Article pubs.acs.org/molecularpharmaceutics
Sinking-Magnetic Microparticles Prepared by the Electrospray Method for Enhanced Gastric Antimicrobial Delivery Shilei Hao, Yazhou Wang, and Bochu Wang* Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, China
ABSTRACT: A targeted oral drug delivery system is useful to improve the treatment of gastrointestinal diseases. A high density sinking dosage form can sink to the bottom of the stomach near the pylori sections to enhance gastric retention. However, it is difficult to achieve a high density sinking system using the traditional technology. In the current study, novel stomach-specific sinking magnetic microparticles (SMMPs) were prepared via the monoaxial electrospray method for enhanced gastric antimicrobial delivery. The size of SMMPs was approximately 5 μm, and the Fe3O4 nanoparticles were observed in the SMMPs by transmission electron microscopy (TEM). The density of SMMPs increased as the concentration of Fe3O4 nanoparticles in the electrospray inlet flow increased, with the maximum true density of approximately 3.52 g/cm3. The SMMPs displayed strong magnetism in vitro and in vivo. They can settle down in water within 120 s in vitro, and the settling time decreased to 20 s under a magnetic field. Furthermore, an in vivo γ scintigraphy study demonstrated that 131I labeled SMMPs were retained in the stomach for over 8 h, and an external permanent magnet can increase their gastric retention time even further. Using Helicobacter pylori as a model bacterium, amoxicillin-loaded SMMPs exhibited a significantly greater eradication of H. pylori compared to the free drug, in vivo. Our results suggested that electrospray is an effective technique to prepare the high density gastroretentive dosage forms. We have shown that stomach-specific SMMPs can supply better treatment for H. pylori infections and have the potential to be used in clinical practice. KEYWORDS: high density, magnetic micropraticles, electrospray, SPECT/CT, Helicobacter pylori
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INTRODUCTION
develop the low density stomach-specific systems, and few articles focused on the development of sinking stomach-specific systems because it is difficult to achieve a high density for a stomach-specific system by traditional technology.10 The sinking system can sink to the bottom of the stomach near the pylori sections to enhance the gastric retention time and control the release rate of drugs.5,11 High density excipient and entrapment efficiency are necessary for the fabrication of sinking systems. Fe 3O4 nanoparticles have the property of high density.12 The bulk density of magnetic nanoparticles is approximately 0.8 g/cm3, and their true density is as high as approximately 5.0 g/cm3.
Delivery of the therapeutic agents to target sites and maintaining high drug concentration for a long time via controlled release is useful to improve the effectiveness of treatment,1 and several controlled release drug delivery systems have been used for gastric antimicrobial delivery. A successful example is the stomach-specific delivery system, which can solve some problems of oral drug administration because of gastric emptying, including the limited gastric retention time, incomplete drug release from the device in the stomach, and low effectiveness of the administered dose when the drug presents in an absorption window.2−4 Various stomach-specific drug delivery systems have been designed, including a floating (low density) system, a sinking (high density) system, a bioadhesive system, a swelling and expanding system, and a magnetic system, on the basis of different mechanisms.5−9 However, many researchers have made considerable efforts to © 2014 American Chemical Society
Received: Revised: Accepted: Published: 1640
January 14, 2014 March 26, 2014 March 28, 2014 March 28, 2014 dx.doi.org/10.1021/mp5000339 | Mol. Pharmaceutics 2014, 11, 1640−1650
Molecular Pharmaceutics
Article
Preparation of AMO-Loaded SMMPs. The blank SMMPs (without drug) were first prepared by emulsion electrospray method at room temperature (relative humidity: 50 ± 15%). The emulsion was prepared to improve the homogeneous distribution of Fe3O4 nanoparticles in electrospray fluid. The water-in-oil (W/O) emulsions were prepared as follow: 9% ERS (w/v) and 1% PVP were dissolved in dichloromethane, and different concentrations of Fe3O4 nanoparticles (from 10% to 30%) were added into the ERS solution as the oil phase. The W/O emulsions were formed by addition of 0.5 mL of aqueous solution to the 10 mL of the oil phase with sonication under ice cooling for 30 s at 400 W. The ultrasonication was supplied by an ultrasonic cell disruption system (JY92- II, Scientz, China). The spray solution was supplied by a syringe pump (TJ-3A, Longer, China) that sprayed from the grounded nozzle (inner diameter 0.88 mm, outer diameter 1.27 mm). The nozzle was connected to a positive electrode (+10 kV) of a high voltage power supply (DW-P503−1AC, Dongwen, China). The flow rate of the solution was 1 500 μL/h, and aluminum foil was placed perpendicular to the nozzle as a collector. The distance between the nozzle and aluminum foil was 100 mm. In addition, the AMO-loaded SMMPs were prepared by addition of AMO to the water phase, and the effects of different AMO concentrations (from 0.25% to 0.40%) on the characterization of drug-loaded SMMPs were investigated. Characterizations. Viscosity Measurement. The viscosities of electrospray solutions with different concentrations of Fe3O4 nanoparticles (from 10% to 30%) were detected using a rotary viscometer (NDJ-8S, Hengping, China). The smallest rotor was selected with rotation rate maintained at 60 rpm. All operations were performed at room temperature. Settling Kinetics Measurement. The settling kinetics of AMO-loaded SMMPs were recorded by a camera. SMMPs were placed into the water solution under or without magnetic field. Morphology. The surface morphology of the AMO-loaded SMMPs with different concentrations of Fe3O4 nanoparticles were observed by transmission electron microscope (TEM) and scanning electron microscope (SEM). A piece of aluminum foil loading with SMMPs was coated with gold metal under vacuum and then examined by SEM (EVOLS25, Zeiss, Germany). The SMMP suspensions were dropped on copper grids, natively stained with phosphotungstic acid and dried at room temperature for the TEM observation (Tecnai G2 20, FEI, USA). In addition, the morphology of Fe3O4 nanoparticles was also observed by TEM. Particle Size and ζ Potential Measurements. The particle size distribution and polydispersity index (PDI) of the SMMPs were measured using a laser light scattering particle size analyzer (3500S, Microtrac Inc., USA). The zeta potentials of the SMMPs were also analyzed using a Zetasizer (Nano ZS90, Malvern, UK). The samples were diluted to appropriate concentrations with deionized water. In addition, the 0.1 N HCl solution (pH 1.2) was prepared as simulated gastric fluid (SGF), and the samples were also diluted with the SGF to determine the zeta potential. Loading Capacity Measurement. The loading capacity and entrapment efficiency of the AMO-loaded SMMPs (30% Fe3O4 nanoparticles) were determined by following method. SMMPs were incubated in dichloromethane to dissolve the SMMPs, PBS buffer solution (pH 7.4) was then added to the solution, and the mixed solution was stirring until dichloromethane was volatilized completely. A permanent magnet was used to
