Triamcinolone-loaded glutaraldehyde cross-linked chitosan microspheres: Prolonged release approach for the treatment of rheumatoid arthritis

Figures & data

Table 1.  Percentage of drug loading, encapsulation efficiency of chitosan microspheres.

Glutaraldehyde saturated toluene (ml): Aq. glutaraldehyde ratio (ml)	Drug:Polymer	Amount of drug (g)	Amount of polymer	Yield (g)	Loading %	Encapsulation %
20:1	1:5	0.2	1.0	1.05	9.14	55.2
10:1	1:5	0.2	1.0	1.0	11.0	48
5:1	1:5	0.2	1.0	0.97	10.62	51.6
1:1	1:5	0.2	1.0	0.95	11.16	53.4

Figure 1.  Scanning electron micrograph (SEM) of TA-loaded chitosan microspheres. (a) TA microspheres (10:1), (b) TA microspheres (5:1).

Figure 2.  Differential scanning calorimetry (DSC) results of pure chitosan (a), pure TA (b), and TA-loaded chitosan microspheres (c).

Figure 3.  FT-IR spectrum of pure chitosan (a), pure TA (b), and TA-loaded chitosan microspheres.

Figure 4.  In-vitro release study of various TA microsphere formulations.

Figure 5.  Comparative in-vivo release study of pure TA and TA microsphere formulation.

Table 2.  Paw volume with percentage inhibition and hemoglobin content.

Treatment parameters	Group I		Group II		Group III
	P.V	Hb	P.V	Hb	P.V	Hb
15^th day	0.575 ± 0.032	6 ± 0.288	0.475 ± 0.032	7.83 ± 0.441	0.562 ± 0.012	7.83 ± 0.166
30^th day	0.525 ± 0.032	6.63 ± 0.448	0.3 ± 0.02^ab (42.84)^*	0.83 ± 0.600^ac	80.212 ± 0.023 (59.61)^*	11.8 ± 0.600
60^th day	0.362 ± 0.023	9.12 ± 0.314	0.188 ± 0.0023^ac (48.06)^*	13 ± 0.408^ac	0.235 ± 0.032 (35.08)^*	9.75 ± 0.322

^a p < 0.001 vs Group I by one-way ANOVA with Dunnet’s t-test, ^b p < 0.01; ^c p < 0.001 vs Group III by one-way ANOVA with Dunnet’s t-test. * Percentage inhibition.

P.V, Paw Volume (ml); Hb, Hemoglobin (g/dl).

Group I, CFA+ saline; group II, CFA+TA microspheres; and group III, CFA + pure TA solution.

Table 3.  Total leucocytes count.

Treatment	Total leucocytes count (1000/mm^3)
	Group I	Group II	Group III
15^th day	7067 ± 1179.56	7,550 ± 822.17	5,233 ± 545.61
30^th day	5767 ± 145.4	14,800 ± 1890.30^a	14,500 ± 1040.99
60^th day	7075 ± 378.5	15,700 ± 896.5^ab

^a p < 0.001 vs Group I, by one-way ANOVA with Dunnet’s t-test. ^b p < 0.001 vs Group III, by one-way ANOVA with Dunnet’s t-test.

Group I, CFA+ saline; group II, CFA+TA microspheres; and group III, CFA + pure TA solution.

Table 4.  Differential cell count.

Treatment	Group I				Group II				Group III
	N	L	M	E	N	L	M	E	N	L	M	E
15^th day	70 ± 1.17	24.7 ± 1.76	2.67 ± 1.8	2 ± 1.17	70 ± 3.27	28 ± 2.08	0.667 ± 0.667	1.33 ± 0.88	55 ± 5.88	44.3 ± 4.11	2.33 ± 0.33	1 ± 1.01
30^th day	62.3 ± 1.48	30.3 ± 3.18	2.33 ± 0.33	1.53 ± 0.9	56.3^ab ± 3.82	40.3^cd ± 2.73	1.33 ± 0.676	2 ± 2.03	59.3 ± 5.30	55.3 ± 5.33	2 ± 1.01	2.67 ± 1.8
60^th day	67.5 ± 1.55	30 ± 1.12	1.25 ± 0.75	1.25 ± 0.75	45.5^ab ± 4.99	56.5^ad ± 3.35	1 ± 0.57	1 ± 0.57	60.2 ± 3.32	35 ± 2.86	2 ± 0.48	2.75 ± 0.478

N, Neutrophils; L, Lymphocytes; M, Monocytes; E, Eosinophils.

^a p < 0.001 vs Group II; ^b p < 0.001 vs Group II, ^c p < 0.005 vs Group I, and ^d p < 0.001 vs Group II, by one-way ANOVA with Dunnet’s t-test.

Group I, CFA+ saline; group II, CFA+TA microspheres; and group III, CFA + pure TA solution.

Figure 6.  Radiographs of joint were obtained at the conclusion of the study. Representative radiographs from Wistar rats with normal (a), Arthritic control (b), and TA-loaded chitosan microsphere treated (c).

Figure 7.  Histopathological observation of inflamed tissues. (a and b) arthritic control; (c and d) normal architecture of synovial membrane, (e and f) TA-loaded chitosan microsphere treated group on 30^th day, (g and h) TA-loaded chitosan microsphere treated group on 60^th day.