Chemical stability enhancement and cytotoxicity reduction of papain loaded in PLGA nanospheres

Figures & data

Table 1. The comparison of particle sizes, zeta potential values and encapsulation efficiency (%EE) of PLGA nanospheres loaded with papain prepared by the ESD and ESE methods.

Samples	Method of preparation	Particle size (nm)		Polydispersity index (PDI)		Zeta potential (mV)		%EE
		BFD	AFD	BFD	AFD	BFD	AFD
Unloaded nanospheres	ESD	317	335	0.317	0.539	−37.5 ± 4.61	−34.8 ± 11.7	–
	ESE	220	229	0.106	0.150	−33.7 ± 5.68	−32.8 ± 4.85	–
Papain-loaded PLGA nanospheres	ESD	295	358	0.135	0.293	−36.2 ± 6.82	−31.9 ± 5.89	19.42 ± 0.63
	ESE	229	232	0.052	0.151	−31.1 ± 4.89	−29.6 ± 7.18	43.03 ± 0.15
Notes: BFD = before freeze drying, AFD = after freeze drying and rehydration.
ESD = Emulsion solvent diffusion method in water.
ESE = Water-oil-water (w/o/w) emulsion solvent evaporation method.

Figure 1. The scanning electron (A) and transmission electron (B) microphotographs of PLGA nanospheres (composed of 100 mg PLGA and 10%w/v PVA403) loaded with 43 µg papain/mg PLGA nanospheres prepared by the ESE method (12000X).

Figure 2. Comparision of profile release of papain loaded in PLGA nanospheres prepared by ESD (emulsion solvent diffusion method in water) and ESE (w/o/w emulsion solvent evaporation method) in 0.2 M phosphate buffer (pH 7.0) solution at 27 ± 2°C.

Table 2. Release kinetics of papain from the PLGA nanospheres prepared by the ESD and ESE method in 0.2 M phosphate buffer (pH 7.0) solution at 27 ± 2°C to 48 h.

		Zero order		First order		Higuchi
		r ^2	k 0 (h^−1)	r ^2	k 1 (h^−1)	r ^2	kH (h^−1/2)
ESD method	First phase	0.7578	12.385	0.9047	0.2724	0.9456	34.375
	Second phase	0.8946	0.1678	0.9243	0.0124	0.9566	1.6275
ESE method	First phase	0.7769	9.4899	0.8614	0.1531	0.9554	26.147
	Second phase	0.9409	0.2128	0.9312	0.0067	0.8750	1.9254
Notes: Zero-order equation: C = k 0 t, where k 0 was the zero-order rate constant expressed in units of papain concentration and time, and t was the time in hours.
First-order equation: Log C = Log C 0 − kt /2.303, where C 0 was the initial concentration of papain and k was the first order constant.
Higuchi equation: Q = kHt ^1/2, where Q was the amount of papain released per unit surface area at time (h), and kH was the release rate constant.

Figure 3. The percentages of human skin fibroblast viability by the SRB assay of (A) the unloaded nanospheres and (B) the PLGA nanospheres loaded with papain prepared by the ESD (emulsion solvent diffusion in water) method (19 µg papain/mg PLGA nanosphere) and the ESE (w/o/w emulsion solvent evaporation) method (43 µg papain/mg PLGA nanosphere).

Figure 4. The percentages remaining of the unloaded papain and loaded in PLGA nanospheres prepared by the ESE (w/o/w emulsion solvent evaporation) method stored at different temperatures (25 ± 2, 4 ± 2 and 45 ± 2°C) for 6 weeks; Pn-4: papain loaded in PLGA nanospheres kept at 4°C; Pn-25: papain loaded in PLGA nanospheres kept at 25°C; Pn-45: papain loaded in PLGA nanospheres kept at 45°C; Ps-4: papain solution in 0.2 M phosphate buffer (pH 5.0) kept at 4°C; Ps-25: papain solution in 0.2 M phosphate buffer (pH 5.0) kept at 25°C and Ps-45: papain solution in 0.2 M phosphate buffer (pH 5.0) kept at 45°C.