About

I am an assistant professor at the Department of Computer Science and Engineering of University of Minnesota. Previously, I was a postdoc at DIMACS, and I obtained my PhD from University of Chicago, where I was advised by Prof. Julia Chuzhoy.

Research topics

• Computer Science
• Biophysics
• Combinatorial chemistry
• Chemistry
• Biology
• Organic chemistry
• Biochemistry
• Polymer chemistry
• Materials science
• Nanotechnology

Selected publications

• Cationic Polymers Enable Internalization of Negatively Charged Chemical Probe into Bacteria

  bioRxiv (Cold Spring Harbor Laboratory) · 2023

  • Chemistry
  • Biophysics
  • Combinatorial chemistry

  Abstract The bacterial cell envelope provides a protective barrier that is challenging for small molecules and biomolecules to cross. Given the anionic nature of both Gram-positive and Gramnegative bacterial cell envelopes, negatively charged molecules are particularly difficult to deliver into these organisms. Many strategies have been employed to penetrate bacteria ranging from reagents such as cell-penetrating peptides, enzymes, and metal-chelating compounds, to physical perturbations. While cationic polymers are known antimicrobial agents, their ability to permeabilize bacterial cells without causing high levels of toxicity and cell lysis has not been demonstrated. Here, we evaluate the ability of four cationic polymers, two linear and two micellar (from self-assembled amphiphilic block copolymers), to facilitate the internalization of an anionic ATP-based chemical probe into Escherichia coli and Bacillus subtilis . Not only did we observe the permeabilization of these organisms, but also found that labeled cells were able to continue to grow and divide. In particular, the micelle-based polymers yielded effective internalization of the negatively charged chemical probe better than their linear analogues.

  PublisherOA PDFDOI

• Cationic Polymers Enable Internalization of Negatively Charged Chemical Probes into Bacteria

  ACS Chemical Biology · 2023-09-06 · 4 citations

  articleOpen access

  The bacterial cell envelope provides a protective barrier that is challenging for small molecules and biomolecules to cross. Given the anionic nature of both Gram-positive and Gram-negative bacterial cell envelopes, negatively charged molecules are particularly difficult to deliver into these organisms. Many strategies have been employed to penetrate bacteria, ranging from reagents such as cell-penetrating peptides, enzymes, and metal-chelating compounds to physical perturbations. While cationic polymers are known antimicrobial agents, polymers that promote the permeabilization of bacterial cells without causing high levels of toxicity and cell lysis have not yet been described. Here, we investigate four polymers that display a cationic poly(2-(dimethylamino)ethyl methacrylate (D) block for the internalization of an anionic adenosine triphosphate (ATP)-based chemical probe into Escherichia coli and Bacillus subtilis. We evaluated two polymer architectures, linear and micellar, to determine how shape and hydrophobicity affect internalization efficiency. We found that, in addition to these reagents successfully promoting probe internalization, the probe-labeled cells were able to continue to grow and divide. The micellar structures in particular were highly effective for the delivery of the negatively charged chemical probe. Finally, we demonstrated that these cationic polymers could act as general permeabilization reagents, promoting the entry of other molecules, such as antibiotics.

  PublisherOA PDFDOI

• Efficient Polymer-Mediated Delivery of Gene-Editing Ribonucleoprotein Payloads through Combinatorial Design, Parallelized Experimentation, and Machine Learning

  ACS Nano · 2020 · 130 citations

  • Computer Science
  • Combinatorial chemistry
  • Computer Science

  nonhomologous end-joining. Structure-function correlations underlying editing efficiency, cellular toxicity, and RNP uptake were probed through machine learning approaches to uncover the physicochemical basis of P38's performance. Although cellular toxicity and RNP uptake were solely determined by polyplex size distribution and protonation degree, respectively, these two polyplex design parameters were found to be inconsequential for enhancing editing efficiency. Instead, polymer hydrophobicity and the Hill coefficient, a parameter describing cooperativity-enhanced polymer deprotonation, were identified as the critical determinants of editing efficiency. Combinatorial synthesis and high-throughput characterization methodologies coupled with data science approaches enabled the rapid discovery of a polymeric vehicle that would have otherwise remained inaccessible to chemical intuition. The statistically derived design rules elucidated herein will guide the synthesis and optimization of future polymer libraries tailored for therapeutic applications of RNP-based genome editing.

