ARTICLE pubs.acs.org/IECR
Preparation of Ca-Alginate Microparticles and Its Application for Phenylketonuria Oral Therapy Yueling Zhang,†,^ Xingyuan Jia,‡,§,^ Lianyan Wang,†,* Jingzhong Liu,§,* and Guanghui Ma† †
National Key Lab of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, P. R. China ‡ Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100005, P. R. China § Beijing Chaoyang Hospital Affiliate of Capital Medical University, 100020, P. R. China
bS Supporting Information ABSTRACT: Lactococcus lactis-expressing phenylalanine ammonia-lyase (LLEP) has been used to treat one of the classic genetic diseases, phenylketonuria (PKU). However, the action of stomach fluid and short residence time of LLEP at the site of absorption become the “neck” for the oral administration of LLEP. To solve these problems, pH-sensitive Ca-alginate microparticles designed as an oral administration carrier were prepared by a spray-solidification method in this study. The spray conditions influenced the size of the Ca-alginate microparticles; thus, conditions were optimized to obtain microparticles with smaller particle size for oral administration to mice. Subsequently, LLEP was encapsulated into Ca-alginate microparticles and the activity retention of LLEP released from the microparticles was examined after the microparticles passed through simulated gastric fluid. The results showed that LLEP could be well protected against simulated gastric fluid and that the final activity retention was up to 92.9%. The effects of alginate concentration on the release profile in vitro and the encapsulation efficiency (EE) were studied, and the results revealed that the microparticles possessed the highest EE and a reasonable release rate when the alginate concentration was 1.0 wt %. This alginate concentration, combined with optimized spray conditions, was used to prepare LLEP-encapsulated microparticles, and they were administered orally to mice with phenylketonuria (PKU). Compared with the groups given blank microparticles and nonencapsulated LLEP, the increase of the blood phenylalanine (Phe) level was significantly slowed after a 7 day treatment with LLEPencapsulated microparticles. Consequently, the Ca-alginate microparticle developed by the spray-solidification method is a promising carrier of LLEP for oral administration.
1. INTRODUCTION Phenylketonuria (PKU) is an inherited metabolic disorder caused by mutations in the phenylalanine catabolic enzyme, phenylalanine hydroxylase (PAH).1 PKU patients always respond favorably to treatment with a low-phenylalanine diet;2 however, compliance with dietary treatment is difficult. Life-long compliance, as now recommended by an internationally used set of guidelines, is probably unrealistic.3,4 Development of an alternate treatment such as enzyme therapy has been explored as a means to reduce reliance on dietary restriction. Enzyme therapy with phenylalanine ammonia-lyase (PAL) was selected as a substitute for the native PAH, because PAL, unlike PAH, is inherently stable and does not require a cofactor for activity. Moreover, PAL could convert phenylalanine (L-Phe), by deamination, into harmless trans-cinnamic acid.5 Although there have been reports of successful delivery of PAL therapeutics across injection routes,6-8 the oral route continues to be most used on account of obvious advantages such as ease of administration, good patient acceptance, etc. Similar to for other enzymes, PAL will normally experience proteolytic degradation in the gastric and intestinal lumen. Hence, protections or modifications are required to native PAL if it is to be orally administered. With the development of genetic engineering, Lactococcus lactis, which is a small intestine-beneficial bacterium, was safely used to produce r 2011 American Chemical Society