Therefore, the introduction of magnetic Fe3O4 nanoparticles into sinking systems would increase their density. In addition, Fe3O4 nanoparticles have been widely used in medical diagnostics and targeted drug delivery due to their good magnetic response.13,14 An external magnetic field was used to retain magnetic drug delivery systems at a specific site in the body.15 Therefore, the produced sinking system can also be a magnetic stomach-specific system, while the magnetic system is based on the retention mechanism that the dosage form contains internal magnet materials and a magnet placed on the abdomen over the position of the stomach.11 Electrospray has received considerable attention in polymeric micro-/nanoparticle manufacture recently,16−18 and the two outstanding features of the electrospray method are its speed and high efficiency, i.e. preparation of the solid particles in one step and entrapment of the drug into the particle without loss (100% entrapment efficiency).19 The size of particles can be adjusted by changing the polymer concentration, the applied voltage, the diameter of the nozzle, and the flow rate of the solution. The drug-loaded particles can be fabricated using single, coaxial, and emulsion electrospray. We have prepared the high drug loading capacity pH-sensitive nanoparticles by the electrospray method in the previous study.19 The aim of the present study was to prepare novel Eudragit RS (ERS) sinking-magnetic microparticles (SMMPs) by the electrospray method for the treatment of Helicobacter pylori (H. pylori) infections. H. pylori infects about half of the people in the world, and it is an important cause of duodenal and gastric ulcers.20,21 Amoxicillin (AMO) was selected as a model drug, and it has been widely used as a critical component of combination therapies for H. pylori infection.22 The physicochemical characteristics of produced SMMPs were examined, and single-photon emission computed tomography (SPECT) was used to investigate the in vivo gastric retention behaviors of the SMMPs in rabbits. Furthermore, the in vitro and in vivo H. pylori clearance abilities of AMO-loaded SMMPs were studied, and histological examination was used to detect tissue inflammatory in H. pylori-infected mice models.
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EXPERIMENTAL SECTION Materials. Eudragit RS PO (ERS, Mw = 32 kDa) was a kind gift from Evonik Industries (Essen, Germany). Amoxicillin sodium (AMO) was supplied by the Tuochukangyuan Pharm&Chem Co., Ltd. (Hubei, China). Dichloromethane was purchased from Chuandong Reagent Factory (Chongqing, China). Human gastric epithelial cell line (GES-1) was supplied by Institute of Pathology, Southwest Hospital (Chongqing, China). MTT was supplied by the Amresco (Solon, USA). Iodine-131 (131I) was supplied by Department of Nuclear Medicine, Chongqing Cancer Hospital (Chongqing, China). H. pylori strain 26695 (ATCC 700392) was supplied by Department of Clinical Microbiology and Clinical Immunology, Third Military Medical University (Chongqing, China). All other materials and reagents used in the present study were analytical grade. Method. Preparation of Magnetic Fe3O4 Nanoparticles. The magnetic Fe3O4 nanoparticles were prepared according to a modified method:23 1.83 g of FeCl2·4H2O and 4.35 g of FeCl3·6H2O (1:1.75 molar ratio of Fe2+/Fe3+) were added into 50 mL water, and 100 mL of 0.75 M NH4OH solution was added at 50 ◦C under vigorous stirring in N2 atmosphere. The suspension was stirring for 1 h. Finally, the precipitate was washed 3 times in ethanol. 1641
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In Vitro Cytotoxicity of the AMO-Loaded SMMPs. The GES-1 cells were used to test the cytotoxicity of AMO-loaded SMMPs (30% Fe3O4 nanoparticles). For the concentrationdependent cytotoxicity experiments, the SMMPs suspension was diluted with culture medium to different concentrations (from 25 to 800 mg/L). The cells were incubated with the AMO-loaded SMMPs suspension for 24 h. For the timedependent cytotoxicity experiments, GES-1 cells were incubated with blank SMMPs and AMO-loaded SMMPs (200 mg/L) for 12, 24, 36, and 48 h. After the addition MTT solution, absorption at 490 nm was measured with a microplate reader (3550, Bio-Rad, USA). The results were expressed as the percentage of reduction in cell growth/viability compared to untreated control wells, whereas, cells that were not incubated with MTT were used as a blank with which to calibrate the spectrophotometer to zero absorbance. In Vivo Evaluation of the Gastric Retention Ability of SMMPs. Iodine-131 (131I) was selected to radiolabel the AMOloaded SMMPs (30% Fe3O4 nanoparticles) for its half-life of 8.02 days.25 Radiolabeling of the SMMPs was carried out by direct labeling method (physical adsorption).5,26−28 AMOloaded SMMPs were put into a tube, and an aliquot of 131I solution equivalent to radioactivity of 5 mci was added into the tube. The labeled SMMPs were recovered by filtration through a filter paper and dried at room temperature overnight. All operations were performed in fume hood. The stability of 131I-labeled SMMPs were tested as follow:29 the 131I-labeled SMMPs (5 mg) were put into three tubes, and different standard buffers solutions (pH 1.2, 6.8 and 7.4) were added individually to those three tubes. Tubes were kept in a water bath at 37 ◦C for 8 h. At predetermined time intervals, 0.2 mL of samples were collected using a pipet attached to a 0.45-μm cellulose acetate filter and at the end of the experiment the SMMPs were recovered, washed and dried. The radioactivities of the SMMPs, the filtrate and the filter were counted in an auto gamma counter (CRC-25R, Capintec, USA). Three adult male New Zealand white rabbits weighing approximately 2−2.5 kg were used in the study. No rabbits were taking any regular medication