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Intestine-targeted delivery potency of O-carboxymethyl chitosan–coated layer-by-layer microcapsules: An in vitro and in vivo evaluation
Materials Science & Engineering C 105 (2019) 110129
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Intestine-targeted delivery potency of O-carboxymethyl chitosan–coated layer-by-layer microcapsules: An in vitro and in vivo evaluation Guo-Qing Huang, Zhi-Kai Zhang, Ling-Yun Cheng, Jun-Xia Xiao
T
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College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China
ARTICLE INFO
ABSTRACT
Keywords: Layer-by-layer assembly Gum Arabic O-carboxymethyl chitosan Intestine-targeted delivery Pharmacokinetics
The intestine-targeted delivery performance of the gum Arabic (GA) – O-carboxymethyl chitosan (OCMC) microcapsules prepared by layer-by-layer (LbL) assembly and genipin crosslinking was evaluated by using an acidsusceptible compound omeprazole as the model. Confocal laser scanning microscope observation revealed that spherical microcapsules with the core-shell structure were successful fabricated. Genipin crosslinking did not affect the microencapsulation yield or drug load, but significantly decreased the particle size and positive charge of the microcapsules, and increased their stability against disintegration in the simulated gastric fluid. Pharmacokinetic analysis indicated that entrapment by GA – OCMC LbL assembly greatly improved the bioavailability of omeprazole and crosslinking by 0.1 mg/mL genipin led to the highest value of 8.76 relative to the control formulation. It was concluded that the GA – OCMC LbL microcapsules could be used for the oral delivery of nutraceuticals and its delivery performance could be tailored by varying the genipin crosslinking degree.
1. Introduction Nutraceuticals are foods and food constituents that provide health benefits beyond basic nutrition. Oral administration is considered to be the most acceptable and preferred route for nutraceuticals as it follows the same natural process of food and nutrient consumption in the body, is non-invasive, and involves neither special techniques nor complex instructions. However, due to the extreme conditions in the gastrointestinal tract, including low stomach pH, digestive enzymes, and alkaline intestine pH, many nutraceuticals have poor oral bioavailability, which significantly lowers their efficacy as disease-preventing agents. Thus, there is a need for delivery systems that could encapsulate, protect, and release susceptible compounds within the food and pharmaceutical industries [1]. Many techniques are now available to deliver cargos to targeted sites [2–7], among which, layer-by-layer (LbL) assembly has attracted particular interests. In this technique, the charged object to be coated is dipped into a solution that contains oppositely charged polyelectrolytes and electrostatic attraction leads to the formation of multilayered emulsions or capsules. With this procedure, laminated coatings with specific functional performances can be designed and fabricated by the careful selection of polyelectrolytes and processing conditions [8]. Using pH-sensitive polyelectrolytes is a recent and promising approach to realize intestine-targeted delivery by LbL assembly [9]. Many
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macromolecules, such as pectin [10], alginate [11], and modified starch [12], have been applied to the outer layer of capsules to impart resistance to the gastric environment, but chitosan has gained special interests due to its natural abundance, biocompatibility, positive charge, and crosslinkability by multiple agents [13,14]. O-carboxymethyl chitosan (OCMC) is a carboxymethylated derivative of chitosan. Compared with its native form, OCMC possesses improved water solubility and more desirable pH sensitivity in addition to other attributes. For these advantages, multiple vehicles, including hydrogels, conjugates, nanoparticles, and microspheres, has been fabricated based on OCMC for the intestine-targeted delivery of nutraceuticals [15]. Its potential as a pH-sensitive LbL coating has been investigated in vitro as well, which found that it could confer better stability against disintegration and controlled release in simulated gastrointestinal fluids [16,17]. However, in vivo evidences are still unavailable to confirm the biological efficacies of OCMC-coated capsules. The authors of this work have systematically investigated the interaction between OCMC and GA as well as the characteristics of resultant complexes [18–20]. Since GA is an excellent emulsifier in the food industry, the application of its combination with OCMC in the formulation of LbL capsules was also investigated. In vitro studies revealed that coating of GA-stabilized oil droplets with OCMC followed by genipin crosslinking conferred enhanced stability against disintegration in the simulated gastric fluid and sustainable release in the
Corresponding author. E-mail address: [email protected] (J.-X. Xiao).
