Development and in vitro/in vivo evaluation of Zn-pectinate microparticles reinforced with chitosan for the colonic delivery of progesterone

Figures & data

Figure 1. (A) Chemical structure of pectin showing its functional groups: (a) carboxyl; (b) amide; (c) ester groups. (B) Chemical structure of chitosan. The indices x and y represent the mole fractions of d-glucosamine and N-acetyl-d-glucosamine moieties, respectively. (C) Schematic illustration of the formation of PG-loaded microparticles. * Indicates continuation of the structure.

Table 1. Preparation conditions of progesterone-loaded Zn-pectinate/chitosan microparticles.

Formula Code	Cross-linking pH	Chitosan concentration (% w/v)	Cross-linking time (min)	Pectin/ PG ratio
F1	4.8	0.5	120	4:1
F2	3	0.5	120	4:1
F3	1.2	0.5	120	4:1
F4	1.2	0.1	120	4:1
F5	1.2	0.25	120	4:1
F6	1.2	0.5	10	4:1
F7	1.2	0.5	30	4:1
F8	1.2	0.5	120	2:1

Figure 2. FT-IR spectra of (A) PG, (B) pectin, (C) chitosan, (D) empty Zn-pectinate/chitosan microparticles and (E) PG-loaded Zn-pectinate/chitosan microparticles.

Figure 3. DSC thermograms of (A) PG, (B) pectin, (C) chitosan, (D) empty Zn-pectinate/chitosan microparticles and (E) PG-loaded Zn-pectinate/chitosan microparticles.

Table 2. Physical characteristics and entrapment efficiency of the prepared Zn-pectinate/chitosan microparticles.

Formula code	Size (µm)	Circularity (C)	Elongation ratio (ER)	Shape	Weight (mg)	Weight loss (%)	Moisture content (%)	%EE
F1	663.7 ± 19.9	0.94 ± 0.01	1.12 ± 0.01	Spherical	27.2 ± 1.3	92.07 ± 0.48	6.09 ± 0.91	76.7 ± 1.4
F2	709.3 ± 19.4	0.96 ± 0.01	1.11 ± 0.02	Spherical	25.1 ± 1.3	93.03 ± 0.12	7.30 ± 1.90	88.4 ± 5.2
F3	718.3 ± 44.4	0.95 ± 0.01	1.14 ± 0.02	Spherical	22.4 ± 1.6	94.08 ± 0.38	9.65 ± 0.77	101.2 ± 4.4
F4	700.7 ± 10.1	0.88 ± 0.03	1. 2 ± 0.09	Non Spherical	27.2 ± 3.4	94.78 ± 0.37	8.26 ± 1.81	101.6 ± 6.3
F5	681.3 ± 41.8	0.88 ± 0.01	1.18 ± 0.08	Non Spherical	23.9 ± 2.6	94.68 ± 0.40	6.21 ± 2.04	97.9 ± 4.5
F6	678.3 ± 60.1	0.93 ± 0.01	1.15 ± 0.01	Spherical	30.6 ± 1.6	93.79 ± 0.43	7.86 ± 1.39	97.8 ± 7.2
F7	708.3 ± 49.5	0.90 ± 0.03	1.15 ± 0.07	Spherical	30.9 ± 5	94.04 ± 0.46	8.35 ± 1.70	98.7 ± 1.2
F8	583.3 ± 30.0	0.88 ± 0.02	1.16 ± 0.07	Non Spherical	11.8 ± 0.6	96.0 ± 0.39	5.88 ± 1.01	99.4 ± 2.37

Mean ± SD of three independent measurements.

Figure 4. (A) Effect of cross-linking pH on the mucoadhesive properties of Zn-pectinate/chitosan MPs prepared at pH 4.8 (F1) or pH 1.2 (F3) (Preparation conditions: chitosan concentration, 0.5%; pectin/PG, 4:1; cross-linking time, 120 min) (B) Effect of pectin/PG ratio on the mucoadhesive properties of pectin/chitosan MPs prepared at pectin/PG of 4:1 (F3) or 2:1 (F8) (Preparation conditions: pH, 1.2; chitosan concentration, 0.5%; cross-linking time, 120 min).

