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Hydroxypropylcellulose as matrix carrier for novel cage-like microparticles prepared by spray-freeze-drying technology
Accepted Manuscript Title: Hydroxypropylcellulose as matrix carrier for novel cage-like microparticles prepared by spray-freeze-drying technology Author: Shaofeng Wei Yueqin Ma Jing Luo Xiaoru He Pengfei Yue Zhiyu Guan Ming Yang PII: DOI: Reference:
S0144-8617(16)31208-5 http://dx.doi.org/doi:10.1016/j.carbpol.2016.10.043 CARP 11663
To appear in: Received date: Revised date: Accepted date:
20-7-2016 14-10-2016 14-10-2016
Please cite this article as: Wei, Shaofeng., Ma, Yueqin., Luo, Jing., He, Xiaoru., Yue, Pengfei., Guan, Zhiyu., & Yang, Ming., Hydroxypropylcellulose as matrix carrier for novel cage-like microparticles prepared by spray-freeze-drying technology.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.10.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
TITLE PAGE
Hydroxypropylcellulose as matrix carrier for novel cage-like microparticles prepared by spray-freeze-drying technology
Shaofeng Wei 1#, Yueqin Ma2#, Jing Luo1, Xiaoru He1, Pengfei Yue1*, Zhiyu Guan1, Ming Yang1
1
Jiangxi University of Traditional Chinese Medicine, 818 MEILINGDADAO Road,
Nanchang 330004, China 2
Department of Pharmaceutics, 94th Hospital of People’s Liberation Army, Nanchang
330000, China
*Corresponding authors at: Jiangxi University of Traditional Chinese Medicine, 818 MEILINGDADAO Road, Nanchang 330004, China E-mail: [email protected]
#
Shaofeng Wei and Yueqin Ma contributed to the work equally as joint first authors.
Highlights:
•A novel cage-like composite particles used HPC as carrier was developed.
•An interesting strategy by means of spray-freeze-drying technique was used.
• The spray-freeze-dried CMs exhibited excellent dissolution-enhanced specialty.
• HPC was demonstrated to be an effective matrix carrier for drug nanocrystals.
ABSTRACT: The objective of this study is to design novel dissolution-enhanced microparticles loaded poorly soluble drug nanocrystals used a low viscosity of hydroxypropylcellulose (HPC) as matrix carrier. An interesting approach combined homogenization and the spray-freeze-drying technique was developed. The results demonstrated that the ratio of HPC to drug played an important role in size-reduction efficiency of drug during homogenization. And the formation of cage-like structure of the composite particles depended on ratio of HPC to drug. The spray-freeze-dried composite particles with HPC ratio of 1:2, 1:1 and 2:1 possessed excellent redispersibility, which attributed to its porous matrix and large surface area (3000m2/g). The dissolution of spray-freeze-dried composite particles with higher ratios of HPC (1:2 and 1:1) was significantly enhanced, which attributed to the particle size reduction of drug. The HPC could immobilize drug nanocrystals in its cage-like structure and prevent it from the subsequent agglomeration during storage. In conclusion, the prepared cage-like microparticles is a promising basis for further formulation development.
Keywords: hydroxypropylcellulose; nanocrystals; cage-like microparticles; spray-freeze-drying; redispersibility
1. Introduction Driven by increasing demands for improving human health, a lot of studies have been carried out to develop innovative pharmaceutical preparations or functional foods by incorporation of all kinds of bioactive ingredients (Velikov and Pelan, 2008; Chen, Seiber and Hotze, 2014). However, many active ingredients are difficultly introduced into pharmaceutical preparations, mainly due to their poor aqueous solubility, which would decrease their oral bioavailability, and hence, limit their use in clinical (Lipinski et al., 2002; Joye et al., 2014). Therefore, it is an urgent need to solve this problem at present. The reduction of drug particle sizes down to the nanometer or submicron scale has been an effective approach of increasing the dissolution rate of poorly soluble drugs (Muller and peter, 1998). Nanocrystals suspension (NS, typically particles smaller than 1 μm) has already been recognized as one of the promising reduction of particles size technologies for poorly soluble drugs in pharmaceutics. NS has some unique advantages that enhance the solubility and dissolution velocity of poorly soluble drug due to their small particle size and high surface area(Jinno et al. 2006; Merisko-Liversidge and Liversidge 2011; Ige et al. 2013), and strengthen the adhesion to biological membrane, prolong adhesion time and detention time (Pawar et al. 2014; Gao et al. 2012). Fine drug NS can usually be produced in liquid media either by “top-down” approaches such as wet media milling (Bilgili et al., 2004; 2006), high-pressure homogenization(Akkar et al., 2004), and jet milling (Vogt et al., 2008) or by “bottom-up” approach such as liquid anti-solvent precipitation (Dalvi and Dave, 2010). Nanocrystals suspensions have a large surface area, leading to significant inter-particle interactions through van der Waals forces, hydrophobic forces, etc. The attractive inter-particle forces can ultimately result in particle aggregation if the suspensions are not properly stabilized. Stabilizing effect can be achieved using polymers, surfactants or a combination of both; however, the particle growth can also occur in the suspension by Ostwald ripening during storage (Wu et al., 2011). Therefore, from a stability perspective as well as patient preference/compliance
perspective, powderization of liquid NS is an effective approach, which can reconstitute into original nanosuspensions states after rehydration (Yue et al., 2012). Spray-drying has been widely applied in powderization of nanosuspensions, because it is advantageous to produce the spherical and size-controlled particles (Figueroa and Bose 2013; Iskandar et al. 2003). However, this technique is not always appropriate for thermolabile or oxidizable ingredients because the spray-drying process requires elevated temperatures. Spray-freeze-drying (SFD) is a novel particle engineering technology (Chow et al., 2007), in which either an aqueous or an aqueous-organic cosolvent solution containing an active ingredient and a pharmaceutical excipient is atomized directly into super-cool phase to form frozen and solid particles. After that these frozen particles will be dried using lyophilization and these processes generally produce fine particles with a larger median particle size, and a significant large specific surface area and high yields (Parsian et al., 2014; Wanning et al., 2015). During the spray-freeze-drying processes, nanoparticles can aggregate into microparticles (particles of size>1μm). However, a key concern with spray-freeze-drying process is that nanocrystals incorporated in composite microparticles may not be recovered to original nanosuspensions states. Therefore, the proper matrix carrier is indispensable for solid nanocrystals after spray-freeze-drying. Hydroxypropylcellulose (HPC, Fig.1) a cellulosic polymer in which some of the hydroxyl groups in the repeating sugar units are hydroxypropylated using propylene oxide (Mezdour et al., 2008). It is an amphiphilic large molecular which is soluble in a range of organic solvents and water. HPC has been frequently used as a carrier of solid dispersion of poorly soluble drug (Sarode et al., 2014; Rehmana et al., 2013). However, the aim of this work is to evaluate the potential of HPC used as a matrix carrier of composite microparticles incorporated with poorly soluble drug nanocrystals. As illustrated in Fig.1, this study elucidated an interesting strategy to combine homogenization with spray-freeze-drying technology to prepare the composite microparticles loaded poorly soluble drug. In this work, a diterpenoid lactone