https://doi.org/10.1016/j.msec.2019.110129 Received 11 August 2018; Received in revised form 18 August 2019; Accepted 23 August 2019 Available online 24 August 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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simulated intestinal fluid [21]. Hence, in vivo data are necessary to further validate the efficacy of this delivery system. Omeprazole is a lipophilic drug that is widely used for the prophylaxis and treatment of gastro-duodenal ulcers. This compound degrades rapidly in the acidic environment of stomach and undergoes hepatic first-pass metabolism [22]. For these reasons, its bioavailability following oral administration is very low, making it an ideal model for evaluating the intestine targeting performance of oral delivery systems. The purpose of this work is to evaluate the intestine-targeted delivery potency of the GA – OCMC LbL microcapsules through in vitro and in vivo assays by using omeprazole as a model. The result could provide valuable evidence for the application of OCMC and the LbL technique in the food and pharmaceutical industries for oral delivery system construction.
aluminum stubs with double-sided tape, coated for 250 s with 15 nm gold, then examined and photographed with a scanning electron microscope (JSM-840 A, Jeol, Ltd., Tokyo, Japan) at an accelerating voltage of 15 kV. 2.5. Omeprazole content determination
2. Materials and methods
The content of omeprazole was determined using a high-performance liquid chromatography (HPLC) method. The sample to be analyzed was filtered through a 0.45 μm membrane and 0.5 μL of the filtrate was injected to a HPLC1200 system equipped with an ZORBAX SBC18 column (Agilent, 250 × 4.6 mm, 5 μm). Elution was carried out isocratically using a mixture of 0.2 mol/L aqueous Na2HPO4 solution and acetonitrile (3:1, v/v) in 1 mL/min and 35 °C with wavelength of 302 nm [23]. The content of omeprazole was calculated by comparing the peak area with the standard curve.
2.1. Materials
2.6. Microencapsulation yield and drug load determination
OCMC with degree of substitution 0.35 was prepared in the same method as mentioned in a previous work [19]. GA and SPAN60 were gifts from Changsha Rongxing Biotechnology Co., Ltd. (Changsha, China). Omeprazole, rhodamine B, and fluorescein isothiocyanate were purchased from Sigma (St. Louis, MO, USA). Genipin was bought from Linchuan Biotechnology Co., Ltd. (Fuzhou, China). Soybean oil was obtained from a local supermarket in Qingdao, China. All other reagents were of analytical or higher grade.
The yield and drug load of the microcapsules were calculated as follows:
Microencapsulation yield (%) Weight of omeprazole in LbL microcapsules = × 100 Total weight of omeprazole added to system
Drug load (mg/g) =
2.2. Preparation of omeprazole-loaded GA – OCMC LbL microcapsules
Weight of omeprazole in LbL microcapsules Weight of LbL microcapsules
The weight of omeprazole in LbL microcapsules was determined indirectly by measuring its residual in the supernatant. After the GA – OCMC LbL microcapsules were separated by centrifugation, the supernatant was collected and subjected to omeprazole content determination in the HPLC method mentioned in section 2.5.
Our previous study revealed that the electrostatic reaction between OCMC and GA could occur in a wide pH range from 2.5 to 7.0 [19]. Since omeprazole is susceptible to destruction by gastric acid, the LbL microcapsules were assembled in pH 6.0 in this work. An amount of 10 g omeprazole was dissolved in 100 mL 0.5% (v/v) aqueous solution of dimethyl sulfoxide to yield a 10% (w/v) solution, which was mixed with soybean oil in volume ratio 1:4 and emulsified at 25 °C and 9000 rpm for 5 min in the presence of 2% SPAN60 (v/v). The resultant primary water-in-oil (W/O) emulsion was poured to four-fold volumes of 5% (w/v) GA solution and emulsified again at 3000 rpm for 3 min. The generated secondary water-in-oil-in-water (W/O/W) emulsion was blended with the same volume of 1% (w/v) OCMC solution to get an OCMC to GA mass ratio 1:4 [19]. The mixture was then adjusted to pH 6.0 with 0.1 mol/L HCl and maintained at 35 °C and 300 rpm magnetic agitation to allow OCMC deposition. Fifteen minutes later, the suspension was centrifuged at 4000 rpm for 10 min and the precipitate was crosslinked by dipping in 0.1 mg/mL, 0.3 mg/mL, or 0.5 mg/mL genipin solution at 30 °C for 6 h. Finally, the crosslinked microcapsules were collected and lyophilized for analysis. Control microcapsules were prepared in the same procedure, except that no crosslinking was applied.
