Egr1/HSP70 Promoter-Driven Activation of Gene Expression for Synergistic Anti-Hepatoma Using PEI-MZF Nanoparticles and Radiation

Figures & data

Figure 1 The flow chart of pHsp70-Egr1-HSVTK construction.

Table

MgCl2	1 µL
5×PCR Buffer	5 µL
dNTPs	0.25 µL
Primers	f/r 0.25~1 µL
cDNA	2 µL
ddH2O	14.5~16µL
Taq Enzyme	0.25 µL

Table

94°C	2min
94°C	30sec
51°C	30 sec	35cycles
72°C	90 sec
72°C	10min
End

Figure 2 (A) HREM image of Mn_0.5 Zn_0.5Fe₂O₄. (B) SEM-EDS analysis of Mn_0.5 Zn_0.5Fe₂O₄. (C) XRD analysis of Mn_0.5 Zn_0.5Fe₂O₄. (D) TEM image of the PEI-Mn_0.5 Zn_0.5Fe₂O₄. (E) FTIR of PEI- Mn_0.5 Zn_0.5Fe₂O₄ and Mn_0.5 Zn_0.5Fe₂O₄. (F) Adsorption of PEI on the surface of nanoparticles under different pH. (G) Scheme of PEI/MZF interaction with DNA.

Table 1 PEI-Mn_0.5Zn_0.5Fe₂O₄ Nanoparticle Toxicity Test MTT (, n=8)

Group	OD Value	Cell Viability %	Cytotoxicity Classification
Negative Group	1.06±0.04	100	0
N/P=1	0.96±0.03	90.8	1
N/P=3	0.87±0.03	85.4	1
N/P=5	0.81±0.02	76.8	1
N/P=7	0.71±0.04	66.6	2
N/P=9	0.69±0.01	65.0	2
N/P=11	0.61±0.02	54.8	2
Positive Group	0.16±0.03	15.2	4

Table 2 PEI/DNA Toxicity Test MTT (, n = 8)

Group	OD Value	Cell Viability %	Cytotoxicity Classification
Negative Group	1.32±0.06	100	0
N/P=1	1.15±0.03	87.1	1
N/P=3	1.08±0.02	80.2	1
N/P=5	0.85±0.03	64.4	2
N/P=7	0.62±0.02	46.8	3
N/P=9	0.53±0.01	40.7	3
N/P=11	0.36±0.02	27.1	3
Positive Group	0.18±0.03	13.6	4

Figure 3 Comparison of PEI-MZF and PEI cytotoxicity under different N/P ratio The N/P ratio of PEI/MZF/DNA and PEI/DNA was 1, 3, 5, 7, 9, 11.

Table 3 Hemolysis Test Results

Group	Absorbance Value (OD)			Average OD	Hemolysis Rate %
	1	2	3
Negative	0.017	0.021	0.025	0.021	0
Experiment	0.024	0.027	0.028	0.026	0.608
Positive	0.834	0.842	0.853	0.843	100

Table 4 Results of Acute Toxicity Test of Nanoparticles

Group	Dosage (g/kg)	Dose Logarithm	n	Mortality	P	Q	p×q
1	1.568	3.195346	10	0	0	1	0
2	2.8	0.447158	10	1	0.1	0.9	0.09
3	5	0.69897	10	3	0.3	0.7	0.21
4	8.92	0.950365	10	4	0.4	0.6	0.24
5	15.94	1.202488	10	9	0.9	0.1	0.09
		I = 0.56		∑p = 1.7

Notes: p is death rate, q is survival rate, n is the number of mice.

Table 5 Micronucleus Test Results

Group	n	Number of PEC	Number of PEC with MN	Micronucleus Formation Rate (‰)
Negative	6	6000	12	2
5.00g/kg	6	6000	11	1.8
2.5g/kg	6	6000	10	1.67
1.25g/kg	6	6000	12	2
0.625g/kg	6	6000	11	1.8
Positive	6	6000	143	23.8

Abbreviation: n, number of mice in each group.

Figure 4 Thermodynamic test of different concentration of PEI- Mn_0.5 Zn_0.5Fe₂O₄in vitro.

Figure 5 Image of agarose gel electro-phoresis of plasmid DNA and complexed with PEI- Mn_0.5 Zn_0.5Fe₂O₄ (Lane1-5(PEI-MZF/DNA w/w): 0:1; 5:1; 10:1; 20:1; 30:1).

