http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.982822
RESEARCH ARTICLE
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Solidification drug nanosuspensions into nanocrystals by freeze-drying: a case study with ursodeoxycholic acid Yue-Qin Ma, Zeng-Zhu Zhang, Gang Li, Jing Zhang, Han-Yang Xiao, and Xian-Fei Li Department of Pharmaceutics, the 94th Hospital of People’s Liberation Army, Nanchang, China
Abstract
Keywords
To elucidate the effect of solidification processes on the redispersibility of drug nanocrystals (NC) during freeze-drying, ursodeoxycholic acid (UDCA) nanosuspensions were transformed into UDCA-NC via different solidification process included freezing and lyophilization. The effect of different concentrations of stabilizers and cryoprotectants on redispersibility of UDCA-NC was investigated, respectively. The results showed that the redispersibility of UDCA-NC was RDI20 C5RDI80 C5RDI196 C during freezing, which indicated the redispersibility of UDCANC at the conventional temperature was better more than those at moderate and rigorous condition. Compared to the drying strengthen, the employed amount and type of stabilizers more dramatically affected the redispersibility of UDCA-NC during lyophilization. The hydroxypropylmethylcellulose and PVPK30 were effective to protect UDCA-NC from damage during lyophilization, which could homogeneously adsorb into the surface of NC to prevent from agglomerates. The sucrose and glucose achieved excellent performance that protected UDCA-NC from crystal growth during lyophilization, respectively. It was concluded that UDCANC was subjected to agglomeration during solidification transformation, and the degree of agglomeration suffered varied with the type and the amounts of stabilizers used, as well as different solidification conditions. The PVPK30-sucrose system was more effective to protect UDCA-NC from the damage during solidification process.
Freeze-drying, nanocrystals, solidification stress, ursodeoxycholic acid
Introduction Nanosuspensions (NS) is generally produced in liquid media in which drug particles size is less than 1 lm and stabilized by surfactants or polymers. NS possess the advantages that enhance the solubility and dissolution velocity of poorly soluble drugs due to their small particle size and high surface area1. However, NS are essentially thermodynamically unstable systems. The enormous surface area and the small size of these particles results in high interfacial tension, which in turn results in an increase in the free energy of the system2. Hence, NS would tend to generate flocculation, aggregation or crystal growth to decrease their free energy. The physical stability problems, which limits the industrialization of NS, are crucial to be solved and recently have been widely investigated. In order to improve the physical stability of liquid NS, NS have to be solidified and then processed further into tablets or capsules. Solid nanosuspensions (SNS) or nanocrystals (NC) (Figure 1) is composed of drug as well as stabilization agent, and can be easily redispersed back to original NS states instantaneously on mild agitation or peristalsis followed by rehydration with aqueous media in vitro or gastrointestinal
Address for correspondence: Yue-Qin Ma and Gang Li, Department of Pharmaceutics, the 94th Hospital of People’s Liberation Army, Nanchang, China. E-mail: [email protected] (Y.-Q. Ma); [email protected] (G. Li)
History Received 8 July 2014 Revised 22 October 2014 Accepted 28 October 2014 Published online 26 November 2014
tract (redispersibility), if they did not go through irreversible aggregation during solidification3,4. Freezing-drying can be used to transform liquid NS into solid NC5–11. The drying process consists on removing water from NS sample by sublimation and desorption under vacuum, or evaporation under low temperature. Nevertheless, this process generates additional thermal stresses (due to heat or freezing for lyophilization), which could inevitably destabilize NC and impact on the redispersibility of NC. For example, if the NS are coated with polymeric surfactants such as poloxamers, drying may lead to crystallization of the polymer, thereby compromising their ability to prevent aggregation. So far, literature about impact of solidification process conditions on redispersibility characteristics of SNS is lacking12, and there are no generally accepted views on the formation of hard agglomerates of NC. In view of these considerations, understanding the impact of solidification processes on redispersibility of solid NC is important. Furthermore, prior to solidification, the cryoprotectant for freeze-drying is often added into the NS, which can be used to protect the NS from solidification damage. Typical cryoprotectants added prior to freeze-drying are water-soluble materials such as sugars (e.g. sucrose, saccharose, and lactose), sugar alcohols (e.g. mannitol, sorbitol)13. However, if the nature of a cryoprotectant or dispersant is inappropriate for drying of drug NC, even excessive amounts are unable to prevent the system from freezing and drying damage. Therefore, it is strongly needed to systematically evaluate the influence of type and concentration
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Figure 1. Schematic illustrations on redispersibility of drug nanocrystals.
of cryoprotectant or dispersants on redispersibility of NC after freeze-drying. Ursodeoxycholic acid (UDCA) is a cholagogue, cholelitholytic and hepatic protector agent and exhibits low bioavailability for oral delivery due to its poor water solubility14. NS can be used to improve the solubility and bioavailability of UDCA. During solidification of NS, it was found that the surface hydrophobicity of drug were responsible for the formability of the SNS during solidification15. But UDCA was a strong hydrophobic drug, so it might be very difficult to transform UDCA-NS into fine UDCANC. The main objective of this artilce is to provide a case study for influence of different solidification conditions on the redispersibility of solid NC. UDCA-NS stabilized by different types of stabilizers were prepared by high pressure homogenization. And the concentration of each stabilizers employed (relative to the weight of UDCA) was 50%, 25% and 10%, respectively. UDCA-NS was transformed into UDCA-NC via freeze-drying applied with different process conditions. And the effect of different series of concentrations of cryoprotectants (sucrose, glucose, trehalose, lactose, manitol, sorbitol and PEG4000) on redispersibility of UDCA-NC was investigated during lyophilization, respectively. The redispersibility of UDCA-NC obtained at predetermined condition was evaluated by means of laser light scattering, scanning electron microscopy and transmission electron microscopy.
Materials and methods Materials UDCA was purchased from Zelang Co. (Nanjing, China). D-a-tocopherol polyethylene glycol 1000 succinate (TPGS) was purchased from Xi’an Healthful Biotechnology Co., Ltd. (Xi’an, China). Poloxamer 188 (P188, LutrolÕ F 68), poloxamer 407(P407, LutrolÕ F 127) and polyoxyethylene hydrogenated castor oil (RH40, CremophorÕ RH 40) were kindly donated by BASF (Ludwigshafen, Germany). Povidone 30 (PK30, PlasdoneÕ K-29/32) were kindly donated by JSP (NJ).
