Albumin pre-coating enhances intracellular siRNA delivery of multifunctional amphiphile/siRNA nanoparticles

Figures & data

Figure 1 Chemical structure of N-(1-aminoethyl)iminobis[N-(oleoylcysteinylhistinyl-1-aminoethyl)propionamide].

Table 1 Conjugation agents and sizes of various EHCO/siRNA nanoparticles

Type of EHCO/siRNA nanoparticle	Conjugation agent	Size range (nm)
Unmodified EHCO/siRNA nanoparticles	–	53 ± 3.3
Pegylated EHCO/siRNA nanoparticles	PEG-Mal	66 ± 2.0
RAD-targeted EHCO/siRNA nanoparticles	RAD-PEG-Mal	58 ± 1.4
RGD-targeted EHCO/siRNA nanoparticles	RGD-PEG-Mal	56 ± 5.4

Note: The sizes of nanoparticles are presented as mean ± standard error (SE) of n = 3 different experiments.

Abbreviations: EHCO, N-(1-aminoethyl)iminobis[N-(oleoylcysteinylhistinyl-1-aminoethyl)propionamide]; siRNA, short-interfering RNA.

Figure 2 Preparation of pegylated targeted EHCO/siRNA nanoparticles.

Abbreviations: EHCO, N-(1-aminoethyl)iminobis[N-(oleoylcysteinylhistinyl-1-aminoethyl)propionamide]; siRNA, short-interfering RNA; PEG, polyethylene glycol.

Figure 3 Cellular uptake of different EHCO/siRNA nanoparticles. The cellular uptake of different nanoparticles was measured using flow cytometric analysis. The transfection was conducted in serum free media (RPMI-1640). AF-647 siGFP is the siRNA used in the preparation of nanoparticles. [AF-647 siGFP] = 100 nM; (a) untreated 4T1 cells; (b) siRNA; (c) PEG/EHCO/siRNA; (d) PEG-RAD/EHCO/siRNA; (e) PEG-RGD/EHCO/siRNA; (f) EHCO/siRNA.

Abbreviations: EHCO, N-(1-aminoethyl)iminobis[N-(oleoylcysteinylhistinyl-1-aminoethyl)propionamide]; siRNA, short-interfering RNA; PEG, polyethylene glycol.

Figure 4 Serum influences size and cellular uptake of targeted EHCO/siRNA nanoparticles. (A) Equal volumes of targeted EHCO/siRNA nanoparticles and FBS in DPBS at indicated concentrations were mixed and the size of the nanoparticles was measured using DLS ([siMIF] = 250 nM). The error bars represent mean ± standard error (SE); n = 3 experiments. *P ≤ 0.15, compared with 0 and 10% FBS in DPBS treatment groups. (B) 4T1-Luc cells were transfected with RGD-targeted EHCO/siRNA nanoparticles for 4 hours in transfection media containing 0, 10%, 25%, and 50% FBS in RPMI-1640 media and cellular uptake of the nanoparticles was analyzed using flow cytometric analysis ([AF-647 siGFP] = 200 nM; (a) untreated cells; (b) 50% FBS in RPMI-1640; (c) 25% FBS in RPMI-1640; (d) 10% FBS in RPMI-1640; (e) 0% FBS in RPMI-1640).

Abbreviations: EHCO, N-(1-aminoethyl)iminobis[N-(oleoylcysteinylhistinyl-1-aminoethyl)propionamide]; siRNA, short-interfering RNA; FBS, fetal bovine serum; DPBS, Dulbecco’s phosphate-buffered saline; DLS, dynamic light scattering.

Figure 5 The effect of BSA on the size, cellular uptake and cytotoxicity of the targeted nanoparticles. (A) The size of targeted EHCO/siRNA nanoparticles measured by DLS indicates that BSA coating increases the size of nanoparticles. ^#P ≤ 0.15, compared with 0 μM BSA treatment group; *P ≤ 0.15, compared with 0, 4.7, and 9.4 μM BSA treatment groups. (B) Cellular uptake of targeted EHCO/siRNA nanoparticles coated with and without BSA in 4T1 cells was measured by flow cytometric analysis ([AF-647-siGFP] = 50 nM, 50% FBS in RPMI-1640). *P ≤ 0.05, compared with all other BSA treatment groups. (C) The viability of 4T1 cells was measured after they were transfected with the nanoparticles coated without and with BSA for 48 hours. BSA coating concentration was 9.4 μM.

Abbreviations: BSA, bovine serum albumin; EHCO, N-(1-aminoethyl)iminobis[N-(oleoylcysteinylhistinyl-1-aminoethyl)propionamide]; siRNA, short-interfering RNA; DLS, dynamic light scattering; FBS, fetal bovine serum.

Figure 6 BSA coating enhanced cellular uptake and gene-silencing efficiency of RGD-targeted EHCO/siRNA nanoparticles.

Notes: 4T1-GLuc cells were transfected with RGD-targeted nanoparticles ([AF-647-siGFP] = 50 nM) coated without and with BSA for 4 hours and then replaced with cell culture growth media. Images were taken using Olympus confocal microscopy at 40× magnification 48 hours later (Transfection media = 50% FBS in RPMI-1640).

Abbreviations: BSA, bovine serum albumin; EHCO, N-(1-aminoethyl)iminobis[N-(oleoylcysteinylhistinyl-1-aminoethyl)propionamide]; GFP, green fluorescent protein; siRNA, short-interfering RNA; FBS, fetal bovine serum.

Figure 7 Illustration of a possible mechanism for improved cellular uptake of targeted EHCO/siRNA nanoparticles coated with BSA.

Abbreviations: EHCO, N-(1-aminoethyl)iminobis[N-(oleoylcysteinylhistinyl-1-aminoethyl)propionamide]; BSA, bovine serum albumin.