Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

Figures & data

Table 1 Formulation of CRS by L9 (3⁴) statistic experiment

Experiment	A (mg/mg)	B (mg/mg)	C (g/100 mL)	D (min)	Particle size (nm)	Zeta potential (mV)	Score
E1	40:60	5:1	0	5	2,422.7	−32.87	−2,488.44
E2	40:60	10:1	1	10	467.9	−27.71	−523.32
E3	40:60	15:1	2	15	103.3	0.29	−102.72
E4	30:70	5:1	1	15	127.8	−17.46	−162.72
E5	30:70	10:1	2	5	1,204.1	−10.07	−1,244.23
E6	30:70	15:1	0	10	2,680.3	−4.06	−2,688.42
E7	0:100	5:1	2	10	122.9	−5.16	−133.22
E8	0:100	10:1	0	15	320.8	36.01	−2,487.8
E9	0:100	15:1	1	5	216.1	6.65	−202.8

Notes: Values are expressed as mean ± standard deviation (n=3). L9 is the code of orthogonal experiment; There are four candidates: A (leci thin:CRS, m/m), B (leci thin + CRS:cholesterol, m/m), C (emulsifying agent, m/v) and D (probe ultrasound, min).

Table 2 Orthogonal analysis of L9 (3⁴) statistic experiment

Parameter	A	B	C	D
K1	−1,380.161	−928.127	−1,808.547	−1,305.160
K2	−1,358.460	−665.447	-296.280	−1,114.987
K3	−194.933	-997.980	−486.727	−171.407
R	1,163.527	332.533	1,512.267	1,133.753

Notes: L9 is the code of orthogonal experiment; There are four candidates: A (leci thin:CRS, m/m), B (leci thin + CRS:cholesterol, m/m), C (emulsifying agent, m/v) and D (probe ultrasound, min). E₁–E₃ (K₁), E₄–E₆ (K₂), E₇–E₉ (K₃) were three levels used in this experiment, and R was used for comprehensive comparison of three levels.

Figure 1 The agarose gel electrophoresis of VEGF-siRNA and CRS with different concentrations.

Notes: (A) 100% CRS, (B) 75% CRS, (C) 50% CRS and (D) 25% CRS (n=3).
Abbreviation: siRNA, small interfering RNA.

Figure 2 Faraday-Tyndall effect of control and CRS (25%, 50%, 75%, 100%).

Note: A, B, C, D and E are control, 25%, 50%, 75% and 100% of CRS, respectively.

Figure 3 Nano-properties of CRS and VEGF-siRNA/CRS.

Notes: (A) Particle size and (B) ζ potential of various CRS concentrations (n=3).
Abbreviation: siRNA, small interfering RNA.

Figure 4 TEM (A–D) and SEM (E–H) images of VEGF-siRNA/CRS.

Abbreviations: TEM, transmission electron microscopy; SEM, scanning electron microscopy; siRNA, small interfering RNA.

Figure 5 Confocal image (×10⁴~10⁵) of in vitro cellular uptakes of VEGF-siRNA/CRS at 2 h and 4 h.

Abbreviation: siRNA, small interfering RNA.

Figure 6 Release of VEGF-siRNA and CRS from VEGF-siRNA/CRS. Note: Data are presented as mean ± SD (n=3).

Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

Figure 7 In vitro inhibition of HeLa cells treated by VEGF-siRNA/CRS (n=3).

Abbreviations: NC, negative control; siRNA, small interfering RNA.

Figure 8 ELISA results for VEGF protein expression of HeLa cells treated with VEGF-siRNA/CRS.

Note: Data are presented as mean ± SD (n=3).
Abbreviations: NC, negative control; siRNA, small interfering RNA; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation.

Figure 9 Gene-silencing efficiency of VEGF-siRNA/CRS on HeLa cells.

Note: Data are presented as mean ± SD (n=3).
Abbreviations: NC, negative control; siRNA, small interfering RNA; SD, standard deviation.

Figure 10 The coronal sections of the tumor from the right armpit of mice.

Note: The tissue of the right armpit of each mouse is shown in gray, and the tumors are shown with the blue circles.

Figure 11 Imaging of mice from animal fluorescence system.

Notes: (A) 0 min; (B) 15 min; (C) 30 min; (D) 60 min; (E) 120 min; (F) 240 min. (a) Cy3-CRS injected and (b) Cy3 injected.

Figure 12 In vivo antitumor effect of VEGF-siRNA/CRS.

Note: Data are presented as mean ± SD (n=10).
Abbreviations: DOX, doxorubicin; NS, normal saline; SD, standard deviation; siRNA, small interfering RNA.

Scheme 1 Synthesis of 1-methyl-β-caboline-3-carboxylic acid.

Notes: Steps: ①, CH₃CHO/H⁺; ②, SOCl₂/CH₃OH; ③, KMnO₄/CH₃COCH₃ and ④, NaOH. By the Pictet–Spengler reaction, l-Trp (I) was initially turned into (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (II) by condensation with CH₃CHO/H⁺, and compound II was esterified into (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid methyl ester (III). The oxidation reaction of compound III was catalyzed by KMnO₄, and (1S,3S)-1-methyl-β-carboline-3-carboxylic acid methyl ester (IV) was synthesized. Compound IV was saponified into 1-methyl-β-caboline-3-carboxylic acid (V).

Scheme 2 Synthesis of RGDS peptide.

Scheme 3 Synthesis of 1-methyl-β-caboline-3-RGDS with carboxyl and amino terminal.

Notes: Steps: ⑤, DCC/HOBt/NMM; ⑥, TFA/TfOH and ⑦, HCl-EtOAc. Following the method in Scheme 1 (steps 1–4), compound V was turned to 1-methyl-β-caboline-3-RGDS (VI) by linking with HCl·Arg(Tos)-Gly-Asp(OBzl)-Ser-OBzl. The 1-methyl-β-caboline-3-RGDS with carboxyl and amino terminal (VII) precipitated out during acidolysis.
Abbreviations: DCC, dicyclohexylcarbodiimide; HOBt, 1-hydroxybenzotriazole; NMM, N-methylmorpholine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; EtOAc, ethyl acetate.