  PublisherDOI

• Efficient Polymer-Mediated Delivery of Ribonucleoprotein Payloads Through Combinatorial Design & Parallelized Experimentation

  2020

  • Computer Science
  • Chemistry
  • Combinatorial chemistry

  Genome editing is almost completely reliant on viral delivery to achieve therapeutic goals, hindering widespread clinical adoption. Chemically defined delivery vehicles such as cationic polymers are versatile alternatives to engineered viruses, but their clinical translation hinges on rapidly exploring vast chemical design spaces and deriving structure-function relationships governing delivery performance. Here, we discovered a polymer for efficient ribonucleoprotein (RNP) delivery through combinatorial polymer design and parallelized experimental workflows. A chemically diverse library of 43 statistical copolymers was synthesized via combinatorial RAFT polymerization, realizing systematic variations in physicochemical properties. We selected cationic monomers that varied in their pKa values (8.1 to 9.2) as well as in the steric bulk and lipophilicity of their alkyl substituents. We also incorporated co-monomers of varying hydrophilicity and elucidated the roles of protonation equilibria and hydrophobic-hydrophilic balance. We screened our multiparametric vector library through image cytometry and rapidly uncovered a hit polymer (P38), which outperforms state-of-the-art commercial transfection reagents, achieving nearly 60\% editing efficiency via non-homologous end-joining. Structure-function correlations underlying editing efficiency, cellular toxicity, and RNP uptake were probed through unbiased statistical learning approaches to uncover the physicochemical basis of P38's performance. Although cellular toxicity and RNP uptake were solely determined by polyplex size distribution and protonation degree respectively, these two polyplex design parameters were found to be inconsequential during RNP delivery. Instead, polymer hydrophobicity and the Hill coefficient, a parameter describing cooperativity-enhanced polymer deprotonation, were identified as the critical determinants of RNP delivery. Our unconventional approach not only discovered a novel polymeric vehicle that may have remained inaccessible to chemical intuition, but also yielded statistically derived design rules to guide the synthesis of future polymer libraries.

  PublisherOA PDFDOI

• Efficient Polymer-Mediated Delivery of Ribonucleoprotein Payloads Through Combinatorial Design & Parallelized Experimentation

  ChemRxiv · 2020-09-29

  preprintOpen access

  Genome editing is almost completely reliant on viral delivery to achieve therapeutic goals, hindering widespread clinical adoption. Chemically defined delivery vehicles such as cationic polymers are versatile alternatives to engineered viruses, but their clinical translation hinges on rapidly exploring vast chemical design spaces and deriving structure-function relationships governing delivery performance. Here, we discovered a polymer for efficient ribonucleoprotein (RNP) delivery through combinatorial polymer design and parallelized experimental workflows. A chemically diverse library of 43 statistical copolymers was synthesized via combinatorial RAFT polymerization, realizing systematic variations in physicochemical properties. We selected cationic monomers that varied in their pK<sub>a</sub> values (8.1 to 9.2) as well as in the steric bulk and lipophilicity of their alkyl substituents. We also incorporated co-monomers of varying hydrophilicity and elucidated the roles of protonation equilibria and hydrophobic-hydrophilic balance. We screened our multiparametric vector library through image cytometry and rapidly uncovered a hit polymer (P38), which outperforms state-of-the-art commercial transfection reagents, achieving nearly 60\% editing efficiency via non-homologous end-joining. Structure-function correlations underlying editing efficiency, cellular toxicity, and RNP uptake were probed through unbiased statistical learning approaches to uncover the physicochemical basis of P38's performance. Although cellular toxicity and RNP uptake were solely determined by polyplex size distribution and protonation degree respectively, these two polyplex design parameters were found to be inconsequential during RNP delivery. Instead, polymer hydrophobicity and the Hill coefficient, a parameter describing cooperativity-enhanced polymer deprotonation, were identified as the critical determinants of RNP delivery. Our unconventional approach not only discovered a novel polymeric vehicle that may have remained inaccessible to chemical intuition, but also yielded statistically derived design rules to guide the synthesis of future polymer libraries.

  PublisherOA PDFDOI

• Efficient Polymer-Mediated Delivery of Ribonucleoprotein Payloads Through Combinatorial Design & Parallelized Experimentation

