Freeze thaw: a simple approach for prediction of optimal cryoprotectant for freeze drying

Abstract

The present study evaluates freeze thaw as a simple approach for screening the most appropriate cryoprotectant. Freeze-thaw study is based on the principle that an excipient, which protects nanoparticles during the first step of freezing, is likely to be an effective cryoprotectant. Nanoparticles of rifampicin with high entrapment efficiency were prepared by the emulsion-solvent diffusion method using dioctyl sodium sulfosuccinate (AOT) as complexing agent and Gantrez AN-119 as polymer. Freeze-thaw study was carried out using trehalose and fructose as cryoprotectants. The concentration of cryoprotectant, concentration of nanoparticles in the dispersion, and the freezing temperature were varied during the freeze-thaw study. Cryoprotection increased with increase in cryoprotectant concentration. Further, trehalose was superior to fructose at equivalent concentrations and moreover permitted use of more concentrated nanosuspensions for freeze drying. Freezing temperature did not influence the freeze-thaw study. Freeze-dried nanoparticles revealed good redispersibility with a size increase that correlated well with the freeze-thaw study at 20% w/v trehalose and fructose. Transmission electron microscopy revealed round particles with a size approximately 400 nm, which correlated with photon correlation spectroscopic measurements. Differential scanning calorimetry and X-ray diffraction suggested amorphization of rifampicin. Fourier transfer infrared spectroscopy could not confirm interaction of drug with AOT. Nanoparticles exhibited sustained release of rifampicin, which followed diffusion kinetics. Nanoparticles of rifampicin were found to be stable for 12 months. The good correlation between freeze thaw and freeze drying suggests freeze-thaw study as a simple and quick approach for screening optimal cryoprotectant for freeze drying.

Fig. 1

Stresses involved in freeze drying and the interventions to prevent against such stresses

Fig. 2

Effect of RFM/AOT ratio on entrapment efficiency and particle size of rifampicin nanoparticles

Fig. 3

Effect of RFM/Gantrez AN-119 ratio on entrapment efficiency of rifampicin nanoparticles at RFM/AOT molar ratio of 1:1.85

Fig. 4

Structures of RFM, Gantrez AN-119, AOT, and Gantrez AN-119 Ester

Fig. 5

Structures of rifampicin, trehalose, and fructose

Fig. 6

Transmission electron microscopic image of rifampicin nanoparticle

Fig. 7

DSC thermograms of RFM, rifampicin nanoparticles, and excipients

Fig. 8

a X-ray powder diffractograms of RFM, b X-ray powder diffractograms of rifampicin nanoparticles, c X-ray powder diffractograms of trehalose, d X-ray powder diffractograms of Gantrez AN-119, e X-ray diffractograms of AOT

Fig. 9

a FTIR spectrum of RFM, b FTIR spectrum of rifampicin nanoparticles, c FTIR spectrum of AOT, d FTIR spectrum of Gantrez AN-119

Fig. 10

a In vitro release and stability of rifampicin nanoparticles at 30 ± 2°C/65 ± 5% RH. b In vitro release and stability of rifampicin nanoparticles at 40 ± 2°C/75 ± 5% RH