2.7. Particle size and zeta potential determination Lyophilized microcapsules were dispersed in 25% (v/v) isopropyl alcohol and vortexed for 3 min in the presence of 0.05% (w/v) Tween 80 [24]. Then, the particle size and zeta potential were measured at 25 °C using a Zetasizer Nano ZS (Malvern, Worcestershire, England). 2.8. Swelling behavior in simulated gastrointestinal fluids Lyophilized microcapsules were weighed (W0) and immersed in the simulated gastric (pH 1.2 HCl solution), intestinal (pH 6.8 PBS solution), and colonic (pH 7.4 PBS solution) fluids at 37 °C in sequence. The incubation in each simulated fluid lasted 3 h and the swollen microcapsules were transferred to the next solution at the end of each incubation. At selected intervals, the swollen microcapsules were taken out, blotted on filter paper, and weighed (Wt). The swelling ratio was then calculated using the following equation:
2.3. Confocal laser scanning microscopy (CLSM)
Swelling ratio =
OCMC and GA were labeled with fluorescein isothiocyanate and rhodamine B respectively in exactly the same methods as mentioned in a previous report [21]. After the remaining dyes were washed away with ethanol, the labeled polyelectrolytes were lyophilized and subjected to omeprazole-loaded microcapsule preparation in the same procedure described above. CLSM images of the samples were then taken by a LEICA TCS SP5 II system (Leica Microsystems, Solms, Germany).
Wt
W0 W0
2.9. In vivo delivery performance evaluation A total of 210 SPF-grade Kunming mice weighed 18–22 g were purchased from Qingdao Drug Inspection Institute, China. Laboratory animal handling and experimental procedures were performed in strict compliance with the requirements of Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation. After acclimatization to standard laboratory conditions for 1 week, the animals were fasted for 24 h, but allowed free access to water during this period. The mice were then randomly divided into 5 groups, including one control group and four experimental groups, with
2.4. Scanning electron microscopy (SEM) The freeze-dried microcapsule powders were mounted on circular 2
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42 mice in each group. Animals in the control group were intragastrically administered with omeprazole in dosage 10 mg/kg·body weight as the primary W/O emulsion mentioned in section 2.2 and those in the other four experimental groups with omeprazole-loaded LbL microcapsules that were not crosslinked or crosslinked by 0.1 mg/mL, 0.3 mg/mL, or 0.5 mg/mL genipin for 6 h in the same equivalent dosage as the control group. At 0 h, 0.5 h, 1.0 h, 1.5 h, 2.0 h, 3.0 h, and 8.0 h post drug administration, 6 mice were randomly selected from each group and 0.5 mL blood was collected from eye ground vein to heparinized tubes. The blood samples were centrifuged at 9000 rpm and 4 °C for 10 min and the plasma was collected and stored in −80 °C until use. Upon omeprazole content determination, the plasma was thawed and mixed with the same volume of acetonitrile – water mixture (9:1, v/v). The suspension was vortexed for 1 min, then allowed to settle while refrigerated at 4 °C. Twenty minutes later, the samples were vortexed for another 20 s and centrifuged at 9000 rpm and 4 °C [25]. The supernatant was then collected for omeprazole content determination in the HPLC method mentioned in section 2.5. The plasma concentration of omeprazole was plotted versus time and three pharmacokinetic parameters, including maximal plasma concentration (Cmax), time taken to reach Cmax (tmax), and area under the concentration-time curve from time 0 to 8 h (AUC), were estimated by using the DAS 2.0 program (Drug and Statistics, Anhui, China). Furthermore, the relative bioavailability of omeprazole was calculated by comparing the AUC of the LbL formulations with that of control.