(code-named as AGP) was used as model drug. AGP has been demonstrated to have a wide range of biological activities including anti-inflammatory and anti-virus. Unfortunately, AGP has a relative low bioavailability (1.9%) after oral administration, which is associated with its high lipophilicity (log P = 2.6) and low aqueous solubility (3.3µg/mL), and it is also a temperature-sensitive drug. AGP nanocrystals suspension (AGP-NS) stabilized by HPC were prepared by homogenization technology and further converted into the AGP microparticles with cage-like structure (AGP-CM) via spray-freeze-drying. Morphology of the spray-freeze-dried AGP-CM was visualized by scanning electronic microscopy (SEM). Characteristics of AGP in the AGP-CM were analyzed by powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC). The redispersibility, dissolution and stability evaluation were performed to investigate the feasibility of HPC as a carrier for AGP-CM. 2. Materials and methods 2.1 Materials AGP was obtained from Zelan Co. (Nanjing, China). Hydroxypropylcellulose (HPC, Klucel® EF) was kindly donated by Chineway Pharmaceutical Technology Co., LTD (SHANGHAI, China). 2.2 Homogenization of AGP-NS AGP-NS was prepared by high pressure homogenization. Before producing AGP-NS, AGP coarse powder (1%, w/v) was dispersed in different concentration of HPC solutions. The amount of HPC to drug was varied from 1:20, 1:10, 1:5, 1:2 to 1:1, and 2:1(w/w). The resultant mixture of AGP and HPC solution was dispersed into coarse suspension by a high shear homogenizer (FLUKO® FA25, Essen, Germany) at 19,000 rpm for 2 min. And then the gained coarse suspension was homogenized into AGP-NS at predetermined pressure using a high-pressure homogenizer with cooling system (AH-1000D, ATS Engineering Inc., Seeker, Canada). The cooling temperature was fixed at 10 ◦C. The AGP formulations with different compositions of HPC and AGP during homogenization were showed in Table 1. 2.3 Spray-freeze-drying of AGP-CM The resultant AGP-NS with various compositions of HPC (Table 1) were
converted into AGP-CM by means of a modified Büchi B-290 mini spray dryer (Büchi, Switzerland) by removing its drying chamber (Cheow et al., 2011). A polypropylene vessel containing 400 mL of liquid nitrogen (-196 ◦C) was placed beneath the spray dryer’s two-fluid atomizer under constant stirring at 500 rpm. The distance between the atomizer and the polypropylene vessel was fixed at 10 cm to minimize the loss of the sprayed droplets due to impaction at the vessel wall. The atomization was performed at feed rate of 10 mL/min, atomizing air flow at 30 mmHg, and feed solid concentration of 1% (w/v). After the atomization, the frozen AGP-NS were dried by means of freeze-dryer (FreezeZone Stoppering Tray Dryers, LABCONCO Corporation, Kansas, USA). The applied cycle conditions were as follows: freezing was performed at -40◦C for 60min. The shelf temperature ramp rates from the freezing step in to the primary drying step were 1◦C/min. Primary drying and secondary drying was performed at -20 ◦C and 10 ◦C for 12 h, respectively. The shelf temperature ramp rates from the primary-drying step in to the secondary drying step were 1 ◦C/min. 2.4 Particle size and distribution assay of AGP-NS The particles size determination of AGP-NS was performed on a Mastersizer Micro Plus (Malvern Instruments Limited, Worcestershire, UK). All the measurements were carried out in triplicate. The average 10%, 50% and 90% volume percentile (D10, D50 and D90) were determined. And span = (D10-D90)/D50, which was used to evaluate the particle size distribution of AGP-NS. 2.5 TEM Morphology of AGP-NS One drop of the AGP-NS with different ratios of HPC and AGP (Table 1) was placed on a copper grid and stained with 2% phosphotungstic acid solution for 5 min. The grid was dried at room temperature for 12 h and examined using Transmission electron microscope (JEM-1200EX, Japan). 2.6 Redispersibility index (RDI) of AGP-CM after rehydration The redispersibility of AGP-CM was evaluated as described in Dan et al, 2016: RDI D
D0
Where D0 represents the volume-weighed mean particle size of the freshly prepared AGP-NS directly prior to spray-freeze-drying and D represents the corresponding value of reconstituted AGP-NS after spray-freeze-drying. An RDI of near 1 would therefore mean that AGP-CM can be completely reconstituted into the original AGP-NS after rehydration. 2.7 Scanning electron microscopy (SEM) Morphological evaluation of the spray-freeze-dried AGP-CM samples with different ratios of HPC and AGP (Table 1) and coarse AGP were performed by means of scanning electron microscope (SEM) (Hitachi X650, Tokyo, Japan). All samples were examined on a brass stub using carbon double-sided tape. AGP-CM samples were glued and mounted on metal sample plates. The samples were gold coated (thickness ≈ 15-20 nm) with a sputter coater (Fison Instruments, 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. 2.8 Differential scanning calorimetry (DSC) Thermograms of coarse AGP powder, HPC, and AGP-CM with different compositions of HPC were performed using a DSC (Diamond DSC, Perkin-Elmer, USA) equipped with an intercooler. The samples were analyzed in open aluminum pans and scanned under a nitrogen purge with a heating rate of 10 ◦C /min from 20 ◦C to 260 ◦C, with an empty pan as a reference. 2.9 X-ray powder diffraction (XPRD) The characterizations of coarse AGP powder, HPC, and AGP-CM with different compositions of HPC were analyzed by a powder X-ray diffractometer (D8ADVANCE, BRUKER AXS GMBH, German) with Cu source of radiation. Measurements were carried out at a voltage of 40 kV and 25 mA. The scanned angle was performed from 5°≤2θ≤90° and the scanning rate was 2 °/min. The measurement was carried out in triplicate. 2.10 Specific surface area (SSA) The specific surface area of the AGP-CM was measured by a surface area analyzer (Nova-1000, Quantachrome Instruments, Boynton Beach, USA) through
argon gas sorption process. The surface area per powder unit weight was calculated based on the fitting of the adsorption data to the BET equation. 2.11 Viscosity determination Viscosity (30 ◦C) was determined for HPC dispersing system with LVDV-II + Pro viscosimeter (Brookfield Engineering Laboratories, Inc., Middleboro, USA). Measurements were performed in triplicate and the average was calculated. 2.12 In vitro dissolution evaluation of AGP-CM The dissolution characterization of AGP-NS, AGP-CM and physical mixture of AGP and HPC, containing the same amount of AGP (50 mg), was evaluated, respectively. According to the Ch.P 2015 paddle method, a dissolution apparatus (RC-8, Tianjin Guoming Medicine and Equipment Co., Inc., China) was used. 900 ml of pure water was used as a dissolution medium at 37 ◦C. The rotation speed of the paddles was set at 100 rpm. At predetermined time intervals (0.5, 1, 1.5, 4, 6, 10, 20 and 30 min), 2 ml samples were withdrawn and filtered through 0.22μm filter membrane immediately. Simultaneously, equal blank medium was compensated immediately after the withdrawal. The amount of dissolved AGP in the sample solution was determined by HPLC method. The chromatographic separation was achieved on Hypersil ODS2 (250 mm×4.6 mm, 5μm) with a security guard column (10 × 4.6 mm, 5 μm). The mobile phase consisted of methanol–water (65:35, v/v). The flow rate was 1 mL/min. The detection wavelength was set at 225 nm. 2.13 Short term stability of AGP-CM The mean particle sizes of AGP-CM were monitored at storage. AGP-CM were placed in tightly sealed screw-cap vials immediately after preparation and kept at 4, 25 and 40 ◦C for six month. Particle size measurements were determined at regular time intervals over the storage period by Mastersizer Micro Plus (Malvern Instruments Limited, Worcestershire, UK). And redispersibility index (RDI) and dissolution of AGP-CM at predetermined time intervals was evaluated in order to evaluate its stability. 3. Results and discussion 3.1 Influences of HPC on characterization of AGP-NS during homogenization
