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

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.

SUBMITTER: Grigsby CL

PROVIDER: S-EPMC3818651 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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

Grigsby Christopher L CL Ho Yi-Ping YP Lin Chao C Engbersen Johan F J JF Leong Kam W KW

Scientific reports 20131106

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 ...[more]

PMID: 24193511

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Project description:Nucleic acid amplification tests (NAATs) are a crucial diagnostic and monitoring tool for infectious diseases. A key procedural step for NAATs is sample preparation: separating and purifying target nucleic acids from crude biological samples prior to nucleic acid amplification and detection. Traditionally, sample preparation has been performed with liquid- or solid-phase extraction, both of which require multiple trained user steps and significant laboratory equipment. The challenges associated with sample preparation have limited the dissemination of NAAT point-of-care diagnostics in low resource environments, including low- and middle-income countries. We report on a paper-based device for purification of nucleic acids from whole blood using isotachophoresis (ITP) for point-of-care NAATs. We show successful extraction and purification of target nucleic acids from large volumes (33 µL) of whole human blood samples with no moving parts and few user steps. Our device utilizes paper-based buffer reservoirs to fully contain the liquid ITP buffers and does not require complex filling procedures, instead relying on the natural wicking of integrated paper membranes. We perform on-device blood fractionation via filtration to remove leukocytes and erythrocytes from our sample, followed by integrated on-paper proteolytic digestion of endogenous plasma proteins to allow for successful isotachophoretic extraction. Paper-based isotachophoresis purifies and concentrates target nucleic acids that are added directly to recombinase polymerase amplification (RPA) reactions. We show consistent amplification of input copy concentrations of as low as 3 × 103 copies nucleic acid per mL input blood with extraction and purification taking only 30 min. By employing a paper architecture, we are able to incorporate these processes in a single, robust, low-cost design, enabling the direct processing of large volumes of blood, with the only intermediate user steps being the removal and addition of tape. Our device represents a step towards a simple, fully integrated sample preparation system for nucleic acid amplification tests at the point-of-care.

Dataset Information

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

Publications

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

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