Design, optimization and characterizations of chitosan fortified calcium alginate microspheres for the controlled delivery of dual drugs

Figures & data

Figure 1. (a) Ishikawa fishbone diagram illustrating CFV and DRV involved in fabrication of CS-Ca-SA microspheres and (b) steps involved in formulation CS-Ca-SA microspheres.

Table 1. Fabrication variables and dependent variables associated with microsphere formulation.

Critical fabrication variables	Low (−)level	High (+)level
Concentration of sodium alginate (SA) (%w/v)	2	5
Concentration of chitosan (CS) (%w/v)	2	4
Concentration of calcium carbonate (CaC) (%w/v)	2.5	5
Cross-linking time (CT) (min)	30	60
Agitation speed (AS) (rpm)	500	2000
Surfactant concentration (SC) (%w/v)	0.5	1
Phase ratio (aqueous:oil) (PR)	1:2	1:6
Dependent response variable
Particle size (PS) (μm)
Average entrapment efficiency (AvEE) (%)
Average burst release (AvB)(%)
Average time for 80% cumulative drug release (avT80%)

Table 2. Plackett-Burman factorial design for fabrication of CS-Ca-SA microspheres.

Run	SA	CS	CaC	SC	CT	AS	PR	PS^a (µm)	AvEE^b (%)	AvB^b (%)	AvT80%^b (%)
1	(+)	(−)	(+)	(−)	(−)	(+)	(−)	283 ± 16.98	83.92	17.01	69.40
2	(+)	(+)	(−)	(−)	(−)	(−)	(+)	377 ± 22.62	75.06	14.78	73.76
3	(−)	(−)	(−)	(+)	(+)	(−)	(+)	158 ± 9.48	62.45	26.85	52.73
4	(+)	(−)	(+)	(−)	(+)	(−)	(+)	461 ± 27.66	83.92	14.32	68.66
5	(−)	(−)	(+)	(+)	(−)	(+)	(+)	74 ± 4.44	69.39	29.97	58.38
6	(−)	(−)	(−)	(−)	(−)	(−)	(−)	195 ± 11.70	66.76	25.01	55.78
7	(−)	(+)	(−)	(−)	(+)	(+)	(−)	108 ± 6.48	65.55	28.20	56.62
8	(+)	(+)	(−)	(+)	(−)	(+)	(+)	252 ± 15.12	68.60	20.11	72.89
9	(−)	(+)	(+)	(−)	(+)	(+)	(+)	134 ± 8.04	67.14	34.63	63.76
10	(+)	(+)	(+)	(+)	(+)	(−)	(−)	349 ± 20.94	86.20	12.21	76.78
11	(−)	(+)	(+)	(+)	(−)	(−)	(−)	172 ± 10.32	68.92	26.76	59.87
12	(+)	(−)	(−)	(+)	(+)	(+)	(−)	236 ± 14.16	77.61	21.74	67.78

^a The values are mean ± SD (n = 3).

^bThe values are average value obtained for both drugs.

Figure 2. Demonstration of Pareto plots for the selection of CFV having significant effect on desired DRVs, viz. particle size (PS), average encapsulation efficiency (avEE), average burst release (avB) and average time for 80% cumulative drug release (avT_80%).

Figure 3. Contour and corresponding surface plots describing most influencing factors on particle size (PS), average encapsulation efficiency (avEE), average burst release (avB) and average time for 80% cumulative drug release (avT_80%).

Table 3. Summary of ANOVA results and regression parameters.

Dependent responsevariables	Actual R^2 (%)	S-value	p-value	Adjusted R^2 (%)	F-value	Predicted R^2 (%)	Degrees offreedom (DF)	Adj. meansquare	Adj. sumof squares
PS	98.65	22.5703	.001*	96.28	41.65	87.82	7	21215	148505
AvEE	97.39	2.1772	.005*	92.83	21.34	76.53	7	101.170	708.187
AvB	94.64	2.7086	.021*	85.26	10.09	51.78	7	74.046	518.320
AvT80%	93.61	3.2722	.029*	82.44	8.38	42.52	7	89.690	627.830

* p-value < .05 indicates model is significant.

