Efficacy and mechanism of tanshinone IIA liquid nanoparticles in preventing experimental postoperative peritoneal adhesions in vivo and in vitro

Figures & data

Figure 1 Chemical structure of tanshinone IIA.

Table 1 Grading of adhesions in rats according to the criteria of Nair et al²⁷

Grade	Description	Classification
0	No adhesions	Insubstantial adhesions
1	Single band of adhesions between viscera or from one viscus to the abdominal wall	Insubstantial adhesions
2	Two bands between viscera or between viscera and abdominal wall	Substantial adhesions
3	More than two bands between viscera or between viscera and abdominal wall, or the entire intestine forming a mass adhering to the abdominal wall to the abdominal wall	Substantial adhesions
4	Viscera directly attached to the abdominal wall, regardless of number or extent of bands	Substantial adhesions

Figure 2 Illustration of experimental postoperative peritoneal adhesions in rats.

Notes: (A) Creation of ischemic buttons to peritoneal sidewall. (B) Adhesions to the buttons at postoperative day 7.

Table 2 Particle sizes, zeta potentials, and polydispersity index values of various NPs (n=3)

Samples	Particle size (nm)	Zeta potential (mV)	Polydispersity index
Blank NPs	211.03±2.81	−37.6±3.2	0.11±0.013
Tan IIA-NPs	217.25±5.68	−31.2±2.7	0.17±0.035

Note: Data are presented as mean ± standard deviation.

Abbreviations: NPs, nanoparticles; Tan IIA-NPs, tanshinone IIA liquid NPs.

Figure 3 Characterization of Tan IIA-NPs and blank NPs.

Notes: Transmission electron microscopy images of (A) blank NPs ×25.0 k and (B) Tan IIA-NPs ×25.0 k. Malvern Zetasizer 3000 HSA size measurement of (C) blank NPs and (D) Tan IIA-NPs. Malvern Zetasizer 3000 HSA zeta potential measurement of (E) blank NPs and (F) Tan IIA-NPs.

Abbreviations: NPs, nanoparticles; Tan IIA-NPs, tanshinone IIA liquid NPs.

Table 3 Characterization of Tan IIA-NPs stored under 60°C and room temperature with 4,500±500 lx for 10 days (n=3)

Storage condition	Time point (days)	Particle size (nm)	Zeta potential (mV)	pH value	Drug content (%)a
60°C	0	217.25±5.68	−31.2±2.7	7.04±0.27	97.86±0.23
	5	228.46±8.27	−33.4±1.9	6.45±0.35	95.72±0.50
	10	241.39±10.33	−34.8±4.1	6.20±0.14	94.03±0.87
Room temperature with 4,500±500 lx	0	217.25±5.68	−31.2±2.7	7.04±0.27	97.86±0.23
	5	219.03±6.60	−30.8±3.0	6.68±0.32	85.62±1.33
	10	230.61±8.58*	−31.9±2.2	6.14±0.08*	66.79±0.96*

Notes: Data are presented as mean ± standard deviation.

a Drug content = actual amount of Tan IIA/labeled amount of Tan IIA ×100%.

* P<0.05 compared with the group at day 0 under the condition of room temperature with 4,500±500 lx.

Abbreviations: Tan IIA, tanshinone IIA; Tan IIA-NPs, Tan IIA liquid nanoparticles.

Table 4 Characterization of Tan IIA-NPs stored at 25°C±2°C away from light during 12 months (n=3)

Time (month)	Particle size (nm)	Zeta potential (mV)	pH value	Drug content (%)a
0	217.25±5.68	−31.2±2.7	7.04±0.27	97.86±0.23
3	219.74±7.02	−31.9±1.6	6.96±0.10	97.86±0.42
6	224.17±10.58	−32.8±2.0	6.81±0.22	96.01±0.39
9	224.66±9.26	−30.0±2.7	6.54±0.41	95.72±0.44
12	231.70±11.44	−30.6±3.2	6.63±0.18	95.01±0.36

Notes: Data are presented as mean ± standard deviation.

a Drug content = actual amount of Tan IIA/labeled amount of Tan IIA ×100%. There was no significant difference of the above parameters compared with the group at month 0.

Abbreviations: Tan IIA, tanshinone IIA; Tan IIA-NPs, Tan IIA liquid nanoparticles.

Table 5 The content of bacterial endotoxin in each batch (n=10)

Batch	Content of bacterial endotoxin (EU/mg)	Bacterial endotoxin limited value (EU/mg)a
1	0.067±0.013*	2.272±0.064
2	0.042±0.009*
3	0.071±0.024*

Notes: Data are presented as mean ± standard deviation.

a The bacterial endotoxin limited value was evaluated following the formula L=K/M, where L is the bacterial endotoxin limited value (EU/mg), K is a threshold pyrogenic dose of endotoxin per kilogram of body weight, and M is equal to the maximum recommended bolus dose of product per kilogram of body weight.

* P<0.05 compared with the bacterial endotoxin limited value.

