(743g) Pnipmaam Based Core/Shell Systems for Improved Retention of Hydrophobic Chemotherapeutics

Chemotherapeutic drug delivery demands localization to the tumor site do to deleterious side effects that occur when they are allowed to spread throughout the body.  The issue with delivering these therapeutics is twofold, a lack of solubility and systemic circulation.  One solution to this is the development of externally triggerable theranostic vehicles that are capable of delivering these devastating agents to the site of disease, preventing off site effects.  These systems, composed of a temperature sensitive coating around a stimuli responsive core, are questioned for a number of reasons.  One major issue is the premature release of their payload, leaving little improvement over systemic delivery. This is particularly problematic for hydrogel based carriers, whose hydrated neutral state leaves little compatibility with, what are commonly, hydrophobic drugs, such as chemotherapeutics.

The premature release of the chemotherapeutics can be addressed by taking advantage of the low solubility of the chemotherapeutics in physiological conditions.  Carrier design that focusses on the polymer-drug interactions can take advantage of partitioning resulting in retention of the chemotherapeutic payload.  In the past, the use of N-Isopropyl acrylamide has demanded the incorporation of hydrophilic comonomers in order to raise the lower critical solution temperature (LCST) above physiological temperature.  This can be avoided by the use of other N-Alkyl substituted acrylamides such as N-Isopropylmethacrylamide (NIPMAAm); which has an LCST of about 45°C.  This allows for copolymerization with hydrophobic comonomers for the depression of the LCST increasing the partitioning of the hydrophobic drug in the polymer.  This can again be improved upon by developing a carrier that has a coating of a drug compatible shell.  This provides a hydrophobic sink that provides a high partitioning coefficient of the drug in the carrier.         

Nanogels were synthesized via an emulsion precipitation polymerization.  This involves the dissolution of NIPMAAm, a cross linker (N’N’-methylene bisacrylamide (MBAM)), and surfactants in water heated to 70°C and nitrogen purged.  The reaction is then initiated by the injection of ammonium persulfate.  For core/shell systems, after 6 hours the shell monomer; phenyl methacrylate (PhMA), tert-butyl methacrylate (TBMA), and ethylene glycol or phenyl acrylate (EGPhA), is injected and the reaction is allowed to proceed overnight.  The resulting mixture is then dialyzed against 18.2 MΩ water and freeze dried.

The hydrogel systems are then characterized by dynamic scanning calorimetry (DSC), nuclear magnetic resonance (NMR) spectroscopy, and dynamic light scanning (DLS) to confirm the development of core/shell systems.  Loading was performed by mixing nanogels with fluorescein or Doxorubacin hydrochloride (DOX) and allowed to equilibrate overnight.  The free drug is then separated by centrifugation filtration and washed several times with water.  Drug loaded is measured by fluorescence and thermogravimetric analysis.          

The nature of the drug in the polymer is further analyzed by comparing DSC curves of free drug to that of drug encapsulated in the nanogels.  The drug loaded nanogels were then also assessed for their biocompatibility in vitro with RAW 264.7 macrophage cells. 

Release was characterized by dissolving drug loaded nanoparticles in phosphate buffered saline (PBS).  The particles were initially allowed to reach equilibrium at 37°C, at 6 hours.  After the 6 hour timepoint the temperature is raised to 45°C and the drug released is measured for another 2 hours. 

The NMR spectrographs show the characteristic peaks of the shell material alone, this is due to the fact that NMR of crosslinked systems in aqueous environments only display the peaks of the solubilized polymer chains on the surface.  DSC theromograms of core/shell materials show multiple glass transition temperatures (T_g) compared to that of PNIPMAAm.  This demonstrates multiple distinct regimes of different polymer networks.  These two facts lend to the conclusion that we successfully synthesized core/shell structures with a polymer compatible shell and a temperature sensitive core.  DLS swelling curves and DSC curves in water demonstrate that these gels still maintain the characteristic swelling response of PNIPMAAm, without depression due to copolymerization.  Showing that the shell has little impact on the responsiveness of the nanogel. 

Loading studies show an increase in the drug loaded into the core/shell particles.  These studies reveal a significant increase in the partitioning of the drug into the nanogels.  This is further observed in the release studies.  While PNIPMAAm nanogels release a large portion of their payload at 37°C.  EGPhA showed mild, but no significant increase in partitioning.  The hydrophobic systems, based on PhMA and TBMA, show undetectable release at 37°C.  After heating to 45°C burst release is achieved, and a large portion of the drug payload is released.  Higher levels of drug release is attained by pulsatile release achieved by cooling back down to 37°C and heating back up to 45°C. 

The synthesis of core/shell nanogels is possible through the sequential addition of comonomers.  These novel structures utilize a core and a shell that are completely separate domains.  These systems have been shown to improve both the loading and release due to increased partitioning of common model chemotherapeutics.  These results lend themselves to application in vivio as it provides extra control over these composite systems.  This increased control leads to increased localization of the chemotherapeutic pay load to the site of the disease.  This should allow the avoidance of the deleterious side effects that are common with standard chemotherapy.