or had a history of gastrointestinal disorders. All animal experiments were approved by the Animal Ethical and Experimental Committee of the Third Military Medical University. After fasting for 24 h to the commencement of experiment, the 131I-labeled SMMPs (30 μci) were orally administered through a gastric tube with the aid of 20 mL water. And the rabbits were not allowed to eat or drink during the imaging period. Scintigrams of test preparation were recorded by a single-photon emission computed tomography (SPECT) apparatus with variable angle dualdetector units (Symbia T2, Siemens, USA). At predetermined time intervals, the rabbits underwent anterior whole-body static scintigraphy for 2 min. In addition, another study also carried out to detect the in vivo magnetic effect of SMMPs, and a permanent magnet was put on the stomach of rabbits during the gamma scintigraphy studies. The biodistribution of 131I-labeled SMMPs in vivo was scanned after the gamma scintigraphy studies using a hybrid scanner comprising a two-row spiral CT and a SPECT camera (Symbia T2, Siemens, USA). Immediately after the SPECT data was acquired, the raw data were reconstructed into transverse slices using a Siemens’ powerful syngo-software-based e.sof t workstation (Siemens Medical Solutions). During both the SPECT and the CT scans, the rabbits were lying stably in a supine position. The processed SPECT and CT images were
precipitate the free Fe3O4 nanoparticles. The content of AMO in the solution was detected using a UV spectrophotometer at 230 nm (Lambda 900, PerkinElmer, USA). The loading capacity and entrapment efficiency were calculated by following eq 1 and eq 2: loading capacity =
wt of drug in SMMPs × 100% wt of SMMPs
entrapment efficiency loading capacity of SMMPs = × 100% loading capacity of SMMPs in theory
(1)
(2)
Density Measurement. The bulk density and tap density of the SMMPs with different concentrations of Fe3O4 nanoparticles (from 10% to 30%) were measured as described previously.24 The weight of the SMMPs filling a 1 mL graduated syringe was recorded to calculate the bulk density. The tap density of the SMMPs was evaluated by tapping on a hard bench until there was no further change in the volume of the SMMPs. The resultant volume was recorded to calculate the tap density. In addition, the true density of SMMPs was measured by Gas Pycnometer Helium True Density Analyzer (Gold APP Instruments, G-DenPyc2900, China). Each measurement was performed at least ten times. Magnetic Measurements. A vibrating sample magnetometer (MPMSXL-7, Quantum design, USA) was used to characterize the magnetic properties of the Fe3O4 nanoparticles and SMMPs with different concentrations of Fe3O4 nanoparticle. The hysteresis of the magnetization was obtained by changing H between +2 and −2 T. These measurements were carried out at room temperature. FT-IR Studies. The chemical structure and complex formation of AMO, ERS, Fe3O4 nanoparticles, AMO-loaded SMMPs (30% Fe3O4 nanoparticles) were analyzed by FT-IR (5DX/550, Nicolet, USA). The samples used for the FT-IR spectroscopic characterization were prepared by grinding the dry specimens with KBr and pressing them to form disks. XRD Studies. The XRD experiments were conducted using an X-ray diffractometer (6000X, Shimadzu, Japan). AMO, ERS, Fe 3O 4 nanoparticles, AMO-loaded SMMPs (30% Fe 3O4 nanoparticles), and physical mixture containing AMO (40 mg), Fe3O4 nanoparticles (3.0 g) and ERS were analyzed in the 2θ ranging from 5 degrees to 45 deg with a step width of 0.04 degree and a count time of 2 s. In Vitro Release Studies. The in vitro release study of AMO from SMMPs (30% Fe3O4 nanoparticles) was carried out in the present study. The SMMPs and 3 mL SGF (pH 1.2) were put into a dialysis tube (MWCO: 12 000), and the dialysis tube was placed in 30 mL SGF at 37 ◦C and maintained under shaking at 100 rpm. At specific time intervals, medium (1 mL) was taken and replaced with fresh SGF. A permanent magnet was used to precipitate the free Fe3O4 nanoparticles. The concentration of AMO was determined using a UV spectrophotometer. The influence of different concentrations of AMO on the release profiles was studied. Furthermore, the morphology of AMOloaded SMMPs after incubation in SGF at different times (1, 2, 4, 6, and 8 h) was also observed by SEM. The protocol used for the incubation of the SMMPs in SGF was similar to that used in the in vitro drug release studies. At specific time intervals, the SMMPs in dialysis tube were transferred to a piece of aluminum foil, and then the SMMPs were lyophilized (230, Modulyod, USA) and examined. 1642
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Table 1. Effect of Different Concentrations of Fe3O4 Nanoparticles on the Characteristics of Blank SMMPs (mean ± SD, n = 3)a ζ potential (mV)
size
a
density (g/cm3)
Fe3O4 conc (%)
μm
PDI
SGF
water
bulk
tap
true
viscosity (mPa·s)
10 20 30
5.50 ± 0.03 5.09 ± 0.01 5.13 ± 0.01
0.10 ± 0.03 0.24 ± 0.01 0.20 ± 0.02
43.5 ± 2.1 40.2 ± 2.9 35.1 ± 1.9
30.2 ± 2.2 27.3 ± 2.1 25.7 ± 2.1
0.53 ± 0.01 0.56 ± 0.01 0.62 ± 0.02
0.97 ± 0.01 1.15 ± 0.02 1.36 ± 0.01
2.25 ± 0.02 2.91 ± 0.01 3.52 ± 0.00
34.5 ± 0.2 36.6 ± 0.1 40.1 ± 0.4
b
The concentrations of ERS and PVP were 9% and 1%, respectively. bPDI: polydispersity index.
Narrow strips of tissue were surgically removed from the greater curvature of the stomach, from the duodenum to the gastric cardia and fixed in 10% buffered formalin and paraffin embedded, sectioned, and stained with hematoxylin and eosin by Institute of Pathology, Southwest Hospital. (Chongqing, China). The gastric tissue was evaluated blindly for the extent, depth, and character of inflammation at the antral-fundic junction. The stomach was opened and dissected into two tissue fragments. The fragments were homogenized in 1 mL of BHI using a tissue homogenizer (Precellys 24, Bertin, France). Serial 10-fold dilutions of the homogenate were plated on BHI plates under microaerobic conditions. When present, H. pylori bacteria were confirmed by their characteristic colony morphology, Gram stain and catalase, oxidase, and urease activities. After 5 days of culture, colonies were counted and the number of CFU per gram of stomach was calculated. Statistical Analysis. All of the measurements were performed at least in triplicate, and the data are presented as the means ± standard deviation (S.D.). For selected evaluation tests, the means of all tested formulations were compared with each other through one-way ANOVA with the one-tailed Student’s t- test. The statistical analyses were performed with the SPSS v 17.0 software. The statistical significance level (P) was set to p < 0.05.