Figure 5. Swelling behavior of Zn-pectinate/chitosan MPs (F3) in enzyme-free simulated gastric fluid (SGF) and enzyme-free simulated small intestinal fluid (SSIF) as a function of time.

Figure 6. (A) In vitro release profiles of PG powder and PG-loaded Zn pectinate/chitosan MPs prepared at a cross-linking pH of 4.8 (Formula F1) or pH 1.2 (Formula F3). (B) In vitro release profiles of PG powder and PG-loaded Zn pectinate/chitosan MPs cross-linked for 10 min (Formula F6) or 120 min (Formula F3). (C) In vitro release profiles of PG powder and PG-loaded Zn pectinate/chitosan MPs prepared at a chitosan concentration of 0.1% w/v (Formula F4) or 0.5% w/v (Formula F3). Release medium: enzyme-free SGF (pH 1.2) for the initial 2 h, followed by enzyme-free SSIF (pH 6.8) for 0.5 h and then enzyme-free SSIF (pH 7.4) until the end of release study (30 h).

Table 3. Kinetic assessment of drug release data from different Zn-pectinate/chitosan MPs according to various kinetic models.

	Correlation coefficient (R^2)
Formula code	Zero order	First order	Higuchi-diffusion	Baker–Lonsdale	Hixson–Crowell	Korsmeyer–Peppas	Korsmeyer–Peppas release exponent (n)
F1	0.806	0.917	0.906	0.955	0.881	0.953	1.32
F2	0.807	0.885	0.903	0.936	0.859	0.957	1.20
F3	0.795	0.865	0.895	0.924	0.841	0.963	1.51
F4	0.788	0.882	0.890	0.933	0.850	0.953	1.81
F5	0.794	0.877	0.895	0.933	0.849	0.969	2.19
F6	0.807	0.881	0.904	0.941	0.856	0.941	1.54
F7	0.786	0.854	0.887	0.914	0.831	0.947	1.58
F8	0.712	0.724	0.833	0.765	0.720	0.924	0.88

Figure 7. Scanning electron microscope photographs (×75 and ×1000 magnifications) of different formulations of PG-loaded Zn pectinate/chitosan MPs. (A) Formula F1, (B) Formula F3 and (C) Formula F4. Left panels are taken at magnification ×75 and right panels at ×1000.

Figure 8. Mean plasma concentration-time profiles of PG after oral administration of selected Zn-pectinate/chitosan microparticle formula (F3) to healthy rabbits compared to the micronized PG powder.

Table 4. Pharmacokinetic parameters of PG after oral administration of micronized PG powder and Zn-pectinate/chitosan microparticles (formula F3) to rabbits.

Pharmacokinetic parameters	Micronized PG powder	PG microparticles (Formula F3)
Cmax (µg/mL)	1.606 ± 0.037	1.319 ± 0.008
Tmax (h)^a	6	12
Ka (h^−1)	0.296 ± 0.081	0.201 ± 0.052
t½a (h)	2.433 ± 0.668	3.564 ± 0.913
Kel (h^−1)	0.063 ± 0.001	0.050 ± 0.004
AUC0–∞ (µg h/mL)	19.36 ± 0.845	32.56 ± 0.723
t½ (h)	10.92 ± 0.173	13.89 ± 0.984
MRT (h)^b	8.30 ± 0.106	18.98 ± 0.312
ClT (mL/min)	16.81 ± 0.497	11.07 ± 0.971
FR (%)^c	–	168.18

^aT_max, time of maximum drug plasma concentration, observed from the plasma concentration-time profile.

^bMean residence time (h) calculated as AUMC/AUC.

^cRelative bioavailability (%) calculated as AUC_0–∞ of microparticles × 100/AUC_0–∞ of PG powder.