Novel applications of poly(ethylene glycol)-bl-poly(propylene sulfide) block copolymers

Block copolymers are comprised of repeating chemical groups (blocks) which commonly contain at least one hydrophilic block coupled to at least one hydrophobic block, forming an amphipathic macromolecule. This molecular arrangement drives the self assembly of these materials in water similar to lipids and low molecular weight surfactants. Unlike these conventional materials, however, block copolymers can typically form a wide variety of morphologies in water such as vesicles, worm like micelles, y-junctions, blackberry micelles, and micelles. This is due to the fact that the large hydrophobic blocks generally display a low mobility inside the core of the aggregate and the initially formed morphologies can be considered as "frozen" structures. Thus, while lipids and other low molecular weight surfactants are largely controlled by thermodynamics, block copolymers are governed by kinetic effects. A large body of work has been produced regarding the physical behavior of block copolymers over the past few decades. Various parameters have been explored including the composition of the dispersing media, the chemical composition of the repeating units of the blocks, the molar fraction of the blocks in solution, the relative and absolute block composition, the polydispersity of the block copolymer, temperature, pressure, and the compatibility of the polymer to the dispersing media. These have been extensively explored both theoretically and empirically for a wide variety of block copolymer systems. Block copolymers of poly(ethylene glycol)-bl-poly(propylene sulfide,) (PEG-PPS,) have recently emerged as a new and interesting block copolymer. This is due both to the low Tg, Tm, and relatively high hydrophobicity of the PPS block. These block copolymers can form polymeric vesicles (polymersomes,) worm like micelles, micelles, or hybrid structures, which are dependent on the relative block lengths of PEG and PPS (∞PEG.) However, the stability of the formed morphologies is directly related to the absolute PPS degree of polymerization, as the hydrophobic effect is the main driving factor towards self assembly. So while morphology is determined by the relative block composition, the stability of the formed aggregates on dilution is determined by the absolute molecular weight of the hydrophobic block. In order to exploit these materials for use as drug delivery vehicles to encapsulate hydrophilic compounds, we have explored the behavior of PEG-PPS by modifying the various parameters described above. We discovered that block copolymers displaying relatively low ∞PEG values are able to form micelles from solvent dispersion using tetrahydrofuran, a good solvent for both blocks, into water. These frustrated micelles, in which the PPS block is tightly packed, can then relax when heated in the presence of a solvent which is capable of swelling the hydrophobic block. This transition, highly dependent on temperature, the nature of the solvent, and the time exposed at elevated temperatures, allows the hydrophobic block to relax in the aqueous solution, and stretch to a more thermodynamically favorable morphology. In this case, we have observed a micelle to vesicle transition, with a wide variety of intermediate structures formed during the relaxation time. There are distinct phases during the transition which are related to critical aggregation numbers, and represent an Ostwald like ripening where vesicles are formed at the cost of micelles and other morphologies. These transitions, elucidated using cryo-TEM and optical density measurements, can be considered to be either 2D, where micelles fuse to form worm like micelles, or 3D where the fusion of micelles leads to inversion or "flip-flop" and directly form vesicles. After a certain period of time, we observe a final, stable state in which all initial and transitional morphologies become an isotropic dispersion of polymeric vesicles. We have exploited this phenomena to encapsulate a biologically active hydrophilic peptide at a high concentration of PEG-PPS micelles (10% wt/vol,) where we observed high encapsulation efficiencies (> 50%.) In a somewhat similar manner, we have also explored the formulation of PEG-PPS with excipients of an amphipathic salt (1,8-Diazabicyclo[5.4.0]undec-7-ene HCl,) (DBU-HCl,) and polyethylene glycol dimethyl ether Mw 500. We discovered that upon heating, PEG-PPS forms a melt with the excipient. When water or a protein solution is added, the mixture forms a milky polymersome dispersion in solution. In the case of adding a protein solution, we observed high encapsulation efficiencies owing to the very high concentration of the block copolymer in solution, and resultant reduced free aqueous volume outside of the polymersomes compared to the thin film hydration method. By characterizing this system using a wide variety of techniques including differential scanning calorimetry, optical density, dynamic light scattering, cryo-TEM, and others, we have been able to show that the direct hydration method is a type of modified solvent dispersion technique where the PEG-PPS is dissolved into the excipient at elevated temperature. When the mixture is allowed to cool to room temperature, and when water is slowly added, the molar fraction of the salt or PEG excipient changes and the mixture becomes more polar. When this occurs the block copolymer is driven towards self assembly where the PPS block is in a relaxed state in the matrix, forming polymeric vesicles. During this process, a substantial portion of the added aqueous solution is subsequently trapped in the core of the vesicles. Because we are forming the vesicles close to the optimal concentration of the block copolymer, a value defined as the sponge or hexagonal packing phase, we observe elevated encapsulation efficiencies compared to conventional methods such as thin film hydration. We also observed that the activity of an enzyme, β-Galactosidase, was not affected by the processing steps. In addition to exploring the behavior and exploitation of PEG-PPS as drug delivery vehicles, we have also produced a set of functionalized micelle forming PEG-PPS block copolymers for either targeted drug delivery, or as adjuvants for vaccine development. By forming mixed micelles with the similar poly(ethylene glycol)-bl-poly(propylene oxide)-bl-poly(ethylene glycol,) or Pluronic block copolymers, we were able to display a variety of surface chemistries by modifying the Pluronic block copolymer. The modification of Pluronic block copolymers is relatively straightforward, as they display PEG terminal primary hydroxyl groups. We have demonstrated that sulfated Pluronic F-127, when blended with PEG-PPS, can target Collagen I, a primary component of the extracellular matrix. Although Pluronics are known to display relatively high critical micellar concentrations (CMC,) the mixed micelles displayed a substantially lower value compared to the Pluronic alone. PEG-PPS has also been demonstrated to be particularly useful for the encapsulation and release of amphipathic and hydrophobic drugs. By quantifying the encapsulation efficiency and release of the immunosuppressant drug Sirolimus, we have proven the concept that mixed micelles of Pluronics and PEG-PPS could be used as novel drug delivery systems for targeting the extracellular matrix. To modify PEG-PPS itself, we have had to develop novel chemistries. We wanted to form block copolymer analogs to the Pluronic F-127 PPS core nanoparticles developed by our group previously. The Pluronic F-127 PPS core nanoparticles have been demonstrated to act as adjuvants for vaccine development. This was accomplished by producing nanoparticles small enough to be transported into the lymph nodes passively via interstitial flow into the lymphatic capillaries. Dendritic cells in the lymph node then are able to internalize these nanoparticles in large numbers, leading to maturation, and processing of antigens on the nanoparticle surface via the alternative complement pathway. To mimic these nanoparticles, we developed block copolymers of PEG-PPS which display either HO or H3CO on the PEG chain terminus and either SH, H2N, phthalimide, or benzyl on the PPS chain terminus. In this way we have fluorescently labeled the block copolymer covalently, and performed a wide variety of experiments to explore the relationship of surface chemistry to complement activation both in vitro and in vivo. In this last chapter, we have shown the transport of PEG-PPS micelles to the lymph nodes and subsequent uptake into lymph node dendritic cells in vivo, and the release of C3a and the maturation of bone marrow dendritic cells in vitro in a surface chemistry dependent manner. In conclusion, PEG-PPS represents an interesting new block copolymer system which we have only begun to explore. The following chapters attempt to contribute to the growing body of knowledge regarding block copolymers and their application as therapeutic drug carriers. The final chapter provides for both a look back at the accomplishments in this thesis, and possible directions for future research.