Pharmaceutical Nanotechnology
A pilot study of freeze drying of poly(epsilon-caprolactone) nanocapsules stabilized by poly(vinyl alcohol): Formulation and process optimization

https://doi.org/10.1016/j.ijpharm.2005.10.003Get rights and content

Abstract

A common limitation of using polymeric nanoparticles in aqueous suspension is due to their poor chemical and physical stability when conserved for a long time. Therefore, freeze drying of these colloidal systems is an alternative method to achieve long-term stability. Nanocapsules have thin and fragile shell structure, which may not resist to the stress of such process. The aim of this study is to investigate the formulation and process parameters in order to ensure the stability of polycaprolactone nanocapsules (PCL NC) by freeze drying.

In this paper, we studied the freeze drying of PCL NC prepared by the emulsion–diffusion method and stabilized by poly(vinyl alcohol) (PVA). Different parameters have been tested throughout the freeze–thawing study including PVA and PCL concentration, cooling rate, cryoprotectant concentrations, nature of encapsulated oil and NC purification. On the other hand, nanocapsules have been freeze dried both before and after purification. Freeze dried purified PCL NC were characterized by particle size measurement, collapse temperature, Tg determination, scanning electron microscope observation, environmental scanning electron microscope imaging and residual humidity quantification. Finally, the effect of annealing on the NC stability and the sublimation rate has been well explored.

The results suggest that PCL NC could be freeze dried without a cryoprotectant if the concentration of PVA stabilizer is sufficient (5%), while for the purified NC the addition of 5% of cryoprotectant seems to be necessary to ensure the stability of NC. The type of cryoprotectants had practically negligible effects on the size and the rehydration of freeze dried nanocapsules. The annealing process could accelerate the sublimation with the conservation of nanocapsules size.

Introduction

Submicronic colloidal vectors have gained a considerable interest in the last few years because of their ability to ensure a specific drug targeting by both the oral route (Ponchel and Irache, 1998) and the parenteral route (Marty et al., 1978, Soppimath et al., 2001).

Such particulate systems have been widely investigated for gene delivery to cells and tissues (Panyam and Labhasetwar, 2003) as in the delivery of anti-sense oligonucleotides (Lambert et al., 2001) and also in cancer therapy and diagnosis (Brigger et al., 2002).

Among these vectors, liposomes and nanoparticles have special advantages with regard to the modulation of an active ingredient distribution within the human body (Soppimath et al., 2001).

Nanoparticles can be classified into nanospheres and nanocapsules. Nanocapsules are vesicular systems in which the drug is confined to an oily or aqueous cavity surrounded by a unique polymeric membrane while nanospheres are matrix systems in which the drug is physically and uniformly dispersed (Soppimath et al., 2001).

The major obstacle that limits the use of such vectors is their instability in an aqueous medium (Chacon et al., 1999). Aggregation and particle fusion are frequently noticed after a long period of storage of such systems (Auvillain et al., 1989). Furthermore, drug leakage out of the nanocapsules and non-enzymatic polymer hydrolysis can happen. Thus, the stabilization of colloidal vectors is deeply explored in order to reach a shelf-life of several years.

Freeze drying, also termed lyophilization, is an industrial process of drying by freezing and sublimation of ice; it is used to convert solutions of labile materials into solids of sufficient stability for distribution and storage (Franks, 1998). This technique was considered as a good method to conserve the integrity of colloidal vectors. In literature, there are many papers that investigate in detail the stabilization by freeze drying of liposomes (Crowe et al., 1986, Crowe et al., 1994, Anchordoguy et al., 1987) and nanospheres (De Chasteigner et al., 1996, Schwarz and Mehnert, 1997, Chacon et al., 1999, Saez et al., 2000;) but few researchers, to the best of our knowledge, studied the lyophilization of nanocapsules which have a very thin and fragile envelope that may not withstand the mechanical stress of freezing (Auvillain et al., 1989, De Chasteigner et al., 1995, Schaffazick et al., 2003, Choi et al., 2002).