Hydroxypropylmethylcellulose (HPMC, Methocel E15LV Premium EPÕ, Colorcon, Dartford, UK), Sodium dodecyl sulfate (SDS, SHANHE, Anhui, China), Tween 80 (TW80, SHANHE, Anhui, China), polyethylene glycol 4000 (PEG4000, SHANHE, Anhui, China) trehalose (Asahi KASEI, Chiyoda-Ku, Japan), sodium carboxymethyl starch (CMS-Na, SHANHE, Anhui, China) and Microcrystalline cellulose and carboxymethyl cellulose sodium (MC, CeolusÔ RC-A591NF, Asahi KASEI, Chiyoda-Ku, Japan) were commercially obtained. Glucose, sucrose, manitol, sorbitol and lactose were obtained from DAMAO Chemical Co., Ltd. (Tianjin, China). Nanosuspensions production UDCA-NS was prepared by high-pressure precipitation tandem homogenization technology16. UDCA coarse powder was completely dissolved in the proper amount of ethanol as organic phase. Different concentrations of stabilizers were dispersed in 100-ml water as water phase. The organic phase loaded 12.5% concentrations of UDCA (w/v) was pumped at a fixed flow rate of 10 ml/min using an injection pump into the inflow reservoir of piston-gap high pressure homogenizer (AH-1000D, ATS Engineering Inc., Seeker, Canada) containing the anti-solvent (100 ml aqueous stabilizer solution). Homogenization was performed at 300–700 bar for 20 cycles as high pressure precipitation step. The NS was subjected to homogenizing for another 20 cycles under 600–1000 bar in continuous mode as high pressure homogenization step. A cold water bath system was used to hold temperature during the whole homogenization process.
Solidification transformation Freezing process The UDCA-NS stabilized by different polymeric dispersants were frozen at different freezing temperature conditions generated by different temperatures. The UDCA-NS (3 mL) in a 10-mL vial
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Table 1. The applied freezing process conditions with different stresses. Temperature conditions
Conventional stress
20 C for12 h
Freezing
Moderate stress
Rigorous stress
196 C for 2 h
80 C for 6 h
Table 2. The applied lyophilization process cycle with different stress conditions. Lyophilization
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Cycle condition
Pre-freezing
Ramp rate
Primary drying
Ramp rate
Secondary drying
Conventional stress
40 C for 60 min
1 C /min
0.05 C /min
10 C for 6 h
Moderate stress
40 C for 60 min
1 C /min
0.2 C /min
10 C for 6 h
Rigorous stress
40 C for 60 min
1 C /min
20 C for 8 h; 10 C for 12 h 10 C for 10 h; 0 C for 8 h 0 C for 12 h
0.8 C /min
10 C for 8 h
were frozen according to the process temperature conditions in Table 1. Then, the system was thawed at room temperature. The average particle sizes were determined. Measurements were made in triplicate for all the measurement runs.
(freezing and lyophilization) and D represents the corresponding value of reconstituted UDCA-NS post-solidification. An RDI of near 1 would therefore means that UDCA-NC powder obtained by solidification transformation can be completely reconstituted back to the original particle size after rehydration.
Lyophilization process The UDCA-NS stabilized by different stabilizers were dried by lyophilization. Each UDCA-NS (3 mL) was freeze-dried in a 10 mL vial using freeze-dry system (FreezeZoneÕ Stoppering Tray Dryers, LABCONCO Corporation, Kansas City, MO). The applied cycle conditions were as follows: freezing was performed at 40 C for 60 min. The shelf temperature ramp rates from the freezing step into the primary drying step were 1 C/min for all cycles performed. Three sets of primary drying conditions were employed according to Table 2. Lyophilization process with cryoprotectant The different amount (100%, 200% and 400%, relative to the weight of UDCA) of cryoprotectants (sucrose, glucose, trehalose, lactose, manitol, sorbitol and PEG4000) was, respectively, added into UDCA-NS stabilized by 10% concentration (relative to the weight of UDCA) of TPGS or PVPK30, respectively. UDCANS were freeze-dried in a 10mL vial using freeze-dry system (FreezeZoneÕ Stoppering Tray Dryers, LABCONCO Corporation, KS). The applied cycle conditions were as follows: freezing was performed at 40 C for 60 min. Primary drying was performed at 20 C for 8 h; 10 C for 6 h; and 0 C for 5 h. The shelf temperature ramp rates from the freezing step into the primary drying step were 1 C/min. The secondary drying was performed at 10 C for 6 h. The shelf temperature ramp rates from the freezing step into the primary drying step were 0.5 C/min. Laser diffractometry (LD) LD was performed on a Mastersizer Micro Plus (Malvern Instruments Limited, Worcestershire, UK). Analysis of the diffraction patterns was done using the Mie model. From the resulting volume distributions, the median was calculated ( ¼ 50% volume percentile, D50). All measurements were performed in triplicate.
Transmission electron microscope (TEM) The morphology of UDCA-NS was observed by TEM (JEM1200EX, Japan). One drop of drug NS was placed on a copper grid and stained with 2% phosphotungstic acid solution for 5 min. The grid was dried at room temperature and was evaluated with the electron microscope. Scanning electron microscopy (SEM) Morphological evaluation of representative samples of UDCANC powder subjected to different solidification conditions was performed and compared against each other under scanning electron microscope( SEM) (Hitachi X650, Tokyo, Japan). All samples were examined on a brass stub using carbon double-sided tape. UDCA-NC samples were glued and mounted on metal sample plates. The samples were gold coated (thickness&15–20nm) with a sputter coater (Fison Instruments, Manchester, UK) using an electrical potential of 2.0 kV at 25 mA for 10 min. An excitation voltage of 20 kV was used in the experiments. Atomic force microscopy (AFM) The surface morphology on three-dimension of re-dispersed UDCA-NS after freeze-drying was investigated by atomic force microscope (AFM) using non-contact mode. Prior to analysis, the surface of mica slice was cleaned with coated fabric to avoid analysis artifacts. With the help of a micropipette, the diluted samples were dropped onto the mica slice and blew into lamellar by nitrogen gas in super-clean bench, following drying at ambient temperature for 2 d. Imaging was performed on Nanoscope IIIA (Model-Veeco Bioscope II, Veeco Instruments Ins, Plainview, NY).