Table 4. Comparison of predicted and experimental value for validation of model.

Responses	Predicted value	Experimental value	Bias (%)
PS (μm)	241.667	257 ± 0.56^a	5.9661
AvEE (%)	81.119	79.89^b	−1.5383
AvB (%)	13.88	14.36^b	3.3426
AvT80% (h)	74.1	72.43^b	−2.3056

^a Values are mean ± SD (n = 3).

^b The values are average value for both drugs.

Figure 4. Overlay spectrum: (a) FTIR spectra of ornidazole, doxycycline, chitosan, alginate, physical mixture, placebo CS-Ca-SA and drug-loaded CS-Ca-SA microspheres; (b) DSC graphs of ornidazole, doxycycline, alginate, chitosan and CS-Ca-SA microspheres; and (c) XRD pattern of ornidazole, doxycycline, chitosan, alginate and CS-Ca-SA microspheres.

Figure 5. Microscopic view of microspheres. Optical microscopic images of (a) w/o microemulsion alginate drops dispersed in oil phase and (b) calculation of sphericity factor of microspheres. SEM images of (c) ornidazole powder, (d) doxcycline hyclate, (e, f, g) CS-Ca-SA microspheres and (h) surface view of microspheres.

Figure 6. In-vitro dissolution profiles of ornidazole (OZ) and doxycycline hyclate (DX) from optimized CS-Ca-SA microspheres in phosphate buffer pH 6.8. Vertical bars indicate mean ± SD.

Table 5. Drug release kinetics of ornidazole (OZ) and doxycycline hyclate (DX) from CS-Ca-SA and Ca-SA microspheres.

Microspheres	Drugs	Zero-order model		First-order model		Higuchi model		Hixson–Crowell model		Korsmeyer–Peppas model			Diffusioncoefficient (D)
		R^2	K (mgh/ml)	R^2	K (h^−1)	R^2	K (h^−1/2)	R^2	K (h ^−1/3)	R^2	K (h^−^n)	n
CS-Ca-SA	OZ	0.902	1.977	0.755	0.024	0.977	13.251	0.945	0.045	0.971	1.343	0.560	8.66 × 10^−4
	DX	0.894	1.923	0.739	0.025	0.972	12.914	0.935	0.042	0.963	0.511	0.586	3.29 × 10^−4
Ca-SA	OZ	0.889	5.794	0.734	0.068	0.969	21.807	0.937	0.132	0.963	3.072	0.496	15.73 × 10^−4
	DX	0.898	5.874	0.742	.0721	0.975	22.074	0.942	0.132	0.968	1.132	0.528	6.07 × 10^−4

Figure 7. Comparative (a) swelling curve and (b) erosion curve of optimized batches of Ca-SA and CS-Ca-SA microspheres in simulated saliva pH 6.8. Vertical bars show mean ± SD.

Figure 8. Zone of inhibition of dissolution study samples against S. aureus and E. coli. Vertical bars indicate mean ± standard error mean.

Figure 9. (a) Percentage cell viabilities of microsphere samples, A (0.01 g/ml), B (0.05 g/ml), C (0.1 g/ml) and control, prepared by incubating in DMEM media for 120 h against L929 cell lines. Vertical bars represent mean ± standard error mean (n = 6); (b) incubation time vs. IC₅₀ plot; and (c) fluorescent microscopic images showing cytocompatibility of various microsphere samples (A, B, C and control (C1)) with L929 cell lines.

Figure 10. Stability study of optimized batch of microspheres at different storage conditions, viz. Storage 1 (refrigeration), Storage 2 (room temperature) and Storage 3 (high temperature). (a) Histograms representing stability parameters. Vertical bars indicate mean ± SD. (b) Shelf-life plots. LS: lower specification (90%).