In the absence of stabilizers, the nanoparticles would aggregate due to high attractive inter-particle forces (Van der Waals, hydrophobic forces, etc.) (Bhakay et al., 2011). The polymer could be used as stabilizer of drug nanosuspensions in the absence of surfactants (Bhakay et al., 2011; Van Eerdenbrugh et al., 2009). In order to evaluate the stabilizing effect of HPC on AGP-NS during homogenization, the minimum concentration of HPC to AGP was fixed to 1:20 (w/w) in this study. The amount of HPC to AGP was stepwise varied, so that a series of formulations having different ratios of AGP to HPC were investigated. The particle sizes of AGP-NS during homogenization were exhibited in Fig. 2A. The results indicated that the particle size of AGP-NS was remarkably related with homogenization pressure. The AGP-NS could be completely disintegrated to nano-sized particles (<1 µm) at pressure 1200 bar for 30 cycles (except for 1:20 concentration of HPC to AGP), compared with other pressures. The results also illustrated that the concentration of HPC to AGP played important role in size-reduction efficiency of AGP during homogenization. The minimum concentration of HPC to AGP (1:20) resulted in insufficient size reduction (D50 >1.5µm). As the concentration of HPC increased from 1:10 to 2:1, the mean particle size of AGP-NS was entirely shifted to submicron range (<1 µm) and its particle size distribution was gradually getting narrow (Fig.2B). And span of AGP-NS with 1:2 and 1:1 ratios of HPC to AGP were acceptable (< 2.5). Furthermore, SEM image of coarse AGP and AGP-NS with different ratios of HPC to AGP were showed in Fig.3. The TEM results also demonstrated that, the coarse AGP had be completely disintegrated to nanocrystals particles (<1 µm) in terms of HPC at ratio of 1:2, 1:1 and 2:1(Fig.3A-F). But the AGP-NS with 2:1 ratio of HPC to AGP seemed to be adhesive appearance (Fig.3G), because the HPC could be relatively excessive. The increase of amount of HPC (1:5 and 1:2) in the system might promote the stabilizing effect, leading to effective pulverization. But large increase of HPC concentration (1:1 and 2:1) resulted in slightly dropping the homogenization efficiency, which might be related with excess HPC dispersed in water. The viscosity of nanosuspensions gradually increased from 2.1 mPa·s (HPC to AGP =1:20) to 12.3
mPa·s (HPC to AGP = 2:1). It was the reason that, the amount of HPC adsorbed onto surface of drug particles might become saturated, the extra amount of HPC (2:1) would substantially increase the viscosity of the dispersing medium, which could result in weakened collision power of cavitations during homogenization. It was assumed that the steric barrier effect of HPC was responsible for stability of AGP-NS during homogenization (Fig.3H). HPC molecules could adsorb onto surface of the disintegrated drug particles in the suspension and effectively prevent from the aggregation between primary particles as steric barriers (Pongpeerapat et al., 2008). 3.2 Influences of HPC on the redispersibility of the spray-freeze-dried AGP-CM Our previous report demonstrated that, the effect of cryo/thermal strength from freeze-drying process on the redispersibility of drug nanocrystals was systemically investigated, which demonstrated that the redispersibility of the frozen nanocrystals at the aggressive temperature (-196 ◦C, meant higher freezing rate) was better than those at the conservative temperature conditions (-80 ◦C and -20 ◦C, meant lower freezing rate). It is concluded that the rapid freezing rate could minimize the crystallization and phase separation of drug (Yue et al., 2014). The spray-freeze-drying technique was used to convert AGP-NS into AGP-CM because of the flash freezing rate. In this study, the influences of HPC on the redispersibility of the spray-freeze-dried AGP-CM were investigated. Fig. 4A showed that the redispersibility of the spray-freeze-dried AGP-CM with different ratios of HPC to AGP. The results revealed that the RDI of spray-freeze-dried AGP-CM decreased with increase of ratio of HPC to AGP (Fig.4A). The spray-freeze-dried AGP-CM with HPC ratio of 1:2, 1:1 and 2:1 possessed excellent redispersibility (RDI near to 1), which indicated that AGP-CM could completely redispersed into the original nanosuspensions prior to spray-freeze-drying. But the spray-freeze-dried AGP-CM with low ratios of HPC (1:20, 1:10 and 1:5) exhibited poor redispersibility (RDI > 1.5), which indicated that HPC could be too little
to
adequately
prevent
from
aggregation
of
nanocrystals
during
spray-freeze-drying. Fig4.B showed that the specific surface area (SSA) of the AGP-CM with different ratios of HPC to AGP. The results illustrated that the SSA of
AGP-CM was increased with increase of ratio of HPC to AGP. The AGP-CM with HPC ratio of 1:2, 1:1 and 2:1 possessed larger surface area (3000m2/g), compared to those with HPC ratio of 1:20, 1:10 and 1:5. The large surface area seemed to be responsible for redispersibility of the AGP-CM with HPC ratio of 1:2, 1:1 and 2:1. In view of disadvantage of increasing viscosity of HPC ratio of 2:1 during homogenization, 1:2 and 1:1 ratio of HPC was relative suitable for preparation of AGP-CM. Fig.5 illustrated SEM photographs of the spray-freeze-dried AGP-CM with different ratios of HPC to AGP. The results demonstrated that microstructure of spray-freeze-dried AGP-CM with HPC ratio of 1:2 and 1:1 was the “cage-like” structure with porous characterization (Fig.5 D, E), in which nano-sized AGP nanocrystals were embedded (Fig.5 F). The formation of pores in “cage-like” composite particles could be related with sublimation of the ice crystals in the spray-frozen droplets. And these could explain that the spray-freeze-dried AGP-CM with HPC ratio of 1:2 and 1:1 possessed the large surface. But the spray-freeze-dried AGP-CM with HPC ratio of 1:20, 1:10 and 1:5 were found to possess some hybrid structure characterizations (Fig.5 A, B, C), which was seemed to agglomerated granules from the large drug fragments due to insufficiency of HPC. Therefore, the amount of HPC played an important role in formation of “cage-like” matrix structure of spray-freeze-dried AGP-CM. 3.3 Solid state characterization of AGP-CM In order to elucidate the influences of formulation processes on AGP-CM properties, the solid state of bulk AGP, the AGP-CM with different HPC ratios (1:20, 1:10, 1:5, and 1:1) was investigated using DSC and XRD techniques, respectively. Fig.6 displayed the XRD diffractograms of HPC, raw AGP, and the AGP-CM with different HPC ratio (1:20, 1:10, 1:5, and 1:1). HPC did not have the obvious characteristic peak observed between 10° and 90° (Fig.6 A). Raw AGP exhibited characteristic crystalline peaks at 2θ of 10.9, 12.1, 13.7, 15.2 and 26.8° (Fig.6 B). For the AGP-CM with different HPC ratio (1:20, 1:10, 1:5, 1:1), the distinct AGP XRD peaks were did not disappear with increase of amount of HPC (Fig. 6 C, D, E, F). But
the peak intensities of the AGP-CM were somewhat reduced with increase of amount of HPC. The differences in peak relative intensities were probably due to the size reduction of AGP nanocrystals. Thermograms of HPC, raw AGP, and the AGP-CM with different HPC ratio (1:20, 1:10, 1:5, 1:1) between 20 and 260 ◦C were presented in Fig. 7 A-F, respectively. HPC did not display the obvious thermograms characterization peaks (Fig. 7 A). Raw AGP exhibited a melting peak at 230 ◦C (Fig. 7 B). For the spray-freeze-dried AGP-CM with different HPC ratio (1:20, 1:10, 1:5, and 1:1), the melting peak was at 205-215 ◦
C (Fig. 7 C, D, E, F). The slight shift in the endothermic peak of AGP-CM toward a