Results and discussion Preparation of ursodeoxycholic acid nanosuspensions
Redispersibility index (RDI) RDI ¼ D0 =D where D0 represents the volume-weighed mean particle size of the freshly prepared UDCA-NS directly prior to solidification
Mean particle size (d50) and average span values of UDCA-NS, respectively, stabilized by different concentrations of stabilizers were listed in Figures 2 and 3. The particle size of NS was in rang of 600–900 nm. These results demonstrated that the coarse UDCA were completely disintegrated to nano-sized particles by means of
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Figure 2. The particle size of freshly prepared UDCA-NC with different concentrations of stabilizers.
Figure 3. The span of freshly prepared UDCA-NC with different concentrations of stabilizers.
high pressure homogenization technology, and successfully formed the different UDCA-NCs in terms of different stabilizers. Effect of freezing process on redispersibility of ursodeoxycholic acid nanosuspensions The freezing step is more significant than the subsequent sublimation step for redispersibility of drug NS during freeze-drying17. Hence, before sublimation step, different freezing processes were employed to investigate the influence of different freezing process on the redispersibility of NS. The freezing conditions and the RDI of NS stabilized by different concentration of stabilizers, respectively, after freeze-thawing were gathered in Figure 4. The TEM images of three representative UDCA-NS reconstructed after freeze-thawing were showed in Figure 5. The results indicated that UDCA-NS reconstructed after freeze-thawing had some aggregations or crystals growth, compared with that of freshly prepared NS.
The redispersibility of UDCA-NC depends on not only the freezing strength but also the type of stabilizers. Figure 4 showed that RDI of UDCA-NC, respectively, stabilized by different concentrations of stabilizers after freezing at three conditions was more than 1. For instance, RDI20 C, RDI80 C and RDI196 C of UDCA-NC stabilized by 10% concentration of PVPK30 was 2.98, 9.01 and 14.16, respectively, but those of UDCA-NC stabilized by 50% concentration of PVPK30 was 1.16, 5.32 and 9.65, respectively. It indicated that the different freezing temperature could induce the irreversible aggregation of UDCA-NC during freezing step. It can also be observed that the redispersibility of the frozen UDCA-NC at three conditions were RDI20 C5RDI80 C5RDI196 C. These results meant that the redispersibility of frozen UDCA-NC at the conventional freezing temperature (meant low freezing rate) was better than those at the moderate and rigorous temperature conditions(high freezing rate). Besides, the type and amounts of stabilizers at an equivalent freezing process have dramatically different protection effect on
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Figure 4. The redispersibility index (RDI) of UDCA-NC with different concentrations of stabilizers (relative of drug weight, m/m) after freezing at three stress conditions of ‘‘Conventional stress’’, ‘‘Moderate stress’’ and ‘‘Rigorous stress’’, respectively.
Figure 5. TEM images of three representative UDCA-NC stabilized by P188, TPGS and HPMC, respectively. A, B, C represent TEM images of UDCA-NS stabilized by P188, TPGS and HPMC, respectively. 1, 2, 3 represent ‘‘Conventional stress’’, ‘‘Moderate stress’’ and ‘‘Rigorous stress’’ conditions (20 C, 80 C and 196 C), respectively.
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Figure 6. Schematic illustration of particle separation in unfrozen phase dependent on freeze rate, diffusion rate of nanocrystals and stabilizer molecule.
UDCA-NC. And the higher was the concentration of stabilizers, the more near to 1% was RDI of UDCA-NC among the three temperature conditions. It meant that redispersibility of UDCANC treated by 50% concentration of surfactants or polymers stabilizers became much better than those with 25% and 10% concentration. The freezing temperature (determine the freezing rate (f) of ice) could be responsible for aggregation or even fusion of drug NS occurred during freezing process. As illustrated in Figure 6, with increase of freezing rate, water molecules excluded the UDCA-NC particles and lead them approach each other and aggregate eventually, simultaneously the activity of stabilizers was no longer effective due to separation of drug crystals and stabilizer molecule in the unfrozen concentrated phase17–19. That is, if a stabilizer polymer chain might be considered to be a particle and there is no interaction among particles, the size of UDCA-NC was at least one order of magnitude larger than that of stabilizer, and diffusive rate(s) of stabilizer might be larger than diffusive rate(n) of UDCA-NC(data not shown). So at conservative conditions (20 C), the freezing rate f, was smaller than s and n, then both the HAR-NC and stabilizer would be rejected by the growing ice crystal phase and form a homogeneous concentrated phase. But at moderate and aggressive conditions (80 C and 196 C), the freezing rate f was between s and n, if UDCA crystal possessed low diffusion, it will be trapped in ice crystals forming their own phases. If the diffusion rate of stabilizer molecule was lower, it would be trapped in ice crystals forming their own phase. Therefore, it was speculated that the redispersibility of UDCA-NC during freezing was most likely dependent on freezing rate(f) yielded from different freezing temperature and the diffusion characteristics of the drug crystals (n) as well as stabilizer molecule (s).
The importance of lyophilization process on redispersibility of UDCA nanocrystals Lyophilization process after freezing can inevitably destabilize the NC and impact on the redispersibility of solid NC, due to additional thermal stresses (heat for lyophilization). So the influence of different lyophilization process on the redispersibility of UDCA-NC was investigated.