reviewed for adequacy of quality, attenuation correction, registration, and fusion by a nuclear medicine specialist. A region of interest (ROI, a circle with 100 mm diameter) was drawn to determinate the radioactivity in the stomach at different time points. ROI quantification was performed with the syngo MI Acquisition Workplace (Siemens Medical Solutions). The radioactivity in the ROI at 0 h was taken as the control and designated as 100%. In Vitro H. pylori Growth Inhibition Study. The standard strain of H. pylori 26695 (ATCC 700392) was maintained on rain−heart infusion (BHI) plates containing 10% rabbit blood and antibiotics under microaerobic conditions (4% O2, 11% CO2, and 85% N2) in a variable atmosphere incubator at 37◦C. After 2 to 3 days of culture, typical colony was collected and suspended in BHI, to a final concentration of 1 × 107 colony forming units (CFU)/mL. The in vitro growth inhibition studies were carried out as follows: bacterial suspensions (30 mL) and the test samples (free drug and AMO-loaded SMMPs) were incubated together for 12 h with shaking at 220 rpm. The samples were put into a dialysis tube (MWCO: 12 000). To simulate the in vivo elimination of drug, bacteria were harvested by centrifugation of 5 000 rpm at 4◦C for 5 min every 2 h. Half of the bacterial suspension was removed and replaced with fresh BHI. At predetermined time intervals, bacterial suspensions (0.5 mL) were taken and serial dilutions (50 μL) were plated on BHI plates. After 5 days of culture under microaerobic conditions, the viable bacterial count was calculated by counting the number of colonies on the BHI plates. In Vivo Clearance of H. pylori. SPF female C57BL/6 mice aged 6−8 weeks were purchased from the Experimental Animal Center of the Third Military Medical University. After an overnight fast, the mice were then orally infected with H. pylori 26695, via the inoculation of 0.3 mL of broth containing approximately 109 CFUs of H. pylori per mL into the stomach of each mouse using an oral feeding needle. This dose was repeated twice daily for 3 consecutive days. Four weeks after the initial infection, the infected mice were randomly divided into ten different groups (n = 4 per group). Different drug regimens were used to treat the H. pylori infection for 3 consecutive days. AMO-loaded SMMPs (30% Fe3O4 nanoparticles) and free drug were orally administered. The frequency of SMMPs was once daily (QD), and free drug groups were administered with QD and twice daily (BID). In addition, the three different doses (low, middle and high) of regimens were studied, which were on the basis of different AMO doses with 15, 35, and 55 mg/kg. Normal saline was orally administered (QD) in the same manner as the control group. The mice were continued to feed 1 week after administration of the final dose, and then the mice were sacrificed, their stomachs were removed and subjected to the following tests.
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RESULTS Preparation of AMO-Loaded SMMPs. Table 1 shows the effect of different concentrations of Fe3O4 nanoparticles on the characterization of blank SMMPs (without drug). The zeta potential decreased and the density increased with an increase in the concentration of Fe3O4 nanoparticles. The zeta potential values were bigger than 30 mV in the SGF, which were higher than that in the water. And the maximum true density was approximately 3.52 g/cm3, which was higher than the density of gastric content (≈1.004 g/cm3).30 In addition, the viscosity of spray fluid also increased with an increase in the concentration of Fe3O4 nanoparticles. However, the sizes of SMMPs were not influenced by the concentration of Fe3O4 nanoparticles, which were approximately 5 μm with a narrow distribution. Table 2 shows the influence of different concentrations of AMO on the loading capacities of AMO-loaded SMMPs. The loading capacity of AMO increased from 0.62 to 0.99% with an increase in the concentration of AMO, but the entrapment efficiencies were maintained at approximately 100%. Characterizations. Morphology. The morphology of AMO-loaded SMMPs were observed by SEM and TEM with different concentrations of Fe3O4 nanoparticles. Figure 1A shows TEM images of SMMPs with different concentrations of Fe3O4 nanoparticles (from 10% to 30%). The shape of SMMPs was spherical in shape, and the amount of Fe3O4 nanoparticles observed in the SMMPs increased with an increase in 1643
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SMMPs, and the density of some SMMPs is not big enough to settle down to the bottom immediately. However, most of SMMPs deposited on the bottom of bottle within 120 Sec. Figure 2B shows the settling kinetics of SMMPs in the water under magnetic field. The SMMPs settled down quickly compared with that without the magnetic field and deposited on the bottom within 20 Sec., which indicated that the produced SMMPs had good sinking and magnetic properties. Magnetic Measurements. Figure 3A shows the magnetization curves of naked magnetic Fe3O4 nanoparticles and SMMPs with different concentrations of Fe3O4 nanoparticles. The saturation magnetization (σs) of Fe3O4 nanoparticle was approximately 59.75 emu/g. The coercivity was approximately 18.99 emu/g, and no remainance was found, indicating that magnetic Fe3O4 nanoparticles are superparamagnetic. And the saturation magnetization of SMMPs decreased from 24.72 to 12.36 emu/g as the concentration of Fe3O4 nanoparticles decreased from 30% to 10%, which should be attributed to the presence of ERS coating on the surface of Fe3O4 nanoparticles. FTIR Studies. Figure 3B depicts the FT-IR spectra of AMO, ERS and AMO-loaded SMMPs. The character bands of ERS including 2 955 cm−1 and 1 450 cm−1 (CHX vibrations), 1 730 cm−1 (CO ester vibration), 1 240 cm−1 and 1 151 cm−1 (−COOR stretching) and character bands of Fe3O4 nanoparticles including 3 380 cm−1 (−OH stretching) and 597 cm−1 (Fe−O vibration) were observed in the spectra of AMO-loaded SMMPs,31 and the characteristic bands of AMO were not found in the spectra of AMO-loaded SMMPs. This results may be due to the low content of drug in the AMO-loaded SMMPs. In addition, new peaks and shifting of peaks were not observed in the spectra of AMO-loaded SMMPs, the results indicated that there were no interaction among AMO, ERS and Fe3O4 nanoparticles. XRD Studies. XRD studies of AMO, ERS, AMO-loaded SMMPs and physical mixture of drug, high density excipient and polymer were also investigated in the current study (Figure 3C). The specific peaks of ERS, AMO, and Fe3O4 nanoparticles were found in the diffractogram of physical mixture. However, the character peaks of AMO were not observed in the diffractograms of AMO-loaded SMMPs. This difference may be due to the low drug loading in the SMMPs, and the characteristic peaks of AMO may be overlapped with the noise of the coated polymer itself.32
Table 2. Effect of Different Concentrations of AMO on the Loading Capacities of AMO-Loaded SMMPs (mean ± S. D., n = 3)a AMO conc (%) 0.25 0.30 0.35 0.40
LC (%)b
EE (%)c
± ± ± ±
100.05 ± 4.01 99.49 ± 2.11 99.87 ± 1.11 100.14 ± 2.89
0.62 0.74 0.87 0.99
0.10 0.10 0.11 0.11
a
The concentrations of ERS, PVP, and Fe3O4 nanoparticles in the formulation were 9%, 1%, and 30%, respectively. bLC: loading capacity. cEE: entrapment efficiency.