smoother and the matrix became more intensified (Fig. 2b–d), indicating that genipin crosslinking increased the compactness of the microcapsules. 3.3. Microcapsule properties Zeta potential measurement helps to confirm the polyelectrolyte deposition through LbL assembly. As shown in Table 1, all the microcapsules were positively charged, indicating successful coating of the GA-emulsified oil droplets by OCMC. This result was consistent with the CLSM observation in Fig. 1 and an earlier report that coating with carboxymethyl chitosan reversed the zeta potential of hyaluronic acidcoated aminated mesoporous silica nanoparticles from negative to positive [28]. Besides, the net charge of the microcapsules decreased with genipin concentration rise. This was because that the crosslinking reaction occurred in the –NH2 groups of OCMC [29]. Higher genipin concentration meant more –NH2 consumption by crosslinking and consequently fewer positive charges of the microcapsules. The microencapsulation yield of uncrosslinked microcapsules was around 50%. Genipin crosslinking increased this parameter, but the differences were not significant. The variation of drug load exhibited the similar pattern. Such results indicated that genipin crosslinking slightly decreased the permeability of the OCMC layer and increased the retention of omeprazole in the microcapsules during the entrapment process. Genipin crosslinking significantly decreased the particle size of the microcapsules and 0.5 mg/mL genipin caused a particle size of 89 μm compared with 155 μm of the uncrosslinked counterpart. This result was consistent with the SEM observation (Fig. 2) that crosslinking increased the compactness of the microcapsules and similar works that crosslinking by tea polyphenol decreased the size of OCMC-coated zein nanoparticles [16] and crosslinking by formaldehyde reduced the size of chitosan microspheres in a dose-dependent manner [26]. The particle size of microcapsules is extremely important for the release rates of carried bioactive compounds and ultimately how much is absorbed into the body and hence the overall efficacy of the compounds [30]. Therefore, genipin crosslinking might be an effective variable to tailor the delivery potency of the GA - OCMC LbL microcapsules.
2.10. Statistical analysis All the experiments except otherwise specified were performed on triplicate samples and values were expressed as means ± SD. Differences between mean values were compared using the one-way analysis of variance (ANOVA) by SPSS 16.0 software. Differences were statistically significant at p < 0.05. 3. Results and discussion Crosslinking is an effectively way to fabricate the properties of coatings [1,26] and genipin crosslinking degree has been found greatly affect the in vitro delivery potency of OCMC-coated LbL microcapsules [21]. Hence, the characteristics and delivery potency of the GA – OCMC LbL microcapsules were evaluated as a function of genipin concentration in this work.
3.4. In vitro swelling behavior As can be seen in Fig. 3, the swelling ratio of the uncrosslinked microcapsules approached zero immediately after contact with the simulated gastric fluid, indicating that the microcapsules disintegrated rapidly in the medium due to the nature of electrostatic interaction. The same phenomenon was also seen in the native chitosan – GA polyelectrolyte complex [31] and the possible reasons had been detailed by Abbaszad Rafi and Mahkam [32]. Genipin crosslinking obviously increased the stability of the microcapsules against disintegration in the simulated gastric fluid as evidenced by their measurable swelling ratios, which was quite beneficial for the delivery of acid-susceptible compounds. It should be noted that crosslinking by higher genipin concentrations led to greater swelling and the ratios decreased gradually as the incubation in the simulated gastrointestinal fluids proceeded. This was because that disintegration and water adsorption occurred simultaneously to the microcapsules during the incubations. For the microcapsules with a lower crosslinking degree, though they were easier in water adsorption due to their looser network structure, they suffered greater disintegration and hence exhibited lower swelling ratios; while for the microcapsules with a higher crosslinking degree, though they were more resistant to water absorption, they possessed better stability against disintegration and consequently displayed higher swelling ratios. As the incubation in the simulated fluids continued, disintegration dominated and the swelling ratios of the microcapsules decreased gradually as a result. The gradual disintegration of the microcapsules in
