My Research

What I Do

Addressing human health and healthcare challenges with nanomedicine

Research Directions

Can we revolutionize delivery techniques by engineering molecular assembly?

Besides chemical structures, the way therapeutic molecules assemble with carrier materials into nanoparticles plays vital roles in protecting the payloads during transportation and releasing them in a programmed manner. The assembly features of therapeutic nanoparticles, including the size, surface, and internal molecular arrangements, direct their interactions with biological systems on the macroscopic scale, synergistically acting with the microscopic, molecular-level interactions. This branch of my research focuses on the study of thermodynamics and kinetics mechanisms that govern intermolecular assembly processes in nanoparticle formation, and how this knowledge can be used for rational design of therapeutic nanoparticles with the most optimal chemical-biological interfaces.

What therapeutic tools can we harness when exploring biological diversity?

The incredible diversity in biology warrants limitless discoveries of useful tools that we can engineer into drugs. The Nobel-winning discovery of CRISPR gene editing system is a great example on how a defense mechanism of bacteria against virus was developed into a powerful therapeutic platform. This branch of my research explores the great tool box of biological diversity and uses genetic and chemical engineering approaches to turn naturally existed biological mechanisms into useful tools to combat diseases, even when they are only remotely related to the target.

Can we make safer and more effective vaccines?

The effectiveness of mRNA vaccine during the COVID-19 pandemic was impressive, but... can we do even better? How can we further boost the immunogenicity of the vaccine to provide more prolonged protection, while minimizing undesired side effects (or reactogenicity)? What does the next generation of vaccine look like after the Nobel-winning technology of mRNA vaccine? Can we use vaccines to prevent cancer and genetic diseases? This branch of my research uses state-of-art engineering approaches and understandings of the immune system to boost vaccine developments, with an ultimate goal of redefining how vaccines can be administered, how vaccines can collaborate with the immune system, and how powerful vaccines can be.

Active Translational Research

Shelf-stable and on-bench stable DNA particles for large-scale transfection to produce viral vectors
A scalable plasmid supramolecular assembly technology

2020 to 2022, Scientific developments
We collaborated with pioneers in viral gene therapy bluebird bio, Inc. and 2seventy bio, Inc. to develop shelf-stable, highly efficient DNA particles for large-scale transfection of HEK293 production cell lines in bioreactors. In this critical manufacturing step of viral vectors, we for the first time discovered that the size of DNA particles assembled with the transfection reagent poly(ethylenimine) (PEI) is one of the most critical parameters for transfection efficiency, and the optimal size resides between 400 to 500 nm. We therefore developed a DNA supramolecular assembly strategy that allows scalable production of such particles, and verified their stability both during long-term storage and on the bench as an off-the-shelf product. This platform technology was first published in 2021 in Nano Letters, an American Chemical Society (ACS) journal, and received recognitions through an oral presentation at the 2022 Annual Meeting for American Society of Gene and Cell Therapy (ASGCT).
2022 to 2023, Translational developments
In collaboration with 2seventy bio, Inc., we developed scale-up modalities and procedures for the continuous production of 400-nm DNA transfection particles through stepwise electrostatic supramolecular assembly. We delivered a batch production of 5,300 mL of 400-nm DNA/PEI particles at a production rate of 1,000 mL/min, at a high DNA concentration of 50 μg/mL. Shelf stability of at least 1 year under -80°C was verified, while the on-bench stability under ambient temperature and transfection efficiency of these particles to produce lentiviral vectors were verified in the laboratory of 2seventy bio. Of important note, there is no theoretical limit on the lot size of the processes developed, which has cleared the way for this technology to be used for production of viral vectors at commercially relevant scales. The paper describing this process development was published in 2024 in the prestigious journal Molecular Therapy—Methods & Clinical Development by ASGCT, American Society for Gene and Cell Therapy.
The Mao Laboratory continues to collaborate with gene therapy companies worldwide to translate this technology.

