New publication
May 20, 2024

There have been extensive efforts both in the academia and industry to optimize RNA lipid nanoparticles (LNP) to make them as uniform as possible. In this context, "uniformity" most of the time refers to "size uniformity", and a uniform preparation is usually characterized by a low polydispersity index (PDI) from dynamic light scattering (DLS) or cryogenic transmission electron microscopy (cryo-TEM).

However, with the rise of our single-nanoparticle profiling technique, cylindrical illumination confocal spectroscopy (CICS) accessing the composition of individual LNPs, we ask a fundamental question with curiosity: Does size uniformity spontaneously mean uniform distribution of payloads (i.e., loading uniformity)?

To answer this question, we coupled the original CICS platform with single-nanoparticle free solution hydrodynamic separation (SN-FSHS) technique, a chromatographical size characterization method. Adding a fourth readout of hydrodynamic size on top of the loading level of three fluorescently tagged LNP components (siRNA payload, helper lipid, and PEG lipid) of a formulation that shares the same composition as the clinically approved drug ONPATTRO, we analyzed the detailed loading-size correlations across a wide size distribution of around 30 to 200 nm. We found that LNPs of like size may have significantly different loading density, which means that loading heterogeneity can co-exist with size uniformity (not so surprisingly!). Through in-depth analyses into the the entire manufacturing process of these RNA LNPs, including the initial complexation at a relatively low pH and the subsequent dialysis towards physiological pH, we concluded that the loading heterogeneity might be a result of intrinsic diffusivity differences between assembly components and kinetics-driven assembly and stabilization during the pH shift. We expect these findings can inspire further discussions and exploration by the LNP community, not only to verify these findings independently, but to think about methods to optimize LNPs for better loading uniformity, which may facilitate a higher efficiency per unit mass of payload on LNPs.

This study was a collaborative effort by teams led by Prof. Hai-Quan Mao and Prof. Tza-huei Wang. We welcomed Prof. Tine Curk from the Department of Materials Science and Engineering of Johns Hopkins University to join the team, who contributed instrumental analyses from a molecular dynamics perspective in understanding the assembly behaviors of RNA LNPs. Our paper describing these findings has now been published with open access and can be accessed by everyone for free: Li and Hu, et al., ACS Nano, 2024.

New publication
Jan 23, 2024

In 2021, we discovered that the size of plasmid DNA/poly(ethylenimine) (PEI) particles is one of the most critical parameters that determine the yield of viral vectors during transfection of production cell lines, and the optimal size is 400 to 500 nm. In our proof-of-concept study, we developed a supramolecular assembly technique that allows production of uniform 400-nm DNA particles with shelf and on-bench stability, and benchmarked their performances against conventional transfection procedures.

Today, we show that this technology is scalable. In a collaboration with a team led by Ting Guo and Brendan Eder at 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. As a milestone for this translational effort, we delivered a single batch production of 5,300 mL of 400-nm DNA/PEI particles at a production rate of 1,000 mL/min, and 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, Inc. 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 is now online in the prestigious journal Molecular Therapy—Methods & Clinical Development by ASGCT, American Society for Gene and Cell Therapy.

Viral vector-mediated gene therapy has been clinically successful and now approved by regulatory agencies worldwide, however, its accessibility is limited by challenges in its manufacturing processes. This work focusing on an important upstream step to produce viral vectors contributes to further enhancing the reliability and reproducibility of multi-plasmid transfection. Together with other industrial partners, I myself, with the team led by Prof. Hai-Quan Mao at Johns Hopkins University, will continue to work towards addressing the scalability and efficiency challenges of transfection in gene therapy.

Nov 17, 2023

I was selected as one of the 2023-2024 class of Convergence Scholars, a program founded by the MIT Marble Center for Cancer Nanomedicine and the MIT Center for Precision Cancer Medicine (CPCM), that is designed to enhance the career development of aspiring independent scientists with diverse interests across academia, industry, science communication, and STEM outreach.

Sep 17, 2023

I won the best poster award in the 21st Nanomedicine and Drug Delivery Symposium held at MIT. The poster summarizes the last project of mine during my Ph.D. study at Johns Hopkins University, a collaboration between the laboratories of Prof. Hai-Quan Mao and Prof. Jordan J. Green in developing a first-in-class technology for targeted mRNA delivery to certain subtypes of immune cells for cancer immunotherapy.

The manuscript describing this work is currently under review.

Apr 07, 2023

My paper last year in Nature Communications, titled "Payload distribution and capacity of mRNA lipid nanoparticles" (Link), co-authored with Sixuan Li, Prof. Tza-huei Wang and Prof. Hai-Quan Mao, was recently listed in Top 25 Life and Biological Sciences Articles of 2022 (Link). 

