
Assembly of micelle into aqueous solutions,
•
Efficient loading of hydrophobic drug and nanoparticles into the core of the polymeric micelle,
•
Investigation on the drug release of model drug at physiological and elevated temperatures to mimic magnetic field heating,
•
Attachment of a targeting ligand to specifically bind micelle to cancer cells.
MATERIALS AND METHODS Diblock-Copolymer Synthesis
NIH-PA Author Manuscript
Several formulations of amphiphillic diblock-copolymers have been synthesized from hydrophilic ethylene glycol and hydrophobic ε-caprolactone as described in a separate paper [8]. A specific PEG42PCL19 diblock with an M̄n of 4000 exhibits a melting point in the appropriate range as confirmed by differential scanning calorimetry. This formulation was used to load pyrene and triamterene, model hydrophobic drugs, to study thermally-activated release. Magnetic Nanoparticle Synthesis and Characterization
NIH-PA Author Manuscript
Magnetite (Fe3O4) nanoparticles were synthesized following previously published techniques [9]. Maghemite (γ-Fe2O3) nanoparticles were synthesized in a two-step fashion, first creating an iron-oleate complex by reacting iron chloride with sodium oleate in a mixture solvent of hexane/ethanol/water at 70°C for four hours. In the second step, oleic acid was added to the iron-oleate complex in an octadecene solvent and heated to 320°C under reflux for two hours. Magnetic nanoparticles were characterized by transmission electron microscopy (TEM) for size distribution and x-ray diffraction (XRD) to determine crystalline structures within the nanoparticles. The dispersed nanoparticles were introduced to an AC magnetic field produced by a custom unit that allows a magnetic field intensity range of 0–5kW and a frequency range of 50–450 kHz (Induction Atmospheres, Rochester, NY). Temperature data was collected using a FLIR ThermaCAM infrared camera (FLIR, North Billerica, MA). Initial temperature for heating profiles was kept at 37°C by jacketing the sample with water channeled from a water bath. Incorporation of Drugs into Self-Assembling Micelles Model drugs, pyrene and triamterene, are very hydrophobic with low water solubility and mimic the properties of doxorubicin, a drug commonly used in cancer chemotherapy. To encapsulate the drug, we utilized a solvent evaporation technique.10.1 grams of the PEG42PCL19 diblock copolymer, and 1 mg of pyrene or triamterene was dissolved in 2mL tetrahydrofuran THF. The resulting mixture was added dropwise to 20mL of deionized water under vigorous stirring. The solution was allowed to equilibrate overnight before residual THF was removed through rotary evaporation concentrating the solution to 5 mL– 10mL. The resulting solution was then filtered through a 0.45 μm filter to remove large
PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 3
particles and ultrafiltered through a 50 kD MWCO centrifugal filter device to remove any free pyrene.
NIH-PA Author Manuscript
Drug Release Study at Physiological Temperature and Hyperthermia Conditions A 1-mL solution of pyrene or triamterene loaded micelles was placed inside a Float-A-Lyzer G2 cellulose ester 50 kD MWCO dialysis tube (Spectrum Laboratories, Rancho Dominguez, CA) which floated in a 100 mL dialysate. The temperature was maintained at 37°CC using a water bath. After 20 minutes, the dialysis device was moved to 100 mL of a new dialysate at 47°C to mimic magnetic heating and cause melting of the micelle core. Dialysate samples (0.5 mL) were taken in 4 minute increments. Triamterene concentrations were monitored by UV/Vis absorbance at 270nm. Pyrene concentrations were monitored using a fluorescence spectrophotometer with an excitation wavelength of 317 nm and emission peak at 392 nm.