CICS platform for single-nanoparticle size and composition characterizations in nanomedicine
A high-throughput spectroscopic system that strengthens quality control and fundamental research

2021 to 2022, Scientific developments
Based on the cylindrical illumination confocal spectroscopy (CICS) technique developed by single-molecule dynamics experts Sixuan Li and Prof. Tza-huei "Jeff" Wang at the Johns Hopkins University Department of Mechanical Engineering, we pioneered in analyzing payload features of therapeutic nanoparticles for both quality control purposes and scientific exploration, as the single-nanoparticle composition information revealed at high throughput by CICS has direct implications on the dynamic molecular assembly processes of the nanoparticles. Our proof-of-concept study for the first time quantitatively characterized the mRNA copy per nanoparticle, empty nanoparticle ratio, and lipid loading level within mRNA lipid nanoparticle (LNP) formulations, and revealed their fluidity along the purification step governed by a kinetics-driven mechanism. This study, titled "Payload distribution and capacity of mRNA lipid nanoparticles", was published in 2022 in Nature Communications, and was one of the Top 25 Life and Biological Sciences Articles of 2022 in the journal.
2022 to 2024, Scientific developments
Based on the CICS platform, we further implemented a chorographical size characterization method, single-nanoparticle free solution hydrodynamic separation (SN-FSHS), for simultaneous size and composition quantifications on the single-nanoparticle level. In our proof-of-concept study, this upgraded SN-FSHS-CICS platform successfully achieved high-throughput profiling of siRNA lipid nanoparticles (LNPs) in terms of their size and loading of up to three payloads (siRNA, helper lipid, and PEGylated lipid). Through in-depth analyses of these data, we reveal loading heterogeneity among LNPs and its dependence on size as well as each step in the manufacturing processes. The study serves as an excellent showcase on how high-dimensional nanoparticle characterization data can direct research of the assembly kinetics of complex systems. This study, titled "Single-particle spectroscopic chromatography reveals heterogeneous RNA loading and size correlations in lipid nanoparticles", was published in 2024 in ACS Nano, a prestigious American Chemical Society journal.
The inventor of the CICS platform, Sixuan Li, is leading the commercialization efforts of this technology.

More underway...

Stay tuned for many more exciting research directions!

Past efforts on translational research

From 2017 to 2019, together with Professors Il Minn and Martin G. Pomper from the Johns Hopkins University School of Medicine, we collaborated with start-up company Cancer Targeting Systems, Inc. to develop DNA nanoparticles that target metastatic lung cancer. This IND-enabling preclinical project aimed for proof-of-concept experiments to demonstrate the efficacy of a cancer-specific DNA promotor combining IL-12 for immunotherapy, and scale-up manufacturing of DNA nanoparticles with tightly controlled parameters and optimized vehicle sizes for highest lung delivery efficiency. The outcomes from this research have been published in ACS Nano in 2019 (Link), and in Scientific Reports in 2021 (Link).

My Ph.D. thesis: Kinetics-based nanoparticle size engineering of gene delivery vehicles

Intracellular gene delivery has significant values in treating genetic and acquired diseases in vivo, and in engineering cells ex vivo or in vitro for therapeutic applications. Compared to viral approaches, gene-loaded non-viral nanoparticles formed with polymeric carriers have the advantages of lower immunogenicity, easier manufacturing, and chemical versatility to render desired vehicle properties. For such vehicles, their colloidal size is a central property that determines the payload capacity of the therapeutic cargo (Li, Hu et al., Nature Communications, 2022), and more importantly, determines their interactions with biological systems such as blood circulation, immune system, target tissues, and target cells.

In summary, my thesis for the first time enabled size control of polymeric nucleic acid nanoparticles within the full nano-to-micro size range: covering 30 to 200 nm via diffusion-controlled assembly in turbulent mixing (Hu et al., ACS Nano, 2019) and 200 to 1000 nm via electrostatic supramolecular assembly (Hu, Zhu et al., Nano Letters, 2019; Hu, Tzeng et al., PNAS, 2024), precisely and even in a scalable manner (Hu et al., Molecular Therapy—Methods & Clinical Development, 2024). Size screening is necessary from a quality control perspective for clinical translation, and improves the efficacy and biocompatibility of gene delivery vehicles in a variety of applications, including large-scale in vitro manufacturing of viral vectors, ex vivo cellular reprogramming, and in vivo therapeutic delivery and immunotherapy, for which different optimal sizes were identified for each application. In addition, analyzing single-nanoparticle size and loading at the same time offers a fresh angle to study the molecular assembly structures of nanomedicine (Li, Hu et al., ACS Nano, 2024).