3.1. CLSM The CLSM images of the LbL microcapsules that were not crosslinked or crosslinked by 0.5 mg/mL genipin were taken as representatives to see if the desired core-shell structure was formed. As shown in Fig. 1, obvious spheres could be seen in both the micrographs and the red-labeled droplets (corresponding to GA) were surrounded by green fluorescence (corresponding to OCMC), indicating that OCMC had deposited onto the surface of GA-emulsified omeprazole droplets and microcapsules of the core-shell structure were produced successfully. 3.2. SEM The microstructures of the omeprazole-loaded LbL microcapsules were illustrated in Fig. 2. The freeze-dried microcapsules existed as clusters and only very few individual microcapsules could be identified. The same problem also observed in the vanilla oil-loaded chitosan – GA microcapsules and had been ascribed to the high viscosity of chitosan that hindered the separation of microcapsules from the matrix [27]. The surface of the uncrosslinked microcapsules (Fig. 2a) was rough with many wrinkles. After crosslinking by genipin, the surfaces became 3
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Fig. 1. CLSM images of the omeprazole-loaded GA – OCMC LbL microcapsules that were not crosslinked (a) or crosslinked by 0.5 mg/mL genipin for 6 h. The green and red fluorescence in the images is emitted by OCMC and GA respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the simulated intestinal and colonic fluids might favor the sustainable release of omeprazole during transit in the gastrointestinal tract. The different swelling and disintegration behavior of the formulations depicted in Fig. 3 implied that the microcapsules might possess diverse delivery performances.
levels beyond 2 h, indicating that the drug was quickly metabolized by mice. For the uncrosslinked LbL microcapsules, the omeprazole plasma level displayed similar profile, but showed significantly higher values, implying that coating the primary emulsion droplets with OCMC improved the adsorption of omeprazole. Crosslinking by the three genipin concentrations further boosted the plasma omeprazole level and the drug was still detectable 2 h post administration, suggesting the sustainable release potency of the microcapsules. Crosslinking by 0.1 mg/ mL genipin caused the highest peak omeprazole level of 14.1 μg/mL compared with 6.6 μg/mL and 5.6 μg/mL of 0.3 mg/mL and 0.5 mg/mL genipin respectively at 0.5 h; as the metabolism proceeded, its plasma omeprazole level showed a much sharper decrease and lower values
3.5. Pharmacokinetic studies Fig. 4 is the plasma concentration versus time profile after the intragastric administration of omeprazole at a single dose of 10 mg/ kg·body weight. As for the control formulation, the omeprazole level in plasma increased rapidly during the first 1 h, then declined to very low
Fig. 2. SEM micrographs of the omeprazole-loaded GA – OCMC microcapsules that were not crosslinked (a) or crosslinked by 0.1 mg/mL (b), 0.3 mg/mL (b), and 0.5 mg/mL genipin for 6 h (× 100). 4
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Table 1 Microencapsulation yield, drug load, particle size, and zeta potential of omeprazole-loaded GA – OCMC LbL microcapsules. Sample
Microencapsulation yield (%)
OCMC GA Uncrosslinked 0.1 mg/mL 0.3 mg/mL 0.5 mg/mL
49.53 54.69 56.05 56.60
± ± ± ±
Drug load (mg/g)
a
5.48 2.7a 5.35a 2.85a
495.26 546.94 564.98 566.02
± ± ± ±
a
54.82 27.02a 53.48a 58.55a
Particle size (μm)
Zeta potential (mV)
3936.33 ± 105.58 414.1 ± 45.77 154.76 ± 18.72a 115.47 ± 8.43b 102.65 ± 9.8b 89.15 ± 8.28c
16.37 ± 0.4 −10.12 ± 0.3 3.1 ± 0.51a 2.89 ± 0.09ab 2.24 ± 0.22bc 1.76 ± 0.18c
Note: Values of the same column without same letter(s) indicate significant difference at p < 0.05.
