Table 3 ∣.

Troubleshooting table.

Steps	Problem	Possible reason	Solution
10	No product	G0-C14 is stuck on the silica gel	Increase the percentage of MeOH to increase the polarity of the eluent
14	Low yield of G0-C14 product	The amines of G0 are partially oxidized, resulting in a yellow G0 solution	Always store the G0 solution in a 4 °C fridge and flush the bottle with argon after each use to protect the amines from oxidization.
24	Low yield of DSPE-PEG-S2P product	The peptide S2P is not completely dissolved.	Warm up the solution at 37 °C in a water bath or add more HEPES buffer.
42	Precipitation of siRNA	The vigorous mixing process changes the homogenous state of the siRNA/G0-C14 solution	Mixing the siRNA/G0-C14 solution by gentle pipetting. Do not vortex the solution
57	Poor reproducibility of DLS results	The siLuc NPs are too diluted and below the DLS detection limit or the extreme dilution destabilizes the NPs.	Adjust the dilution factor according to the detection limit of the DLS instrument and do not dilute too much.
68	Poor contrast of TEM images	The volume of staining solution is not large enough or incubation time is too short	Increase the volume of the staining solution or increase the incubation time
	Very few samples in TEM images	The sample is too diluted	Increase the concentration of the sample, or repeat the sample loading procedure several times
	The background is too dark in TEM images	The incubation time for the staining solution is too long	Decrease the incubation time of the staining solution
91	The gel bands are too weak	The siRNA concentration is too low	Before gel electrophoresis, concentrate the siRNA samples using Amicon tubes (MWCO, 10 kDa)
101	Low siRNA EE	Fail to transfer all the Dy647-siLuc NPs from the Amicon tube	Pipette the Dy647-siLuc NPs solution vigorously before transferring; Also, wash the tube one or two times with a small volume of PBS.
121	Low fluorescence signals of Dy647	The concentration of Dy647-siLuc NPs is too low or the cell density is too high	Raise the concentration of Dy647-siLuc NPs or decrease the cell density.
131	Low bioluminescence signal	The bioluminescence signal decays	Prepare and warm up the imaging system before adding the substrate. Upon adding the substrate, finish the measurement within 10 min.
	Low gene silencing efficiency	G0-C14 is degraded	The G0-C14 can be easily degraded after dissolution in the solvent. Aliquot the G0-C14 solution and store aliquots at −20 °C. Do not use the same batch more than three times.
152	All the samples are PI positive	The PI staining process is too long	Shorten the PI staining process; Finish the measurement within 1 h after the PI staining
165	The centrifugation takes too much time	The concentration of siRNA NPs is too high, making the ultrafiltration difficult	Split the samples into more Amicon tubes for washing, combine and transfer all the samples to one Amicon tube to adjust the concentration of NPs for in vivo injection
166	The volume of the NPs is too large for injection	The siRNA concentration is too low	Add less PBS to redisperse the NPs during the preparation of NPs
176	Weak fluorescence signal of the sections	Low activity of the primary antibody or the primary antibody concentration is too low	Handle the primary antibodies on ice to retain their optimal activity; Decrease the dilution factor of the primary antibodies.