Effects of Microfluidic Shear on the Plasmid DNA Structure: Implications for Polymeric Gene Delivery Vectors
Bibliographic record
Abstract
Microfluidic manufacturing of advanced gene delivery vectors necessitates consideration of the effects of microfluidic shear forces on the structural integrity of plasmid DNA (pDNA). In this paper, we expose pDNA to variable shear forces in a two-phase, gas–liquid microfluidic reactor and apply gel electrophoresis to analyze the products of on-chip shear-induced degradation. The effects of shear rate, solvent environment, pDNA size, and copolymer complexation on shear-induced degradation are investigated. We find that small naked pDNA (pUC18, 2.7 kb) exhibits shear rate-dependent shear degradation in the microfluidic channels in a mixed organic solvent (dioxane/water/acetic acid; 90/10/<0.1 w/w/w), with the extents of both supercoil isoform relaxation and complete fragmentation increasing as the maximum shear rates increase from 4 × 10 5 to 2 × 10 6 s –1 . However, over the same range of shear rates, the same pDNA sample shows no evidence of microfluidic shear-induced degradation in a pure aqueous environment. Quiescent control experiments in the same mixed organic solvent prove that a combination of solvent and shear forces is involved in the observed shear-induced degradation. Furthermore, we show that shear degradation effects in mixed organic solvents can be significantly attenuated by complexation of pDNA with the block copolymer polycaprolactone- block -poly(2-vinylpyridine) prior to exposure to microfluidic shear. Finally, we demonstrate that medium (pDSK519, 8.1 kb) and large (pRK290, 20 kb) naked pDNA are more sensitive to shear-induced microfluidic degradation in the mixed organic solvent environment than small pDNA, with both plasmids showing complete fragmentation even at the lowest shear rate, although we found no evidence of shear-induced damage in water for the largest investigated naked pDNA even at the highest flow rate. The resulting understanding of the interplay of the solvent and shear effects during microfluidic processing should inform microfluidic manufacturing routes to new gene therapy formulations.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".