15
Table 2 Pharmacokinetic parameters estimated after a single dose of 10 mg/kg·bw omeprazole formulated in emulsion or omeprazole-loaded GA - OCMC LbL microcapsules.
Uncrosslinked Crosslinked by 0.1 mg/mL genipin Crosslinked by 0.3 mg/mL genipin Crosslinked by 0.5 mg/mL genipin
13
Swelling ratio
11 9 7 5 3 1
1
2
3
4
5
6
7
8
tmax (h)
Cmax (μg/mL)
AUC (h·μg/mL)
Relative bioavailability
Control Uncrosslinked 0.1 mg/mL 0.3 mg/mL 0.5 mg/mL
0.83 ± 0.29 0.83 ± 0.27 0.5 ± 0 1 ± 0.5 0.5 ± 0
0.29 ± 0.09 0.94 ± 0.32 14.11 ± 4.14 6.14 ± 0.27 5.41 ± 2.60
1.56 ± 0.12 4.57 ± 3.32 13.67 ± 2.83 7.52 ± 0.35 7.10 ± 1.56
1 2.93 8.76 4.82 4.55
to 13.67 h·μg/mL was recorded in 0.1 mg/mL genipin. As the crosslinker concentration increased to 0.3 mg/mL and 0.5 mg/mL, the AUC decreased sharply to 7.52 and 7.10 h·μg/mL, but were still higher than the control formulation, indicating that excessive crosslinking retarded the release of omeprazole from the microcapsules due to their compact network structure. Consequently, the relative bioavailabilities of omeprazole in the four LbL formulations with crosslinking degree from 0 (no crosslinking) to the highest were 2.93, 8.76, 4.82, and 4.55 by comparing with the control formulation. Owning to the improved water solubility and more desirable pHsensitivity, OCMC has been regarded as an attractive alternative to chitosan for the development of oral delivery systems [15]. Nanoparticles prepared by crosslinking OCMC alone with Ca2+ showed certain pH-sensitivity [33]. The application of OCMC as a pH-sensitive coating has been reported by few researchers as well. It was reported that deposition of OCMC to zein nanoparticle followed by crosslinking with tea polyphenols [16] or Ca2+ [17] greatly reduced the release of loaded β-carotene or vitamin D3 and provided excellent stability in simulated gastrointestinal conditions. Compared with chitosan, OCMC coating created better controlled release for zein nanoparticles in gastric fluid, because OCMC could form a gel layer in contact with acidic medium that could function as barrier against the diffusion of loaded cargo, while chitosan is highly soluble under the pH of gastric fluid and might decompose quickly. For these reasons, the OCMC-coated zein nanoparticles have been proposed as a promising carrier for the intestine-targeted delivery of bioactive compounds [17,34]. The in vivo results of the current work further confirmed that coating with OCMC could create the intestine-targeted delivery potency for resultant microcapsules. Crosslinking by genipin further enhanced the delivery performance of the microcapsules, which depended greatly on the degree of crosslinking and a moderate crosslinking could yield a relative bioavailability of up to 8.76. Since GA is a common emulsifier and genipin is a safe and natural crosslinker [35], the GA – OCMC LbL system could be used for the oral delivery of nutraceuticals in the food and pharmaceutical industries.
-1 0
Sample
9
Time (h) Fig. 3. Swelling behavior of the omeprazole-loaded GA - OCMC LbL microcapsules as a function of genipin concentration in simulated gastric fluid (0–3 h, pH 1.2), intestinal (3–6 h, pH 6.8), and colonic (6–9 h, pH 7.4) fluids.
