Preparation and in vivo absorption evaluation of spray dried powders containing salmon calcitonin loaded chitosan nanoparticles for pulmonary delivery

Figures & data

Table 1 Effect of mannitol concentration and inlet air temperature on percentage yield and particle size of spray dried powders (mean ± SD, n = 3)

Formulation	Mannitol (%)	Inlet air temperature (°C)	Spray dried powders yield (%)	Particle sizes D(0.5) (μm)	Span
F1	90	110	46	5.70 ± 1.53	3.42 ± 0.11
F2	80	110	41	5.42 ± 2.34	3.33 ± 0.19
F3	70	110	38	5.01 ± 1.54	3.01 ± 0.11
F4	90	130	57	4.67 ± 2.12	2.74 ± 0.19
F5	80	130	48	4.03 ± 1.34	2.55 ± 0.18
F6	70	130	42	3.89 ± 1.48	2.10 ± 0.20
F7	90	150	68	4.04 ± 1.47	2.22 ± 0.22
F8	80	150	57	3.86 ± 2.05	1.88 ± 0.16
F9	70	150	51	3.74 ± 1.21	1.99 ± 0.17

Abbreviation: SD, standard deviation.

Figure 1 Scanning electron micrographs of sCT-CS-NPs spray dried powders using different nanoparticles/mannitol ratios (NPs/M) and inlet temperatures. (A) NPs/M = 10/90, (B) NPs/M = 20/80, (C) NPs/M = 30/70 all prepared at 110°c, (D) NPs/M = 10/90, (E) NPs/M = 20/80, (F) NPs/M = 30/70 all prepared at 130°c and (G) NPs/M = 10/90, (H), NPs/M = 20/80, (I) NPs/M all prepared at 150°c.

Abbreviations: M, mannitol; sCT-CS-NPs, salmon calcitonin loaded chitosan nanoparticles.

Table 2 Densities and aerodynamic diameter of dry powders prepared using different mannitol concentrations and inlet air temperatures (mean ± SD, n = 3)

Formulation	Mannitol concentrations (%w/w)	Inlet air temperatures (°C)	True density (g/cm^3)	Tapped density (g/cm^3)	CI (%)	daer (μm)
F1	90	110	1.45 ± 0.02	0.36 ± 0.05	27.88 ± 0.12	4.70 ± 0.07
F2	80	110	1.46 ± 0.04	0.32 ± 0.02	25.71 ± 0.08	4.12 ± 0.04
F3	70	110	1.48 ± 0.03	0.28 ± 0.06	20.56 ± 0.10	3.81 ± 0.10
F4	90	130	1.49 ± 0.06	0.35 ± 0.03	24.85 ± 0.06	4.37 ± 0.09
F5	80	130	1.55 ± 0.05	0.28 ± 0.01	23.35 ± 0.08	3.83 ± 0.13
F6	70	130	1.59 ± 0.03	0.25 ± 0.04	19.58 ± 0.13	2.89 ± 0.06
F7	90	150	1.57 ± 0.07	0.30 ± 0.05	22.67 ± 0.11	4.04 ± 0.03
F8	80	150	1.59 ± 0.02	0.23 ± 0.07	20.57 ± 0.07	3.76 ± 0.11
F9	70	150	1.58 ± 0.01	0.15 ± 0.02	18.15 ± 0.02	2.51 ± 0.07

Abbreviations: CI, Carr index; d_aer, aerodynamic diameter; SD, standard deviation; w, weight.

Figure 2 (A) SDS-PAGE analysis of l1: sCT standard marker, L2: unprocessed sCT (0.1 μg/ml), L3, L4, and L5: sCT recovered from sCT-CS-NPs spray dried powders (NPs/M = 30/70), prepared at the inlet temperatures of 110°c, 130°c, and 150°c, respectively, L6: blank spray dried powder, and L7: sample buffer. (B) SDS-PAGE analysis of L1: sCT standard marker, L2 and L3: unprocessed sCT (0.2 and 0.1 μg/mL), L4, L5, and L6: sCT recovered from sCT-CS-NPs spray dried powders with NPs/M = 10/90, NPs/M = 20/80, and NPs/M = 30/70, respectively, when prepared using an inlet temperature of 130°C, L7: blank spray dried powder and l8: sample buffer.

