We have invented a new flash nanocomplexation (FNC) process for scalable production of polyelectrolyte complex nanoparticles (US patent issued in 2019); and applied this method to kinetically control the packaging of various type of nucleic acids, protein/peptide therapeutics, and small molecular weight drugs. More recently, we extended this method to assemble uniform, stable, and off-the-shelf particles within the size range of 200 to 900 nm to enhance the co-delivery efficiency of different virus packaging plasmids and improve the lentivirus production efficiency.

We applied a complex coacervation method for preparing DNA nanoparticles using natural polymers including chitosan and tailor-designed biodegradable polycations, block or graft copolymers with improved control over their self-assembly with plasmid DNA or siRNA forming micellar nanoparticles. We also developed approaches to tune the shapes of DNA nanoparticle with diameters in 20 – 100 nm range and provided insights in the mechanism in shape regulation during the process. We applied these gene therapy strategies for mucosal gene delivery and genetic immunization, to enhance liver-targeted gene delivery of DNA nanoparticles via retrograde intrabiliary infusion (RII) method and probed the DNA transport kinetics of DNA nanoparticles using a SPECT/CT imaging method. More recently, we developed a lipid nanoparticle screening platform to identify effective and cell-type specific compositions for the delivery of plasmid DNA and other nucleic acid therapeutics and employed these lipid nanoparticles for liver-specific gene delivery and oral vaccination.

We have developed an injectable nanofiber-hydrogel composite material (US patent issued in 2019) that retains needed structural integrity while maintaining sufficient porosity to enable host cell infiltration and vascularization, and that provides biostimulatory conditioning of the tissue environment without relying on exogenous growth factors (Li, et al, 2019). This composite material combines high porosity of hyaluronic acid hydrogel with tunable stiffness and structural reinforcement feature of the composite by incorporating electrospun poly(ε-caprolactone) (PCL) nanofiber fragments that are covalently conjugated to the HA network. The covalent interfacial bonding in the composite structure enables mechanical properties mimicking those of native soft tissues while retaining an open, porous architecture encouraging host tissue infiltration and remodeling for soft tissue repair (Henn, et al, 2020) and spinal cord regeneration (Li, et al, 2020; Haggerty, et al, 2022).

We have developed electrospun nanofibrous matrix to mimic the biochemical and topographical characteristics of the basement membrane to synergistically improve the self-renewal and proliferation, while maintaining the phenotype of human hematopoietic stem/progenitor cells (HSPCs) (Chua et al, 2006, 2007, Jiang et al, 2011), and demonstrated the potential of nanofiber-expanded cells for angiogenic therapies in treating heart and limb ischemia and osteoporosis. We have developed nanofiber matrix as an extrinsic cue to promote preferential differentiation of stem cells and generate functional progenitors and tissue cells for regenerative therapies, including neural stem cells (Christopherson, et al, 2009; Lim, et al, 2010; Mahairaki, et al, 2011), human embryonic stem cell (ESC)-derived neural crest stem cells (Ren, et al, 2012), and human ESC-derived retinal ganglion cells (Slush et al, 2015). We have developed a patented electrostretching method to generate hydrogel microfiber bundles with embedded nanofiber topography using aqueous solutions of natural polymers (Zhang, et al, 2014), and demonstrated its utility as a 3D scaffold with internal alignment cue to induce organized cell/tissue regeneration: electrospun fibrin fibers for arterial vascular engineering (Barreto-Ortiz, 2013; 2015; Elliot, et al, 2019) and skeletal muscle myofiber regeneration (Gilbert-Honick, et al, 2018, 2020; Somers, et al, 2019; Morrissette-McAlmon, et al, 2020).

Using nanoparticles as an artificial antigen presentation cell (aAPC), with surface-conjugated antigen in the context of an MHC molecule and co-stimulating molecules (Signals 1 and 2), we developed the first quantitative analysis of particle size effect on aAPCs with both Signals 1 and 2 based on T cell biology and identified an optimal size range for higher efficiently. Recently, we developed a hyaluronic acid hydrogel-based artificial T-cell stimulation matrix that incorporates matrix stiffness cue and T cell proliferation Signal 3 to synergize with the Signals 1 and 2 for ex vivo and in vivo T cell activation and proliferation.

We developed nanovaccines to potentiate immune responses by site-specific drainage to lymph nodes. Using a nanovaccine encapsulating VP1 protein antigen from enterovirus 71, which causes hand-foot-mouth disease (HFMD) and tumor necrosis factor-α (TNFα) or CpG as an adjuvant, we showed rapid drainage to proximal and distal lymph nodes and subsequently induced strong immune activation and conferred effective protection against lethal virus challenge. We have also developed similar nanoparticles for plasmid DNA encoding antigen and microparticles for prolonged release of antigen, working towards a single dose vaccine.