Lactococcus lactis-expressing phenylalanine ammonia-lyase (LLEP).9 The Lactococcus lactis organism could provide a protective environment for the enzyme from the action of digestive proteases. Nevertheless, the oral administration of LLEP continued to be a challenge due to the action of stomach fluid and short residence time of LLEP at the site of absorption. Therefore, it is necessary to improve the protection of LLEP in the stomach and prolong residence time in the intestine. Microparticles as a potential oral delivery system for enzyme have attracted great interest because they represent a possible strategy to overcome some problems in oral enzyme administration, such as pH degradation, controlled release, or sustained release.10 Alginate was chosen as the carrier material to prepare microparticles because of its excellent biocompatibility, mucoadhesiveness, pH sensitivity, and mild gelation conditions.11 A spray-solidification technique was developed by directly spraying alginate solution into CaCl2 solution to prepare the Ca-alginate microparticles in our work. To meet the requirement of animal oral administration, the size of microparticle should be far smaller Received: September 26, 2010 Accepted: February 3, 2011 Revised: January 31, 2011 Published: February 28, 2011 4106
dx.doi.org/10.1021/ie101973h | Ind. Eng. Chem. Res. 2011, 50, 4106–4112
Industrial & Engineering Chemistry Research than the diameter of oral administration device (1.5 mm). Hence, the spray conditions were at first systematically investigated to obtain the suitable size of microparticles for oral administration. Then the LLEP was encapsulated into Ca-alginate microparticles, and the effects of alginate concentrations on the EE were studied. The experiment in vitro was conducted in the simulated stomach (pH 1.2) and intestinal (pH 7.8) fluids to reveal the relationship between release profile and alginate concentration. According to the results under optimized conditions, experiments in vivo were conducted to evaluate the therapeutic effects on the PKU mice.
2. EXPERIMENTAL SECTION 2.1. Materials. Sodium alginate was purchased from Acros Organics (Morris Plains, NJ). Calcium chloride (anhydrous) was ordered from Beijing Chemical Works (Beijing, China), Lactococcus lactis-expressing PAL was constructed by our research group. Cinnamic acid was from Sigma-Aldrich Inc. (Germany). L-Phenylalanine was from Beijing XinJingKe Biotechnology Co., Ltd. (China), and other reagents were analytically pure. 2.2. Mice and Diets. The PKU model mice, which were offspring from hybridization of BTBR-Pahenu2 mice and C57BL/ 6J mice,12 were bred in the Laboratory of Animal Sciences, Peking University Health Center. Animals were maintained and bred by standard methods of mouse husbandry, using mouse and rat formula feed according to GB 14924.3-2001. The low-Phe diet contained all essential amino acids except Phe (prepared as a formula of the Teklad Research Diet #TD90368). The PKU model mice were fed with a low-Phe diet for 20 days before the therapy, and then each mouse was put on a regular diet of 3 g/day during the therapy. All procedures were reviewed and approved by the Animal Care Committee, Peking University Health Center. 2.3. Preparation of Blank and LLEP-Encapsulated CaAlginate Microparticles. The experimental setup is shown in Figure 1. In detail, 6 g of alginate solution (1.0 wt %) was introduced into the system by a pump, and droplets were produced by nitrogen gas passing through the nozzle. Then the droplets dropped into a large beaker containing 200 mL of CaCl2 solution (0.5 mol/L) to be directly solidified. The Caalginate microparticles containing LLEP were prepared by dispersing 1 g of LLEP into 6 g of alginate solution (1.0 wt %). The preparation procedure was as the same as that mentioned above. The blank or Ca-alginate microparticles containing LLEP were separated by centrifugation after solidification and washed twice with physiological saline to remove the residual CaCl2 on the surface. 2.4. Sample Characterization. An optical microscope (XSZH3, ChongQing Opitical & Electrical Instrument Co., Ltd., P. R.
Figure 1. A schematic presentation of the experimental setup.
