Preparation, Characterization and in vivo Study of Borneol-Baicalin-Liposomes for Treatment of Cerebral Ischemia-Reperfusion Injury

Figures & data

Table 1 Factors and Responses in Response Surface-Central Composite Design

Factors	Levels
	−1	0	1
A	5	10	15
B	3	4	5
C	4	7	10

Table 2 Screening Results of Preparation Methods

Results	EE (%)	DL (%)
Thin film dispersion	31.55	6.13
Reverse evaporation	42.28	5.65
Ethanol injection	38.97	3.90

Table 3 Box–Behnken Design and Results

No.	Independent Variables			Dependent Variables
	A	B	C	Y1	Y2
1	5	5:1	7:1	30.41	5.61
2	15	4:1	10:1	32.64	3.17
3	5	4:1	10:1	31.51	6.51
4	10	4:1	7:1	40.78	3.84
5	15	3:1	7:1	31.98	8.36
6	15	5:1	7:1	28.32	4.51
7	5	3:1	7:1	38.82	6.77
8	10	5:1	10:1	38.48	4.89
9	10	5:1	4:1	32.7	3.56
10	5	4:1	4:1	34.05	7.03
11	10	3:1	10:1	31.38	6.49
12	10	4:1	7:1	43.69	6.72
13	10	3:1	4:1	25.54	4.10
14	15	4:1	4:1	36.69	5.04
15	10	4:1	7:1	40.78	3.79
16	10	4:1	7:1	41.88	3.25
17	10	4:1	7:1	40.08	3.52

Notes: Independent variables: A, the dosage of BO (mg); B, the ratio of phospholipid to drug (w/w); C, the ratio of phospholipid to cholesterol (w/w). Dependent variables: Y1: EE(%); Y2: DL(%).

Table 4 Variance Analysis of EE Regression Model

Source	Sum of Squares	df	Mean Square	F Value	P value Prob>F
Model	288.82	9	32.09	1.44	0.0048
A	3.33	1	3.33	0.15	0.7109
B	0.6	1	0.6	0.027	0.8745
C	3.16	1	3.16	0.14	0.7178
AB	5.64	1	5.64	2.60	0.6306
AC	5.70E-01	1	5.70E-01	2.60E-02	0.8775
BC	9.00E-04	1	9.00E-04	4.03E-05	0.9951
A^2	57.05	1	57.05	2.56	0.1539
B^2	121.8	1	121.8	5.46	0.0522
C^2	68.67	1	68.67	3.08	0.0229
Residual	156.25	7	22.32
Lack of Fit	148.27	3	49.42	24.78	0.3233
Pure Error	7.98	4	1.99
Cor Total	445.07	16	445.07

Table 5 Variance Analysis of DL Regression Model

Source	Sum of Squares	df	Mean Square	F Value	P value Prob>F
Model	22.71	9	2.52	0.98	0.0205
A	2.93	1	2.93	1.44	0.3206
B	6.39	1	6.39	2.49	0.1583
C	0.22	1	0.22	0.086	0.7775
AB	1.81	1	1.81	0.71	0.4286
AC	4.60E-01	1	4.60E-01	1.80E-01	0.6859
BC	2.80E-04	1	2.80E-04	1.10E-01	0.0003
A^2	8.05	1	8.05	3.14	0.0196
B^2	2.1	1	2.1	0.82	0.3959
C^2	0.12	1	0.12	0.047	0.8342
Residual	17.94	7	2.56
Lack of Fit	9.93	3	3.31	1.65	0.3124
Pure Error	8.01	4	2
Cor Total	40.65	16

Figure 1 Three-dimensional effect surface diagrams of encapsulation efficiency. (A) BO dosage to phospholipid–drug ratio (EE). (B) BO dosage to phospholipid–cholesterol ratio (EE). (C) Phospholipid–drug ratio to phospholipid–cholesterol ratio (EE). (D) BO dosage to phospholipid–drug ratio (DL). (E) BO dosage to phospholipid–cholesterol ratio (DL). (F) Phospholipid–drug ratio to phospholipid–cholesterol ratio (DL).

Table 6 The Results of Verification of the Predictive Model Test

No.	EE (%)	DL (%)
1	41.23	7.15
2	40.39	7.00
3	41.17	7.14
Measured average values	40.39	7.09
Predicted value	41.49	7.15
Deviation%	1.35	0.84

Figure 2 Characterization of BO-BA-LP. (A) The size distribution of BO-BA-LP. (B) The transmission electron microscope photograph of BO-BA-LP.

Table 7 The Results of Stability Test

Time/d	1		2		3
	EE%	DL%	EE%	DL%	EE%	DL%
0	41.32	7.16	41.56	7.21	40.92	7.10
5	41.37	7.17	40.97	7.10	42.23	7.32
10	40.63	7.05	41.35	7.17	41.26	7.16
15	41.22	7.15	40.63	7.05	40.15	6.96
20	37.49	6.50	38.22	6.63	37.03	6.42
30	30.57	5.30	32.49	5.63	29.59	5.13

Figure 3 In vitro release profiles of BA and BO-BA-LP.

Table 8 Main Plasma Pharmacokinetic Parameters of BA and BA-LP After i.v. Administration to MCAO Rats at the Dosage of 25 Mg/Kg (n=5, mean±SD)

Parameters	Units	BA	BA-BO-LP
Cmax	μg/mL	230.14±37.58	282.72±56.59*
t1/2	min	17.05±1.81	128.81±16.16**
AUC0-t	μg/mL*min	2233.72±44.10	14,718.50±2368.97**
AUC0-∞	μg/mL*min	2264.09±35.78	15,674.28±2697.39
MRT0-t	min	12.65±0.89	114.23±16.03**
MRT0-∞	min	14.03±1.34	146.94±25.23

Notes: Data were presented as mean±SD. *Significantly different from the BA group at P < 0.05, **Extremely significant different from the I/R group at P < 0.01.

Figure 4 The plasma concentration-time profile of BA after i.v. administration of BA and BO-BA-LP (25 mg/kg).

Table 9 Neurological Function Score and Infarction Rate in Different Groups (n=5)

Groups	Neurological Function Score	Average Infarct Volume Ratio (%)
Sham	–	–
I/R	3.10±0.32	25.08±3.46
BA	2.30±0.46*	19.14±2.28*
BA-LP	1.70±0.67*	15.86±1.37*
BA/BO-LP	1.30±0.90*	12.67±1.48*^#

Notes: Data were presented as mean ± SD. All animals were scored, 10 rats per group, and half of them in the determination of cerebral infarction. Differences were considered significant at P< 0.05. *Significantly different from the I/R group at P < 0.05; ^#Significantly different from the BA-LP group at P < 0.05.

Figure 5 The results of the TTC staining in each groups. (A) I/R group; (B) MCAO model with BA (25mg/kg); (C) MCAO model with BA-LP (25mg/kg); (D) MCAO model with BO-BA-LP (25mg/kg). The infarct area exhibited white, whereas the non-infarct areas were stained red.

Figure 6 The HE staining of brain tissue slices with the treatment of sham (A), I/R (B), BA group (25 mg/kg, (C), BA-LP group (25 mg/kg; (D) and BO-BA-LP (25 mg/kg; (E) under 200-fold light microscope.

Figure 7 The Nissl staining of brain tissue slices with the treatment of sham (A), I/R (B), BA group (25 mg/kg, (C), BA-LP group (25 mg/kg; (D) and BO-BA-LP group (25 mg/kg; (E) under 200-fold light microscope. ** Extremely significant different from the I/R group at P < 0.01, ## Extremely significant different from the BA-LP group at P < 0.01.