  ChemRxiv · 2020-11-02

  preprintOpen access

  Genome editing is almost completely reliant on viral delivery to achieve therapeutic goals, hindering widespread clinical adoption. Chemically defined delivery vehicles such as cationic polymers are versatile alternatives to engineered viruses, but their clinical translation hinges on rapidly exploring vast chemical design spaces and deriving structure-function relationships governing delivery performance. Here, we discovered a polymer for efficient ribonucleoprotein (RNP) delivery through combinatorial polymer design and parallelized experimental workflows. A chemically diverse library of 43 statistical copolymers was synthesized via combinatorial RAFT polymerization, realizing systematic variations in physicochemical properties. We selected cationic monomers that varied in their pK a values (8.1 to 9.2) as well as in the steric bulk and lipophilicity of their alkyl substituents. We also incorporated co-monomers of varying hydrophilicity and elucidated the roles of protonation equilibria and hydrophobic-hydrophilic balance. We screened our multiparametric vector library through image cytometry and rapidly uncovered a hit polymer (P38), which outperforms state-of-the-art commercial transfection reagents, achieving nearly 60\% editing efficiency via non-homologous end-joining. Structure-function correlations underlying editing efficiency, cellular toxicity, and RNP uptake were probed through unbiased statistical learning approaches to uncover the physicochemical basis of P38's performance. Although cellular toxicity and RNP uptake were solely determined by polyplex size distribution and protonation degree respectively, these two polyplex design parameters were found to be inconsequential during RNP delivery. Instead, polymer hydrophobicity and the Hill coefficient, a parameter describing cooperativity-enhanced polymer deprotonation, were identified as the critical determinants of RNP delivery. Our unconventional approach not only discovered a novel polymeric vehicle that may have remained inaccessible to chemical intuition, but also yielded statistically derived design rules to guide the synthesis of future polymer libraries.

  PublisherDOI

• Block Polymer Micelles Enable CRISPR/Cas9 Ribonucleoprotein Delivery: Physicochemical Properties Affect Packaging Mechanisms and Gene Editing Efficiency

  Macromolecules · 2019-10-21 · 69 citations

  article1st author

  Gene editing with CRISPR/Cas9 is revolutionizing biotechnology and medical research, yet affordable, efficient, and tailorable delivery systems are urgently needed to advance translation. Herein, a series of monodisperse amphiphilic block polymers poly[ethylene oxide-b-2-(dimethylamino) ethyl methacrylate-b-n-butyl methacrylate] (PEO-b-PDMAEMA-b-PnBMA) that housed three PEO lengths (2, 5, and 10 kDa) and a variant lacking PEO (PDMAEMA-b-PnBMA) were synthesized via controlled radical polymerization and assembled into well-defined spherical cationic micelles. The cationic micelles were complexed via electrostatic interactions with Cas9 protein/guide RNA ribonucleoproteins (RNPs) that exhibit anionic charges due to the overhanging RNA. The resulting micelleplex formulations in both phosphate-buffered saline (PBS) and water were screened via high content analysis for gene editing efficiency. The micelle variant with the 10 kDa PEO block offered the highest gene editing performance and was advanced for in-depth characterization. For the first time, quantitative static and dynamic light scattering characterization and cryogenic transmission electron microscopy images of Cas9 protein/guideRNA RNP loading into well-defined micelleplex nanoparticles are revealed, where the formulation solvent was found to play a major role in the physicochemical properties and biological performance. In PBS, the solutions containing the micelles (63 triblock polymers per micelle) were assembled with the Cas9 protein/guideRNA RNP payloads offering uniform loading of 14 RNPs per micelleplex and moderate editing efficiency; this homogeneous system offers promise for future in vivo/preclinical applications. Interestingly, when the uniform micelles were formulated with the RNP payloads in water, larger multimicelleplex nanoparticles were formed that offered double the editing efficiency of Lipofectamine 2000 (40% gene editing) due to the rapid sedimentation kinetics of the larger colloids onto adherent cells, offering promising in vitro, ex vivo, and/or cell therapy applications. This work presents the first quantitative demonstration of tailorable block polymer micelle formulations for advancing CRISPR/Cas9 RNP delivery and fundamental correlation of the solutions physics to biological performance.

  PublisherDOI

• Cationic Polymers and Polymeric Micelles as Plasmid DNA and CRISPR-Cas9 Ribonucleoprotein Delivery Vehicles

  University of Minnesota Digital Conservancy (University of Minnesota) · 2019-11-01

  dissertationOpen access1st authorCorresponding

  University of Minnesota Ph.D. dissertation. November 2019. Major: Chemistry. Advisor: Theresa Reineke. 1 computer file (PDF); xvi, 188 pages.