RESULTS AND DISCUSSION Magnetic Heating
NIH-PA Author Manuscript
Solutions of magnetic nanoparticles in hexane subjected to an AC magnetic field exhibited effective magnetic induction heating. Four concentrations of magnetite in hexane were each placed under a magnetic field with the same intensity (700 Gauss); their behavior is summarized in Table 1. Heating was related to magnetic nanoparticle concentration. Melting the core of the micelle to trigger drug release will likely be dependent on the concentration of magnetic nanoparticles loaded within the core. Externally-tunable parameters governing the magnetic field also greatly influence heating. Our magnetic heat induction unit enables the manipulation of voltage, frequency and capacitance to control the magnetic field. Figure 2 shows the effect of field intensity on heating profiles. The highest intensity (700G) produced the most efficient heating. Reaching higher temperatures quicker would minimize the time a potential patient is exposed to hyperthermia conditions to activate chemotherapy. Triamterene Release
NIH-PA Author Manuscript
Triamterene loaded micelles allow us to investigate controlled release with a model drug easily detectible in the UV/Vis range. Triamterene was observed in the dialysate immediately upon insertion into the 37 °C medium, possibly due to incomplete separation of the micelles from free unencapsulated triamterene. Upon heating to 47°C, triamterene release increased for a short time, theoretically due to the melting of the PCL micelle core. Further experiments are on-going to determine the capacity of PEG-PCL micelles to encapsulate hydrophobic drugs including triamterene and pyrene, as well as doxorubicin, and determine the release behavior when subjected to heating.
CONCLUSIONS A magnetothermally-triggered polymeric micelle drug delivery system requires a sophisticated design, but by providing targeting, controlled release, and hyperthermia, potential for improved cancer therapy with minimized side-effects is possible. Here, magnetic heating has been proven for magnetite nanoparticles, and the self-assembly of PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 4
PEG-PCL diblock copolymers around hydrophobic drugs has been confirmed with release of triamterene modulated by an increase in temperature.
NIH-PA Author Manuscript
Acknowledgments The project described was supported by Award Number R21CA141388 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.
References
NIH-PA Author Manuscript
1. Allen TM, Cullis PR. Science. 2004; 303(5665):1818–22. [PubMed: 15031496] 2. Schiffelers RM, Koning GA, ten Hagen TL, Fens MH, Schraa AJ, Janssen AP, Kok RJ, Molema G, Storm G. J Control Release. 2003; 91(1–2):155–22. 3. Choi Y, Thomas T, Kotlyar A, Islam MT, Baker JR Jr. Chem Biol. 2005; 12(1):41–51. 4. Brigger I, Dubernet C, Couvreur P. Adv Drug Deliv Rev. 2002; 54(5):631–51. [PubMed: 12204596] 5. Pankhurst QA, Connolly J, Jones SK, Dobson J. Journal of Magnetism and Magnetic Materials Proceedings of the Fifth International Conference on Scientific Clinical Applications of Magnetic Carriers. 2003; 293(1):514–9. 6. Rosensweig RE. J Magn Magn Mat. 2002; 252:370–374. 7. Hergt R, Andra W, d’Ambly CG, Hilger I, Kaiser WA, Ricter U, Schmidt HG. IEEE trans Magnetics. 1998; 34(5):3745–3754. 8. Glover AL, Bennett JB, Nikles SM, Nikles JA, Brazel CS, Nikles DE. Polymer Prepr. 2011; (105) 9. Sun S, Zeng H, Robinson DB, Raoux S, Rice PM, Wang SX, Li G. J Am Chem Soc. 2004; 126(1): 273–279. [PubMed: 14709092]
NIH-PA Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 5
NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 1.
Schematic of a magnetothermally-triggered drug delivery vehicle. A hyrdrophobic crystalline polymer poly(caprolactone) (red) forms a core that encapsulates the cancer drug (blue) and magnetic nanoparticles (m). A targeting moiety such as an RGD-peptide can be attached to the hydrophilic block poly(ethylene glycol) (green) to specifically bind the drug carrier to cancer cells. Upon magnetic heating, drug is released.
NIH-PA Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 6
NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 2.
Magnetic heating profiles for 8.4 g/L Fe3O4 nanoparticles in hexane at three magnetic field intensities and a frequency of 266kHz.
