Development of a thermally responsive peptide for sustained deliver of solyble TNF receptor II to attenuate inflammatory events associated with radiculopathys
Bibliographic record
Abstract
Background: Tumor necrosis factor alpha (TNFα) is a cytokine that may mediate inflammatory histopathology of the dorsal root ganglion following lumbar disc herniation.1 Soluble TNF receptor II (sTNFRII) competitively binds TNFa with clinical value for painful radiculopathy.2 Bioactive peptides expressed with elastin-like polypeptides (ELP) fusion partners gain a thermally responsive domain, by which they can undergo hydrophobic collapse and separate from solution to aggregate at physiological temperatures.3 Protein release from such a depot may locally sustain drug presence, an effect demonstrated for non-fusion ELP after intra-articular injection.4 Methods: We expressed sTNFRII fused to ELP to demonstrate potential bidomain functionality. Protein Expression. A gene encoding ELP-(VPGVG)60 was subcloned adjacent to the sTNFRII and transformed into E.coli for expression.5 Protein Safety. Endotoxin content of purified fusion protein was evaluated using a limulus amebocyte lysate endpoint assay and compared to non-fusion ELP using a two-tailed Student’s t-test. Thermal Responsiveness. Dynamic light scattering evaluated the inverse thermal phase transition behaviour of ELP-sTNFRII, and absorbance spectrophotometry quantified the in vitro depot release at 37°C. Fusion Domain Function. Anti-TNFα bioactivity was assessed by the in vitro inhibition of TNFα-induced glutamate production by microglia. Single-factor ANOVA analyzed treatment differences for ELP-sTNFRII, commercial sTNFRII (positive control), and non-fusion ELP (negative control). A 44 kDa recombinant fusion protein was expressed from E. coli and purified by inverse transition cycling. Results: Measured endotoxin content for ELP-sTNFRII was comparable to ELP alone (p < 0.01), well below FDA levels for biomedical implants. The fusion protein underwent a thermally-induced phase transition and formed observable aggregates of ~240 nm upon heating to physiological temperatures (Tt = 32°C). Slow release was observed from this depot with a time constant of 21 ± 3 hours. The fusion protein demonstrated anti-TNFα activity in vitro by attenuating TNFα-induced microglial glutamate production, albeit requiring a greater concentration than the free antagonist to achieve the same effect.(p < 0.01). Conclusion: Fusion of a sTNFRII protein to an ELP can serve to generate a thermally-induced drug depot that may sustain anti-cytokine activity of agents delivered locally to a nerve region. Further directions may involve studying in vivo biodistribution after perineural delivery of ELP and in vivo disease modifying activity of this agent.
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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 | 0.000 |
| 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 | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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".