Formulation, optimization, and pharmacodynamic evaluation of chitosan/phospholipid/β-cyclodextrin microspheres

Figures & data

Table 1 The factors and levels discussed in the orthogonal design

Levels	Factors
	Tinlet (°C)	Feeding rate (mL/min)	Air flow rate (L/h)	CTS/PL/β-CD ratio
1	190	12	700	1:1:0.33
2	170	8	400	2:1:0.33
3	150	14	300	3:1:0.33

Abbreviations: Tinlet, inlet temperature; CTS, chitosan; PL, phospholipid; β-CD, β-cyclodextrin.

Table 2 Results of the orthogonal design

Run number	A (°C)	B (mL/min)	C (L/h)	D	DL (%)	EE (%)
1	190	12	700	1/1/0.33	3.07±0.13	7.15±0.46
2	190	8	400	2/1/0.33	2.70±1.78	3.39±1.13
3	190	14	300	3/1/0.67	2.44±1.31	3.77±1.23
4	170	12	400	3/1/0.67	0.21±0.04	0.50±0.13
5	170	8	300	1/1/0.33	3.14±2.31	5.01±1.45
6	170	14	700	2/1/0.33	0.42±0.12	0.71±0.22
7	150	12	300	2/1/0.33	4.93±0.16	8.71±0.23
8	150	8	700	3/1/0.67	4.03±0.57	7.78±0.32
9	150	14	400	1/1/0.33	5.08±0.15	9.53±0.69

Notes: A, inlet temperature; B, feeding rate; C, air flow rate; and D, CTS/PL/β-CD ratio. Data in the “DL” and “EE” columns presented as mean ± standard deviation.

Abbreviations: CTS, chitosan; PL, phospholipid; β-CD, β-cyclodextrin; DL, drug loading; EE, encapsulation efficiency.

Figure 1 The SEM of spray-drying microspheres.

Notes: The magnification is 3,000×. (A) 190°C, 12 mL/min, 700 L/h, 1:1:0.33; (B) 190°C, 8 mL/min, 400 L/h, 2:1:0.33; (C) 190°C, 14 mL/min, 300 L/h, 3:1:0.67; (D) 170°C, 12 mL/min, 400 L/h, 3:1:0.67; (E) 170°C, 8 mL/min, 300 L/h, 1:1:0.33 (F) 170°C, 14 mL/min, 700 L/h, 2:1:0.33; (G) 150°C, 12 mL/min, 300 L/h, 2:1:0.33; (H) 150°C, 8 mL/min, 700 L/h, 3:1:0.67; (I) 150°C, 14 mL/min, 400 L/h, 1:1:0.33; and (J) 150°C, 8 mL/min, 300 L/h, 1:1:0.33.
Abbreviation: SEM, scanning electron microscope.

Figure 2 The FTIR spectra of (A) CTS, (B) PL, (C) CTS/PL MS, and (D) CTS/PL/β-CD MS.

Abbreviations: FTIR, Fourier transform infrared; CTS, chitosan; PL, phospholipid; MS, microspheres; β-CD, β-cyclodextrin.

Figure 3 The DSC thermograms of (A) CTS/PL/β-CD microspheres, (B) CTS, (C) PL, and (D) PL/β-CD mixture.

Abbreviations: DSC, differential scanning calorimetry; CTS, chitosan; PL, phospholipid; β-CD, β-cyclodextrin.

Figure 4 The moisture content of microspheres under different fabrication conditions.

Figure 5 The swelling ratio of microspheres under different fabrication conditions.

Figure 6 The tapped density of microspheres under different fabrication conditions.

Figure 7 The moisture absorption rates of microspheres under different fabrication conditions.

Table 3 Effects of microspheres on escape latency in rats

Groups	Preoperative (t/s)	Postoperative 14 days (t/s)	Postoperative 30 days (t/s)
Control group	21.04±5.75	17.45±5.47	11.78±2.66b
Model group	20.99±2.86	40.92±3.83a	37.98±7.14
Low-dose group	20.13±6.97	38.73±2.48a	17.82±1.66b
Medium-dose group	21.70±8.32	37.47±2.89a	16.86±3.12b
High-dose group	22.21±3.92	36.94±2.94a	15.75±1.59b

Notes:

a P<0.01 versus control group;

b P<0.01 versus model group. Data (mean ± SD, n=10) obtained after the same treatment day are significantly different.

Abbreviations: SD, standard deviation; t/s, time/second.

Table 4 Effects of microspheres on the numbers of crossings over the former location of the platform by rats

Groups	Preoperative (time)	Postoperative 14 days (time)	Postoperative 30 days (time)
Control group	4.10±0.66	4.80±0.63	4.65±0.58b
Model group	4.20±0.54	1.10±0.21a	1.30±0.48
Low-dose group	4.15±0.58	1.15±0.24a	3.40±0.46b
Medium-dose group	4.05±0.68	1.35±0.47a	3.90±0.39b
High-dose group	4.25±0.68	1.30±0.42a	4.20±0.63b

Notes:

a P<0.01 versus control group;

b P<0.01 versus model group. Data (mean ± SD, n=10) obtained after the same treatment day are significantly different.

Abbreviation: SD, standard deviation.

Table 5 Effects of microspheres on the expression of PKC-δ and Iba1

Groups	PKC-δ	Iba1
Control group	15.0±3.68a	16.2±3.32b
High-dose group	25.9±3.17a	33.0±5.20b
Medium-dose group	30.9±3.69a	39.4±4.25b
Low-dose group	34.5±3.77a	45.3±7.14b
Model group	41.8±4.23	56.1±7.07

Notes:

a P<0.05 versus model group;

b P<0.05 versus model group. Data (mean ± SD, n=10) are obtained on the same day of treatment.

Abbreviations: SD, standard deviation; PKC-δ, protein kinase C-δ; Iba1, ionized calcium-binding adaptor molecule-1.

Figure 8 Effects of CTS/PL/β-CD microspheres on the quantities of PKC-δ in the hippocampus by immunohistochemistry staining (light microscope, ×400).

Notes: (A) Control group, (B) high-dose group, (C) medium-dose group, (D) low-dose group, and (E) model group.
Abbreviations: CTS, chitosan; PL, phospholipid; β-CD, β-cyclodextrin; PKC-δ, protein kinase C-δ.

Figure 9 Effects of CTS/PL/β-CD microspheres on the quantities of Iba1 in the hippocampus by immunohistochemistry staining (light microscope, ×400).

Notes: (A) Control group, (B) high-dose group, (C) medium-dose group, (D) low-dose group, and (E) model group.
Abbreviations: CTS, chitosan; PL, phospholipid; β-CD, β-cyclodextrin; Iba1, ionized calcium-binding adaptor molecule-1.