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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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Related in: MedlinePlus

Cellular internalization and intracellular unpacking.(A) Labeling scheme to detect uptake and unpacking using QD-FRET. Biotinylated pDNA was labeled with QD energy donors, while the polymer was functionalized with the Cy5 QD-FRET acceptor. QD-FRET emission is detected while polyplexes are intact. Following unpacking, QD-FRET signal is lost and separate donor and acceptor emissions are recovered. (B), (C) Cellular internalization was quantified by measuring the fluorescence signal of QD-labeled pDNA in cells at different time points using flow cytometry. No significant difference was observed in the rates of uptake by cell number of normalized geometric mean fluorescence. (D) Intracellular unpacking was quantified by measuring the QD-FRET signal in cells at different time points using flow cytometry. Bulk controls unpacked more rapidly than MAC polyplexes (p < 0.05 for all timepoints after 300 min).
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f5: Cellular internalization and intracellular unpacking.(A) Labeling scheme to detect uptake and unpacking using QD-FRET. Biotinylated pDNA was labeled with QD energy donors, while the polymer was functionalized with the Cy5 QD-FRET acceptor. QD-FRET emission is detected while polyplexes are intact. Following unpacking, QD-FRET signal is lost and separate donor and acceptor emissions are recovered. (B), (C) Cellular internalization was quantified by measuring the fluorescence signal of QD-labeled pDNA in cells at different time points using flow cytometry. No significant difference was observed in the rates of uptake by cell number of normalized geometric mean fluorescence. (D) Intracellular unpacking was quantified by measuring the QD-FRET signal in cells at different time points using flow cytometry. Bulk controls unpacked more rapidly than MAC polyplexes (p < 0.05 for all timepoints after 300 min).

Mentions: To elucidate the mechanistic relationship between the physical attributes of MAC polyplexes and their improved performance, we focused on two of the primary rate-limiting barriers of nonviral gene delivery: cellular uptake and intracellular unpacking. Although the optimal dimensions of particles for cellular uptake remain a topic of debate, endocytosis is believed to be a size-dependent process29. It follows that the most straightforward means for MAC to improve transfection would be through an increase in cellular uptake due to the smaller size of MAC polyplexes. We measured cellular uptake in a high-throughput manner using flow cytometric detection of internalized QD-labeled pDNA. Bulk and MAC polyplexes were delivered to HEK293 cells, which were fixed to arrest endocytosis at defined timepoints and washed with heparin to remove any remaining membrane-associated complexes. Flow cytometry was used to detect the percentage of cells containing the labeled plasmid at each point, as well as the mean fluorescence signal that correlates with the total mass of internalized plasmid. We observed no difference in uptake between bulk and MAC prepared polyplexes over the time course of a typical transfection period by either metric (Figure 5B and 5C). While the size of MAC polyplexes is reduced, the difference may not be sufficient to alter the rates or modes of endocytosis. If increased uptake is not responsible for the functional benefits of MAC polyplexes, the improvements likely arise from a subsequent process.


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

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

Cellular internalization and intracellular unpacking.(A) Labeling scheme to detect uptake and unpacking using QD-FRET. Biotinylated pDNA was labeled with QD energy donors, while the polymer was functionalized with the Cy5 QD-FRET acceptor. QD-FRET emission is detected while polyplexes are intact. Following unpacking, QD-FRET signal is lost and separate donor and acceptor emissions are recovered. (B), (C) Cellular internalization was quantified by measuring the fluorescence signal of QD-labeled pDNA in cells at different time points using flow cytometry. No significant difference was observed in the rates of uptake by cell number of normalized geometric mean fluorescence. (D) Intracellular unpacking was quantified by measuring the QD-FRET signal in cells at different time points using flow cytometry. Bulk controls unpacked more rapidly than MAC polyplexes (p < 0.05 for all timepoints after 300 min).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Cellular internalization and intracellular unpacking.(A) Labeling scheme to detect uptake and unpacking using QD-FRET. Biotinylated pDNA was labeled with QD energy donors, while the polymer was functionalized with the Cy5 QD-FRET acceptor. QD-FRET emission is detected while polyplexes are intact. Following unpacking, QD-FRET signal is lost and separate donor and acceptor emissions are recovered. (B), (C) Cellular internalization was quantified by measuring the fluorescence signal of QD-labeled pDNA in cells at different time points using flow cytometry. No significant difference was observed in the rates of uptake by cell number of normalized geometric mean fluorescence. (D) Intracellular unpacking was quantified by measuring the QD-FRET signal in cells at different time points using flow cytometry. Bulk controls unpacked more rapidly than MAC polyplexes (p < 0.05 for all timepoints after 300 min).
Mentions: To elucidate the mechanistic relationship between the physical attributes of MAC polyplexes and their improved performance, we focused on two of the primary rate-limiting barriers of nonviral gene delivery: cellular uptake and intracellular unpacking. Although the optimal dimensions of particles for cellular uptake remain a topic of debate, endocytosis is believed to be a size-dependent process29. It follows that the most straightforward means for MAC to improve transfection would be through an increase in cellular uptake due to the smaller size of MAC polyplexes. We measured cellular uptake in a high-throughput manner using flow cytometric detection of internalized QD-labeled pDNA. Bulk and MAC polyplexes were delivered to HEK293 cells, which were fixed to arrest endocytosis at defined timepoints and washed with heparin to remove any remaining membrane-associated complexes. Flow cytometry was used to detect the percentage of cells containing the labeled plasmid at each point, as well as the mean fluorescence signal that correlates with the total mass of internalized plasmid. We observed no difference in uptake between bulk and MAC prepared polyplexes over the time course of a typical transfection period by either metric (Figure 5B and 5C). While the size of MAC polyplexes is reduced, the difference may not be sufficient to alter the rates or modes of endocytosis. If increased uptake is not responsible for the functional benefits of MAC polyplexes, the improvements likely arise from a subsequent process.

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