Figure 1. (A) TEM images of SMMPs with different concentrations of Fe3O4 nanoparticles. (B) SEM images of SMMPs (30% Fe3O4 nanoparticles). (C) TEM image of Fe3O4 nanoparticles.
concentration of Fe3O4 nanoparticles. Figure 1B shows the SEM images of SMMPs (30% Fe3O4 nanoparticles). The surfaces of SMMPs were not smooth, and the sizes of SMMPs were approximately 5 μm. In addition, Figure 1C shows the TEM image of Fe3O4 nanoparticles, the mean size of magnetic nanoparticles was approximately 20 nm. Particle Settling Experiment. Figure 2A displays the settling kinetics of SMMPs in the water. The SMMPs settled down to the bottom of bottle as time goes on, but the particle suspension was observed at 60 Sec., which may be due to the inhomogeneous distribution of Fe3O4 nanoparticles in the
Figure 2. (A) Settling kinetics of SMMPs in water. (B) Settling kinetics of SMMPs in water under magnetic field. 1644
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Figure 3. (A) Magnetic hysteresis curves of naked Fe3O4 nanoparticles and SMMPs with different concentrations of Fe3O4 nanoparticles. (B) FTIR spectra of AMO, ERS, Fe3O4 nanoparticles, and AMO-loaded SMMPs (30% Fe3O4 nanoparticles). (C) XRD patterns of AMO, ERS, Fe3O4 nanoparticles, AMO-loaded SMMPs, and a physical mixture of AMO, Fe3O4 nanoparticles, and ERS (30% Fe3O4 nanoparticles).
In Vitro Release Studies. The release profiles of AMO from AMO-loaded SMMPs were investigated in SGF. Figure 4A shows the release profiles of AMO from SMMPs with
2 h incubation period, the SMMPs became swelling and conglutination. Increasing number of SMMPs showed fusion with the extension of incubation time, and the obvious fused SMMPS can be observed at 6 h. In Vitro Cytotoxicity Studies. The GES-1 cells were used to evaluate the cytotoxicity of AMO-loaded SMMPs using MTT tests. A decrease in the cell viability was noted with an increase in the concentration of SMMPs. But a significant decrease in the cell viability can be found when the concentration of AMO-loaded SMMPs increased to 200 mg/ L (Figure 5A). The cell viability of AMO-loaded SMMPs and blank SMMPs after incubation with cells for 12, 24, 36, and 48 h were investigated in the subsequent studies. Both the blank SMMPs and AMO-loaded SMMPs can decrease the cell viability, and the cytotoxicity of SMMPs increased as the extension of incubation time (Figure 5B). The cytotoxicity of blank SMMPs would result from the toxicity of Fe3O4 nanoparticles, and AMO would also cause the cytotoxicity. The minimum cell viability in the present study was higher than 70% after treatment with AMO-loaded SMMPs, illustrating that the in vitro cytotoxicity of the prepared SMMPs was slight.33 In Vivo Evaluation of the Gastric Retention Ability of SMMPs. First, the stability of nuclide is important for the quantitative determination of radioactivity in the ROI. So a long half-life nuclide of 131I (8.02 days) was selected for labeling the SMMPs in the current study, and only approximately 2% 131 I was decayed within 8 h. Furthermore, the stabilities of the 131 I-labeled SMMPs in standard buffer solutions with pH values of 1.2, 6.8, and 7.4 were also tested to confirm that the 131I remained bound to the SMMPs during the study. The activity released from 131I-labeled SMMPs were approximately 5.19 ± 0.13% in pH 1.2, 5.47 ± 0.29% in pH 6.8, and 5.73 ± 0.06% in pH 7.4 after 8 h. Figure 6A shows the γ scintigraphic images of the fasted rabbit after oral administration of 131I labeled AMO-loaded SMMPs under magnetic field. The outline of the rabbit was drawn manually and based on the actual rabbit to observe the transformation of hotspot visually. We can see the hotspots locating on the stomach, and the hotspots could be found in the intestine after 2.5 h, which was due to the peristalsis of stomach. In addition, the hotspot also appeared in the rabbit’s thyroid gland, as a result of free 131I. But an amount of hotspots can be seen in the stomach after 8 h. Figure 6B shows the
Figure 4. (A) In vitro release profiles of AMO from SMMPs (30% of Fe3O4 nanoparticles) with different concentratizons of AMO in SGF. (B) SEM images of AMO-loaded SMMPs (30% Fe3O4 nanoparticles) after incubation in SGF for different times.
different concentrations of AMO (from 0.25% to 0.40%), and the release rate of AMO slightly increased with an increase in the concentration of AMO. Furthermore, the sustained release profiles of AMO were observed in acidic medium, and the release rate of AMO is fast compared to some long-circulating particles, the reason could be that the addition of PVP could increase the hydrophilicity of SMMPs. The proportion of AMO released from SMMPs within 8 h was approximately 80%. Furthermore, Figure 4B shows the morphology of AMOloaded SMMPs after incubation different times in SGF. After a 1645
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Figure 5. (A) Cell viability after treatment with different concentrations of AMO-loaded SMMPs (30% Fe3O4 nanoparticles) after 24 h. (B) Cell viability after treatment with blank SMMPs and AMO-loaded SMMPs (200 mg/L) after incubation for different times. (**P ≤ 0.01, *P ≤ 0.05 compared with control group).