It was observed that RDI of UDCA-NC, respectively, stabilized by 10% concentration of stabilizers after lyophilization was more than 2, but by 50% concentrations of polymer stabilizers was less than 2 (Figure 7). For example, RDIconventional, RDImoderate and RDIrigorous of UDCA-NC stabilized by 10% concentration of HPMC after lyophilization at three conditions was 1.31, 3.42 and 2.56, respectively, but those of UDCA-NC stabilized by 50% concentration of HPMC was 1.24, 1.28 and 1.16, respectively. It showed that the types and amounts of stabilizers played an important role on the redispersibility of UDCA-NC. It can be seen that UDCA-NC stabilized by HPMC did not form some aggregation or crystals growth at predetermined conditions, but UDCA-NC, respectively, stabilized by RH40 and Tween80 had some aggregation or crystals growth at three conditions. The polymeric stabilizer HPMC possessed better effect on RDI of UDCA-NC than other stabilizers, and the higher the concentration of stabilizers used was, the better protection effect of stabilizers displayed at high concentration condition. Figure 8(A) showed that the SEM image of UDCA-NC stabilized by HPMC after lyophilization at high concentration condition. As a technique complementary to TEM, AFM was another important technology for the characterization of drug NC. Figure 8(B) presented the AFM surface morphology of the UDCA-NC. It was found that no aggregation of UDCA-NC could be observed after rehydration in water. When the amounts for all the employed stabilizers (such as TPGS, HPMC and PVPK30) were up to 50% (relate to the weight of drug), the RDI of UDCA-NC was more nearer to 1, compared to those of 25% and 10%. The results demonstrated that compared to the drying temperature, the employed amount and type of stabilizers more dramatically affected the redispersibility of UDCA-NC during lyophilization. The aggregation of drug NC was inevitable during freezedrying. During drying, capillary pressure enabled drug NC to approach each other and form crystal bridges, and then these crystal bridges combined and large agglomerates could be formed20. However, the aggregation tendency of NS can be counterbalanced by the steric barrier effect of stabilizer, such as HPMC and PVPK30. The polymer stabilizers were effective enough to protect the NS from damage came from drying process, which could homogeneously absorb into the surface of NC and form layer to prevent from agglomerates during lyophilization21.
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Figure 7. The redispersibility index (RDI) of UDCA-NC with different concentration (relative to the drug weight, m/m) of stabilizers after lyophilization at three stress conditions of ‘‘Conventional stress’’, ‘‘Moderate stress’’ and ‘‘Rigorous stress’’, respectively.
Figure 8. SEM(A) and AFM(B) morphology of UDCA-NC with 10% concentration (relative to the drug weight, m/m) of HPMC after lyophilization at rigorous stress condition.
Role of cryoprotectants on redispersibility of UDCA nanocrystals To prevent the irreversible aggregation and maintain the redispersibility of UDCA-NC, cryoprotectants such as sucrose and lactose are often used to fill the gaps between the NC after the removal of water during lyophilization22. Therefore, the protection effects provided by different concentration of cryoprotectants during solidification were scientifically investigated. It was observed that RDI of UDCA-NC, respectively, added into 10% concentration of cryoprotectants (such as lactose, manitol) after lyophilization was more than 2, but 400% concentrations of cryoprotectants (e.g. sucroses, sorbitol) was
less than 2 (Figure 9). The types and concentrations of cryoprotectants played an important role in maintaining good redispersibility features of UDCA-NC, and the higher the concentration of cryoprotectants used was, the better protection effect of cryoprotectants was at three concentration conditions. It can be seen that cryoprotectant sucrose had a better performance on RDI of UDCA-NC than the other cryoprotectants, but the manitol had a worst performance for UDCA-NC, among of all the seven cryoprotectants. It was also found from Figure 9 that, respectively, used a 10% concentration of TPGS and PVPK30 (10%) as stabilizer, RDI of UDCA-NC, respectively, added into high concentration (relate to the weight of drug, 400%) of sucrose and glucose was more nearer
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Figure 9. The redispersibility index (RDI) of UDCA-NC with different concentration (relative to the drug weight, m/m) of cryoprotectants after lyophilization, respectively.
Figure 10. The morphology of UDCA-NC with 400% concentration (relative to the drug weight, m/m) of cryoprotectants after lyophilization, respectively. (A) SEM of UDCA-NC with TPGS-sucrose system; (B) SEM of UDCA-NC with TPGS-glucose system; (C) SEM of UDCA-NC with TPGS-lactose system; (D) AFM of UDCA-NC with TPGS-sucrose system; (E) AFM of UDCA-NC with TPGS-glucose system; (F) AFM of UDCA-NC with TPGS-lactose system.
to 1, compared to the one of 200% and 100%. The surface morphology of UDCA-NC added into cryoprotectants after lyophilization also demonstrated that sucrose and glucose (Figure 10A and B) possessed better performance that protected
UDCA-NC from crystal growth during lyophilization compared with other cryoprotectants (Figure 10C), respectively. These could be demonstrated by AFM morphology of UDCA-NC (Figure 10D, E and F). Furthermore, PVPK30-sucrose and
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DOI: 10.3109/10837450.2014.982822
PVPK30-glucose protection systems used at 100% concentration condition possessed better performance that protected UDCA-NC from crystal growth during lyophilization, compared with other systems, respectively. It was thought that the redispersibility of NC might be related with a synergistic effect relationship between polymer stabilizer and cryoprotectants. It was well understood that during freezing, the cryoprotectants solution became freezeconcentrated and then became a stable glass with a high viscosity and low mobility, prevented NC fusion/aggregation and protected from damage by ice crystals, finally, the hydrogen bonds of sugars reduced the interactions between the water and UDCA-NC and then replaced the water during lyophilization. Furthermore, the interactions between the hydrogen bonds sugars and polymer stabilizer head groups reduced the entanglement of polymer chains in the dry state and prevented the steric barrier effect of polymer stabilizer from damage23.
Conclusion Solid NC were novel drug delivery system to improve the physical and chemical stability of liquid NS. The solidification process was known to be the crucial role for solidification formability of NS during freeze-drying. The UDCA-NC as a model case was investigated for influence of different solidification transformation on the redispersibility of drug NS. The results demonstrated that the freezing condition was a crucial factor for redispersibility of UDCA-NC during freezing, and the redispersibility of UDCANC at conventional temperature was better more than those at the moderate and rigorous conditions. The polymer stabilizers were effective enough to protect the NS from damage came from the various during lyophilization, which could homogeneously absorb into the surface of NC to prevent from agglomerates during lyophilization. The types and concentrations of cryoprotectants played an important role in maintaining the redispersibility features of UDCA-NC. The sucrose and glucose achieved excellent performance that protected UDCA-NC from crystal growth during lyophilization. However, the in-depth mechanism behind the phenomenon is not yet well-understood in this study. This further systematically elucidates protection mechanism for drug NC during solidification.
Declaration of interest The authors report no conflicts of interest.
