Injectable liposome-containing click hydrogel microparticles for release of macromolecular cargos

Abstract

Hydrogel microparticles ranging from 0.1-100 μm, referred to as microgels, are attractive for biological applications afforded by their injectability and modularity, which allows facile delivery of mixed populations for tailored combinations of therapeutics. Significant efforts have been made to broaden methods for microgel production including via the materials and chemistries by which they are made. Via droplet-based-microfluidics we have established a method for producing click poly-(ethylene)-glycol (PEG)-based microgels with or without chemically crosslinked liposomes (lipo-microgels) through the Michael-type addition reaction between thiol and either vinyl-sulfone or maleimide groups. Unifom spherical microgels and lipo-microgels were generated with sizes of 74 ± 16 μm and 82 ± 25 μm, respectively, suggesting injectability that was further supported by rheological analyses. Super-resolution confocal microscopy was used to further verify the presence of liposomes within the lipo-microgels and determine their distribution. Atomic force microscopy (AFM) was conducted to compare the mechanical properties and network architecture of bulk hydrogels, microgels, and lipo-microgels. Further, encapsulation and release of model cargo (FITC-Dextran 5 kDa) and protein (equine myoglobin) showed sustained release for up to 3 weeks and retention of protein composition and secondary structure, indicating their ability to both protect and release cargos of interest.

Fig. 1. Overview of microgel fabrication (A) Thiol-Michael addition between PEG-4-vinyl-sulfone (5 kDa) (PEG-VS₄) and PEG-4-thiol (5 kDa) (PEG-SH₄) was used to form hydrogel networks. (B) Flow-focusing water-in-oil microfluidics was utilized to form monodisperse hydrogel microparticles (microgels). Pressure pumps control the flow rate of the hydrogel forming solutions entering the microfluidic device and a syringe pump controls the oil flow rate. (C) Design of microfluidic chip for formation of PEG microgels; (i) oil stream; (ii) PEG-SH₄ and PEG-VS₄ streams; (iii) buffer stream. (D) Schematic of PEG Microgels and PEG Lipo-microgels where grey depicts polymer and teal depicts FITC-dextran.

Fig. 2. Formation of microgels and lipo-microgels (A) Confocal image of microgels containing FITC-Dextran and histogram of diameters (B) Confocal image of lipo-microgels containing Cy5-labeled liposomes and histogram of diameters. (Scale bar = 100 μm).

Fig. 3. Visualization of liposomes in microgels (A) xy plane obtained on LSM 880 from a z-stack of a microgel containing AF594 tagged liposomes (B) IMARIS 3D rendering of the microgel containing AF594 tagged liposomes.

Fig. 4. Mechanical properties of microgels and microgel suspensions (A) Young's modulus determined via atomic force microscopy for bulk hydrogels, lipo-microgels, and microgels (B) Young's modulus determined via atomic force microscopy of microgels stored dry at room temperature (Dry RT), in 1x PBS at 4 °C (Hydro 4 °C), and in 1x PBS at room temperature (Hydro RT) over 4 weeks. No significant differences observed in all cases. Scale bar represents the mean ± standard deviation n = 5.

Fig. 5. Injectability of microgel suspensions (A) Steady shear viscosity of microgel suspensions at increasing concentrations (B) Microgel diameter and brightfield images of trypan blue stained microgels before and after injection through a 27 G syringe. Data represents the mean ± standard deviation. (Scale bar = 100 μm).

Fig. 6. Model release (A) release of FITC-Dextran over 3 weeks from both microgels and lipo-microgels. Each data point represents the mean ± standard deviation for n = 3 (B) confocal microscopy of FITC-dextran microgels and lipo-microgels over 1 h. Scale bars = 100 μm.