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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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Transfection efficiency in four cell types.(A) Fluorescence micrographs of four cell types transfected with bulk or MAC polyplexes loaded with 1 μg of either pDNA or mRNA encoding GFP. Scale bar = 50 μm (B) Quantification of GFP transfection efficiency with flow cytometry (p < 0.05 in each case) (C) Transfection efficiency in HEK293 cells across a range of doses confirms that the increased transfection by MAC polyplexes is not dose-dependent. (D) Quantification of total transgene expression following transfection with pLuc DNA complexes.
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f4: Transfection efficiency in four cell types.(A) Fluorescence micrographs of four cell types transfected with bulk or MAC polyplexes loaded with 1 μg of either pDNA or mRNA encoding GFP. Scale bar = 50 μm (B) Quantification of GFP transfection efficiency with flow cytometry (p < 0.05 in each case) (C) Transfection efficiency in HEK293 cells across a range of doses confirms that the increased transfection by MAC polyplexes is not dose-dependent. (D) Quantification of total transgene expression following transfection with pLuc DNA complexes.

Mentions: The most critical aspect of polyplex performance is transfection efficiency. We tested both DNA and RNA polyplexes in four cell types that spanned the most common target classes for gene delivery applications. Primary mouse embryonic fibroblast (PMEF) cells were selected for their prevalence in the cellular reprogramming and induced pluripotent stem cell fields. Human mesenchymal stem cells (hMSC) were included to represent primary human adult stem cells. HepG2 human hepatocellular carcinoma cells were tested due to their characteristic low transfectability and the importance of hepatocytes as a destination for gene delivery systems designed to target the liver. Lastly, HEK293 human embryonic kidney cells were chosen to compare with the large body of prior work. At 24 hours following delivery to the four different cell types, the transfection efficiency polyplexes loaded with 1 μg pDNA or mRNA encoding GFP was assessed with fluorescence microscopy and quantified by flow cytometry. In each case, transfection with MAC polyplexes resulted in a larger fraction of cells expressing the GFP reporter protein (Figure 4A and 4B). The gains ranged from 6 to 31 percent more cells transfected, and were particularly significant in difficult-to-transfect cell types such as HepG2 and hMSC. Several different doses of polyplexes were delivered to HEK293 cells to verify that this result was not dose-dependent, and the improvement persisted across the range of doses (Figure 4C). In some applications, the total level of transgene produced may be more important than the number of individual cells transfected. To measure gross transgene expression, 1 μg doses of pDNA encoding luciferase was delivered to each of the four cell types. In each case, transfection with MAC polyplexes resulted in 1.9 to 6.8-fold higher total expression as quantified by luminescence detection (Figure 4D). Cytotoxicity comparisons are not shown, as poly(CBA-ABOL) is effectively non-toxic at single dose levels (Supplementary Figure 3).


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

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

Transfection efficiency in four cell types.(A) Fluorescence micrographs of four cell types transfected with bulk or MAC polyplexes loaded with 1 μg of either pDNA or mRNA encoding GFP. Scale bar = 50 μm (B) Quantification of GFP transfection efficiency with flow cytometry (p < 0.05 in each case) (C) Transfection efficiency in HEK293 cells across a range of doses confirms that the increased transfection by MAC polyplexes is not dose-dependent. (D) Quantification of total transgene expression following transfection with pLuc DNA complexes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Transfection efficiency in four cell types.(A) Fluorescence micrographs of four cell types transfected with bulk or MAC polyplexes loaded with 1 μg of either pDNA or mRNA encoding GFP. Scale bar = 50 μm (B) Quantification of GFP transfection efficiency with flow cytometry (p < 0.05 in each case) (C) Transfection efficiency in HEK293 cells across a range of doses confirms that the increased transfection by MAC polyplexes is not dose-dependent. (D) Quantification of total transgene expression following transfection with pLuc DNA complexes.
Mentions: The most critical aspect of polyplex performance is transfection efficiency. We tested both DNA and RNA polyplexes in four cell types that spanned the most common target classes for gene delivery applications. Primary mouse embryonic fibroblast (PMEF) cells were selected for their prevalence in the cellular reprogramming and induced pluripotent stem cell fields. Human mesenchymal stem cells (hMSC) were included to represent primary human adult stem cells. HepG2 human hepatocellular carcinoma cells were tested due to their characteristic low transfectability and the importance of hepatocytes as a destination for gene delivery systems designed to target the liver. Lastly, HEK293 human embryonic kidney cells were chosen to compare with the large body of prior work. At 24 hours following delivery to the four different cell types, the transfection efficiency polyplexes loaded with 1 μg pDNA or mRNA encoding GFP was assessed with fluorescence microscopy and quantified by flow cytometry. In each case, transfection with MAC polyplexes resulted in a larger fraction of cells expressing the GFP reporter protein (Figure 4A and 4B). The gains ranged from 6 to 31 percent more cells transfected, and were particularly significant in difficult-to-transfect cell types such as HepG2 and hMSC. Several different doses of polyplexes were delivered to HEK293 cells to verify that this result was not dose-dependent, and the improvement persisted across the range of doses (Figure 4C). In some applications, the total level of transgene produced may be more important than the number of individual cells transfected. To measure gross transgene expression, 1 μg doses of pDNA encoding luciferase was delivered to each of the four cell types. In each case, transfection with MAC polyplexes resulted in 1.9 to 6.8-fold higher total expression as quantified by luminescence detection (Figure 4D). Cytotoxicity comparisons are not shown, as poly(CBA-ABOL) is effectively non-toxic at single dose levels (Supplementary Figure 3).

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