Table 6 Effects on hematology, liver function, and kidney function parameters in healthy BALB/c mice determined by acute toxicity study (n=20)

	Parameter	Tan IIA-NPs	Drug-free NPs	Saline
Hematology	WBCs (G/L)	5.04±0.62	4.96±0.85	5.10±0.92
	RBCs (T/L)	8.27±0.97	8.04±0.75	8.01±0.89
	PLTs (G/L)	989.6±98.42	1,025.7±131.77	1,037.2±140.54
	Lymphocytes (%)	65.7±2.74	66.1±3.02	66.4±3.85
Liver function	AST (U/L)	103±4.80	110±6.24	107±3.94
	ALT (U/L)	47.2±5.71	46.9±7.41	45.1±4.80
	TBIL (μmol/L)	1.02±0.045	1.09±0.072	1.07±0.078
Kidney function	BUN (mmol/L)	4.07±0.96	4.12±0.75	4.23±0.69
	CR (μmol/L)	8.3±1.01	8.2±0.88	8.3±0.85
	UA (μmol/L)	16.7±3.31	16.4±2.96	16.0±3.75
	HCO3 (mmol/L)	2.43±0.47	2.39±0.88	2.46±0.65

Notes: Data are presented as mean ± standard deviation. There was no significant difference of the above parameters compared with the saline group (P>0.05).

Abbreviations: AST, aspartate aminotransferase; ALT, alanine transaminase; BUN, blood urea nitrogen; CR, creatinine; NPs, nanoparticles; PLTs, platelets; RBCs, red blood cells; Tan IIA-NPs, tanshinone IIA liquid NPs; TBIL, total bilirubin; UA, uric acid; WBCs, white blood cells.

Figure 4 The change of body weight (g) of mice before and after administration of corresponding solutions over 14 days.

Notes: Body weight (g) of mice in each group slowly increased during the 2-week period. There was no significant difference in body weight compared with the saline group.

Abbreviations: NPs, nanoparticles; Tan IIA-NPs, tanshinone IIA liquid NPs.

Figure 5 Histological staining of excised organs from healthy BALB/c mice following intravenous administration three times within 24 hours with (A) 0.9 g/dL natrium chloride, (B) drug-free NPs, or (C) Tan IIA-NPs.

Note: Representative images of organ sections were at 400× magnification.

Abbreviations: NPs, nanoparticles; Tan IIA-NPs, tanshinone IIA liquid NPs.

Table 7 Intra-abdominal adhesion scores of groups (n=15)

Grading of adhesion	Score	HD Tan IIA-NPs	MD Tan IIA-NPs	LD Tan IIA-NPs	Blank NPs	Control
0	1	12	7	3	0	0
1	2	2	2	5	0	0
2	3	1	2	1	1	0
3	4	0	4	1	4	5
4	5	0	0	5	10	10
Mean ± SD		1.27±0.59*,#	2.20±1.32*,#	3.00±1.64*,#	4.60±0.63	4.67±0.49

Notes: Data are presented as mean ± SD.

* P<0.05 compared with the control group;

# P<0.05 compared with the blank NP group. The HD group received 10 mg/kg Tan IIA-NPs; the MD group 5 mg/kg Tan IIA-NPs; and the LD group 2.5 mg/kg Tan IIA-NPs.

Abbreviations: HD, high-dose; LD, low-dose; MD, medium-dose; NPs, nanoparticles; SD, standard deviation; Tan IIA-NPs, tanshinone IIA liquid NPs.

Figure 6 Effects of tPA activity in intraperitoneal lavage fluid.

Notes: High, medium and low dose groups were administered with 10, 5 and 2.5 mg/kg Tan IIA-NPs via tail vein injection, respectively. High- (n=15), medium(n=15), and low-dose (n=15) groups showed a significant increase in tPA activity in peritoneal fluid compared with the control, blank, and drug-free NP groups. *P<0.05 compared with blank; ^#P<0.05 compared with control; ^§P<0.05 compared with drug-free NPs.

Abbreviations: NPs, nanoparticles; tPA, tissue-type plasminogen activator.

Figure 7 tPA mRNA expression (A), PAI-1 mRNA expression (B), and the ratio of protein expression level of tPA/PAI (C) in peritoneal tissues.

Notes: High, medium and low dose groups were administered with 10, 5 and 2.5 mg/kg Tan IIA-NPs via tail vein injection, respectively. Relative expression levels of tPA and PAI-1 mRNAs are normalized against β-actin. The ratio of protein expression level of tPA/PAI was detected by enzyme-linked immunosorbent assay and the ratio was evaluated using the following formula: ratio of tPA/PAI = (level of tPA protein/total protein)/(level of PAI protein/total protein). Results are expressed as a percentage of controls and shown as mean ± standard deviation (n=5). *P<0.05 compared with blank; ^#P<0.05 compared with control; ^§P<0.05 compared with drug-free NPs.

Abbreviations: NPs, nanoparticles; tPA, tissue-type plasminogen activator.

Figure 8 TGF-β1 expression in the peritoneal tissues after 7 days of treatment, detected by immunohistochemistry.

