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Polymeric Nanoparticles as Drug Carriers for the Treatment of Malignant Glioma BME 4310 Alexander Paul and Joel Pepper Significance Malignant Glioma • Originates in the brain or spinal cord from glial cells • Makes up about 80% of all malignant brain tumors • Median survival is about 1 year • Local tumor recurrence is virtually universal Significance Widely Used Solutions • Chemotherapy • Drawback: High cytotoxicity, difficult to target at tumor • Surgery • Drawback: High risk of human error, very expensive • Ionizing Radiation: • Radiation which destroys cells by via ionization of their components • Drawback: High cytotoxicity, difficult to target at tumor Significance Improvements • Temporary Opening of the BBB • Improves intracranial delivery • Drawbacks: Increases toxicity to normal tissues as well • • There’s a reason the BBB exists Convection-Enhanced Delivery (CED) • Direct delivery that uses a hydrostatic pressure gradient to distribute macromolecules through the BBB via an implanted catheter • Greatly increases volume of distribution of macromolecules • Drawbacks: Highly unpredictable delivery of agent • Losses infusate into ventricles, subarachnoid space and/or the anisotropic tissue surrounding a tumor Significance Delivery Vectors • • Viral • Most widely studied • Drawbacks: Potential toxicity, difficult to produce large scale, cannot incorporate MRI contrast agents Non-Viral • • Liposomes • Can incorporate contrast agents, can hold both polar and nonpolar payloads • Drawbacks: Very large, oftentimes not much help with BBB Nanoparticles (NP’s) • 1-100 nm in size, much more viable for BBB permeation • Drawbacks: Many are toxic, many cannot carry tracers, not yet well studied/utilized (although everyone in CBEC seems to be trying) Approach This Study • Polymeric NP’s • Biocompatibility • Can delivery therapeutic payload and incorporate contrast agents • Production • • Iron oxide for tracing • Payload is Temozolomide (TMZ), an anti-glioma agent Fabricated specifically for CED • Hence the importance of iron oxide for MRI tracking Approach Animals and Cells • 6-7 week-old male Lewis or Athymic Nude mice • Human glioblastoma cell line (U87) Approach Polymeric magnetite-bearing NP’s (PMNP) 1. NP formulations in organic solutions containing PMNP precursor components produced using a proprietary electrohydrodynamic process. 2. Resulting solution included a. PMNP’s sans their payload or fluorescent agent b. PMNP’s with the agent c. PMNP’s with both agent and TMZ payload 3. Characterizations performed: a. UV-vis b. Dynamic Light Scattering Analysis c. TEM d. Magnetic susceptibility e. In vitro release kinetics (more on next slide) Approach PMNP In Vitro Release Kinetics • TMZ only stable at acidic pH • Release history would be masked if TMZ in solution is included in the analysis • Fix: Separate the remaining PMNP’s from solution, then analyze their TMZ content • Once PMNP’s are separated, their polymers were disrupted with 100 µl of 0.1 N HCl • Next a timelapsed UV-vis analysis at 330 nm was performed • t=0 => Total agent uptaken by PMNP’s • Calculating Release • After a given amount of time, separated PMNP’s from solution, disrupt, then measure TMZ concentration • TMZ in PMNP’s = Total TMZ (i.e. when t=0) - Amount TMZ @ given t • Analysis performed at 4, 22 & 37°C Approach Measuring uptake of PMNPs • U87 cells were pretreated with endocytosis inhibitors • PMNPs were fluorescently plated overnight and added to cells in media • Uptake was measured under microscope • Catheter was used to infuse PMNPs in the brains of rats • CED and bolus injection methods were compared Approach In Vivo MRI Studies • PMNPS’s/NP’s infused via either CED or bolus (injection) • Animals placed in MRI within 1 hour of injection • Acquired both normal and fast spin images • Animals were sacrificed immediately following imaging Approach Histology • Brains removed and fixed in OCT • Sectioned at 20 µm • Fluorescently tagged NP’s • • Low power FITC filter and ImageJ General brain histology and iron oxide distribution • Formalin-fixed brains sectioned and stained Approach Calculation of Vd/Vi • Volume of distribution / Volume of infusion • Used to compare bolus with CED • Take section of image in Imagej, calculate area using known section thickness • Sum across serial sections to obtain Vd • Vi is known • Backflow was excluded from measurements Approach In Vivo Glioma Studies • Hind limb experiment • U87 cells were inoculated into the hind limb of nude mice and tumor size was measured daily • When tumors reached size of 200 mm3, PMNP formulations were injected at days 0,3, and 5 • • Treatment groups included: untreated control, blank-NPs, TMZ loaded NPs, and the supernatant acquired after filtration Intracranial experiment • U87 cells were inoculated in the brains of nude mice • On days 4 and 7 after tumor grafting, PMNP formulations were infused by CED Results Characterizations • Polymer: Combination of PEG, PLA, PCL • Enabled <100-nm particle creation with (-) surface charge • • • • “Robust convection through EC space of brain parenchyma” Size verified by DLS and TEM Tracer: Magnetite • Superparamagnetic iron oxide • 11% Fe3O4 optimal mix of polymer content and magnetic susceptibility • Charge verified by DLS and TEM (ζ=-7.75 mV) Drug Delivery • At 37°C, TMZ payload completely released after 20 hours • Lower temperatures => slower release • Physiological temperature => PMNPs release payload more quickly Results Efficiency of Bolus vs CED infusion • CED showed a significantly higher Vd/ Vi compared to bolus injection • Examined via fluorescent microscopy in a rat model • Examined via MRI in vivo • Bolus injection showed PMNP backflow along catheter tract • No gross parenchymal changes seen after CED infusion • • Convection of PNMPs results in minimal brain damage Staining for iron oxide with prussian blue dye shows that iron oxide is present far from injection site Results Results Cellular Uptake • • • Fluorescently tagged PMNP’s noticeably uptaken within 2 hours of exposure • Concentration dependent, 0.5 µL of PMNP was used • Imaging showed uptake specifically in cytosol Endocytosis inhibition showed decrease in uptake • Sucrose and chlorpromazine used to inhibit • Proves that PMNP’s uptaken at least in part via endocytosis TMZ-PMNP’s decreased the survival of glioma cells • • • Blank NP’s and free TMZ had no effect Post TMZ-PMNP uptake endocytosis inhibition reversed damage to colony • Applied sucrose after TMZ was successfully delivered • Sucrose does not affect integrity of PMNP’s themselves Conclusion: PMNP’s must be actively uptaken in order to work Results Cytotoxic Efficacy • • U87 glioma xenografts in hindlimb of athymic nude mice • Single intra-tumoral injection of TMZ-PMNP’s, blank PMNP’s and free TMZ • TMZ-PMNP’s significantly reduced tumor growth compared to other injections • Conclusion: Free TMZ has no effect, must be encapsulated U87 xenografts in striatum of athymic nude mice • • TMZ-PMNP’s significantly extended survival of mice compared to blank PMNP’s/free TMZ Conclusion: TMZ-PMNP’s can be used to safely deliver a chemotherapeutic agent intracranially and positively treat malignant glioma Results Discussion Potential Pitfalls • Although CED showed a higher Vd than bolus injection, it is still less than 1 • This is adequate for an animal model, but clinical tumors can possess characteristics that will further inhibit the ability of the drug to reach the tumor • Some clinical trials using CED have shown disappointing results • The potential toxicity of iron oxide must be considered • Uncoated particles have been shown to induce reactive oxygen production, inflammation and DNA damage • Biocompatibility is improved after coating iron oxide particles with polymers such as PEG Discussion Future Directions • Although only TMZ was used, a broad range of therapeutics or toxins could be encapsulated for drug delivery • The use of MRI to visualize drug delivery could allow for real time adjustments or post operative confirmation of successful delivery • Targeting moieties such as ligands or anti-bodies could be incorporated into the particle to improve delivery efficiency • Further examination in higher order animals that have spontaneous tumors is required before determining potential for clinical use Conclusions • PEG-PLA-PCL copolymer can be used to create <100 nm, charged PMNP’s • 11% Fe3O4 is optimal • Physiological temperature results in relatively rapid TMZ delivery • CED is more effective than Bolus for PMNP delivery • TMZ-PMNP’s must be actively endocytosed to be effective • TMZ must be in a carrier to be effective • TMZ-PMNP’s delivered via CED are an effective treatment for glioma