Abbreviations: CS, chitosan; M, mannitol; NPs, nanoparticles; sCT, salmon calcitonin loaded; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 3 sCT circular dichroism spectra in acetate buffer pH 4.4 of: (◆) native sCT, (■) sCT-CS-NPs compared to sCT-CS-NPs spray dried powders containing different sCT loaded CS-NPs/mannitol ratios of (▲) NPs/M = 30/70; (•) NPs/M = 20/80; and (X) NPs/M = 10/90 when using an inlet air temperature of 130°c.

Abbreviations: CS, chitosan; M, mannitol; NPs, nanoparticles; sCT, salmon calcitonin loaded.

Figure 4 FTIR spectra of: (A) native sCT, (B) chitosan, (C) mannitol, and sCT-CS-NPs spray dried powders of (D) NPs/M = 30/70, (E) NPs/M = 20/80, (F) NPs/M = 10/90 prepared using an inlet temperature of 130°c.

Abbreviations: CS, chitosan; FTIR, Fourier transform infrared; M, mannitol; NPs, nanoparticles; sCT, salmon calcitonin loaded; T, transmittance.

Table 3 Size and zeta potential of freshly prepared sCT-CS-NPs and nanoparticles recovered from spray dried powders (mean ± SD, n = 3)

Formulation	Size (nm)	Polydispersity index (PDI)	Zeta potential (mV)
Freshly prepared sCT-CS-NPs	210 ± 0.12	0.109 ± 0.14	+24 ± 0.08
sCT-CS-NPs spray dried powders	215 ± 0.15	0.115 ± 0.11	+28 ± 0.16

Abbreviations: sCT-CS-NPs, salmon calcitonin loaded chitosan nanoparticles; SD, standard deviation.

Figure 5 TEM of (A) freshly prepared nanoparticles and (B) recovered nanoparticles from spray dried powder (NPs/M = 30/70) prepared at an inlet temperature of 130°C.

Abbreviations: M, mannitol; NPs, nanoparticles; TEM, transmission electron microscopy.

Figure 6 In vitro release profile of sCT-CS-NPs and sCT-CS-NPs spray dried powders (NPs/M = 30/70) prepared using an inlet air temperature of 130°C.

Abbreviations: M, mannitol; sCT-CS-NPs, salmon calcitonin loaded chitosan nanoparticles.

Table 4 Mass median aerodynamic diameter, fine particle fraction, emitted dose and dispersibility of sCT-CS-NPs spray dried powders determined with ACI (mean ± SD, n = 3)

Formulation	MMAD (μm)	FPF (%)	ED (%)	Dispersibility (%)
sCT-CS-NPs powder	3.45	18.67	22.43	48.71
sCT-CS-NPs spray dried powders (NPs/M = 30/70)	2.72	63.50	71.17	78.58

Abbreviations: ACI, Andersen Cascade Impactor; ED, emitted dose; FPF, fine particle fraction; MMAD, mass median aerodynamic diameter; M, mannitol; sCT-CS-NPs, salmon calcitonin loaded chitosan nanoparticles; SD, standard deviation.

Table 5 Pharmacokinetic parameters (mean ± SD, n = 6)

Sample	Cmax (pg · mL^−1)	Tmax (min)	AUC0–360 (min · pg · mL^−1)	Frel
Subcutaneous injection (Miacalcin®)	68 ± 5.14	30	10,014.60 ± 125.47	2.00
Native sCT	25 ± 3.03	60	5,004.60 ± 87.64	1.00
sCT-CS-NPs	47 ± 4.45	60	8,068.80 ± 105.18	1.61
sCT-CS-NPs spray dried powders (NPs/M = 30/70)	56 ± 4.56	45	8,578.80 ± 98.63	1.71
CS-NPs spray dried powders (blank, without sCT loading)	0.34 ± 1.23	360	0.697 ± 24.38	0.0083

Abbreviations: AUC_0–360, area under the curve; CS, chitosan; C_max, maximum plasma concentration; F_rel, relative bioavailability; M, mannitol; NPs, nanoparticles; T_max, time to reach maximum concentration; sCT, salmon calcitonin loaded; SD, standard deviation.

Figure 7 Plasma sCT concentration versus time profiles following intratracheal administration of native sCT, sCT-CS-NPs, sCT-CS-NPs spray dried powders and CS-NPs spray dried powders (blank, without loading sCT) compared to subcutaneous administration in rats; mean ± SD (n = 6).

Abbreviations: CS, chitosan; M, mannitol; NPs, nanoparticles; sCT, salmon calcitonin loaded; SD, standard deviation.