Within 6 months since its publication, the article has been downloaded more than 22,000 times, and cited 8 times. It also received an independent recommendation on Faculty Opinions. Following the publication, we received numerous emails from both academia and industry with requests for discussions and collaborations. In March, upon an invitation from Dr. Zongming Fu, Sixuan gave a seminar at GSK Global Vaccines R&D Center on this paper, followed by in-depth conversations regarding the findings by our study. We are glad and greatly encouraged to see these interests in the technique and the fundamental sciences behind this paper.

Jan 02, 2023

I am glad to announce that I have joined the laboratory of Professor Daniel G. Anderson at David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology as a postdoctoral associate to carry out research on vaccine technologies.

New publication
Sep 23, 2022

It raised my great interest when I got to know that the exact molecular structure of mRNA lipid nanoparticles, those vehicles used in the proven successful COVID-19 vaccine, is still unknown to scientists. Though it does not have implication with potency or safety, this unsatisfaction in characterizing these promising nanoparticles motivated me to carry out deeper investigations. With great support from my advisor Prof. Hai-Quan Mao, we established collaboration with two distinguished experts of single-molecule dynamics, Sixuan Li and Tza-huei Wang, Ph.D. student and Louis M. Sardella Professor from Department of Mechanical Engineering, Johns Hopkins University, to study the fundamentals of mRNA lipid nanoparticles.

With extraordinary leadership by Sixuan, we set up a multi-color cylindrical illumination confocal microscopy (CICS) platform that can assess cargo and carrier content of delivery vehicles at single-nanoparticle level. The technique features in-line examination in a flow set-up, 100% mass detection efficiency and single-fluorophore sensitivity owing to a 1-D laser deformation design. When multiple fluorophores are used to tag different molecular components, CICS identifies and differentiates distinct populations within a lipid nanoparticle formulation, including free mRNA, mRNA-loaded lipid nanoparticles, and surprisingly, empty lipid nanoparticles that do not contain an mRNA molecule. It then quantitatively resolve single-nanoparticle fluorescence to give a distribution of mRNA and lipid payload level within the formulation.

We asked two questions in this study: (1) Are all lipid nanoparticles loaded with mRNA molecules (is the myth about empty nanoparticles true)? (2) How many mRNA molecules can a single lipid nanoparticle load? The findings turned out to be extremely interesting beyond delivering convincing, quantitative answers to these two questions. We also found out that these nanoparticles are fluid during the manufacturing processes, meaning that both input formulation conditions and purification procedures shape the final payload profiles. Though their behaviors showed a great degree of complexity, we were able to extract some basic kinetic rules. If you are as curious as we were about the payload features of mRNA lipid nanoparticles, you may find the open-access paper in this link: Li and Hu et al., Nature Communications, 2022.

I feel so glad that our study has furthered the fundamental understandings of these promising nanoparticles. We believe it will promote investigations about how these vehicles can be effective to deliver genes, and eventually lead to rational design strategies that can aid optimization of them for diverse applications.

Also in the news coverage by Johns Hopkins University: Molecular detection platform provides new insights into gene medicine manufacturing

May 18, 2022

I delivered two talks, titled "Size-optimized and shelf-stable plasmid DNA particles for production of viral vectors", and "Particle size engineering to enhance mRNA delivery efficiency via biodegradable carriers in vivo" in the 25th annual meeting of American Society of Gene and Cell Therapy (ASGCT) at Washington D.C. The full abstracts were published in the journal Molecular Therapy (#74 and #861).

The talks showcased our approach to optimize gene delivery vehicles for in vitro and in vivo applications through particle size and assembly engineering. Benefits of delivery efficiency and biocompatibility were achieved in both plasmid DNA and mRNA delivery systems. Both systems are being further implemented into industrial practices, and carefully examined in pre-clinical studies, respectively. We will share the exciting results in our future publications in a timely manner.

The founders award keynote session this year featured Professor Francis Collins, director of the National Institute of Health. His inspirational talk about the future of gene and cell therapy highlighted the mission of the society to develop accessible therapies for people around the world with different economical backgrounds. I am so proud that our research directions in the lab of Professor Hai-Quan Mao, with a focus on translational nanomedicine and tissue engineering, are all-out towards this mission.

April 30, 2022

It felt so good to attend my first in-person conference since the start of the pandemic and saw so many familiar faces at the Annual Meeting and Exposition of Society for Biomaterials (SFB). I delivered two talks, titled "Payload distribution and capacity of mRNA lipid nanoparticles", and "Supra-molecular assembly of nucleic acids in polymer carriers and therapeutic opportunities within the sub-micron size range" in the sessions held by Drug Delivery Special Interest Group. Both showed unpublished data that drew a lot of attention. We hope to publish them very soon!

March 21, 2022

Today I successfully defended my thesis titled "Kinetics-based Particle Size Engineering of Polymeric Gene Delivery Vehicles". A big thank-you to my advisor Prof. Hai-Quan Mao, my committee, all my collaborators, my lab colleagues, my friends and my family! It was such a wonderful journey at Hopkins.