NIH-PA Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 7
NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 3.
Release of triamterene from PEG42PCL19 micelles subjected to a temperature increase at 20 minutes.
NIH-PA Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 8
Table 1
NIH-PA Author Manuscript
Temperature Maximums after 3 Minutes of Magnetic Induction Heating for Various Concentrations of Fe3O4 Nanoparticles in Hexane Fe3O4 Concentration (g/L)
Maximum T3 min (°C)
8.4
51.2
4.2
44.6
1.05
41.3
0.503
40.0
NIH-PA Author Manuscript NIH-PA Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
NIH-PA Author Manuscript
Published in final edited form as: PMSE Preprints. 2011 ; 105: 953–954.
Magnetothermally-triggered Drug Delivery Using Temperatureresponsive Polymeric Micelles James B. Bennett1, Amanda L. Glover2, David E. Nikles2, Jacqueline A. Nikles3, and Christopher S. Brazel1 1Department
of Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, Alabama 35487-0203 USA
2Department
of Chemistry, The University of Alabama, Tuscaloosa, Alabama 35487-0336 USA
3Department
of Chemistry, The University of Alabama at Birmingham, Birmingham, Alabama 35294-1240 USA
NIH-PA Author Manuscript
INTRODUCTION
NIH-PA Author Manuscript
The development of nanoscale systems with medicinal applications has accelerated in the last several years. Emerging developments in cancer research include polymer-based drug delivery systems, (immuno-)liposomes, and dendrimers [1,2,3]. Many proposed drug carriers incorporate a combination of targeting, imaging, and therapeutic moieties to improve diagnostics and while minimizing side effects. Magnetic nanoparticles are another developing technology gaining interest for their potential in clinical cancer therapy and MRI contrast agents [4,5]. Magnetic fluid hyperthermia has been used in Europe to improve patient response when combined with traditional localized radiation and chemotherapy [6, 7]. This project explores the design of a drug delivery system that utilizes similar superparamagnetic nanoparticles which can be localized within human tissue and heated using an AC magnetic field applied externally. The drug carrier is a targeted micelle made of a diblock-copolymer which self-assembles to encapsulate the magnetic nanoparticles and anticancer drug into temperature-responsive semi-crystalline core as depicted in Figure 1. The core material exhibits a positive thermal response so that a phase change occurs when a temperature just above physiological temperature is reached. When the system is subjected to the AC magnetic field, the incorporated magnetic nanoparticles heat the core triggering a phase change designed to allow the release of the therapeutic drug. Here we report the ability of custom-synthesized nanoparticles to heat modulate drug release of the polymeric micelles in response to temperature increase. For the application potential of our drug-delivery system to be realized, there are several important foci in our work including: •
Use of a biocompatible diblock-copolymer system which exhibits a phase-change around 40–45 °C,
•
Synthesis and characterization of magnetic nanoparticles that heat efficiently using an AC magnetic field,
Bennett et al.
Page 2
NIH-PA Author Manuscript
•
Optimizing the magnetic nanoparticles heating profile by tuning nanoparticle concentration, AC field intensity and frequency,
•
Assembly of micelle into aqueous solutions,
•
Efficient loading of hydrophobic drug and nanoparticles into the core of the polymeric micelle,
•
Investigation on the drug release of model drug at physiological and elevated temperatures to mimic magnetic field heating,
•
Attachment of a targeting ligand to specifically bind micelle to cancer cells.
