Anhydrous reverse micelle nanoparticles: new strategy to overcome sedimentation instability of peptide-containing pressurized metered-dose inhalers

Figures & data

Table 1. The TBA volume and lecithin concentration in each formulation and the detected particle size and PdI of the obtained original lipid vesicles.

Formulation	TBA volume (μL)	Lecithin concentration (%, w/v)	Particle size (nm)	PdI
1	150	5	174.8	0.290
2	200	5	161.9	0.202
3	250	5	145.5	0.084
4	150	10	241.9	0.360
5	200	10	176.5	0.244
6	250	10	166.0	0.138
7	150	20	266.2	0.375
8	200	20	206.1	0.267
9	250	20	177.9	0.264

Table 2. Results of estimated marginal means of the particle size and PdI.

				95% Confidence interval
Parameter	Formulation compositionc	Mean	Std. error	Lower bound	Upper bound
Particle size	TBA volume (μL)
	150	227.6	10.2	199.2	256.1
	200	181.5	10.2	153.1	209.9
	250	163.1	10.2	134.7	191.6
	Lecithin concentration (%)
	5	160.7	10.2	132.3	189.2
	10	194.8	10.2	166.4	223.2
	20	216.7	10.2	188.3	245.1
PdI	TBA volume (μL)
	150	0.342	0.021	0.285	0.399
	200	0.239	0.021	0.182	0.296
	250	0.162	0.021	0.105	0.219
	Lecithin concentration (%)
	5	0.192	0.021	0.135	0.249
	10	0.248	0.021	0.191	0.305
	20	0.303	0.021	0.246	0.360

Figure 1. Effect of stirring rate on the vesicle size (A) and PdI (B) of original lipid vesicles. Data represent mean (± SD) of three preparations, *p < 0.05 with each group.

Figure 2. TEM micrographs of the samples: (A) Original lipid vesicles; (B) ARM-NPs; (C) reconstructed liposomes.

Table 3. The particle size and PdI of original lipid vesicles, ARM-NPs and reconstructed liposomes.

Sample	Particle size (nm)	PdI
Original lipid vesicles	151.1 ± 1.0	0.119 ± 0.013
ARM-NPs	147.7 ± 2.0	0.152 ± 0.021
Reconstructed liposomes	181.0 ± 4.9	0.102 ± 0.029

Figure 3. FT-IR and CD results. (A) Comparison of FT-IR spectra: lyophilized complexes, pure sCT, atomized sCT from pMDIs and freeze-dried free sCT; (B) Fitting curves of sCT amide I band of pure sCT; (C) Fitting curves of sCT amide I band of freeze-dried free sCT; (D) Fitting curves of sCT amide I band of atomized sCT from pMDIs; (E) Comparison of sCT CD spectra under different conditions: aqueous solutions of SCT with SDS, pure sCT, atomized sCT from pMDIs and the lyophilized complexes.

Figure 4. Results of formulation characterization and stability evaluation. (A) The aerosolization properties of the optimized sCT-containing ARM-NPs for pMDIs formulation. Data represent mean (± SD) of three preparations; (B) The aerosolization properties of the optimized sCT-containing ARM-NPs for pMDIs formulation 12-week during storage under 4–6 °C condition. Data represent mean (± SD) of three preparations; (C) Content of drug per press during 12-week storage under 4–6 °C condition. Data represent mean (± SD) of three preparations.

Table 4. Characterization of particle size and PdI of sCT containing pMDIs during 10-day storage under different conditions: 4, 20 and 30 °C.

	Particle size (nm)			PdI
Temperature (°C)	Day 0	Day 5	Day 10	Day 0	Day 5	Day 10
4	160.4 ± 6.6	164.9 ± 5.2	164.2 ± 9.1	0.024 ± 0.002	0.102 ± 0.023	0.171 ± 0.015
20	147.3 ± 0.4	152.9 ± 9.0	150.9 ± 5.5	0.051 ± 0.005	0.178 ± 0.014	0.107 ± 0.011
30	165.1 ± 0.5	163.4 ± 6.0	169.9 ± 9.3	0.065 ± 0.018	0.160 ± 0.028	0.176 ± 0.023

Figure 5. DSC thermograms of TBA/water 1:4, TBA/water 1:4 + lecithin, lipid vesicle suspension, TBA/water 3:2 and TBA/water 3:2 + lecithin.

Table 5. The four kinds of frozen crystallization behavior of TBA/water system.

Type	TBA% (w/w)	Phase I (high temperature)	Phase II (moderate temperature)	Phase III (low temperature)
A	0–20	l.s.	Ice + l.s.	Ice + eutectic A
B	20–70	l.s.	TBA hydrate + l.s.	TBA hydrate + eutectic A
C	70–90	l.s.	TBA hydrate + l.s.	TBA hydrate + eutectic B
D	90–100	l.s.	TBA + l.s.	TBA + eutectic B

Note: l.s., liquid solution.

Figure 6. The schematic diagram about the forming process of ARM-NPs. (A) The TBA/water 1:4 system; (B) the TBA/water 3:2 system; (1) before freeze-drying; (2) during freeze-drying; (3) after adding ethanol absolute. Larger balls without tails and smaller balls with tails represented sCT and lecithin, respectively. A1, A2, B1 and B2 referred to aqueous environment while A3 and B3 depicted the addition of ethanol absolute.