(475x) Protein Encapsulation into Thermo-Responsive Biodegradable Nanospheres

Polysuccinimide (PSI) synthesized by acid catalized polycondensation of L-aspartic acid was allowed to react with isopropylamine in order to create novel thermo-responsive polymer possessing biodegradability and biocompatibility. Resultant isopropylamine modified poly succinimide (IPA-PSI, Poly[α, β(DL-asparate isopropyl amide)-co-succinimide]) has same side chain structure as that of one of the most famous thermo-responsive polymer, poly(N-isopropyl acrylamide) (PNIPAAm). In spite of the same side chain structure, IPA-PSI with mole fraction of ca. 45 % aspartate isopropyl amide unit in repeating units shows much different thermo-responsivity from PNIPAAm. PNIPAAm and other thermo-responsive polymer dissolve in water at low temperature and insolubilize at high temperature. Generally, this phase transition induced by temperature change is reversible. On the other hand, though IPA-PSI dissolve in cold water and lose water solubility at high temperature, the colloid formed by heating didn't recover their water solubility enen at very low temperature. This colloid can be collected by centrifugation and can be dispersed to water again. From this result, it is indicated that the thermo-induced phase change of IPA-PSI was not liquid-liquid phase separation but liquid-solid phase transition. From dynamic light scattering measurement of this colloid, it was found that polymeric nanoparticles with 100 ? 200 nm in diameter were formed by this thermo-induced phase transition. Furthermore, the result of scanning electron microscopy observation of dried IPA-PSI colloid was consistent with that of dynamic light scattering measurement. The phase transition temperature of IPA-PSI was greatly decreased with increase of polymer concentration while that of PNIPAAm don't have much polymer concentration dependency. This result indicate that intermolecular interaction between polymer molecules is significant for the phase transition of IPA-PSI. Nanoparticles of IPA-PSI redissolve by addition of base such as sodium hydroxide, resulting in cleavage of imide ring in IPA-PSI molecules. Therefore, nanoparticles of IPA-PSI may be stabilized by imide ring ? imide ring interaction even at low temperature. From these results, the mechanism of this irreversible phase transition is shown below. First of all, IPA-PSI molecules dissolve in cold water by forming hydrogen bonding between hydrophilic amide bond and water molecules. Then, IPA-PSI molecules lose water solubility by dehydration with increase of temperature. Finally, hydrophobic IPA-PSI molecules aggregate with each other by intermolecular hydrophobic interaction and synchronize with stabilization by imide ring ? imide ring interaction. Aqueous IPA-PSI solutions dissolving protein were heated in order to prepare nanoparticles encapsulating protein. Hemoglobin used as model protein was encapsulated efficiently by this treatment. The pH of aqueous IPA-PSI solution is about 5.5 and IPA-PSI charge negatively because of carboxylic group of IPA-PSI. Then, hemoglobin whose isoelectric point is about 7.0 charged positively in aqueous IPA-PSI solution. Therefore, the driving force of encapsulation is likely to be electrostatic force. Actually, the zeta potential of nanoparticles decrease with increase of amount of encapsulated hemoglobin. The spectrum of circular dichroism of hemoglobin released from nanoparticles was compared with that of hemoglobin without this treatment. From this measurement, it is confirmed that encapsulated hemoglobin was not denatured by this treatment.