Fig. 4. Plasma concentration of omeprazole versus time after a single oral administration of the omeprazole-loaded GA - OCMC LbL microcapsules crosslinked by different genipin concentrations.
than the other two genipin concentrations. This variation pattern indicated that a lower crosslinking degree could provide quicker omeprazole release due to the greater disintegration of the microcapsules in the gastrointestinal tract, but a higher crosslinking degree yielded better sustainable release owing to the compacter structure of the microcapsules. This trend was consistent with the swelling behavior of the microcapsules depicted in Fig. 3. Table 2 summarized the pharmacokinetic parameters deduced from the plasma level versus time profile of different omeprazole formulations. When omeprazole was fed as an emulsion (control formulation), the AUC was around 1.56 h·μg/mL. Upon entrapment by GA – OCMC assembly, the AUC increased markedly to 4.57 ± 3.32 h·μg/mL. Genipin crosslinking further increased the index and the highest value of up
4. Conclusion The intestine-targeted delivery performance of GA – OCMC LbL microcapsules was evaluated by using acid-susceptible omeprazole as a model. CLSM observation confirmed the successful preparation of LbL 5
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microcapsules with the core-shell structure. Genipin crosslinking did not affect the microencapsulation yield or drug load, but significantly decreased the particle size and positive charge of the microcapsules, and increased their stability against disintegration in the simulated gastric fluid. Entrapment by GA - OCMC LbL assembly greatly improved the oral bioavailability of omeprazole and its delivery performance could be tailored by varying the genipin crosslinking degree. Hence, the GA – OCMC LbL system could be a promising vehicle for the oral delivery of nutraceuticals.
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Declaration of competing interest None declared. Acknowledgement This work was supported by the National Science Foundation of China (grant number 31571890), the Key R&D Project (Medical Foods) of Shandong Province (grant number 2018YYSP013), and the Major Fundamental Research Project of Shandong Province (grant number ZR2018ZC0945). References [1] D.J. McClements, Y. Li, Structured emulsion-based delivery systems: controlling the digestion and release of lipophilic food components, Adv. Colloid Interf. Sci. 159 (2010) 213–228. [2] M.C. Braithwaite, C. Tyagi, L.K. Tomar, P. Kumar, Y.E. Choonara, V. Pillay, Nutraceutical-based therapeutics and formulation strategies augmenting their efficiency to complement modern medicine: an overview, J. Funct. Foods 6 (2014) 82–99. [3] M. Yao, D.J. McClements, H. Xiao, Improving oral bioavailability of nutraceuticals by engineered nanoparticle-based delivery systems, Curr. Opin. Food Sci. 2 (2015) 14–19. [4] Y.D. Livney, Nanostructured delivery systems in food: latest developments and potential future directions, Curr. Opin. Food Sci. 3 (2015) 125–135. [5] K.P. Velikov, E. Pelan, Colloidal delivery systems for micronutrients and nutraceuticals, Soft Matter 4 (2008) 1964–1980. [6] D.J. McClements, Enhanced delivery of lipophilic bioactives using emulsions: a review of major factors affecting vitamin, nutraceutical, and lipid bioaccessibility, Food Funct. 9 (2018) 22–41. [7] D.J. McClements, The future of food colloids: next-generation nanoparticle delivery systems, Curr. Opin. Colloid Interface Sci. 28 (2017) 7–14. [8] A.P.R. Johnston, C. Cortez, A.S. Angelatos, F. Caruso, Layer-by-layer engineered capsules and their applications, Curr. Opin. Colloid Interface Sci. 11 (2006) 203–209. [9] E. Guzmán, J.A. Cavallo, R. Chuliá-Jordán, C. Gómez, M.C. Strumia, F. Ortega, R.G. Rubio, pH-Induced changes in the fabrication of multilayers of poly(acrylic acid) and chitosan: fabrication, properties, and tests as a drug storage and delivery system, Langmuir 27 (2011) 6836–6845. [10] F. Comert, A.J. Malanowski, F. Azarikia, P.L. Dubin, Coacervation and precipitation in polysaccharide–protein systems, Soft Matter 12 (2016) 4154–4161. [11] W. Liu, J. Liu, W. Liu, T. Li, C. Liu, Improved physical and in vitro digestion stability of a polyelectrolyte delivery system based on layer-by-layer self-assembly alginate–chitosan-coated nanoliposomes, J. Agric. Food Chem. 61 (2013) 4133–4144. [12] B. Zhang, Y. Pan, H. Chen, T. Liu, H. Tao, Y. Tian, Stabilization of starch-based microgel-lysozyme complexes using a layer-by-layer assembly technique, Food Chem. 214 (2017) 213–217. [13] A. Bepeyeva, J.M.S. de Barros, H. Albadran, A.K. Kakimov, Z.K. Kakimova, D. Charalampopoulos, V.V. Khutoryanskiy, Encapsulation of Lactobacillus casei into calcium pectinate-chitosan beads for enteric delivery, J. Food Sci. 82 (2017)