Auvillain et al. (1989) found that the freeze drying of polycaprolactone nanocapsules is possible but 30% trehalose as cryoprotectant was necessary to preserve the integrity of nanocapsules. In such a case, rapid freezing has been favourable and the conservation of encapsulated oil in liquid state during the freezing process improved the success of lyophilization.

De Chasteigner et al. (1995) have reported the lyophilization of polylactide nanocapsules with 10% glucose or sucrose but the produced lyophilizates showed a two-fold increase in their size after redispersion in water. The authors explained this result by a clustering of nanocapsules.

Schaffazick et al. (2003) reported the lyophilization of polycaprolactone and eudragit nanocapsules upon the addition of colloidal silicon dioxide, however such an addition makes intravenous administration of the nanocapsules impossible.

Finally, Choi et al. (2002) freeze dried nanocapsules of polycaprolactone with pluronic F68 as a stabilizer without a cryoprotectant. These authors found that the freeze process can break the nanocapsules and promote leakage of their contents. They concluded that the nanocapsules may not have been broken by water crystallization in the external phase but by the solidification of the oil (miglyol) in the internal phase.

The aim of this study is to investigate the factors which can influence the nanocapsules stability during the different steps of lyophilization. Different parameters have been tested throughout the freeze–thawing study including PVA concentration, polymer concentration, cooling rate, cryoprotectant concentration, nature of encapsulated oil and nanocapsules purification. Nanocapsules have been freeze dried both before and after purification. Freeze dried purified nanocapsules were characterized by particle size measurement, collapse temperature and Tg determination, scanning electron microscope observation, environmental scanning electron microscope imaging and residual humidity quantification. Finally, the effect of annealing on the nanocapsules stability and the sublimation rate has been well explored. A successful nanocapsules lyophilization requires a good formulation and optimal conditions of freezing and desiccation. Such process must produce an acceptable non-collapsed cake, which can rehydrate immediately with the conservation of nanocapsules properties.

Section snippets

Materials

Poly(epsilon-caprolactone) (PCL) (Mw: 14,000 g/mol) and hydroxypropyl beta cyclodextrin (HPβCD) were obtained from Sigma–Aldrich (France). Poly(vinyl alcohol) (PVA) Moviol 4-88 (88% hydrolyzed, Mw: 31,000 g/mol) was purchased from Clariant (France). Miglyol 810 and miglyol 829 were supplied from Condea chemie (Germany). Ethyl acetate was obtained from Carlo Erba (France). d(+)Sucrose from prolabo (France). d(+)Anhydrous glucose, d(+)trehalose and d-mannitol from Flucka biochemika (Switzerland).

Influence of cooling rate

Nanocapsules were prepared in this study by the emulsification diffusion method which is capable of preparing nanocapsules in a simple, efficient and reproducible manner. This method was preferred to the other techniques of nanocapsules preparation such as emulsification evaporation of solvent and nanoprecipitation. Emulsification evaporation technique requires working with toxic solvents, whereas nanoprecipitation method produces low yields and poor entrapment efficacy (Quintanar-Guerrero et

Discussion

PCL NC prepared by emulsion–diffusion method produces NC with a mean size of 294–401 nm in a reproducible and an efficient way. In this present study, PVA was used as stabilizer for preparing PCL NC. This polymer is one of the most frequently used stabilizers to produce stable nanoparticles, since it enhances the production of stable particles with a small size and narrow size distribution (Zambaux et al., 1998, Sahoo et al., 2002). Many papers have mentioned that a fraction of PVA used in the

Conclusion

The results of this study demonstrate that polycaprolactone nanocapsules stabilized by PVA and prepared using the emulsion–diffusion method can be freeze dried without a cryoprotectant when the concentration of PVA is sufficient to prevent the NC from aggregation during freezing. However, for purified NC, the addition of cryoprotectant at a concentration of 5% seems necessary. The type of cryoprotectants had practically negligible effects on the size and the rehydration of freeze dried

Acknowledgement

The authors are grateful to Annie Rivoire from “Le Centre Technologique des Microstructures de l’Université Lyon 1” for her technical assistance with the SEM and ESEM imaging.

References (36)

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