Figure 6 (A) PCR gel electrophoresis of Egr1-HSVTK transfected (lane 1: Marker IV (each band in turn from top to bottom is 7 K, 5.5 K, 3.5 K, 2 K, 1 K, 500 bp); lane 1: Egr1-HSVTK). (B) Sequencing and alignment of Egr1-HSVTK (sequence 0: HSVTK; sequence 1:Egr1-HSVTK-Egr1-f.ab1; software: Dnassit 2.0).

Figure 7 (A) PCR gel electrophoresis of Hsp70-Egr1-EGFP transfected (lane 1: Marker IV each band in turn from top to bottom is 7 K, 5.5 K, 3.5 K, 2 K, 1 K, 500 bp); lane 1–4: Hsp70-Egr1-EGFP①-④). (B) Sequencing and alignment of Hsp70-Egr1-EGFP (sequence 0: HSP70; sequence 1: Hsp70-Egr1-EGFP-f.ab1; software: Dnassit 2.0).

Figure 8 (A) Egr1-Hsp70- HSVTK was identified by EcoRI and HindIII dual restriction enzyme digestion (lane 1: Marker IV each band in turn from top to bottom is 7 K, 5.5 K, 3.5 K, 2 K, 1 K, 500 bp;lane 1–3: pCDNA3.1-Egr1- Hsp70-HSVTK①-③digested by EcoRI and HindIII.). (B) Egr1- Hsp70-HSVTK was identified by MluI restriction enzyme digestion (lane 1: Marker IV each band in turn from top to bottom is 7 K, 5.5 K, 3.5 K, 2 K, 1 K, 500 bp;lane 1–2: Hsp70-Egr1- HSVTK ①-②digested by MluI.).

Figure 9 TK expression induced by radiation at different time point. Compared the TK expression at 48h with that in other groups, P < 0.05. Compared the TK expression at 6h with that at other time point, P < 0.05.

Figure 10 TK expression induced by different dose of radiation. Compared the TK expression with 0Gy radiation with that in other groups P < 0.05. Compared the TK expression after 2Gy radiation with that in other groups P < 0.05.

Figure 11 TK expression induced by different methods. Compared the TK expression without treatment, with that in other groups P < 0.05. Compared the TK expression with radiation and thermo, with that in other groups P < 0.05.

Figure 12 Expression of HSV-TK in SMC7721cells induced by different methods. 1 100bp Marker. 2 Negative control (not transfected). 3 Radiation group (not transfected). 4 Hyperthermia group (not transfected). 5 TK transfection by radiation induction group. 6 TK transfection by heat induction group. 7 TK transfection by radiation and heat shock induction group.

Table 6 The Cell Viability of SMMC-7721 cells After Treatment (, n = 6)

Group	A Value	Cell Viability (%)
Negative group	1.19±0.012	100
Radiation group	1.027±0.015*	86.31
Radiation-gene group	0.946±0.015^Δ	79.24
Heat group	0.696±0.011^Δ	58.33
Heat-gene group	0.537±0.023^Δ	44.96
Combined treatment group	0.204±0.029^Δ	17.12

Notes: *Compared with negative group P > 0.05, ^ΔCompared with negative group P < 0.05.

Figure 13 The cell viability of SMC7721 cells after treatment.

Table 7 Flow Cytometry of Cell Cycles and Apoptosis

Group	Apoptosis (%)	G1 Phase	G2 Phase	S Phase
Negative group	0.12%	54.22%	33.78%	11.99%
Radiation group	8.68%	22.89%	45.77%	31.34%
Radiation-gene group	15.16%	30.89%	44.79%	24.31%
Heat group	20.22%	41.16%	37.72%	21.13%
Heat-gene group	27.94%	45.19%	33.66%	21.14%
Combined treatment group	34.87%	38.72%	15.91%	45.37%

Figure 14 (A) Cell cycle results of SMMC7721 cells after treatment. (B) Flow cytometry results of SMMC7721 cells after treatment.

Figure 15 (A) Inhibitory rate of weight and mass in xenograft tumor after treatment. (B) Tumor morphological appearance after treatment. 1 Negative group. 2 Radiation group. 3 Radiation-gene group. 4 Mn_0.5Zn_0.5Fe₂O₄ hyperthermia group. 5 Heat-gene group. 6 The combined group. (C) The histo-morphology of tumors after treatment (100×). Negative group: The cells are tightly arranged, with rich cytoplasm, large and deeply stained nucleus, and active proliferation. Combined group: The cells are obviously necrotic, and magnetic material deposition can be seen between the tissue. (D) The histo-morphology of organs after treatment (100×). A lung B heart C kidney D liver E spleen.