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RESEARCH ARTICLE
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Solidification drug nanosuspensions into nanocrystals by freeze-drying: a case study with ursodeoxycholic acid Yue-Qin Ma, Zeng-Zhu Zhang, Gang Li, Jing Zhang, Han-Yang Xiao, and Xian-Fei Li Department of Pharmaceutics, the 94th Hospital of People’s Liberation Army, Nanchang, China
Abstract
Keywords
To elucidate the effect of solidification processes on the redispersibility of drug nanocrystals (NC) during freeze-drying, ursodeoxycholic acid (UDCA) nanosuspensions were transformed into UDCA-NC via different solidification process included freezing and lyophilization. The effect of different concentrations of stabilizers and cryoprotectants on redispersibility of UDCA-NC was investigated, respectively. The results showed that the redispersibility of UDCA-NC was RDI20 C5RDI80 C5RDI196 C during freezing, which indicated the redispersibility of UDCANC at the conventional temperature was better more than those at moderate and rigorous condition. Compared to the drying strengthen, the employed amount and type of stabilizers more dramatically affected the redispersibility of UDCA-NC during lyophilization. The hydroxypropylmethylcellulose and PVPK30 were effective to protect UDCA-NC from damage during lyophilization, which could homogeneously adsorb into the surface of NC to prevent from agglomerates. The sucrose and glucose achieved excellent performance that protected UDCA-NC from crystal growth during lyophilization, respectively. It was concluded that UDCANC was subjected to agglomeration during solidification transformation, and the degree of agglomeration suffered varied with the type and the amounts of stabilizers used, as well as different solidification conditions. The PVPK30-sucrose system was more effective to protect UDCA-NC from the damage during solidification process.
Freeze-drying, nanocrystals, solidification stress, ursodeoxycholic acid
Introduction Nanosuspensions (NS) is generally produced in liquid media in which drug particles size is less than 1 lm and stabilized by surfactants or polymers. NS possess the advantages that enhance the solubility and dissolution velocity of poorly soluble drugs due to their small particle size and high surface area1. However, NS are essentially thermodynamically unstable systems. The enormous surface area and the small size of these particles results in high interfacial tension, which in turn results in an increase in the free energy of the system2. Hence, NS would tend to generate flocculation, aggregation or crystal growth to decrease their free energy. The physical stability problems, which limits the industrialization of NS, are crucial to be solved and recently have been widely investigated. In order to improve the physical stability of liquid NS, NS have to be solidified and then processed further into tablets or capsules. Solid nanosuspensions (SNS) or nanocrystals (NC) (Figure 1) is composed of drug as well as stabilization agent, and can be easily redispersed back to original NS states instantaneously on mild agitation or peristalsis followed by rehydration with aqueous media in vitro or gastrointestinal
Address for correspondence: Yue-Qin Ma and Gang Li, Department of Pharmaceutics, the 94th Hospital of People’s Liberation Army, Nanchang, China. E-mail: [email protected] (Y.-Q. Ma); [email protected] (G. Li)
History Received 8 July 2014 Revised 22 October 2014 Accepted 28 October 2014 Published online 26 November 2014
tract (redispersibility), if they did not go through irreversible aggregation during solidification3,4. Freezing-drying can be used to transform liquid NS into solid NC5–11. The drying process consists on removing water from NS sample by sublimation and desorption under vacuum, or evaporation under low temperature. Nevertheless, this process generates additional thermal stresses (due to heat or freezing for lyophilization), which could inevitably destabilize NC and impact on the redispersibility of NC. For example, if the NS are coated with polymeric surfactants such as poloxamers, drying may lead to crystallization of the polymer, thereby compromising their ability to prevent aggregation. So far, literature about impact of solidification process conditions on redispersibility characteristics of SNS is lacking12, and there are no generally accepted views on the formation of hard agglomerates of NC. In view of these considerations, understanding the impact of solidification processes on redispersibility of solid NC is important. Furthermore, prior to solidification, the cryoprotectant for freeze-drying is often added into the NS, which can be used to protect the NS from solidification damage. Typical cryoprotectants added prior to freeze-drying are water-soluble materials such as sugars (e.g. sucrose, saccharose, and lactose), sugar alcohols (e.g. mannitol, sorbitol)13. However, if the nature of a cryoprotectant or dispersant is inappropriate for drying of drug NC, even excessive amounts are unable to prevent the system from freezing and drying damage. Therefore, it is strongly needed to systematically evaluate the influence of type and concentration
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Figure 1. Schematic illustrations on redispersibility of drug nanocrystals.
of cryoprotectant or dispersants on redispersibility of NC after freeze-drying. Ursodeoxycholic acid (UDCA) is a cholagogue, cholelitholytic and hepatic protector agent and exhibits low bioavailability for oral delivery due to its poor water solubility14. NS can be used to improve the solubility and bioavailability of UDCA. During solidification of NS, it was found that the surface hydrophobicity of drug were responsible for the formability of the SNS during solidification15. But UDCA was a strong hydrophobic drug, so it might be very difficult to transform UDCA-NS into fine UDCANC. The main objective of this artilce is to provide a case study for influence of different solidification conditions on the redispersibility of solid NC. UDCA-NS stabilized by different types of stabilizers were prepared by high pressure homogenization. And the concentration of each stabilizers employed (relative to the weight of UDCA) was 50%, 25% and 10%, respectively. UDCA-NS was transformed into UDCA-NC via freeze-drying applied with different process conditions. And the effect of different series of concentrations of cryoprotectants (sucrose, glucose, trehalose, lactose, manitol, sorbitol and PEG4000) on redispersibility of UDCA-NC was investigated during lyophilization, respectively. The redispersibility of UDCA-NC obtained at predetermined condition was evaluated by means of laser light scattering, scanning electron microscopy and transmission electron microscopy.
Materials and methods Materials UDCA was purchased from Zelang Co. (Nanjing, China). D-a-tocopherol polyethylene glycol 1000 succinate (TPGS) was purchased from Xi’an Healthful Biotechnology Co., Ltd. (Xi’an, China). Poloxamer 188 (P188, LutrolÕ F 68), poloxamer 407(P407, LutrolÕ F 127) and polyoxyethylene hydrogenated castor oil (RH40, CremophorÕ RH 40) were kindly donated by BASF (Ludwigshafen, Germany). Povidone 30 (PK30, PlasdoneÕ K-29/32) were kindly donated by JSP (NJ).