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China) installed with a picture capturer (wv-CP230, Panasonic Co., Ltd., Japan) was used to monitor the blank and encapsulated microparticles. The samples were dried at ambient temperature (20-25 C), and the surface morphology was observed by scanning electron microscopy (SEM, JEM-6700F, Japan). The size distribution was analyzed by a Mastersizer 2000 laser particle analyzer. Polydispersity was determined by the SPAN factor expressed as SPAN ¼ ½Dðv, 90Þ - Dðv, 10Þ=Dðv, 50Þ where D (v, 90), D (v, 10), and D (v, 50) are volume size diameters at 90%, 10%, and 50% of the cumulative volume, respectively. A high value of SPAN indicates a wide distribution in size and a high polydispersity. 2.5. Evaluation of the LLEP Encapsulation Efficiency and Relative Activity Retention. LLEP encapsulated into microparticles was examined by destroying the structure of microparticles. The pH-dependent behavior of alginate can be exploited to release the encapsulated material. In detail, an exact weight (5 g) of Ca-alginate microparticles containing LLEP was first dispersed into an acidic solution (pH 1.2) for 30 min. In such a simulated gastric environment, the calcium alginate is converted into an insoluble alginic acid. Then the microparticles were separated by centrifugation at 3000 rpm (Anke TGL-16G, Anting Co., China) from the hydrochloric acid solution and washed twice to remove HCl and CaCl2. Afterward, the microparticles were transferred into a phosphate buffer solution (pH = 8) for 2 h with magnetic stirring at room temperature. Once passed into the higher pH, the alginic acid is soluble and results in the release of LLEP. After being centrifuged at 5000 rpm for 5 min and washed twice with deionized water, LLEP in the bottom was collected and dried completely in a vacuum drying oven (DZ-2BC, Tianjin Taisite Instrument Co. Ltd., China) and then weighed accurately. The encapsulation efficiency (EE) was calculated according to the following equation: EE ¼ Wt=Wi 100% where Wt is the total amount of LLEP encapsulated into microspheres, and Wi represents the initial amount of LLEP added in the preparation process. To evaluate the protection of the Ca-alginate microparticle from an acidic environment, the relative activity retention of original LLEP and encapsulated LLEP were determined as follows: original LLEP (0.03 g) was dispersed into an acidic solution (pH 1.2) for 30 min and then transferred into 5 mL of sodium borate buffer (0.1 mol/L) at pH 8.5 (pH 8.5 was the optimal pH for PAL activity13). Five milliliters of Phe solution (0.02 mol/L, sodium borate buffer) was added immediately. Furthermore, LLEP-encapsulated microparticles were subjected to the same treatment. As mentioned above, LLEP expressed the active PAL, which could convert Phe to cinnamic acid. Consequently, with the addition of Phe, the cinnamic acid content began to increase due to the deamination reaction between LLEP and Phe. Meanwhile, as a basis for comparison, another sample of original LLEP (0.03 g) was directly dispersed into the sodium borate buffer (0.1 mol/L) at pH 8.5 but Phe was also added. Samples were withdrawn at 0, 30, 60, 90, 120, 150, and 180 min, and the supernatants were separated by centrifugation (10 000g/ 5 min). The absorbance values of cinnamic acid in the supernatants 4107
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were measured (290 nm, Ultrospec 2100 Pro, UV-Vis spectrophotometer, Amersham Biosciences Ltd. Co., U.K.). The LLEP relative activity (RA) retention was calculated according to the following equation: RA ¼ Ae =Ao 100% where Ae = (absorbance value at the end - absorbance value at the beginning) of the encapsulated LLEP or original LLEP; Ao= (absorbance value at the end - absorbance value at the beginning) of the original LLEP in control. 2.6. LLEP Release Profile in Vitro. The release of LLEP from microspheres under simulated pH conditions in gastric fluid followed by intestinal fluid was investigated. A detailed experiment was carried out by dipping a defined amount of encapsulated microparticles (equivalent to 0.03 g of LLEP) into 10 mL of simulated gastric fluid (hydrochloric acid buffer at pH 1.2) and incubated at 37 C for 2 h. At appropriate intervals, 0.2 mL of sample was taken and separated from the microspheres by centrifugation (5000g/5 min) to obtain the supernatant for LLEP determination. Then fresh medium was added to maintain a constant volume. To simulate the process of microparticles moving from the stomach into the intestine, the microparticles were transferred after 2 h to 10 mL of simulated intestinal fluid (sodium borate buffer at pH 7.8) and incubated at 37 C for an additional 2 h. Supernatant samples were withdrawn at appropriate time intervals and replaced by fresh medium. The LLEP content in the supernatant was measured by the Micro BCA Protein Assay (Beckman Coulter, Inc., Brea, CA). Cumulative LLEP release at different time intervals was calculated. 