  Publisher

• Polycation Architecture and Assembly Direct Successful Gene Delivery: Micelleplexes Outperform Polyplexes via Optimal DNA Packaging

  Journal of the American Chemical Society · 2019-09-25 · 97 citations

  article1st author

  Cellular delivery of biomacromolecules is vital to medical research and therapeutic development. Cationic polymers are promising and affordable candidate vehicles for these precious payloads. However, the impact of polycation architecture and solution assembly on the biological mechanisms and efficacy of these vehicles has not been clearly defined. In this study, four polymers containing the same cationic poly(2-(dimethylamino)ethyl methacrylate) (D) block but placed in different architectures have been synthesized, characterized, and compared for cargo binding and biological performance. The D homopolymer and its diblock copolymer poly(ethylene glycol)-block-poly(2-(dimethylamino) ethyl methacrylate) (OD) readily encapsulate pDNA to form polyplexes. Two amphiphilic block polymer variants, poly(2-(dimethylamino)ethyl methacrylate)-block-poly(n-butyl methacrylate) (DB) and poly(ethylene glycol)-block-poly(2-(dimethylamino)ethyl methacrylate)-block-poly(n-butyl methacrylate) (ODB), self-assemble into micelles, which template pDNA winding around the cationic corona to form micelleplexes. Micelleplexes were found to have superior delivery efficiency compared to polyplexes and detailed physicochemical and biological characterizations were performed to pinpoint the mechanisms by testing hypotheses related to cellular internalization, intracellular trafficking, and pDNA unpackaging. For the first time, we find that the higher concentration of amines housed in micelleplexes stimulates both cellular internalization and potential endosomal escape, and the physical motif of pDNA winding into micelleplexes, reminiscent of DNA compaction by histones in chromatin, preserves the pDNA secondary structure in its native B form. This likely allows greater payload accessibility for protein expression with micelleplexes compared to polyplexes, which tightly condense pDNA and significantly distort its helicity. This work provides important guidance for the design of successful biomolecular delivery systems via optimizing the physicochemical properties.

  PublisherDOI

• Nonviral Gene Delivery with Cationic Glycopolymers

  Accounts of Chemical Research · 2019-04-17 · 163 citations

  article

  The field of gene therapy, which aims to treat patients by modulating gene expression, has come to fruition and has landed several landmark FDA approvals. Most gene therapies currently rely on viral vectors to deliver nucleic acid cargo into cells, but there is significant interest in moving toward chemical-based methods, such as polymer-based vectors, due to their low cost, immunocompatibility, and tunability. The full potential of polymer-based delivery systems has yet to be realized, however, because most polymeric transfection reagents are either too inefficient or too toxic for use in the clinic. In this Account, we describe developments in carbohydrate-based cationic polymers, termed glycopolymers, for enhanced nonviral gene delivery. As ubiquitous components of biological systems, carbohydrates are a rich class of compounds that can be harnessed to improve the biocompatibility of non-native polymers, such as linear polyamines used for promoting transfection. Reineke et al. developed a new class of carbohydrate-based polymers called poly(glycoamidoamine)s (PGAAs) by step-growth polymerization of linear monosaccharides with linear ethyleneamines. These glycopolymers were shown to be both efficient and biocompatible transfection reagents. Systematic modifications of the structural components of the PGAA system revealed structure-activity relationships important to its function, including its ability to degrade in situ. Expanding upon the development of step-growth glycopolymers, monosaccharides, such as glucose, were functionalized as vinyl-based monomers for the formation of diblock copolymers via radical addition-fragmentation chain-transfer (RAFT) polymerization. Upon complexation with plasmid DNA, the glucose-containing block creates a hydrophilic shell that promotes colloidal stability as effectively as PEG functionalization. An N-acetyl-d-galactosamine variant of this diblock polymer yields colloidally stable particles that show increased receptor-mediated uptake by liver hepatocytes in vitro and promotes liver targeting in mice. Finally, the disaccharide trehalose was incorporated into polycationic structures using both step-growth and RAFT techniques. It was shown that these trehalose-based copolymers imparted increased colloidal stability and yielded plasmid and siRNA polyplexes that resist aggregation upon lyophilization and reconstitution in water. The aforementioned series of glycopolymers use carbohydrates to promote effective and safe delivery of nucleic acid cargo into a variety of human cells types by promoting vehicle degradation, tissue-targeting, colloidal stabilization, and stability toward lyophilization to extend shelf life. Work is currently underway to translate the use of glycopolymers for safe and efficient delivery of nucleic acid cargo for gene therapy and gene editing applications.

  PublisherDOI

Frequent coauthors

• Theresa M. Reineke

  University of Minnesota System

  33 shared

• Mary E. Brown

  28 shared

• Ngoc Le

  Vietnam National University Ho Chi Minh City

  26 shared

• Shan Jiang

  26 shared

• Ramya Kumar

  Colorado School of Mines

  10 shared

• Christian J. Grimme

  University of Minnesota

  2 shared

• Erin E. Carlson

  2 shared

• Hannah K. Lembke

  University of Minnesota

  2 shared

Education

• PhD, Chemistry

  University of Minnesota

  2019

• B.S., Chemistry

  Fudan University

  2014