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Microfluidic preparation of polymer-nucleic acid nanocomplexes improves nonviral gene transfer.

Grigsby CL, Ho YP, Lin C, Engbersen JF, Leong KW - Sci Rep (2013)

Bottom Line: Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation.These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines.Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, 136 Hudson Hall, Box 90281, Durham, NC 27708, USA.

ABSTRACT
As the designs of polymer systems used to deliver nucleic acids continue to evolve, it is becoming increasingly apparent that the basic bulk manufacturing techniques of the past will be insufficient to produce polymer-nucleic acid nanocomplexes that possess the uniformity, stability, and potency required for their successful clinical translation and widespread commercialization. Traditional bulk-prepared products are often physicochemically heterogeneous and may vary significantly from one batch to the next. Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation. The intracellular integrity of nanocomplexes prepared with this microfluidic method is significantly prolonged, as detected using a high-throughput flow cytometric quantum dot Förster resonance energy transfer nanosensor system. These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines. Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

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Design of microfluidic chip and gene carriers.(A) A microfluidic cross-flow droplet generator chip is used to produce emulsified aqueous droplets containing the polymeric gene carrier and nucleic acids. While confined to these ~ 100 pL droplets, the polyions self-assemble into nanocomplexes. Following collection and disruption of the droplets, the polyplexes are collected and used directly. Channel dimensions are 50 μm (width) × 35 μm (height) (B) Chemical structure of p(CBA-ABOL) (C) Chemical structure of p(CBA-ABOL90/BDA10).
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f1: Design of microfluidic chip and gene carriers.(A) A microfluidic cross-flow droplet generator chip is used to produce emulsified aqueous droplets containing the polymeric gene carrier and nucleic acids. While confined to these ~ 100 pL droplets, the polyions self-assemble into nanocomplexes. Following collection and disruption of the droplets, the polyplexes are collected and used directly. Channel dimensions are 50 μm (width) × 35 μm (height) (B) Chemical structure of p(CBA-ABOL) (C) Chemical structure of p(CBA-ABOL90/BDA10).

Mentions: We prepared polyplexes loaded with either plasmid DNA or messenger RNA using the bioreducible linear poly(amido amine) poly(CBA-ABOL) in bulk and MAC formats (Figure 1). We selected this gene carrier for its high efficiency, low toxicity, and ability to deliver multiple types of nucleic acids2425. Following a systematic optimization of binding characteristics, DNA and RNA polyplexes were synthesized exclusively at polymer:nucleic acid mass ratios of 45:1 and 60:1, respectively (Supplementary Figure 1). In both cases, preparation with MAC resulted in the production of smaller and more monodispersed polyplexes. The Z-average diameters of MAC polyplexes were 40–50% smaller than those of bulk controls immediately following synthesis (Figure 2A). The width of the size distribution was also significantly reduced, as evidenced by similar reductions in the polydispersity index (PDI). To quantify the propensity of the products to aggregate, we additionally measured changes in polyplex size at five-minute intervals over the course of a typical four-hour transfection period (Figure 2B). Bulk polyplexes began to aggregate immediately, and continued to grow in size throughout the period studied. In contrast, MAC polyplexes exhibited a much higher degree of colloidal stability and remained approximately unchanged in size throughout the measurement period. The surface charge density of the polyplexes, represented by the zeta-potential, was also considered (Figure 2C). MAC polyplexes exhibited lower zeta-potentials, suggesting more complete charge neutralization or the presence of a diminished polymer corona, either of which may contribute to improved colloidal stability by reducing charge imbalances and intraparticle heterogeneity26. The physical profiles of nanoparticles are important, as the putative rate-limiting barriers associated with the low efficiency of nonviral vectors include cellular binding and uptake, endosomal escape, cytosolic transport and unpacking, nuclear entry, and transcriptional processing. Physical particle properties determine the degree to which particles are able to overcome each of these hurdles. Knowing that MAC preparation yields smaller, more monodispersed, and less positively charged DNA and RNA polyplexes, we next examined complex binding stability and the final disposition of the polymer component following the complexation reaction.