lower temperature AGP (Fig.7B) could be the result of nanoization of crystals (Zhang et al., 2007; Ng et al., 2010). 3.4 In vitro dissolution evaluation of AGP-CM
To evaluate the success of the AGP-CM with different HPC ratio, in vitro release evaluation was performed. Fig. 8 showed that dissolution of the spray-freeze-dried AGP-CM with higher ratios of HPC (1:2 and 1:1, Fig8.d, e) was superior to those of the crude AGP (Fig8. a), and lower ratios of HPC (1:10 and 1:5, Fig8. b, c). Within 30 min, only 33.35% concentration of AGP was dissolved from the coarse AGP. Approximate 49.87% and 72.16% concentration of AGP was dissolved from the spray-freeze-dried AGP-CM with lower ratios of HPC (1:10 and 1:5). In contrast to these, the dissolution of AGP-CM with higher ratios of HPC (1:2 and 1:1) was significantly enhanced (p<0.05), which indicated that high ratio of HPC could completely prevent from aggregation of AGP-CM during spray-freeze-drying, compared with low ratio (1:10 and 1:5) of HPC. These were consistent with results of redispersibility of the spray-freeze-dried AGP-CM with low ratio of HPC (Fig.4). The improvement of dissolution of the spray-freeze-dried AGP-CM attributed to the particle size reduction of AGP, the increase of surface area of AGP nanocrystals (Fig.4), and the porous structure of particles (Fig.5). 3.5 Stability evaluation of the spray-freeze-dried AGP-CM
The physical stability of the spray-freeze-dried AGP-CM with higher ratios of HPC (1:2 and 1:1) was studied upon storage at different temperatures for a period up to 6 months. Figure 9 A, B showed the redispersibility and in vitro dissolution of the spray-freeze-drying AGP-CM at storage for six months. The storage temperature conditions significantly impact stability of AGP-CM with HPC ratio of 1:2(Fig.9 a, b). The RDIs of AGP-CM with HPC ratio of 1:2 at 25 ◦C and 40 ◦C were more than 2, and their dissolution were remarkably decreased compared with those prior to storage (Fig8.d), which indicated that AGP nanoparticles significantly aggregated. But the redispersibility and dissolution of AGP-CM with HPC ratio of 1:2 at 4 ◦C was obvious changed (Fig.9 c). For the AGP-CM with HPC ratio of 1:1 at different storage conditions, the RDIs were near to 1(Fig.9 d, e, f), and their dissolution were equal with those prior to storage (Fig8.e). It was attributed that high ratio of HPC could prevent from agglomeration of AGP nanoparticles at storage. As illustrated in Fig9.C, the AGP nanoparticles were “imprisoned” into “cage-like” matrix structure of HPC and reduced the free mobility. The “cage-like” structure of HPC might prevent nanocrystals from particle-particle agglomeration during storage. Therefore, the AGP-CM could easily reconstituted into nanosuspensions after rehydration and improve the dissolution of AGP. 4. Conclusions A kind of cage-like microparticles for poorly soluble drugs used HPC as carrier has been developed, by means of an interesting approach combined homogenization and the spray-freeze-drying technique. The AGP particles were homogenized into the nanosuspensions and the resultant suspension was further processed into composite particles via spray-freeze-drying. It was demonstrated that HPC not only was an effective stabilizer during homogenization, but also a unique matrix carrier for drug nanocrystals. The spray-freeze-dried AGP-CM with 1:1 ratio of HPC could form cage-like matrix structure, in which the drug nanocrystals particles were completely imprisoned and immobilized. Such unique structure could help AGP-CM to reconstitute into the liquid nanosuspensions and significantly improve the dissolution of AGP. The PXRD and DSC results indicated that the AGP crystal state was altered
during homogenization and spray-freeze-drying process. And the redispersibility of the AGP-CM with high ratio of HPC was unaffected upon storage. This study elucidated a dissolution-enhanced formulation strategy for poorly soluble drug used HPC as matrix carrier. Acknowledgements The authors would like to acknowledge the financial support from the Scientific Research Foundation for the National Natural Science Foundation of China (No. 81560656), and Fund of distinguished young scientists of Jiangxi Province and the Natural Science Fund of Jiangxi Province (No. 20161BAB205221). References: Akkar, A., Namsolleck, Blaut, P.M., Muller, R. (2004). Solubilizing poorly soluble antimycoticagents by emulsification via a solvent-free process. AAPS PharmSciTech, 5, 1–6. Bhakay, A.，Davé, R.，Bilgili, E. (2013). Recovery of BCS Class II drugs during aqueous redispersion of core–shell type nanocomposite particles produced via fluidized bed coating. Powder Technology, 236(6), 221-234. Bhakay, A., Merwade, M., Bilgili, E., Dave, R. (2011). Novel aspects of wet milling for the production of microsuspensions and nanosuspensions of poorly water soluble drugs. Drug Development and Industrial Pharmacy, 37, 963-976. Bilgili, E., Hamey, R., Scarlett, B. (2004). Production of pigment nanoparticles using a wetstirred media mill with polymeric media. China Particuology, 2,93–100. Bilgili, E. Hamey, R. Scarlett, B. (2006). Nano-milling of pigment agglomerates using a wetstirred media mill: elucidation of the kinetics and breakage mechanisms. Chemical Engineering Science, 61,149–157. Chen, H., Seiber, J.N., & Hotze, M. (2014). ACS select on nanotechnology in food and agriculture: a perspective on implications and applications. Journal of Agricultural and Food Chemistry, 62, 1209-1212. Cheow, W.S., Ng, M.L.L., Kho, K., Hadinoto, K. (2011). Spray-freeze-drying production of thermally sensitive polymeric nanoparticle aggregates for inhaled drug delivery: Effect of freeze-drying adjuvants. Int. J. Pharm.,404, 289-300.
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delivery. American Pharmaceutical Review, 5, 82–85. Merisko-Liversidge, E., Liversidge, G.G. (2011). Nanosizing for oral and parenteral drug delivery: A perspective on formulating poorly-water soluble compounds using wet media milling technology. Adv Drug Deliver Rev, 63, 427-440. Mezdour, S., Lepine, A., Erazo-Majewicz, P., Ducept, F., & Michon, C. (2008). Oil/water surface rheological properties of hydroxypropylcellulose (HPC) alone and mixed with lecithin: Contribution to emulsion stability. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 331, 76-83. Muller, R., Peters, K. (1998). Nanosuspensions for the formulation of poorly soluble drugs: I. Preparation by a size reduction technique. International Journal of Pharmaceutics, 160, 229–237. Ng, W.K., Kwek, J.W., Yuen, A., Tan, C.L., Tan, R. (2010). Effect of milling on DSC thermogram of excipient adipic acid. AAPS PharmSciTech, 11, 159-167. Pawar, V.K., Singh, Y., Meher, J.G., Gupta, S., & Chourasia, M.K. (2014). Engineered nanocrystal technology: in-vivo fate, targeting and applications in drug delivery. J Control Release, 83, 51-66. Parsian, A.R.，Vatanara, A.，Rahmati, M.R.，Gilani, K.，Khosravi, K.M., Najafabadi, A.R. (2014). Inhalable budesonide porous microparticles tailored by spray freeze drying technique. Powder Technology, 260, 36-41. Pongpeerapat, A., Wanawongthai, C., Tozuka, Y., Moribe, K., Yamamoto, K., 2008. Formulation mechanism of colloidal nanoparticles obtained from probucol/ PVP/SDS ternary ground mixture. Int. J. Pharm. 352, 309-316. Rehmana Rashid, Dong Wuk Kim, Fakhar ud Din, Omer Mustapha, Abid Mehmood Yousaf, Jong Hyuck Park, Jong Oh Kim, Chul Soon Yong, Han-Gon Choi. (2015). Effect of hydroxypropylcellulose and Tween 80 on physicochemical properties and bioavailability of ezetimibe-loaded solid dispersion. Carbohydrate Polymers, 130(5), 26-31. Sarode, Ashish L., Malekar, Swapnil A., Cote Catherine, David R. (2014). Worthen. Hydroxypropyl cellulose stabilizes amorphous solid dispersions of the poorly water soluble drug felodipine. Carbohydrate Polymers, 112(4), 512-519.
