Influence of Lyophilization and Cryoprotection on the Stability and Morphology of Drug-Loaded Poly(ethylene glycol- b-ε-caprolactone) Micelles

Abstract

Polymeric micelles are promising carriers for the delivery of poorly water-soluble drugs, providing enhanced drug solubility, blood circulation times, and bioavailability. Nevertheless, the storage and long-term stability of micelles in solution present challenges requiring the lyophilization and storage of formulations in the solid state, with reconstitution immediately prior to application. Therefore, it is important to understand the effects of lyophilization/reconstitution on micelles, particularly their drug-loaded counterparts. Herein, we investigated the use of β-cyclodextrin (β-CD) as a cryoprotectant for the lyophilization/reconstitution of a library of poly(ethylene glycol-b-ε-caprolactone) (PEG-b-PCL) copolymer micelles and their drug-loaded counterparts, as well as the effect of the physiochemical properties of different drugs (phloretin and gossypol). The critical aggregation concentration (CAC) of the copolymers decreased with increasing weight fraction of the PCL block (f_PCL), plateauing at ~1 mg/L when the f_PCL was >0.45. The blank (empty) and drug-loaded micelles were lyophilized/reconstituted in the absence and presence of β-CD (9% w/w) and analyzed via dynamic light scattering (DLS) and synchrotron small-angle X-ray scattering (SAXS) to assess for changes in aggregate size (hydrodynamic diameter, D_h) and morphology, respectively. Regardless of the PEG-b-PCL copolymer or the use of β-CD, the blank micelles displayed poor redispersibility (<10% relative to the initial concentration), while the fraction that redispersed displayed similar D_h to the as-prepared micelles, increasing in D_h as the f_PCL of the PEG-b-PCL copolymer increased. While most blank micelles displayed discrete morphologies, the addition of β-CD or lyophilization/reconstitution generally resulted in the formation of poorly defined aggregates. Similar results were also obtained for drug-loaded micelles, with the exception of several that retained their primary morphology following lyophilization/reconstitution, although no obvious trends were noted between the microstructure of the copolymers or the physicochemical properties of the drugs and their successful redispersion.

Keywords: cryoprotection; lyophilization; micelles; reconstitution; redispersibility; β-CD.

Figure 1

Ratiometric intensity (I₃₈₄/I₃₇₂) of the pyrene emission as a function of copolymer concentration in high-purity water for the (a) PEG₂PCL_y, (b) PEG₅PCL_y, and (c) PEG₁₀PCL_y copolymer series. (d) CAC of the copolymers as a function of f_PCL.

Figure 2

D_h versus f_PCL as determined from the number PSDs for the (a) PEG₂PCL_Y, (b) PEG₅PCL_Y, and (c) PEG₁₀PCL_Y micelles as prepared (AP), after lyophilization/reconstitution (LR), and in the absence and presence of β-CD (−/+CD). All values are reported as the mean + std. dev. (n = 3).

Figure 3

Representative SAXS scattering profiles complete with power laws to indicate the morphology of the aggregates or micelles: (a) PEG₂PCL_0.4 (red profile), (b) PEG₂PCL_1.1, and (c) PEG₁₀PCL_3.2 as prepared in the absence of β-CD, (a) PEG₅PCL_9.5 (green profile) following lyophilization/reconstitution in the absence of β-CD, and (d) gossypol-loaded PEG₅PCL_9.5 as prepared in the absence of β-CD.

Figure 4

D_h versus f_PCL as determined from number PSDs for (a) gossypol (GP) and (b) phloretin (PH)-loaded PEG₅PCL_1.3, PEG₅PCL_2.4, PEG₅PCL_4.2, and PEG₅PCL_9.5 micelles as prepared (AP), after lyophilization/reconstitution (LR), and in the absence and presence of β-CD (−/+CD). All values are reported as the mean + std. dev. (n = 3).