2.7. Oral Administration and Blood Phenylalanine Concentration Determination. The animal test was approved by Experimental Animal Ethics Committee in Beijing. The following formulations were administered intragastrically to mice (eight mice per group) for 7 days: (1) LLEP (0.5 g) suspended in physiological saline (1 mL), (2) blank Ca-alginate microparticles suspended in physiological saline (1 mL), (3) LLEPencapsulated Ca-alginate microparticles (equivalent to 0.5 g of LLEP) suspended in physiological saline (1 mL). To simulate three meals a day, the formulations were administrated by gavages three times (∼0.3 mL at a time) every day. The blood phenylalanine was measured at predetermined time points (0, 4, 7, 10 days). Animals were fasted overnight before the tail blood samples were taken. Then the samples were centrifuged and the phenylalanine concentration in the serum was measured14 by high performance liquid chromatography (HPLC, System Gold pump-125, UV detector-66, Beckman-Coulter, Brea, CA). 2.8. Statistical Analysis. Each value was expressed as the mean ( SD. The statistical difference was analyzed by using a one-way analysis of variance (ANOVA). For a value of P less than 0.05, the difference was considered significant.
3. RESULTS AND DISCUSSION 3.1. Preparation of Blank and Encapsulated Ca-Alginate Microparticles. In general, the Ca-alginate microparticles or
microspheres were manufactured by electrostatic droplet generation-gelation,15,16 membrane emulsification-gelation,17 or a spray-drying technique.18 However, they have some disadvantages for the encapsulation of LLEP for oral administration to mice. In particular, electrostatic droplet generation-gelation cannot realize large-scale production, and the spray-drying technique easily leads to activity loss of the core material.19
Figure 2. The particle size distribution of blank Ca-alginate microparticles (a) and LLEP-encapsulated Ca-alginate microparticles (b).
Furthermore, considering the size of LLEP (∼1 μm), it is difficult to pass the membrane pores when membrane emulsificationgelation was used. Fortunately, these drawbacks were successfully overcome by employing spray-solidification techniques. In the process of preparation, we found that the spray conditions have significant influence on the size of microparticles. In order to make the microparticles pass the device smoothly for animal tests, smaller microparticles need to be prepared. The corresponding conditions were optimized in this study. According to the pre-experimental results (Supporting Information), with an increase in feed rate and a decrease in gas velocity, the particle size became smaller. Because of the limitation of instrument parameters, the minimum feed rate and the maximum gas velocity were maintained as 0.5 mL/min and 600 L/h, respectively. As a result, the Ca-alginate microparticle diameter prepared by the instrument was 58 μm (d4,3, volume weighted mean diameter). The particle size distribution of asprepared Ca-alginate microparticles is shown in Figure 2a. The span value of Ca-alginate microparticles obtained was 2.949, which suggested that the particle size distribution was a little too broad. In the spray process, the liquid was randomly sheared by the gas stream at high speed to form small droplets. Accordingly, the particles manufactured by the spray technique were usually polydispersed. The as-prepared wet microparticles and dried microparticles were observed by optical microscopy and SEM. Figure 3a shows the Ca-alginate microparticles prepared by this method were nonspherical. This was because the alginate solution was sheared and then dropped into the CaCl2 solution to be solidified in a short time. The droplets kept the original shape from the nozzle and had no time to form spherical particles. Figure 3b demonstrates that most of the blank Ca-alginate microparticles collapsed and fused together after air-drying. This phenomenon might be induced by the unique structure of Ca-alginate gel which contains 99-99.5% water.20 Once the water was extracted during the drying process, the shape could not be retained. Compared to the larger particles, the smaller ones (