Van Eerdenbrugh, B., Vermant, J., Martens, J., Froyen, L., VanHumbeeck, J., Augustijns, P., Mooter, G.. (2009). A screening study of surface stabilization during the production of drug nanocrystals. Journal of Pharmaceutical Sciences, 98, 2091-2103. Velikov, K.P., Pelan, E. (2008). Colloidal delivery systems for micronutrients and Nutraceuticals, Soft Matter, 4, 1964-1980. Vogt, M., Kunath, K., Dressman, J. (2008). Dissolution enhancement of fenofibrate by micronization, cogrinding and spray-drying: comparison with commercial preparations. European Journal of Pharmaceutics and Biopharmaceutics 68, 283–288. Wanning, S., Süverkrüp, R., Lamprecht, A. (2015). Pharmaceutical spray freeze drying. Int J Pharm. 488, 136-53. Wu, L., Zhang, J., Watanabe, W. (2011). Physical and chemical stability of drug nanoparticles. Adv Drug Deliv Rev. 63(6), 456-69. Yue, P.F., Wang, Y., Wan, J., Wu, Z.F., Hu, P.Y., Zheng, Q., Yuan, H.L., Yang, M. (2012). The research progress of preparation methods of solid nanocrystal delivery system. Acta Pharmaceutica Sinica, 47(9), 1120-1127. Yue, P.F., Li, G., Dan, J.X., Wu, Z.F., Wang, C.H., Zhu, W.F., Yang, M. (2014). Study on formability of solid nanosuspensions during solidification: II novel roles of freezing stress and cryoprotectant property. Int J Pharm. 475(1-2), 35-48. Zhang, D., Tan, T., Gao, L., Zhao, W., Wang, P. (2007). Preparation of azithromycin nanosuspensions by high pressure homogenization and its physicochemical characteristics studies. Drug development and industrial pharmacy, 33, 569-575.
Fig.1 Schematic image of design of composite microparticles via combination of homogenization with spray-freeze-drying technology
Fig.2 The particle sizes of AGP-NS with different ratios of HPC during predetermined homogenization pressure (A), and span of AGP-NS with different ratios of HPC during homogenization of 1200bar (B)
Fig.3 SEM image of coarse AGP (A) and TEM images of AGP-NS with different ratios of HPC to AGP (B 1:20, C 1:10, D 1:5, E 1:2, F 1:1, G 2:1), and schematic image of stability mechanism of AGP-NS (H)
Fig.4 The redispersibility (RDI) and specific surface area (SSA) of the spray-freeze-dried AGP-CM with different ratios of HPC to AGP
Fig.5 SEM photographs of the AGP-CM with different ratios of HPC to AGP (A 1:20, B 1:10, C 1:5, D 1:2, E1:1, F 1:1 Magnification)
Fig.6 XRD diffractograms of HPC (A), raw AGP (B), and the AGP-CM with different HPC ratios (C 1:20, D 1:10, E 1:5, and F 1:1)
Fig.7 Thermograms of HPC (A), raw AGP (B), and the AGP-CM with different ratios of HPC (C 1:20, D 1:10, E 1:5, F 1:1)
Fig.8 Dissolution of raw AGP (a) and the AGP-CM with different ratios of HPC (b 1:10, c 1:5, d 1:2, e 1:1)
Fig.9 The redispersibility(A) and in vitro dissolution(B) the AGP-CM at storage for six months, and schematic image of stability of AGP-CM used HPC as carrier(C)
Table 1 Formulation of homogenization and spray-freeze-drying processes with various compositions of HPC and AGP Composition ratios of HPC to AGP Materials Unit
1:20
1:10
1:5
1:2
1:1
2:1
HPC
mg
50
100
200
500
1000
2000
AGP
mg
1000
1000
1000
1000
1000
1000
Water
mL
100
100
100
100
100
100
S0144-8617(16)31208-5 http://dx.doi.org/doi:10.1016/j.carbpol.2016.10.043 CARP 11663
To appear in: Received date: Revised date: Accepted date:
20-7-2016 14-10-2016 14-10-2016
Please cite this article as: Wei, Shaofeng., Ma, Yueqin., Luo, Jing., He, Xiaoru., Yue, Pengfei., Guan, Zhiyu., & Yang, Ming., Hydroxypropylcellulose as matrix carrier for novel cage-like microparticles prepared by spray-freeze-drying technology.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.10.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
TITLE PAGE
Hydroxypropylcellulose as matrix carrier for novel cage-like microparticles prepared by spray-freeze-drying technology
Shaofeng Wei 1#, Yueqin Ma2#, Jing Luo1, Xiaoru He1, Pengfei Yue1*, Zhiyu Guan1, Ming Yang1
1
Jiangxi University of Traditional Chinese Medicine, 818 MEILINGDADAO Road,
Nanchang 330004, China 2
Department of Pharmaceutics, 94th Hospital of People’s Liberation Army, Nanchang
330000, China
*Corresponding authors at: Jiangxi University of Traditional Chinese Medicine, 818 MEILINGDADAO Road, Nanchang 330004, China E-mail: [email protected]
#
Shaofeng Wei and Yueqin Ma contributed to the work equally as joint first authors.
Highlights:
•A novel cage-like composite particles used HPC as carrier was developed.
•An interesting strategy by means of spray-freeze-drying technique was used.
• The spray-freeze-dried CMs exhibited excellent dissolution-enhanced specialty.
• HPC was demonstrated to be an effective matrix carrier for drug nanocrystals.
ABSTRACT: The objective of this study is to design novel dissolution-enhanced microparticles loaded poorly soluble drug nanocrystals used a low viscosity of hydroxypropylcellulose (HPC) as matrix carrier. An interesting approach combined homogenization and the spray-freeze-drying technique was developed. The results demonstrated that the ratio of HPC to drug played an important role in size-reduction efficiency of drug during homogenization. And the formation of cage-like structure of the composite particles depended on ratio of HPC to drug. The spray-freeze-dried composite particles with HPC ratio of 1:2, 1:1 and 2:1 possessed excellent redispersibility, which attributed to its porous matrix and large surface area (3000m2/g). The dissolution of spray-freeze-dried composite particles with higher ratios of HPC (1:2 and 1:1) was significantly enhanced, which attributed to the particle size reduction of drug. The HPC could immobilize drug nanocrystals in its cage-like structure and prevent it from the subsequent agglomeration during storage. In conclusion, the prepared cage-like microparticles is a promising basis for further formulation development.