Figure 6. γ scintigraphic images of 131I-labeled SMMPs (30% Fe3O4 nanoparticles) in rabbit under magnetic field (A) and without magnetic field (B). (C) Reconstruction of the planar SPECT, CT, and SPECT/CT fused images at rabbits’ stomach and bladder into transverse slices. (D) Radioactive counts of 131I-labeled SMMPs under and without magnetic field in the ROI (2 min cumulative amounts).
increase of gastric retention time of SMMPs, which was due to their strong magnetism (Figure 6B). In addition, the release rate of free 131I from SMMPs decreased under magnetic field because of the aggregation of SMMPs in the stomach. An in vivo biodistribution of 131I labeled AMO-loaded SMMPs was also investigated to provide better anatomic localization of hotspots in hybrid imaging using SPECT/CT techniques. We reconstructed the planar SPECT, CT, and SPECT/CT fused images into transverse slices (Figure 6C).
gamma scintigraphic images of the fasted rabbit after oral administration of 131I labeled SMMPs without magnetic field. The hotspots can also be seen in the stomach, but the transport speed of SMMPs from the stomach to the intestine was fast compared with that under the magnetic field, and bigger amount of free 131I released from the SMMPs and gathered in the rabbit’s thyroid gland. These results indicated that prepared SMMPs display strong gastric retention ability in the stomach, and the external magnetic field was more helpful for the 1646
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Figure 7. (A) Sustained antibacterial effects of free drug, blank SMMPs (30% Fe3O4 nanoparticles), and AMO-loaded SMMPs (30% Fe3O4 nanoparticles). Colonization of H. pylori in the stomach after oral administration of free drug and AMO-loaded SMMPs with low dose (B), middle dose (C), and high dose (D). (**p < 0.01, *p < 0.05, compared with control group; QD, once daily; BID, twice daily).
The fused images display that the intensive hotspots are indeed locating on the rabbits’ stomach, and some hotspots were confirmed in the rabbits’ bladder. A ROI (a circle with 100 mm diameter) was drawn manually around the stomach to measure the radioactive counts. Figure 6D displays the radioactive counts of I131-labeled SMMPs in the ROI for a period of 8 h (2 min cumulative amounts). The radioactive counts in the ROI decreased with time during the γ scintigraphy studies, whether under the magnetic field or not. And the decline rate of radioactive counts without magnetic field was faster than that under magnetic field. Approximately 45% radioactive counts remained in the ROI after 8 h under the magnetic field, while the radioactive counts decreased to 40% without magnetic field under the same time. In Vitro H. pylori Growth Inhibition Study. The in vitro H. pylori growth inhibition effects of AMO-loaded SMMPs, free drug, and blank SMMPs were investigated, and the results are shown in Figure 7A. Blank SMMPs displayed slight inhibition effect on the H. pylori growth in vitro, and both the AMOloaded SMMPs and free drug showed strong inhibition effect. However, free drug exhibited stronger inhibition effect than did the SMMPs at early time points (within 6 h). The number of H. pylori decreased quickly after 1 h of treating with the free drug, but the bacterial number began to increase slowly thereafter, while SMMPs displayed a sustained inhibition effect compared with the free drug, which was due to the sustained release of drug. These results suggested that SMMPs in vitro could supply a sustained and more effective inhibition effect on H. pylori growth. In Vivo Clearance of H. pylori. The in vivo clearance of H. pylori infection by treatment with AMO-loaded SMMPs and free drug was evaluated. The AMO-loaded SMMPs (QD) and AMO solution were divided into three groups on the basis of different AMO doses of 15 (low dose, Figure 7B), 35 (middle dose, Figure 7C), and 55 mg/kg (high dose, Figure 7D). The
free drug groups were divided into two groups (QD and BID) according to different administration frequencies. The mean bacterial counts significantly decreased after 3 days of incubation with AMO-loaded SMMPs and free drug (p < 0.01), which also decreased with an increase in the dose of AMO. Furthermore, the clearance effect of free drug on H. pylori increased with increase of the administration frequency from QD to BID. However, there was no significant difference in the clearance of H. pylori between the free drug with BID dosing and AMO-loaded SMMPs (p < 0.05). Meanwhile, the complete clearance of H. pylori was observed when the dosages of AMO-loaded SMMPs and free drug were middle dose and high dose, respectively. The results indicated that the SMMPs can eradicate H. pylori completely with lower dose and administration frequency of AMO compared with the free drug. In addition, we examined the gastric pathological changes of infected mice and drug-treated infected mice, and the gastric tissue biopsy was stained with hematoxylin and eosin for histological examination (Figure 8). Some findings in the tissue biopsy of infected mice were observed, including inflammatory cell infiltration by neutrophils and lymphocytes (blue arrows), superficial damage and atrophic changes in gastric mucosa (black arrows), infiltration of the inflammatory cells from the lamina propria mucosa to submucosa at sites of mucosal damage, and necrosis of the epithelium (red arrows). The symptom of gastritis was improved by treating the AMO-loaded SMMPs and free drug, including the decrease of inflammatory cells and the alleviation of pathological damage on the surface epithelium. However, slight pathological damage of surface epithelium also existed, and the gastric mucosal damage observed in the infected mice after treatment with free drug with QD dosing was more severe than that observed in the mice after treatment with the free drug with BID dosing and AMO-loaded SMMPs. These results indicated that SMMPs 1647
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Figure 8. Histopathological findings in the gastric mucosa of C57BL/6 mice at 1 month after H. pylori inoculation, and treated with low dose, middle dose, and high dose of free drug (QD and BID dosing) and AMO-loaded SMMPs (QD dosing) after staining with hematoxyline eosin. (QD, once daily; BID, twice daily; blue arrows, inflammatory cell infiltration; black arrows, superficial damage and atrophic changes; red arrows, necrosis of the epithelium.).
were more helpful for the healing of superficial damage than free drug under the same dose and administration frequency.