Hydroxypropylmethylcellulose (HPMC, Methocel E15LV Premium EPÕ, Colorcon, Dartford, UK), Sodium dodecyl sulfate (SDS, SHANHE, Anhui, China), Tween 80 (TW80, SHANHE, Anhui, China), polyethylene glycol 4000 (PEG4000, SHANHE, Anhui, China) trehalose (Asahi KASEI, Chiyoda-Ku, Japan), sodium carboxymethyl starch (CMS-Na, SHANHE, Anhui, China) and Microcrystalline cellulose and carboxymethyl cellulose sodium (MC, CeolusÔ RC-A591NF, Asahi KASEI, Chiyoda-Ku, Japan) were commercially obtained. Glucose, sucrose, manitol, sorbitol and lactose were obtained from DAMAO Chemical Co., Ltd. (Tianjin, China). Nanosuspensions production UDCA-NS was prepared by high-pressure precipitation tandem homogenization technology16. UDCA coarse powder was completely dissolved in the proper amount of ethanol as organic phase. Different concentrations of stabilizers were dispersed in 100-ml water as water phase. The organic phase loaded 12.5% concentrations of UDCA (w/v) was pumped at a fixed flow rate of 10 ml/min using an injection pump into the inflow reservoir of piston-gap high pressure homogenizer (AH-1000D, ATS Engineering Inc., Seeker, Canada) containing the anti-solvent (100 ml aqueous stabilizer solution). Homogenization was performed at 300–700 bar for 20 cycles as high pressure precipitation step. The NS was subjected to homogenizing for another 20 cycles under 600–1000 bar in continuous mode as high pressure homogenization step. A cold water bath system was used to hold temperature during the whole homogenization process.
Solidification transformation Freezing process The UDCA-NS stabilized by different polymeric dispersants were frozen at different freezing temperature conditions generated by different temperatures. The UDCA-NS (3 mL) in a 10-mL vial
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DOI: 10.3109/10837450.2014.982822
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Table 1. The applied freezing process conditions with different stresses. Temperature conditions
Conventional stress
20 C for12 h
Freezing
Moderate stress
Rigorous stress
196 C for 2 h
80 C for 6 h
Table 2. The applied lyophilization process cycle with different stress conditions. Lyophilization
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Cycle condition
Pre-freezing
Ramp rate
Primary drying
Ramp rate
Secondary drying
Conventional stress
40 C for 60 min
1 C /min
0.05 C /min
10 C for 6 h
Moderate stress
40 C for 60 min
1 C /min
0.2 C /min
10 C for 6 h
Rigorous stress
40 C for 60 min
1 C /min
20 C for 8 h; 10 C for 12 h 10 C for 10 h; 0 C for 8 h 0 C for 12 h
0.8 C /min
10 C for 8 h
were frozen according to the process temperature conditions in Table 1. Then, the system was thawed at room temperature. The average particle sizes were determined. Measurements were made in triplicate for all the measurement runs.
(freezing and lyophilization) and D represents the corresponding value of reconstituted UDCA-NS post-solidification. An RDI of near 1 would therefore means that UDCA-NC powder obtained by solidification transformation can be completely reconstituted back to the original particle size after rehydration.
Lyophilization process The UDCA-NS stabilized by different stabilizers were dried by lyophilization. Each UDCA-NS (3 mL) was freeze-dried in a 10 mL vial using freeze-dry system (FreezeZoneÕ Stoppering Tray Dryers, LABCONCO Corporation, Kansas City, MO). The applied cycle conditions were as follows: freezing was performed at 40 C for 60 min. The shelf temperature ramp rates from the freezing step into the primary drying step were 1 C/min for all cycles performed. Three sets of primary drying conditions were employed according to Table 2. Lyophilization process with cryoprotectant The different amount (100%, 200% and 400%, relative to the weight of UDCA) of cryoprotectants (sucrose, glucose, trehalose, lactose, manitol, sorbitol and PEG4000) was, respectively, added into UDCA-NS stabilized by 10% concentration (relative to the weight of UDCA) of TPGS or PVPK30, respectively. UDCANS were freeze-dried in a 10mL vial using freeze-dry system (FreezeZoneÕ Stoppering Tray Dryers, LABCONCO Corporation, KS). The applied cycle conditions were as follows: freezing was performed at 40 C for 60 min. Primary drying was performed at 20 C for 8 h; 10 C for 6 h; and 0 C for 5 h. The shelf temperature ramp rates from the freezing step into the primary drying step were 1 C/min. The secondary drying was performed at 10 C for 6 h. The shelf temperature ramp rates from the freezing step into the primary drying step were 0.5 C/min. Laser diffractometry (LD) LD was performed on a Mastersizer Micro Plus (Malvern Instruments Limited, Worcestershire, UK). Analysis of the diffraction patterns was done using the Mie model. From the resulting volume distributions, the median was calculated ( ¼ 50% volume percentile, D50). All measurements were performed in triplicate.
Transmission electron microscope (TEM) The morphology of UDCA-NS was observed by TEM (JEM1200EX, Japan). One drop of drug NS was placed on a copper grid and stained with 2% phosphotungstic acid solution for 5 min. The grid was dried at room temperature and was evaluated with the electron microscope. Scanning electron microscopy (SEM) Morphological evaluation of representative samples of UDCANC powder subjected to different solidification conditions was performed and compared against each other under scanning electron microscope( SEM) (Hitachi X650, Tokyo, Japan). All samples were examined on a brass stub using carbon double-sided tape. UDCA-NC samples were glued and mounted on metal sample plates. The samples were gold coated (thickness&15–20nm) with a sputter coater (Fison Instruments, Manchester, UK) using an electrical potential of 2.0 kV at 25 mA for 10 min. An excitation voltage of 20 kV was used in the experiments. Atomic force microscopy (AFM) The surface morphology on three-dimension of re-dispersed UDCA-NS after freeze-drying was investigated by atomic force microscope (AFM) using non-contact mode. Prior to analysis, the surface of mica slice was cleaned with coated fabric to avoid analysis artifacts. With the help of a micropipette, the diluted samples were dropped onto the mica slice and blew into lamellar by nitrogen gas in super-clean bench, following drying at ambient temperature for 2 d. Imaging was performed on Nanoscope IIIA (Model-Veeco Bioscope II, Veeco Instruments Ins, Plainview, NY).