Keywords: hydroxypropylcellulose; nanocrystals; cage-like microparticles; spray-freeze-drying; redispersibility
1. Introduction Driven by increasing demands for improving human health, a lot of studies have been carried out to develop innovative pharmaceutical preparations or functional foods by incorporation of all kinds of bioactive ingredients (Velikov and Pelan, 2008; Chen, Seiber and Hotze, 2014). However, many active ingredients are difficultly introduced into pharmaceutical preparations, mainly due to their poor aqueous solubility, which would decrease their oral bioavailability, and hence, limit their use in clinical (Lipinski et al., 2002; Joye et al., 2014). Therefore, it is an urgent need to solve this problem at present. The reduction of drug particle sizes down to the nanometer or submicron scale has been an effective approach of increasing the dissolution rate of poorly soluble drugs (Muller and peter, 1998). Nanocrystals suspension (NS, typically particles smaller than 1 μm) has already been recognized as one of the promising reduction of particles size technologies for poorly soluble drugs in pharmaceutics. NS has some unique advantages that enhance the solubility and dissolution velocity of poorly soluble drug due to their small particle size and high surface area(Jinno et al. 2006; Merisko-Liversidge and Liversidge 2011; Ige et al. 2013), and strengthen the adhesion to biological membrane, prolong adhesion time and detention time (Pawar et al. 2014; Gao et al. 2012). Fine drug NS can usually be produced in liquid media either by “top-down” approaches such as wet media milling (Bilgili et al., 2004; 2006), high-pressure homogenization(Akkar et al., 2004), and jet milling (Vogt et al., 2008) or by “bottom-up” approach such as liquid anti-solvent precipitation (Dalvi and Dave, 2010). Nanocrystals suspensions have a large surface area, leading to significant inter-particle interactions through van der Waals forces, hydrophobic forces, etc. The attractive inter-particle forces can ultimately result in particle aggregation if the suspensions are not properly stabilized. Stabilizing effect can be achieved using polymers, surfactants or a combination of both; however, the particle growth can also occur in the suspension by Ostwald ripening during storage (Wu et al., 2011). Therefore, from a stability perspective as well as patient preference/compliance
perspective, powderization of liquid NS is an effective approach, which can reconstitute into original nanosuspensions states after rehydration (Yue et al., 2012). Spray-drying has been widely applied in powderization of nanosuspensions, because it is advantageous to produce the spherical and size-controlled particles (Figueroa and Bose 2013; Iskandar et al. 2003). However, this technique is not always appropriate for thermolabile or oxidizable ingredients because the spray-drying process requires elevated temperatures. Spray-freeze-drying (SFD) is a novel particle engineering technology (Chow et al., 2007), in which either an aqueous or an aqueous-organic cosolvent solution containing an active ingredient and a pharmaceutical excipient is atomized directly into super-cool phase to form frozen and solid particles. After that these frozen particles will be dried using lyophilization and these processes generally produce fine particles with a larger median particle size, and a significant large specific surface area and high yields (Parsian et al., 2014; Wanning et al., 2015). During the spray-freeze-drying processes, nanoparticles can aggregate into microparticles (particles of size>1μm). However, a key concern with spray-freeze-drying process is that nanocrystals incorporated in composite microparticles may not be recovered to original nanosuspensions states. Therefore, the proper matrix carrier is indispensable for solid nanocrystals after spray-freeze-drying. Hydroxypropylcellulose (HPC, Fig.1) a cellulosic polymer in which some of the hydroxyl groups in the repeating sugar units are hydroxypropylated using propylene oxide (Mezdour et al., 2008). It is an amphiphilic large molecular which is soluble in a range of organic solvents and water. HPC has been frequently used as a carrier of solid dispersion of poorly soluble drug (Sarode et al., 2014; Rehmana et al., 2013). However, the aim of this work is to evaluate the potential of HPC used as a matrix carrier of composite microparticles incorporated with poorly soluble drug nanocrystals. As illustrated in Fig.1, this study elucidated an interesting strategy to combine homogenization with spray-freeze-drying technology to prepare the composite microparticles loaded poorly soluble drug. In this work, a diterpenoid lactone
(code-named as AGP) was used as model drug. AGP has been demonstrated to have a wide range of biological activities including anti-inflammatory and anti-virus. Unfortunately, AGP has a relative low bioavailability (1.9%) after oral administration, which is associated with its high lipophilicity (log P = 2.6) and low aqueous solubility (3.3µg/mL), and it is also a temperature-sensitive drug. AGP nanocrystals suspension (AGP-NS) stabilized by HPC were prepared by homogenization technology and further converted into the AGP microparticles with cage-like structure (AGP-CM) via spray-freeze-drying. Morphology of the spray-freeze-dried AGP-CM was visualized by scanning electronic microscopy (SEM). Characteristics of AGP in the AGP-CM were analyzed by powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC). The redispersibility, dissolution and stability evaluation were performed to investigate the feasibility of HPC as a carrier for AGP-CM. 2. Materials and methods 2.1 Materials AGP was obtained from Zelan Co. (Nanjing, China). Hydroxypropylcellulose (HPC, Klucel® EF) was kindly donated by Chineway Pharmaceutical Technology Co., LTD (SHANGHAI, China). 2.2 Homogenization of AGP-NS AGP-NS was prepared by high pressure homogenization. Before producing AGP-NS, AGP coarse powder (1%, w/v) was dispersed in different concentration of HPC solutions. The amount of HPC to drug was varied from 1:20, 1:10, 1:5, 1:2 to 1:1, and 2:1(w/w). The resultant mixture of AGP and HPC solution was dispersed into coarse suspension by a high shear homogenizer (FLUKO® FA25, Essen, Germany) at 19,000 rpm for 2 min. And then the gained coarse suspension was homogenized into AGP-NS at predetermined pressure using a high-pressure homogenizer with cooling system (AH-1000D, ATS Engineering Inc., Seeker, Canada). The cooling temperature was fixed at 10 ◦C. The AGP formulations with different compositions of HPC and AGP during homogenization were showed in Table 1. 2.3 Spray-freeze-drying of AGP-CM The resultant AGP-NS with various compositions of HPC (Table 1) were
converted into AGP-CM by means of a modified Büchi B-290 mini spray dryer (Büchi, Switzerland) by removing its drying chamber (Cheow et al., 2011). A polypropylene vessel containing 400 mL of liquid nitrogen (-196 ◦C) was placed beneath the spray dryer’s two-fluid atomizer under constant stirring at 500 rpm. The distance between the atomizer and the polypropylene vessel was fixed at 10 cm to minimize the loss of the sprayed droplets due to impaction at the vessel wall. The atomization was performed at feed rate of 10 mL/min, atomizing air flow at 30 mmHg, and feed solid concentration of 1% (w/v). After the atomization, the frozen AGP-NS were dried by means of freeze-dryer (FreezeZone Stoppering Tray Dryers, LABCONCO Corporation, Kansas, USA). The applied cycle conditions were as follows: freezing was performed at -40◦C for 60min. The shelf temperature ramp rates from the freezing step in to the primary drying step were 1◦C/min. Primary drying and secondary drying was performed at -20 ◦C and 10 ◦C for 12 h, respectively. The shelf temperature ramp rates from the primary-drying step in to the secondary drying step were 1 ◦C/min. 2.4 Particle size and distribution assay of AGP-NS The particles size determination of AGP-NS was performed on a Mastersizer Micro Plus (Malvern Instruments Limited, Worcestershire, UK). All the measurements were carried out in triplicate. The average 10%, 50% and 90% volume percentile (D10, D50 and D90) were determined. And span = (D10-D90)/D50, which was used to evaluate the particle size distribution of AGP-NS. 2.5 TEM Morphology of AGP-NS One drop of the AGP-NS with different ratios of HPC and AGP (Table 1) was placed on a copper grid and stained with 2% phosphotungstic acid solution for 5 min. The grid was dried at room temperature for 12 h and examined using Transmission electron microscope (JEM-1200EX, Japan). 2.6 Redispersibility index (RDI) of AGP-CM after rehydration The redispersibility of AGP-CM was evaluated as described in Dan et al, 2016: RDI D
