Cryoprotection-lyophilization and physical stabilization of rifampicin-loaded flower-like polymeric micelles

Abstract

Rifampicin-loaded poly(ε-caprolactone)-b-poly(ethylene glycol)-poly(ε-caprolactone) flower-like polymeric micelles display low aqueous physical stability over time and undergo substantial secondary aggregation. To improve their physical stability, the lyoprotection-lyophilization process was thoroughly characterized. The preliminary cryoprotectant performance of mono- and disaccharides (e.g. maltose, glucose), hydroxypropyl-β-cyclodextrin (HPβCD) and poly(ethylene glycol) (PEG) of different molecular weights was assessed in freeze-thawing assays at -20°C, -80°C and -196°C. The size and size distribution of the micelles at the different stages were measured by dynamic light scattering (DLS). A cryoprotectant factor (f(c)) was determined by taking the ratio between the size immediately after the addition of the cryoprotectant and the size after the preliminary freeze-thawing assay. The benefit of a synergistic cryoprotection by means of saccharide/PEG mixtures was also assessed. Glucose (1 : 20), maltose (1 : 20), HPβCD (1 : 5) and glucose or maltose mixtures with PEG3350 (1 : 20) (copolymer:cryoprotectant weight ratio) were the most effective systems to protect 1 per cent micellar systems. Conversely, only HPβCD (1 : 5) cryoprotected more concentrated drug-loaded micelles (4% and 6%). Then, those micelle/cryoprotectant systems that displayed f(c) values smaller than 2 were freeze-dried. The morphology of freeze-dried powders was characterized by scanning electron microscopy and atomic force microscopy and the residual water content analysed by the Karl Fisher method. The HPβCD-added lyophilisates were brittle porous cakes (residual water was between 0.8% and 3%), easily redispersable in water to form transparent systems with a minimal increase in the micellar size, as determined by DLS.

Scheme 1.

Rifampicin structure. The pK_a1 (in water) has been attributed to the hydroxyl moiety at C-8, while the pK_a2 (in water) has been attributed to N-4 of piperazine.

Figure 1.

f_c values for (a) 1% and (b) 4% and 6% RIF-loaded PCL(4500) micelles; (c) 1% and (d) 4% and 6% RIF-loaded PCL(3700) micelles after the freeze–thawing assay. Cryoprotectants were used in different copolymer:cryoprotectant weight ratios (wt : wt). Samples were frozen at −80°C (black bar with white dots) and −196°C (white bars with black dots). Asterisk, PEG3350/glucose (50:50) and PEG3350/maltose (50:50). ND, not determined.

Figure 2.

Lyophilized RIF-loaded PCL(4500) micelles (1% w/v) after redispersion in distilled water (a) without and (b) with the addition of HPβCD (1 : 5). The cryoprotectant effect is given by the complete redispersion of the freeze-dried powder and the translucent appearance of the suspension (b). (Online version in colour.)

Figure 3.

f_c values of RIF-loaded (a) PCL(4500) and (b) PCL(3700) micelles after the freeze-drying assay. HPβCD was used in different copolymer:cryoprotectant weight ratios (wt : wt). Samples were frozen at −80°C (black bar with white dots) and −196°C (white bar with black dots).

Figure 4.

SEM microphotographs of freeze-dried samples of (a) 1% RIF-loaded PCL(4500) micelles without cryoprotection (control), (b) freeze-dried 5% HPβCD solution (control) and (c) 1% RIF-loaded PCL(4500) micelles containing HPβCD (1 : 5 copolymer:cryoprotectant weight ratio). Scale bar, 10 μm. Photo inset: magnification of (c); scale bar, 100 nm. All the samples were frozen by immersion in liquid nitrogen (−196°C), prior to lyophilization.

Figure 5.

Tapping mode AFM phase two-dimensional images of (a) fresh and (b) freeze-dried 1% w/v RIF-loaded PCL(4500) micelles cryoprotected with HPβCD 1 : 5 (copolymer:cryoprotectant weight ratio) and diluted (1 : 20) immediately before the analysis. (c) Same as (b) but three dimensional. (d) AFM height versus diameter profile of the structures (a and b) pointed out in (b,c). The micelle pointed out in (b,c) as (c) presents a height of 17.18 nm and diameter of 68.84 nm. (Online version in colour.)