MATERIALS AND METHODS Diblock-Copolymer Synthesis
NIH-PA Author Manuscript
Several formulations of amphiphillic diblock-copolymers have been synthesized from hydrophilic ethylene glycol and hydrophobic ε-caprolactone as described in a separate paper [8]. A specific PEG42PCL19 diblock with an M̄n of 4000 exhibits a melting point in the appropriate range as confirmed by differential scanning calorimetry. This formulation was used to load pyrene and triamterene, model hydrophobic drugs, to study thermally-activated release. Magnetic Nanoparticle Synthesis and Characterization
NIH-PA Author Manuscript
Magnetite (Fe3O4) nanoparticles were synthesized following previously published techniques [9]. Maghemite (γ-Fe2O3) nanoparticles were synthesized in a two-step fashion, first creating an iron-oleate complex by reacting iron chloride with sodium oleate in a mixture solvent of hexane/ethanol/water at 70°C for four hours. In the second step, oleic acid was added to the iron-oleate complex in an octadecene solvent and heated to 320°C under reflux for two hours. Magnetic nanoparticles were characterized by transmission electron microscopy (TEM) for size distribution and x-ray diffraction (XRD) to determine crystalline structures within the nanoparticles. The dispersed nanoparticles were introduced to an AC magnetic field produced by a custom unit that allows a magnetic field intensity range of 0–5kW and a frequency range of 50–450 kHz (Induction Atmospheres, Rochester, NY). Temperature data was collected using a FLIR ThermaCAM infrared camera (FLIR, North Billerica, MA). Initial temperature for heating profiles was kept at 37°C by jacketing the sample with water channeled from a water bath. Incorporation of Drugs into Self-Assembling Micelles Model drugs, pyrene and triamterene, are very hydrophobic with low water solubility and mimic the properties of doxorubicin, a drug commonly used in cancer chemotherapy. To encapsulate the drug, we utilized a solvent evaporation technique.10.1 grams of the PEG42PCL19 diblock copolymer, and 1 mg of pyrene or triamterene was dissolved in 2mL tetrahydrofuran THF. The resulting mixture was added dropwise to 20mL of deionized water under vigorous stirring. The solution was allowed to equilibrate overnight before residual THF was removed through rotary evaporation concentrating the solution to 5 mL– 10mL. The resulting solution was then filtered through a 0.45 μm filter to remove large
PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 3
particles and ultrafiltered through a 50 kD MWCO centrifugal filter device to remove any free pyrene.
NIH-PA Author Manuscript
Drug Release Study at Physiological Temperature and Hyperthermia Conditions A 1-mL solution of pyrene or triamterene loaded micelles was placed inside a Float-A-Lyzer G2 cellulose ester 50 kD MWCO dialysis tube (Spectrum Laboratories, Rancho Dominguez, CA) which floated in a 100 mL dialysate. The temperature was maintained at 37°CC using a water bath. After 20 minutes, the dialysis device was moved to 100 mL of a new dialysate at 47°C to mimic magnetic heating and cause melting of the micelle core. Dialysate samples (0.5 mL) were taken in 4 minute increments. Triamterene concentrations were monitored by UV/Vis absorbance at 270nm. Pyrene concentrations were monitored using a fluorescence spectrophotometer with an excitation wavelength of 317 nm and emission peak at 392 nm.
RESULTS AND DISCUSSION Magnetic Heating
NIH-PA Author Manuscript
Solutions of magnetic nanoparticles in hexane subjected to an AC magnetic field exhibited effective magnetic induction heating. Four concentrations of magnetite in hexane were each placed under a magnetic field with the same intensity (700 Gauss); their behavior is summarized in Table 1. Heating was related to magnetic nanoparticle concentration. Melting the core of the micelle to trigger drug release will likely be dependent on the concentration of magnetic nanoparticles loaded within the core. Externally-tunable parameters governing the magnetic field also greatly influence heating. Our magnetic heat induction unit enables the manipulation of voltage, frequency and capacitance to control the magnetic field. Figure 2 shows the effect of field intensity on heating profiles. The highest intensity (700G) produced the most efficient heating. Reaching higher temperatures quicker would minimize the time a potential patient is exposed to hyperthermia conditions to activate chemotherapy. Triamterene Release
NIH-PA Author Manuscript
Triamterene loaded micelles allow us to investigate controlled release with a model drug easily detectible in the UV/Vis range. Triamterene was observed in the dialysate immediately upon insertion into the 37 °C medium, possibly due to incomplete separation of the micelles from free unencapsulated triamterene. Upon heating to 47°C, triamterene release increased for a short time, theoretically due to the melting of the PCL micelle core. Further experiments are on-going to determine the capacity of PEG-PCL micelles to encapsulate hydrophobic drugs including triamterene and pyrene, as well as doxorubicin, and determine the release behavior when subjected to heating.