D0
Where D0 represents the volume-weighed mean particle size of the freshly prepared AGP-NS directly prior to spray-freeze-drying and D represents the corresponding value of reconstituted AGP-NS after spray-freeze-drying. An RDI of near 1 would therefore mean that AGP-CM can be completely reconstituted into the original AGP-NS after rehydration. 2.7 Scanning electron microscopy (SEM) Morphological evaluation of the spray-freeze-dried AGP-CM samples with different ratios of HPC and AGP (Table 1) and coarse AGP were performed by means of scanning electron microscope (SEM) (Hitachi X650, Tokyo, Japan). All samples were examined on a brass stub using carbon double-sided tape. AGP-CM samples were glued and mounted on metal sample plates. The samples were gold coated (thickness ≈ 15-20 nm) with a sputter coater (Fison Instruments, 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. 2.8 Differential scanning calorimetry (DSC) Thermograms of coarse AGP powder, HPC, and AGP-CM with different compositions of HPC were performed using a DSC (Diamond DSC, Perkin-Elmer, USA) equipped with an intercooler. The samples were analyzed in open aluminum pans and scanned under a nitrogen purge with a heating rate of 10 ◦C /min from 20 ◦C to 260 ◦C, with an empty pan as a reference. 2.9 X-ray powder diffraction (XPRD) The characterizations of coarse AGP powder, HPC, and AGP-CM with different compositions of HPC were analyzed by a powder X-ray diffractometer (D8ADVANCE, BRUKER AXS GMBH, German) with Cu source of radiation. Measurements were carried out at a voltage of 40 kV and 25 mA. The scanned angle was performed from 5°≤2θ≤90° and the scanning rate was 2 °/min. The measurement was carried out in triplicate. 2.10 Specific surface area (SSA) The specific surface area of the AGP-CM was measured by a surface area analyzer (Nova-1000, Quantachrome Instruments, Boynton Beach, USA) through
argon gas sorption process. The surface area per powder unit weight was calculated based on the fitting of the adsorption data to the BET equation. 2.11 Viscosity determination Viscosity (30 ◦C) was determined for HPC dispersing system with LVDV-II + Pro viscosimeter (Brookfield Engineering Laboratories, Inc., Middleboro, USA). Measurements were performed in triplicate and the average was calculated. 2.12 In vitro dissolution evaluation of AGP-CM The dissolution characterization of AGP-NS, AGP-CM and physical mixture of AGP and HPC, containing the same amount of AGP (50 mg), was evaluated, respectively. According to the Ch.P 2015 paddle method, a dissolution apparatus (RC-8, Tianjin Guoming Medicine and Equipment Co., Inc., China) was used. 900 ml of pure water was used as a dissolution medium at 37 ◦C. The rotation speed of the paddles was set at 100 rpm. At predetermined time intervals (0.5, 1, 1.5, 4, 6, 10, 20 and 30 min), 2 ml samples were withdrawn and filtered through 0.22μm filter membrane immediately. Simultaneously, equal blank medium was compensated immediately after the withdrawal. The amount of dissolved AGP in the sample solution was determined by HPLC method. The chromatographic separation was achieved on Hypersil ODS2 (250 mm×4.6 mm, 5μm) with a security guard column (10 × 4.6 mm, 5 μm). The mobile phase consisted of methanol–water (65:35, v/v). The flow rate was 1 mL/min. The detection wavelength was set at 225 nm. 2.13 Short term stability of AGP-CM The mean particle sizes of AGP-CM were monitored at storage. AGP-CM were placed in tightly sealed screw-cap vials immediately after preparation and kept at 4, 25 and 40 ◦C for six month. Particle size measurements were determined at regular time intervals over the storage period by Mastersizer Micro Plus (Malvern Instruments Limited, Worcestershire, UK). And redispersibility index (RDI) and dissolution of AGP-CM at predetermined time intervals was evaluated in order to evaluate its stability. 3. Results and discussion 3.1 Influences of HPC on characterization of AGP-NS during homogenization
In the absence of stabilizers, the nanoparticles would aggregate due to high attractive inter-particle forces (Van der Waals, hydrophobic forces, etc.) (Bhakay et al., 2011). The polymer could be used as stabilizer of drug nanosuspensions in the absence of surfactants (Bhakay et al., 2011; Van Eerdenbrugh et al., 2009). In order to evaluate the stabilizing effect of HPC on AGP-NS during homogenization, the minimum concentration of HPC to AGP was fixed to 1:20 (w/w) in this study. The amount of HPC to AGP was stepwise varied, so that a series of formulations having different ratios of AGP to HPC were investigated. The particle sizes of AGP-NS during homogenization were exhibited in Fig. 2A. The results indicated that the particle size of AGP-NS was remarkably related with homogenization pressure. The AGP-NS could be completely disintegrated to nano-sized particles (<1 µm) at pressure 1200 bar for 30 cycles (except for 1:20 concentration of HPC to AGP), compared with other pressures. The results also illustrated that the concentration of HPC to AGP played important role in size-reduction efficiency of AGP during homogenization. The minimum concentration of HPC to AGP (1:20) resulted in insufficient size reduction (D50 >1.5µm). As the concentration of HPC increased from 1:10 to 2:1, the mean particle size of AGP-NS was entirely shifted to submicron range (<1 µm) and its particle size distribution was gradually getting narrow (Fig.2B). And span of AGP-NS with 1:2 and 1:1 ratios of HPC to AGP were acceptable (< 2.5). Furthermore, SEM image of coarse AGP and AGP-NS with different ratios of HPC to AGP were showed in Fig.3. The TEM results also demonstrated that, the coarse AGP had be completely disintegrated to nanocrystals particles (<1 µm) in terms of HPC at ratio of 1:2, 1:1 and 2:1(Fig.3A-F). But the AGP-NS with 2:1 ratio of HPC to AGP seemed to be adhesive appearance (Fig.3G), because the HPC could be relatively excessive. The increase of amount of HPC (1:5 and 1:2) in the system might promote the stabilizing effect, leading to effective pulverization. But large increase of HPC concentration (1:1 and 2:1) resulted in slightly dropping the homogenization efficiency, which might be related with excess HPC dispersed in water. The viscosity of nanosuspensions gradually increased from 2.1 mPa·s (HPC to AGP =1:20) to 12.3
mPa·s (HPC to AGP = 2:1). It was the reason that, the amount of HPC adsorbed onto surface of drug particles might become saturated, the extra amount of HPC (2:1) would substantially increase the viscosity of the dispersing medium, which could result in weakened collision power of cavitations during homogenization. It was assumed that the steric barrier effect of HPC was responsible for stability of AGP-NS during homogenization (Fig.3H). HPC molecules could adsorb onto surface of the disintegrated drug particles in the suspension and effectively prevent from the aggregation between primary particles as steric barriers (Pongpeerapat et al., 2008). 3.2 Influences of HPC on the redispersibility of the spray-freeze-dried AGP-CM Our previous report demonstrated that, the effect of cryo/thermal strength from freeze-drying process on the redispersibility of drug nanocrystals was systemically investigated, which demonstrated that the redispersibility of the frozen nanocrystals at the aggressive temperature (-196 ◦C, meant higher freezing rate) was better than those at the conservative temperature conditions (-80 ◦C and -20 ◦C, meant lower freezing rate). It is concluded that the rapid freezing rate could minimize the crystallization and phase separation of drug (Yue et al., 2014). The spray-freeze-drying technique was used to convert AGP-NS into AGP-CM because of the flash freezing rate. In this study, the influences of HPC on the redispersibility of the spray-freeze-dried AGP-CM were investigated. Fig. 4A showed that the redispersibility of the spray-freeze-dried AGP-CM with different ratios of HPC to AGP. The results revealed that the RDI of spray-freeze-dried AGP-CM decreased with increase of ratio of HPC to AGP (Fig.4A). The spray-freeze-dried AGP-CM with HPC ratio of 1:2, 1:1 and 2:1 possessed excellent redispersibility (RDI near to 1), which indicated that AGP-CM could completely redispersed into the original nanosuspensions prior to spray-freeze-drying. But the spray-freeze-dried AGP-CM with low ratios of HPC (1:20, 1:10 and 1:5) exhibited poor redispersibility (RDI > 1.5), which indicated that HPC could be too little
