Based on our experience supporting in-vitro applications of similar conjugates, several well-characterized factors can influence functional knockdown. Consider some key points below, with supporting literature where applicable
1. Serum Binding Can Reduce Peptide–siRNA Uptake Many cell-penetrating peptides (CPPs) and targeting peptides show reduced internalization in the presence of serum due to nonspecific protein binding (e.g., albumin, globulins). This can significantly limit intracellular siRNA delivery when using 10% FBS.
Relevant publications:
- Kirschbrown et al., Molecular Therapy—Nucleic Acids, 2020: demonstrated that serum proteins strongly inhibit uptake of peptide–siRNA conjugates and that reduced-serum incubation improves delivery efficiency.
- Henriques et al., Cellular and Molecular Life Sciences, 2007: reviewed how CPP uptake is negatively affected by serum interactions, impacting downstream biological activity.
For many applications, improved activity is observed when conjugates are incubated initially in 0–2% serum for 4–6 hours, before returning to normal culture conditions.
2. Linker & Conjugate Stability Can Be Affected by Resuspension Conditions Certain linkers (especially disulfide, ester, and acid-sensitive linkages) can undergo hydrolysis or rearrangement in aqueous solutions lacking buffering capacity. This may lead to premature siRNA release or partial degradation, reducing functional delivery.
Relevant publications:
- Huang et al., Journal of Controlled Release, 2016: showed that peptide–nucleic acid conjugates exhibit improved stability when resuspended in buffered solutions rather than pure water.
- Miller et al., Bioconjugate Chemistry, 2018: reported that minimizing freeze–thaw cycles and maintaining neutral pH preserves conjugate integrity.
Although nuclease-free water is generally acceptable, peptide–siRNA conjugates often remain more stable in low-ionic-strength buffers (e.g., 10 mM HEPES pH 7.4 or 1× TE).
3. Dose Optimization Is Essential for Peptide-Conjugated siRNA Compared to lipid-mediated transfection, peptide-mediated delivery may require a broader dose range to achieve knockdown. Many groups report optimal activity in the 5–100 nM range depending on cell type and peptide chemistry.
Supporting publication:
- Ruseska & Zimmer, Advanced Drug Delivery Reviews, 2020: reviewed dose-dependent variability in peptide-based siRNA delivery and emphasized the need for empirical concentration titration in new cell lines.
If you have not already done so, we recommend testing several concentrations spanning this range.
4. siRNA Sequence Potency Must Be Verified Independently Before evaluating delivery efficiency, it is important to confirm that the siRNA sequence itself produces knockdown under standard conditions. Delivering the same siRNA using a lipid-based reagent can verify whether the target sequence is intrinsically active.
Supporting publication:
- Reynolds et al., Nature Biotechnology, 2004: established design rules for potent siRNA sequences and showed that even small deviations can significantly alter knockdown efficiency.
If the siRNA is unvalidated in your specific cell line, this control is essential.
Trouble Shooting Checklist:
- Cell line used and confluence at treatment
- Final working concentration of the conjugate
- Exposure time and assay time points
- Readout type (qPCR, Western, viability, etc.)
- Whether the same siRNA shows activity with a standard lipid transfection
- Storage details after resuspension (temperature, time, freeze–thaw cycles)