(609e) Cationic, Anionic, and Amphoteric Dual pH/Temperature-Responsive Degradable Microgels Via Self-Assembly of Functionalized Oligomeric Precursor Polymers

Stimulus-responsive or ‘smart’ materials are increasingly attracting interest in biotechnology and medicine, especially in the context of drug-delivery systems, given their potential to rapidly switch properties (e.g. size, hydrophobicity, charge, etc.) when exposed to small changes in their environment (i.e. pH and temperature) [1]. These changes are reversible, allowing the system to return to its initial state when the environmental trigger is removed. In particular, “smart” microgels that can change their properties in response to physical (temperature, light, electric and magnetic fields) or chemical (pH, ionic strength, and the presence of chemical or biological compounds) stimuli have been identified to have various applications including sensors [2][3], optics [4] and loading/release applications [5][6]. Temperature-responsive poly(N-isopropylacrylamide) (PNIPAM) microgels are the best-known example [7][8]. PNIPAM microgels have a volume phase transition temperature (VPTT), a direct result of the lower critical solution temperature (LCST) of the PNIPAM polymer (~32°C) [9]; consequently, PNIPAM microgels are swollen at room temperature but, when heated above the VPTT, they discontinuously shrink, inducing tunable changes in diameter, hydrophobicity, pore size and surface charge as a function of temperature.

Conventionally, PNIPAM microgels are fabricated using free-radical precipitation polymerization, with a free radical initiator, a monofunctional monomer (giving the linear segments), a bifunctional monomer (giving crosslinks) [10]. By copolymerizing acidic or basic comonomers, pH-responsiveness can be introduced to create dual responsive microgel systems useful for ion exchange, drug delivery, environmental sorption, and other applications. However, to use such microgels for biomedical applications, degradability remains an issue. Most microgel preparations use a non-degradable crosslinker; even if the crosslinker is degradable, the molecular weights of the degradation products are often not controllable and thus may not be cleared following gel degradation.

In response, we have developed a novel approach to overcome this limitation by creating degradable thermosensitive microgels by the controlled precipitation of well-defined hydrazide and aldehyde-functionalized PNIPAM oligomers [11][12]. This thermally driven self-assembly approach enables the formation of a hydrolytically-labile hydrazone cross-linked network that can break down over time into oligomeric by-products of molecular weights below the renal filtration limit. These self-assembled PNIPAM microgels are degradable, monodisperse, homogeneously crosslinked, and stable over months, while retaining their responsiveness to temperature due to the presence of PNIPAM.

Objectives:

By copolymerizing charged monomers into the precursor polymers used in self-assembly, pH-responsive swelling behaviors can be achieved that may are relevant for a number of applications ranging from sensors to drug delivery. The primary and foremost objective of this work is to synthesize and characterize single-charged and amphoteric PNIPAM microgels prepared using the self-assembly method. Next, the internal structure of these charged microgels are analyzed using small-angle and ultra-small angle neutron scattering experiments. Prior to in vitro drug release studies, the microgels were exposed to C2C12 myoblast cells to evaluate their cytocompatibility. Lastly, moderately hydrophobic drugs (i.e. naproxen sodium) were loaded into these microgels using two methods, and the encapsulation efficiencies and drug release kinetics were analyzed.

Methodology:

Hydrazide and aldehyde-functionalized PNIPAM polymers were synthesized via free radical copolymerization of NIPAM in ethanol using thioglycolic acid as the chain transfer agent and 2,2-azobisisobutyric acid dimethyl ester as the initiator. Cationic and anionic charges are incorporated into the self-assembled PNIPAM microgels by functionalizing either functionalized polymer with anionic (acrylic acid) and cationic (N,N-dimethylaminoethyl methacrylate) comonomers. Titration, NMR and GPC data was collected to characterize each precursor polymer. The hydrazide-functionalized PNIPAM polymer is dissolved and heated to 5+LCST for 2-3 minutes, after which the aldehyde-functionalized PNIPAM polymer is added dropwise at 20 mass%, and the self-assembly is complete after ~15 minutes. Incorporation of pH-ionizable monomers into the precursor polymers, and subsequent self-assembly of those polymers will result in dual pH/temperature-responsive microgels. Charged oligomers are added in different combinations and sequences, enabling precise tuning of the charge density and charge distribution based on the net charge and sequence of assembly of the precursor polymers used (e.g. amphoteric microgels with different charge profiles can be fabricated by mixing cationic and anionic-functionalized precursor polymers at different stages during the self-assembly process). The optimal conditions associated with the production of these pH-responsive PNIPAM microgels were explored and evaluated in terms of size (dynamic light scattering), charge (electrophoretic mobility), internal morphology (small angle neutron scattering, SANS) and degradation (light scattering). Cell compatibility was evaluated using fluorescently labeled C2C12 myoblast cells and performing an MTS assay. Moderately hydrophobic drugs (i.e. naproxen sodium) were co-self-assembled with the polymers to attempt to optimize drug loading by avoiding partitioning-based effects that typically limit drug loading in conventional microgels. Drug release experiments were conducted using membrane devices, with the amount of drug released assessed using high performance liquid chromatography.

Results:

Self-assembled single-charged PNIPAM microgels exhibit similar pH and temperature-responsive particle sizes and zeta potentials as conventional microgels, with the added advantage of degradability into polymers with known molecular weights below the kidney clearance limit. Amphoteric microgels can also be successfully fabricated by mixing cationic and anionic-functionalized precursor polymers during the self-assembly process, reproducing the high pH/low pH parabolic swelling response observed in conventional amphoteric microgels. The internal structure was analyzed using SANS and USANS experimental data and compared to the homogenous internal structure of the uncharged self-assembled PNIPAM microgels. No major structural differences can be observed despite the introduction of charge into the system. Cytotoxicity studies using an MTS assay and C2C12 mouse myoblast cells indicated no cytotoxic effects are observed for the charged microgels or the precursor polymer degradation products (>80% viability). The common drug loading method for conventional precipitation based PNIPAM microgels is through passive diffusion of the drug into the polymer network. This method of drug loading is slow (>24 hours) and typically results in low encapsulation efficiencies due to the incompatibility between the hydrophobic drug and hydrophilic crosslinked networks of the microgels. In contrast, the radical-free self-assembly method also allows for in situ drug loading by directly co-assembling the drug with the prepolymers, facilitating between 3-5 times higher drug loading that is completed within <15 minutes (concurrent with particle fabrication) for moderately hydrophobic drugs (e.g. dexamethasone, naproxen sodium), which preferentially partition inside the hydrophobic nanoaggregate formed during the self-assembly process. In addition, due to the resulting different distribution of drug inside the microgels following in situ versus passive release, no significant burst release was observed for the naproxen-loaded microgels and near zero-order drug release continued over the course of two weeks, significantly longer than achieved with conventional passive drug loading.

Significance:

The significance of this self-assembly approach relative to conventional microgel synthesis is based on (1) the potential for degradation via hydrolysis and (2) the ability to form particles by simple sequential mixing within minutes. Introduction of pH-sensitivity to the already temperature-responsive self-assembled PNIPAM microgels allows for multiple stimuli-responsive properties. Coupling the precise dual responsive swelling responses achievable with the degradability of the hydrazone crosslinks, self-assembled charged PNIPAM microgels offer potential for improved performance in drug delivery applications demanding dual pH/temperature specific delivery (e.g. targeting infection sites or cancer).

References:

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