to
adequately
prevent
from
aggregation
of
nanocrystals
during
spray-freeze-drying. Fig4.B showed that the specific surface area (SSA) of the AGP-CM with different ratios of HPC to AGP. The results illustrated that the SSA of
AGP-CM was increased with increase of ratio of HPC to AGP. The AGP-CM with HPC ratio of 1:2, 1:1 and 2:1 possessed larger surface area (3000m2/g), compared to those with HPC ratio of 1:20, 1:10 and 1:5. The large surface area seemed to be responsible for redispersibility of the AGP-CM with HPC ratio of 1:2, 1:1 and 2:1. In view of disadvantage of increasing viscosity of HPC ratio of 2:1 during homogenization, 1:2 and 1:1 ratio of HPC was relative suitable for preparation of AGP-CM. Fig.5 illustrated SEM photographs of the spray-freeze-dried AGP-CM with different ratios of HPC to AGP. The results demonstrated that microstructure of spray-freeze-dried AGP-CM with HPC ratio of 1:2 and 1:1 was the “cage-like” structure with porous characterization (Fig.5 D, E), in which nano-sized AGP nanocrystals were embedded (Fig.5 F). The formation of pores in “cage-like” composite particles could be related with sublimation of the ice crystals in the spray-frozen droplets. And these could explain that the spray-freeze-dried AGP-CM with HPC ratio of 1:2 and 1:1 possessed the large surface. But the spray-freeze-dried AGP-CM with HPC ratio of 1:20, 1:10 and 1:5 were found to possess some hybrid structure characterizations (Fig.5 A, B, C), which was seemed to agglomerated granules from the large drug fragments due to insufficiency of HPC. Therefore, the amount of HPC played an important role in formation of “cage-like” matrix structure of spray-freeze-dried AGP-CM. 3.3 Solid state characterization of AGP-CM In order to elucidate the influences of formulation processes on AGP-CM properties, the solid state of bulk AGP, the AGP-CM with different HPC ratios (1:20, 1:10, 1:5, and 1:1) was investigated using DSC and XRD techniques, respectively. Fig.6 displayed the XRD diffractograms of HPC, raw AGP, and the AGP-CM with different HPC ratio (1:20, 1:10, 1:5, and 1:1). HPC did not have the obvious characteristic peak observed between 10° and 90° (Fig.6 A). Raw AGP exhibited characteristic crystalline peaks at 2θ of 10.9, 12.1, 13.7, 15.2 and 26.8° (Fig.6 B). For the AGP-CM with different HPC ratio (1:20, 1:10, 1:5, 1:1), the distinct AGP XRD peaks were did not disappear with increase of amount of HPC (Fig. 6 C, D, E, F). But
the peak intensities of the AGP-CM were somewhat reduced with increase of amount of HPC. The differences in peak relative intensities were probably due to the size reduction of AGP nanocrystals. Thermograms of HPC, raw AGP, and the AGP-CM with different HPC ratio (1:20, 1:10, 1:5, 1:1) between 20 and 260 ◦C were presented in Fig. 7 A-F, respectively. HPC did not display the obvious thermograms characterization peaks (Fig. 7 A). Raw AGP exhibited a melting peak at 230 ◦C (Fig. 7 B). For the spray-freeze-dried AGP-CM with different HPC ratio (1:20, 1:10, 1:5, and 1:1), the melting peak was at 205-215 ◦
C (Fig. 7 C, D, E, F). The slight shift in the endothermic peak of AGP-CM toward a
lower temperature AGP (Fig.7B) could be the result of nanoization of crystals (Zhang et al., 2007; Ng et al., 2010). 3.4 In vitro dissolution evaluation of AGP-CM
To evaluate the success of the AGP-CM with different HPC ratio, in vitro release evaluation was performed. Fig. 8 showed that dissolution of the spray-freeze-dried AGP-CM with higher ratios of HPC (1:2 and 1:1, Fig8.d, e) was superior to those of the crude AGP (Fig8. a), and lower ratios of HPC (1:10 and 1:5, Fig8. b, c). Within 30 min, only 33.35% concentration of AGP was dissolved from the coarse AGP. Approximate 49.87% and 72.16% concentration of AGP was dissolved from the spray-freeze-dried AGP-CM with lower ratios of HPC (1:10 and 1:5). In contrast to these, the dissolution of AGP-CM with higher ratios of HPC (1:2 and 1:1) was significantly enhanced (p<0.05), which indicated that high ratio of HPC could completely prevent from aggregation of AGP-CM during spray-freeze-drying, compared with low ratio (1:10 and 1:5) of HPC. These were consistent with results of redispersibility of the spray-freeze-dried AGP-CM with low ratio of HPC (Fig.4). The improvement of dissolution of the spray-freeze-dried AGP-CM attributed to the particle size reduction of AGP, the increase of surface area of AGP nanocrystals (Fig.4), and the porous structure of particles (Fig.5). 3.5 Stability evaluation of the spray-freeze-dried AGP-CM
The physical stability of the spray-freeze-dried AGP-CM with higher ratios of HPC (1:2 and 1:1) was studied upon storage at different temperatures for a period up to 6 months. Figure 9 A, B showed the redispersibility and in vitro dissolution of the spray-freeze-drying AGP-CM at storage for six months. The storage temperature conditions significantly impact stability of AGP-CM with HPC ratio of 1:2(Fig.9 a, b). The RDIs of AGP-CM with HPC ratio of 1:2 at 25 ◦C and 40 ◦C were more than 2, and their dissolution were remarkably decreased compared with those prior to storage (Fig8.d), which indicated that AGP nanoparticles significantly aggregated. But the redispersibility and dissolution of AGP-CM with HPC ratio of 1:2 at 4 ◦C was obvious changed (Fig.9 c). For the AGP-CM with HPC ratio of 1:1 at different storage conditions, the RDIs were near to 1(Fig.9 d, e, f), and their dissolution were equal with those prior to storage (Fig8.e). It was attributed that high ratio of HPC could prevent from agglomeration of AGP nanoparticles at storage. As illustrated in Fig9.C, the AGP nanoparticles were “imprisoned” into “cage-like” matrix structure of HPC and reduced the free mobility. The “cage-like” structure of HPC might prevent nanocrystals from particle-particle agglomeration during storage. Therefore, the AGP-CM could easily reconstituted into nanosuspensions after rehydration and improve the dissolution of AGP. 4. Conclusions A kind of cage-like microparticles for poorly soluble drugs used HPC as carrier has been developed, by means of an interesting approach combined homogenization and the spray-freeze-drying technique. The AGP particles were homogenized into the nanosuspensions and the resultant suspension was further processed into composite particles via spray-freeze-drying. It was demonstrated that HPC not only was an effective stabilizer during homogenization, but also a unique matrix carrier for drug nanocrystals. The spray-freeze-dried AGP-CM with 1:1 ratio of HPC could form cage-like matrix structure, in which the drug nanocrystals particles were completely imprisoned and immobilized. Such unique structure could help AGP-CM to reconstitute into the liquid nanosuspensions and significantly improve the dissolution of AGP. The PXRD and DSC results indicated that the AGP crystal state was altered
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Fig.1 Schematic image of design of composite microparticles via combination of homogenization with spray-freeze-drying technology
Fig.2 The particle sizes of AGP-NS with different ratios of HPC during predetermined homogenization pressure (A), and span of AGP-NS with different ratios of HPC during homogenization of 1200bar (B)
Fig.3 SEM image of coarse AGP (A) and TEM images of AGP-NS with different ratios of HPC to AGP (B 1:20, C 1:10, D 1:5, E 1:2, F 1:1, G 2:1), and schematic image of stability mechanism of AGP-NS (H)
Fig.4 The redispersibility (RDI) and specific surface area (SSA) of the spray-freeze-dried AGP-CM with different ratios of HPC to AGP
Fig.5 SEM photographs of the AGP-CM with different ratios of HPC to AGP (A 1:20, B 1:10, C 1:5, D 1:2, E1:1, F 1:1 Magnification)
Fig.6 XRD diffractograms of HPC (A), raw AGP (B), and the AGP-CM with different HPC ratios (C 1:20, D 1:10, E 1:5, and F 1:1)
Fig.7 Thermograms of HPC (A), raw AGP (B), and the AGP-CM with different ratios of HPC (C 1:20, D 1:10, E 1:5, F 1:1)
Fig.8 Dissolution of raw AGP (a) and the AGP-CM with different ratios of HPC (b 1:10, c 1:5, d 1:2, e 1:1)
Fig.9 The redispersibility(A) and in vitro dissolution(B) the AGP-CM at storage for six months, and schematic image of stability of AGP-CM used HPC as carrier(C)
Table 1 Formulation of homogenization and spray-freeze-drying processes with various compositions of HPC and AGP Composition ratios of HPC to AGP Materials Unit
1:20
1:10
1:5
1:2
1:1
2:1
HPC
mg
50
100
200
500
1000
2000
AGP
mg
1000
1000
1000
1000
1000
1000
Water
mL
100
100
100
100
100
100