Notes: High, medium and low dose groups were administered with 10, 5 and 2.5 mg/kg Tan IIA-NPs via tail vein injection, respectively. Representative images (at 200× magnification) of peritoneal tissues that were stained immunohistochemically with anti-TGF-β1 antibody. (A) Blank control group. (B) Control group. (C) Drug-free group. (D) Low-dose group. (E) Medium-dose group. (F) High-dose group. (G) Quantitative assay of OD from five randomly selected fields. The results are presented as mean ± standard deviation (n=5). *P<0.05 compared with blank; ^#P<0.05 compared with control; ^§P<0.05 compared with drug-free NPs.

Abbreviations: NPs, nanoparticles; OD, optic density; TGF-β1, transforming growth factor-β1.

Figure 9 Collagen I expression in the peritoneal tissues after 7 days of treatment, detected by immunohistochemistry.

Notes: High, medium and low dose groups were administered with 10 mg/kg, 5 mg/kg and 2.5 mg/kg Tan IIA-NPs via tail vein injection, respectively. Representative images (at 200× magnification) of peritoneal tissues that were stained immunohistochemically with anti-collagen I antibody. (A) Blank control group. (B) Control group. (C) Drug-free group. (D) Low-dose group. (E) Medium-dose group. (F) High-dose group. (G) Quantitative assay of OD from five randomly selected fields. The results are presented as mean ± standard deviation (n=5). *P<0.05 compared with blank; ^#P<0.05 compared with control; ^§P<0.05 compared with drug-free NPs.

Abbreviations: NPs, nanoparticles; OD, optic density.

Figure 10 Effect of Tan IIA-NPs on wound healing in the model of anastomotic healing.

Notes: The results are presented as mean ± standard deviation (n=5). High- (n=5), medium- (n=5), and low-dose (n=5) groups showed no significance in burst pressure compared with the control and drug-free NP groups.

Abbreviations: NPs, nanoparticles; Tan IIA-NPs, tanshinone IIA liquid NPs.

Figure 11 Effect of TGF-β1 on the ratio of tPA/PAI-1 in HMrSV5 cells.

Notes: (A) Change of the ratio of tPA/PAI-1 induced by different concentrations of TGF-β1 for 4 hours. (B) Change of the ratio of tPA/PAI-1 induced by 9 pmol/L TGF-β1 at 0.5-, 2-, and 4-hour points. Data are expressed as mean ± standard deviation (n=3). *P<0.05 compared with control.

Abbreviations: PAI-1, plasminogen activator inhibitor-1; TGF-β1, transforming growth factor-β1; tPA, tissue-type plasminogen activator.

Figure 12 Effect of free-Tan IIA (A) and Tan IIA-NPs (B) on the ratio of tPA/PAI-1 in HMrSV5 cells induced by TGF-β1.

Notes: High, medium and low dose groups were treated with 20, 10, and 5 μg/mL Tan IIA-NPs, respectively. And free-Tan IIA group was treated with 20 μg/mL Tan IIA. Data are expressed as mean ± standard deviation (n=3). *P<0.05 compared with control; ^#P<0.05 compared with model; ^§P<0.05 compared with blank.

Abbreviations: NPs, nanoparticles; Tan IIA, tanshinone IIA; Tan IIA-NPs, Tan IIA liquid NPs; PAI-1, plasminogen activator inhibitor-1; TGF-β1, transforming growth factor-β1; tPA, tissue-type plasminogen activator.

Figure 13 Effect of Tan IIA-NPs (A) and free-Tan IIA (B) on the TGF-β1 protein expression in HMrSV5 cells.

Notes: High, medium and low dose groups were treated with 20, 10, and 5 μg/mL Tan IIA-NPs, respectively. And free-Tan IIA group was treated with 20 μg/mL Tan IIA. Data are expressed as mean ± standard deviation (n=3). *P<0.05 compared with blank; ^#P<0.05 compared with control; ^§P<0.05 compared with drug-free NPs.

Abbreviations: NPs, nanoparticles; Tan IIA, tanshinone IIA; Tan IIA-NPs, Tan IIA liquid NPs; TGF-β1, transforming growth factor-β1.

Figure 14 Effect of Tan IIA-NPs (A) and free-Tan IIA (B) on collagen I protein expression in HMrSV5 cells.

Notes: High, medium and low dose groups were treated with 20, 10, and 5 μg/mL Tan IIA-NPs, respectively. And free-Tan IIA group was treated with 20 μg/mL Tan IIA. Data are expressed as mean ± standard deviation (n=3). *P<0.05 compared with blank; ^#P<0.05 compared with control; ^§P<0.05 compared with drug-free NPs.

Abbreviations: NPs, nanoparticles; Tan IIA, tanshinone IIA; Tan IIA-NPs, Tan IIA liquid NP; TGF-β1, transforming growth factor-β1.

NairSKBhatIKAuroraALRole of proteolytic enzyme in the prevention of postoperative intraperitoneal adhesionsArch Surg197410868498534829809

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