Results and discussion Preparation of ursodeoxycholic acid nanosuspensions
Redispersibility index (RDI) RDI ¼ D0 =D where D0 represents the volume-weighed mean particle size of the freshly prepared UDCA-NS directly prior to solidification
Mean particle size (d50) and average span values of UDCA-NS, respectively, stabilized by different concentrations of stabilizers were listed in Figures 2 and 3. The particle size of NS was in rang of 600–900 nm. These results demonstrated that the coarse UDCA were completely disintegrated to nano-sized particles by means of
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Figure 2. The particle size of freshly prepared UDCA-NC with different concentrations of stabilizers.
Figure 3. The span of freshly prepared UDCA-NC with different concentrations of stabilizers.
high pressure homogenization technology, and successfully formed the different UDCA-NCs in terms of different stabilizers. Effect of freezing process on redispersibility of ursodeoxycholic acid nanosuspensions The freezing step is more significant than the subsequent sublimation step for redispersibility of drug NS during freeze-drying17. Hence, before sublimation step, different freezing processes were employed to investigate the influence of different freezing process on the redispersibility of NS. The freezing conditions and the RDI of NS stabilized by different concentration of stabilizers, respectively, after freeze-thawing were gathered in Figure 4. The TEM images of three representative UDCA-NS reconstructed after freeze-thawing were showed in Figure 5. The results indicated that UDCA-NS reconstructed after freeze-thawing had some aggregations or crystals growth, compared with that of freshly prepared NS.
The redispersibility of UDCA-NC depends on not only the freezing strength but also the type of stabilizers. Figure 4 showed that RDI of UDCA-NC, respectively, stabilized by different concentrations of stabilizers after freezing at three conditions was more than 1. For instance, RDI20 C, RDI80 C and RDI196 C of UDCA-NC stabilized by 10% concentration of PVPK30 was 2.98, 9.01 and 14.16, respectively, but those of UDCA-NC stabilized by 50% concentration of PVPK30 was 1.16, 5.32 and 9.65, respectively. It indicated that the different freezing temperature could induce the irreversible aggregation of UDCA-NC during freezing step. It can also be observed that the redispersibility of the frozen UDCA-NC at three conditions were RDI20 C5RDI80 C5RDI196 C. These results meant that the redispersibility of frozen UDCA-NC at the conventional freezing temperature (meant low freezing rate) was better than those at the moderate and rigorous temperature conditions(high freezing rate). Besides, the type and amounts of stabilizers at an equivalent freezing process have dramatically different protection effect on
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DOI: 10.3109/10837450.2014.982822
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Figure 4. The redispersibility index (RDI) of UDCA-NC with different concentrations of stabilizers (relative of drug weight, m/m) after freezing at three stress conditions of ‘‘Conventional stress’’, ‘‘Moderate stress’’ and ‘‘Rigorous stress’’, respectively.
Figure 5. TEM images of three representative UDCA-NC stabilized by P188, TPGS and HPMC, respectively. A, B, C represent TEM images of UDCA-NS stabilized by P188, TPGS and HPMC, respectively. 1, 2, 3 represent ‘‘Conventional stress’’, ‘‘Moderate stress’’ and ‘‘Rigorous stress’’ conditions (20 C, 80 C and 196 C), respectively.
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Figure 6. Schematic illustration of particle separation in unfrozen phase dependent on freeze rate, diffusion rate of nanocrystals and stabilizer molecule.
UDCA-NC. And the higher was the concentration of stabilizers, the more near to 1% was RDI of UDCA-NC among the three temperature conditions. It meant that redispersibility of UDCANC treated by 50% concentration of surfactants or polymers stabilizers became much better than those with 25% and 10% concentration. The freezing temperature (determine the freezing rate (f) of ice) could be responsible for aggregation or even fusion of drug NS occurred during freezing process. As illustrated in Figure 6, with increase of freezing rate, water molecules excluded the UDCA-NC particles and lead them approach each other and aggregate eventually, simultaneously the activity of stabilizers was no longer effective due to separation of drug crystals and stabilizer molecule in the unfrozen concentrated phase17–19. That is, if a stabilizer polymer chain might be considered to be a particle and there is no interaction among particles, the size of UDCA-NC was at least one order of magnitude larger than that of stabilizer, and diffusive rate(s) of stabilizer might be larger than diffusive rate(n) of UDCA-NC(data not shown). So at conservative conditions (20 C), the freezing rate f, was smaller than s and n, then both the HAR-NC and stabilizer would be rejected by the growing ice crystal phase and form a homogeneous concentrated phase. But at moderate and aggressive conditions (80 C and 196 C), the freezing rate f was between s and n, if UDCA crystal possessed low diffusion, it will be trapped in ice crystals forming their own phases. If the diffusion rate of stabilizer molecule was lower, it would be trapped in ice crystals forming their own phase. Therefore, it was speculated that the redispersibility of UDCA-NC during freezing was most likely dependent on freezing rate(f) yielded from different freezing temperature and the diffusion characteristics of the drug crystals (n) as well as stabilizer molecule (s).
The importance of lyophilization process on redispersibility of UDCA nanocrystals Lyophilization process after freezing can inevitably destabilize the NC and impact on the redispersibility of solid NC, due to additional thermal stresses (heat for lyophilization). So the influence of different lyophilization process on the redispersibility of UDCA-NC was investigated.