CONCLUSIONS A magnetothermally-triggered polymeric micelle drug delivery system requires a sophisticated design, but by providing targeting, controlled release, and hyperthermia, potential for improved cancer therapy with minimized side-effects is possible. Here, magnetic heating has been proven for magnetite nanoparticles, and the self-assembly of PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 4
PEG-PCL diblock copolymers around hydrophobic drugs has been confirmed with release of triamterene modulated by an increase in temperature.
NIH-PA Author Manuscript
Acknowledgments The project described was supported by Award Number R21CA141388 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.
References
NIH-PA Author Manuscript
1. Allen TM, Cullis PR. Science. 2004; 303(5665):1818–22. [PubMed: 15031496] 2. Schiffelers RM, Koning GA, ten Hagen TL, Fens MH, Schraa AJ, Janssen AP, Kok RJ, Molema G, Storm G. J Control Release. 2003; 91(1–2):155–22. 3. Choi Y, Thomas T, Kotlyar A, Islam MT, Baker JR Jr. Chem Biol. 2005; 12(1):41–51. 4. Brigger I, Dubernet C, Couvreur P. Adv Drug Deliv Rev. 2002; 54(5):631–51. [PubMed: 12204596] 5. Pankhurst QA, Connolly J, Jones SK, Dobson J. Journal of Magnetism and Magnetic Materials Proceedings of the Fifth International Conference on Scientific Clinical Applications of Magnetic Carriers. 2003; 293(1):514–9. 6. Rosensweig RE. J Magn Magn Mat. 2002; 252:370–374. 7. Hergt R, Andra W, d’Ambly CG, Hilger I, Kaiser WA, Ricter U, Schmidt HG. IEEE trans Magnetics. 1998; 34(5):3745–3754. 8. Glover AL, Bennett JB, Nikles SM, Nikles JA, Brazel CS, Nikles DE. Polymer Prepr. 2011; (105) 9. Sun S, Zeng H, Robinson DB, Raoux S, Rice PM, Wang SX, Li G. J Am Chem Soc. 2004; 126(1): 273–279. [PubMed: 14709092]
NIH-PA Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 5
NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 1.
Schematic of a magnetothermally-triggered drug delivery vehicle. A hyrdrophobic crystalline polymer poly(caprolactone) (red) forms a core that encapsulates the cancer drug (blue) and magnetic nanoparticles (m). A targeting moiety such as an RGD-peptide can be attached to the hydrophilic block poly(ethylene glycol) (green) to specifically bind the drug carrier to cancer cells. Upon magnetic heating, drug is released.
NIH-PA Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 6
NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 2.
Magnetic heating profiles for 8.4 g/L Fe3O4 nanoparticles in hexane at three magnetic field intensities and a frequency of 266kHz.
NIH-PA Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 7
NIH-PA Author Manuscript NIH-PA Author Manuscript
Figure 3.
Release of triamterene from PEG42PCL19 micelles subjected to a temperature increase at 20 minutes.
NIH-PA Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
Bennett et al.
Page 8
Table 1
NIH-PA Author Manuscript
Temperature Maximums after 3 Minutes of Magnetic Induction Heating for Various Concentrations of Fe3O4 Nanoparticles in Hexane Fe3O4 Concentration (g/L)
Maximum T3 min (°C)
8.4
51.2
4.2
44.6
1.05
41.3
0.503
40.0
NIH-PA Author Manuscript NIH-PA Author Manuscript PMSE Preprints. Author manuscript; available in PMC 2014 October 17.