Another limitation for the development of sinking systems is the low drug content.10 The loading capacities of AMO were lower than 1% in the present study, which was due to the low mass ratio of drug to excipients in the formulation, and the solubility of AMO in aqueous solution is limited. The concentration of AMO was 0.40% in the formulation, whereas the concentrations of ERS and Fe3O4 nanoparticles were 10% and 30%, respectively. However, the volume of SMMPs would be small, so it would not cause inconvenience to patients. Figure 1A shows TEM images of SMMPs with different concentrations of Fe3O4 nanoparticles (from 10% to 30%). And the amount of Fe3O4 nanoparticles observed in the SMMPs increased with an increase in the concentration of Fe3O4 nanoparticles. In addition, the homogeneity of Fe3O4 nanoparticles in the spray fluid is good in the current study, which can maintain stability within 6 h when the concentration of Fe3O4 nanoparticles increased to 30%. And emulsion would contribute to the homogeneous distribution of Fe3O4 nanoparticles. However, the concentration of Fe3O4 nanoparticles may influence their homogeneity in the spray fluid, and the emulsion may be not stable if a larger amount of Fe3O4 nanoparticles were added into the emulsion. The aim of the in vitro H. pylori growth inhibition study was to investigate the sustained inhibition effect of AMO-loaded SMMPs, so the choice of drug dose is important. The sustained inhibition effect cannot be observed with a too high or too low
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DISCUSSION Sinking stomach-specific systems have been designed for many years according to the sedimentation mechanisms, which can sink to the bottom of the stomach near the pylori sections to enhance the gastric retention. The submerged dosage form could supply high drug concentration in the stomach, and then is helpful for the treatment of gastric ulcer, gastritis, and bacterial infection. However, few researchers focused on the sinking systems in the past decades due to the limitation of traditional technology. High density excipient and novel technology with high entrapment capacity are necessary for the fabrication of sinking systems. The electrospray method had been used to prepare the high drug loading particles due to its ultrahigh entrapment efficiency in our previous studies.19 And the high density magnetic Fe3O4 nanoparticles were used as the high density excipient in the current study. The true density of Fe3O4 nanoparticles is as high as 5.0 g/cm3. And the true density of produced SMMPs increased from 2.25 to 3.52 g/cm3 as the concentration of Fe3O4 nanoparticles increased from 10% to 30%. Some articles have reported that a density (∼3 g/ cm3) seems necessary for significant prolongation of gastric residence time.10 1648
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chloride by novel solvent evaporation-matrix erosion method. Carbohydr. Polym. 2011, 85 (3), 592−598. (5) Guan, J.; Zhou, L.; Nie, S.; Yan, T.; Tang, X.; Pan, W. A novel gastric-resident osmotic pump tablet: in vitro and in vivo evaluation. Int. J. Pharm. 2010, 383 (1), 30−36. (6) Liu, Y.; Zhang, J.; Gao, Y.; Zhu, J. Preparation and evaluation of glyceryl monooleate-coated hollow-bioadhesive microspheres for gastroretentive drug delivery. Int. J. Pharm. 2011, 413 (1), 103−109. (7) Chen, J.; Blevins, W. E.; Park, H.; Park, K. Gastric retention properties of superporous hydrogel composites. J. Controlled Release 2000, 64 (1), 39−51. (8) Chen, R.-N.; Ho, H.-O.; Yu, C.-Y.; Sheu, M.-T. Development of swelling/floating gastroretentive drug delivery system based on a combination of hydroxyethyl cellulose and sodium carboxymethyl cellulose for Losartan and its clinical relevance in healthy volunteers with CYP2C9 polymorphism. Eur. J. Pharm. Sci. 2010, 39 (1), 82−89. (9) Sauzet, C.; Claeys-Bruno, M.; Nicolas, M.; Kister, J.; Piccerelle, P.; Prinderre, P. An innovative floating gastro retentive dosage system: Formulation and in vitro evaluation. Int. J. Pharm. 2009, 378 (1), 23− 29. (10) Prajapati, V. D.; Jani, G. K.; Khutliwala, T. A.; Zala, B. S. Raft forming systemAn upcoming approach of gastroretentive drug delivery system. J. Controlled Release 2013, 168 (2), 151−165. (11) Bardonnet, P.; Faivre, V.; Pugh, W.; Piffaretti, J.; Falson, F. Gastroretentive dosage forms: Overview and special case of Helicobacter pylori. J. Controlled Release 2006, 111 (1), 1−18. (12) He, H.; Gao, C. Supraparamagnetic, conductive, and processable multifunctional graphene nanosheets coated with high-density Fe3O4 nanoparticles. ACS Appl. Mater. Interfaces 2010, 2 (11), 3201−3210. (13) McCarthy, J. R.; Weissleder, R. Multifunctional magnetic nanoparticles for targeted imaging and therapy. Adv. Drug Delivery Rev. 2008, 60 (11), 1241−1251. (14) Mornet, S.; Vasseur, S.; Grasset, F.; Duguet, E. Magnetic nanoparticle design for medical diagnosis and therapy. J. Mater. Chem. 2004, 14 (14), 2161−2175. (15) Berry, C. C.; Curtis, A. S. Functionalisation of magnetic nanoparticles for applications in biomedicine. J. Phys. D Appl. Phys. 2003, 36 (13), R198. (16) Jung, J. H.; Park, S. Y.; Lee, J. E.; Nho, C. W.; Lee, B. U.; Bae, G. N. Electrohydrodynamic nano-spraying of ethanolic natural plant extracts. J. Aerosol Sci. 2011, 42 (10), 725−736. (17) Lee, Y.-H.; Mei, F.; Bai, M.-Y.; Zhao, S.; Chen, D.-R. Release profile characteristics of biodegradable-polymer-coated drug particles fabricated by dual-capillary electrospray. J. Controlled Release 2010, 145 (1), 58−65. (18) Qiu, K.; He, C.; Feng, W.; Wang, W.; Zhou, X.; Yin, Z.; Chen, L.; Wang, H.; Mo, X. Doxorubicin-loaded electrospun poly (L-lactic acid)/mesoporous silica nanoparticles composite nanofibers for potential postsurgical cancer treatment. J. Mater. Chem. B 2013, 1 (36), 4601−4611. (19) Hao, S.; Wang, Y.; Wang, B.; Deng, J.; Liu, X.; Liu, J. Rapid preparation of pH-sensitive polymeric nanoparticle with high loading capacity using electrospray for oral drug delivery. Mat. Sci. Eng. CMater. 2013, 33 (8), 4562−4567. (20) Harris, A. Current regiments for treatment of Helicobacter pylori infection. Brit. Med. Bull. 1998, 54 (1), 195−205. (21) Misiewicz, J.; Harris, A.; Bardhan, K.; Levi, S.; O’Morain, C.; Cooper, B.; Kerr, G.; Dixon, M.; Langworthy, H.; Piper, D. One week triple therapy for Helicobacter pylori: a multicentre comparative study. Gut 1997, 41 (6), 735−739. (22) Graham, D. Y.; Fischbach, L. Helicobacter pylori treatment in the era of increasing antibiotic resistance. Gut 2010, 59 (8), 1143− 1153. (23) García-Jimeno, S.; Estelrich, J. Ferrofluid based on polyethylene glycol-coated iron oxide nanoparticles: Characterization and properties. Colloids Surf. A 2013, 420, 74−81. (24) Aquino, R. P.; Auriemma, G.; Mencherini, T.; Russo, P.; Porta, A.; Adami, R.; Liparoti, S.; Porta, G. D.; Reverchon, E.; Del Gaudio, P. Design and production of gentamicin/dextrans microparticles by
AMO dose. The MIC50 of AMO on H. pylori isolates is 0.125 mg/L,34 so the doses of AMO in free drug and AMO-loaded SMMPs formulations were at the concentration of 0.125 mg/L. In addition, the in vitro inhibitory effect of the drug would be more sustainable than that in vivo because of the lack of the drug elimination. The in vivo elimination half-times of AMO are about 1−1.3 h,35 and we assumed that the elimination half-time of AMO was 2 h in the present study. So the concentration of AMO in culture solution would be reduced by half every 2 h.