Microfluidic preparation of polymer-nucleic acid nanocomplexes improves nonviral gene transfer.

Grigsby CL, Ho YP, Lin C, Engbersen JF, Leong KW - Sci Rep (2013)

Design of microfluidic chip and gene carriers.(A) A microfluidic cross-flow droplet generator chip is used to produce emulsified aqueous droplets containing the polymeric gene carrier and nucleic acids. While confined to these ~ 100 pL droplets, the polyions self-assemble into nanocomplexes. Following collection and disruption of the droplets, the polyplexes are collected and used directly. Channel dimensions are 50 μm (width) × 35 μm (height) (B) Chemical structure of p(CBA-ABOL) (C) Chemical structure of p(CBA-ABOL90/BDA10).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818651&req=5

f1: Design of microfluidic chip and gene carriers.(A) A microfluidic cross-flow droplet generator chip is used to produce emulsified aqueous droplets containing the polymeric gene carrier and nucleic acids. While confined to these ~ 100 pL droplets, the polyions self-assemble into nanocomplexes. Following collection and disruption of the droplets, the polyplexes are collected and used directly. Channel dimensions are 50 μm (width) × 35 μm (height) (B) Chemical structure of p(CBA-ABOL) (C) Chemical structure of p(CBA-ABOL90/BDA10).
Mentions: We prepared polyplexes loaded with either plasmid DNA or messenger RNA using the bioreducible linear poly(amido amine) poly(CBA-ABOL) in bulk and MAC formats (Figure 1). We selected this gene carrier for its high efficiency, low toxicity, and ability to deliver multiple types of nucleic acids2425. Following a systematic optimization of binding characteristics, DNA and RNA polyplexes were synthesized exclusively at polymer:nucleic acid mass ratios of 45:1 and 60:1, respectively (Supplementary Figure 1). In both cases, preparation with MAC resulted in the production of smaller and more monodispersed polyplexes. The Z-average diameters of MAC polyplexes were 40–50% smaller than those of bulk controls immediately following synthesis (Figure 2A). The width of the size distribution was also significantly reduced, as evidenced by similar reductions in the polydispersity index (PDI). To quantify the propensity of the products to aggregate, we additionally measured changes in polyplex size at five-minute intervals over the course of a typical four-hour transfection period (Figure 2B). Bulk polyplexes began to aggregate immediately, and continued to grow in size throughout the period studied. In contrast, MAC polyplexes exhibited a much higher degree of colloidal stability and remained approximately unchanged in size throughout the measurement period. The surface charge density of the polyplexes, represented by the zeta-potential, was also considered (Figure 2C). MAC polyplexes exhibited lower zeta-potentials, suggesting more complete charge neutralization or the presence of a diminished polymer corona, either of which may contribute to improved colloidal stability by reducing charge imbalances and intraparticle heterogeneity26. The physical profiles of nanoparticles are important, as the putative rate-limiting barriers associated with the low efficiency of nonviral vectors include cellular binding and uptake, endosomal escape, cytosolic transport and unpacking, nuclear entry, and transcriptional processing. Physical particle properties determine the degree to which particles are able to overcome each of these hurdles. Knowing that MAC preparation yields smaller, more monodispersed, and less positively charged DNA and RNA polyplexes, we next examined complex binding stability and the final disposition of the polymer component following the complexation reaction.

Bottom Line: Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation.These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines.Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, 136 Hudson Hall, Box 90281, Durham, NC 27708, USA.

ABSTRACT
As the designs of polymer systems used to deliver nucleic acids continue to evolve, it is becoming increasingly apparent that the basic bulk manufacturing techniques of the past will be insufficient to produce polymer-nucleic acid nanocomplexes that possess the uniformity, stability, and potency required for their successful clinical translation and widespread commercialization. Traditional bulk-prepared products are often physicochemically heterogeneous and may vary significantly from one batch to the next. Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation. The intracellular integrity of nanocomplexes prepared with this microfluidic method is significantly prolonged, as detected using a high-throughput flow cytometric quantum dot Förster resonance energy transfer nanosensor system. These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines. Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

Show MeSH
Related in: MedlinePlus