It was observed that RDI of UDCA-NC, respectively, stabilized by 10% concentration of stabilizers after lyophilization was more than 2, but by 50% concentrations of polymer stabilizers was less than 2 (Figure 7). For example, RDIconventional, RDImoderate and RDIrigorous of UDCA-NC stabilized by 10% concentration of HPMC after lyophilization at three conditions was 1.31, 3.42 and 2.56, respectively, but those of UDCA-NC stabilized by 50% concentration of HPMC was 1.24, 1.28 and 1.16, respectively. It showed that the types and amounts of stabilizers played an important role on the redispersibility of UDCA-NC. It can be seen that UDCA-NC stabilized by HPMC did not form some aggregation or crystals growth at predetermined conditions, but UDCA-NC, respectively, stabilized by RH40 and Tween80 had some aggregation or crystals growth at three conditions. The polymeric stabilizer HPMC possessed better effect on RDI of UDCA-NC than other stabilizers, and the higher the concentration of stabilizers used was, the better protection effect of stabilizers displayed at high concentration condition. Figure 8(A) showed that the SEM image of UDCA-NC stabilized by HPMC after lyophilization at high concentration condition. As a technique complementary to TEM, AFM was another important technology for the characterization of drug NC. Figure 8(B) presented the AFM surface morphology of the UDCA-NC. It was found that no aggregation of UDCA-NC could be observed after rehydration in water. When the amounts for all the employed stabilizers (such as TPGS, HPMC and PVPK30) were up to 50% (relate to the weight of drug), the RDI of UDCA-NC was more nearer to 1, compared to those of 25% and 10%. The results demonstrated that compared to the drying temperature, the employed amount and type of stabilizers more dramatically affected the redispersibility of UDCA-NC during lyophilization. The aggregation of drug NC was inevitable during freezedrying. During drying, capillary pressure enabled drug NC to approach each other and form crystal bridges, and then these crystal bridges combined and large agglomerates could be formed20. However, the aggregation tendency of NS can be counterbalanced by the steric barrier effect of stabilizer, such as HPMC and PVPK30. The polymer stabilizers were effective enough to protect the NS from damage came from drying process, which could homogeneously absorb into the surface of NC and form layer to prevent from agglomerates during lyophilization21.
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DOI: 10.3109/10837450.2014.982822
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Figure 7. The redispersibility index (RDI) of UDCA-NC with different concentration (relative to the drug weight, m/m) of stabilizers after lyophilization at three stress conditions of ‘‘Conventional stress’’, ‘‘Moderate stress’’ and ‘‘Rigorous stress’’, respectively.
Figure 8. SEM(A) and AFM(B) morphology of UDCA-NC with 10% concentration (relative to the drug weight, m/m) of HPMC after lyophilization at rigorous stress condition.
Role of cryoprotectants on redispersibility of UDCA nanocrystals To prevent the irreversible aggregation and maintain the redispersibility of UDCA-NC, cryoprotectants such as sucrose and lactose are often used to fill the gaps between the NC after the removal of water during lyophilization22. Therefore, the protection effects provided by different concentration of cryoprotectants during solidification were scientifically investigated. It was observed that RDI of UDCA-NC, respectively, added into 10% concentration of cryoprotectants (such as lactose, manitol) after lyophilization was more than 2, but 400% concentrations of cryoprotectants (e.g. sucroses, sorbitol) was
less than 2 (Figure 9). The types and concentrations of cryoprotectants played an important role in maintaining good redispersibility features of UDCA-NC, and the higher the concentration of cryoprotectants used was, the better protection effect of cryoprotectants was at three concentration conditions. It can be seen that cryoprotectant sucrose had a better performance on RDI of UDCA-NC than the other cryoprotectants, but the manitol had a worst performance for UDCA-NC, among of all the seven cryoprotectants. It was also found from Figure 9 that, respectively, used a 10% concentration of TPGS and PVPK30 (10%) as stabilizer, RDI of UDCA-NC, respectively, added into high concentration (relate to the weight of drug, 400%) of sucrose and glucose was more nearer
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Figure 9. The redispersibility index (RDI) of UDCA-NC with different concentration (relative to the drug weight, m/m) of cryoprotectants after lyophilization, respectively.
Figure 10. The morphology of UDCA-NC with 400% concentration (relative to the drug weight, m/m) of cryoprotectants after lyophilization, respectively. (A) SEM of UDCA-NC with TPGS-sucrose system; (B) SEM of UDCA-NC with TPGS-glucose system; (C) SEM of UDCA-NC with TPGS-lactose system; (D) AFM of UDCA-NC with TPGS-sucrose system; (E) AFM of UDCA-NC with TPGS-glucose system; (F) AFM of UDCA-NC with TPGS-lactose system.
to 1, compared to the one of 200% and 100%. The surface morphology of UDCA-NC added into cryoprotectants after lyophilization also demonstrated that sucrose and glucose (Figure 10A and B) possessed better performance that protected
UDCA-NC from crystal growth during lyophilization compared with other cryoprotectants (Figure 10C), respectively. These could be demonstrated by AFM morphology of UDCA-NC (Figure 10D, E and F). Furthermore, PVPK30-sucrose and
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DOI: 10.3109/10837450.2014.982822
PVPK30-glucose protection systems used at 100% concentration condition possessed better performance that protected UDCA-NC from crystal growth during lyophilization, compared with other systems, respectively. It was thought that the redispersibility of NC might be related with a synergistic effect relationship between polymer stabilizer and cryoprotectants. It was well understood that during freezing, the cryoprotectants solution became freezeconcentrated and then became a stable glass with a high viscosity and low mobility, prevented NC fusion/aggregation and protected from damage by ice crystals, finally, the hydrogen bonds of sugars reduced the interactions between the water and UDCA-NC and then replaced the water during lyophilization. Furthermore, the interactions between the hydrogen bonds sugars and polymer stabilizer head groups reduced the entanglement of polymer chains in the dry state and prevented the steric barrier effect of polymer stabilizer from damage23.
Conclusion Solid NC were novel drug delivery system to improve the physical and chemical stability of liquid NS. The solidification process was known to be the crucial role for solidification formability of NS during freeze-drying. The UDCA-NC as a model case was investigated for influence of different solidification transformation on the redispersibility of drug NS. The results demonstrated that the freezing condition was a crucial factor for redispersibility of UDCA-NC during freezing, and the redispersibility of UDCANC at conventional temperature was better more than those at the moderate and rigorous conditions. The polymer stabilizers were effective enough to protect the NS from damage came from the various during lyophilization, which could homogeneously absorb into the surface of NC to prevent from agglomerates during lyophilization. The types and concentrations of cryoprotectants played an important role in maintaining the redispersibility features of UDCA-NC. The sucrose and glucose achieved excellent performance that protected UDCA-NC from crystal growth during lyophilization. However, the in-depth mechanism behind the phenomenon is not yet well-understood in this study. This further systematically elucidates protection mechanism for drug NC during solidification.
Declaration of interest The authors report no conflicts of interest.
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