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CONCLUSIONS The AMO-loaded SMMPs were prepared via the electrospray method in the present study to treat H. pylori infections. The SMMPs can settle down in water completely within 120 s, and the settling time can be decreased to 20 s under magnetic field. The drug loading capacity of SMMPs is low because of the addition of high density excipients. However, the administration of produced SMMPs would not be inconvenient to patients because the amount of drug in a certain volume of the system is not low. In addition, an in vitro sustained release of AMO was observed. SMMPs also displayed good gastric retention ability in vivo, as they can be retained in the stomach for over 8 h, and an external magnet field was helpful for the prolongation of their gastric residence time. The in vivo H. pylori clearance studies clearly indicate that AMO-loaded SMMPs displayed a stronger anti-H. pylori effect than free drug, and these results demonstrated that electrospray is an effective technique for the preparation of SMMPs. The produced AMO-loaded SMMPs have the potential to treat H. pylori infections in clinical practice.
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AUTHOR INFORMATION
Corresponding Author
*E-mail address:
[email protected]. Tel./Fax: +86-236511-2840. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Prof. Zou Quanming for the H. pylori strain 26695 and SPF C57BL/6 mice supply (Department of Clinical Microbiology and Clinical Immunology, Third Military Medical University, Chongqing of China) and Dr. Huang Haiping (Department of Pathology, Chongqing Cancer Hospital, Chongqing of China) for help with the histopathological analysis. The authors acknowledge the financial assistance provided by National Basic Research Program of China (973 Program, Grant No. 2014CB541603) and the National Natural Science Foundation of China (Grant No. 31200713).
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REFERENCES
(1) Bae, S. E.; Son, J. S.; Park, K.; Han, D. K. Fabrication of covered porous PLGA microspheres using hydrogen peroxide for controlled drug delivery and regenerative medicine. J. Controlled Release 2009, 133 (1), 37−43. (2) Davis, S. S. Formulation strategies for absorption windows. Drug Discovery Today 2005, 10 (4), 249−257. (3) Goole, J.; Vanderbist, F.; Amighi, K. Development and evaluation of new multiple-unit levodopa sustained-release floating dosage forms. Int. J. Pharm. 2007, 334 (1), 35−41. (4) Saravanan, M.; Anupama, B. Development and evaluation of ethylcellulose floating microspheres loaded with ranitidine hydro1649
dx.doi.org/10.1021/mp5000339 | Mol. Pharmaceutics 2014, 11, 1640−1650
Molecular Pharmaceutics
Article
Supercritical Assisted Atomization for the treatment of wound bacterial infections. Int. J. Pharm. 2013, 440 (2), 188−194. (25) Tanaka, K.; Takahashi, Y.; Sakaguchi, A.; Umeo, M.; Hayakawa, S.; Tanida, H.; Saito, T.; Kanai, Y. Vertical profiles of iodine-131 and cesium-137 in soils in Fukushima prefecture related to the Fukushima Daiichi Nuclear Power Station accident. Geochem. J. 2012, 46 (1), 73− 76. (26) Yao, H.; Yao, H.; Zhu, J.; Yu, J.; Zhang, L. Preparation and evaluation of a novel gastric floating alginate/poloxamer inner-porous beads using foam solution. Int. J. Pharm. 2012, 422 (1), 211−219. (27) Kedzierewicz, F.; Thouvenot, P.; Lemut, J.; Etienne, A.; Hoffman, M.; Maincent, P. Evaluation of peroral silicone dosage forms in humans by gamma-scintigraphy. J. Controlled Release 1999, 58 (2), 195−205. (28) Jain, S. K.; Agrawal, G. P.; Jain, N. K. A novel calcium silicate based microspheres of repaglinide: in vivo investigations. J. Controlled Release 2006, 113 (2), 111−116. (29) Badve, S. S.; Sher, P.; Korde, A.; Pawar, A. P. Development of hollow/porous calcium pectinate beads for floating-pulsatile drug delivery. Eur. J. Pharm. Biopharm. 2007, 65 (1), 85−93. (30) Streubel, A.; Siepmann, J.; Bodmeier, R. Floating matrix tablets based on low density foam powder: effects of formulation and processing parameters on drug release. Eur. J. Pharm. Sci. 2003, 18 (1), 37−45. (31) Sun, J.; Zhou, S.; Hou, P.; Yang, Y.; Weng, J.; Li, X.; Li, M. Synthesis and characterization of biocompatible Fe3O4 nanoparticles. J. Biomed. Mater. Res. A 2007, 80 (2), 333−341. (32) Ji, J.; Hao, S.; Wu, D.; Huang, R.; Xu, Y. Preparation, characterization and in vitro release of chitosan nanoparticles loaded with gentamicin and salicylic acid. Carbohydr. Polym. 2011, 85 (4), 803−808. (33) Shen, Y.; Chen, J.; Liu, Q.; Feng, C.; Gao, X.; Wang, L.; Zhang, Q.; Jiang, X. Effect of wheat germ agglutinin density on cellular uptake and toxicity of wheat germ agglutinin conjugated PEG−PLA nanoparticles in Calu-3 cells. Int. J. Pharm. 2011, 413 (1), 184−193. (34) Seck, A.; Mbengue, M.; Gassama-Sow, A.; Diouf, L.; Ka, M. M.; Boye, C. S.-B. Antibiotic susceptibility of Helicobacter pylori isolates in Dakar, Senegal. J. Infec.t Dev. Countr. 2009, 3 (02), 137−140. (35) Spyker, D. A.; Rugloski, R. J.; Vann, R. L.; O’Brien, W. M. Pharmacokinetics of amoxicillin: dose dependence after intravenous, oral, and intramuscular administration. Antimicrob. Agents Chemother. 1977, 11 (1), 132−141.
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