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Intestine-targeted delivery potency of O-carboxymethyl chitosan–coated layer-by-layer microcapsules: An in vitro and in vivo evaluation Guo-Qing Huang, Zhi-Kai Zhang, Ling-Yun Cheng, Jun-Xia Xiao
T
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College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China
ARTICLE INFO
ABSTRACT
Keywords: Layer-by-layer assembly Gum Arabic O-carboxymethyl chitosan Intestine-targeted delivery Pharmacokinetics
The intestine-targeted delivery performance of the gum Arabic (GA) – O-carboxymethyl chitosan (OCMC) microcapsules prepared by layer-by-layer (LbL) assembly and genipin crosslinking was evaluated by using an acidsusceptible compound omeprazole as the model. Confocal laser scanning microscope observation revealed that spherical microcapsules with the core-shell structure were successful fabricated. Genipin crosslinking did not affect the microencapsulation yield or drug load, but significantly decreased the particle size and positive charge of the microcapsules, and increased their stability against disintegration in the simulated gastric fluid. Pharmacokinetic analysis indicated that entrapment by GA – OCMC LbL assembly greatly improved the bioavailability of omeprazole and crosslinking by 0.1 mg/mL genipin led to the highest value of 8.76 relative to the control formulation. It was concluded that the GA – OCMC LbL microcapsules could be used for the oral delivery of nutraceuticals and its delivery performance could be tailored by varying the genipin crosslinking degree.
1. Introduction Nutraceuticals are foods and food constituents that provide health benefits beyond basic nutrition. Oral administration is considered to be the most acceptable and preferred route for nutraceuticals as it follows the same natural process of food and nutrient consumption in the body, is non-invasive, and involves neither special techniques nor complex instructions. However, due to the extreme conditions in the gastrointestinal tract, including low stomach pH, digestive enzymes, and alkaline intestine pH, many nutraceuticals have poor oral bioavailability, which significantly lowers their efficacy as disease-preventing agents. Thus, there is a need for delivery systems that could encapsulate, protect, and release susceptible compounds within the food and pharmaceutical industries [1]. Many techniques are now available to deliver cargos to targeted sites [2–7], among which, layer-by-layer (LbL) assembly has attracted particular interests. In this technique, the charged object to be coated is dipped into a solution that contains oppositely charged polyelectrolytes and electrostatic attraction leads to the formation of multilayered emulsions or capsules. With this procedure, laminated coatings with specific functional performances can be designed and fabricated by the careful selection of polyelectrolytes and processing conditions [8]. Using pH-sensitive polyelectrolytes is a recent and promising approach to realize intestine-targeted delivery by LbL assembly [9]. Many
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macromolecules, such as pectin [10], alginate [11], and modified starch [12], have been applied to the outer layer of capsules to impart resistance to the gastric environment, but chitosan has gained special interests due to its natural abundance, biocompatibility, positive charge, and crosslinkability by multiple agents [13,14]. O-carboxymethyl chitosan (OCMC) is a carboxymethylated derivative of chitosan. Compared with its native form, OCMC possesses improved water solubility and more desirable pH sensitivity in addition to other attributes. For these advantages, multiple vehicles, including hydrogels, conjugates, nanoparticles, and microspheres, has been fabricated based on OCMC for the intestine-targeted delivery of nutraceuticals [15]. Its potential as a pH-sensitive LbL coating has been investigated in vitro as well, which found that it could confer better stability against disintegration and controlled release in simulated gastrointestinal fluids [16,17]. However, in vivo evidences are still unavailable to confirm the biological efficacies of OCMC-coated capsules. The authors of this work have systematically investigated the interaction between OCMC and GA as well as the characteristics of resultant complexes [18–20]. Since GA is an excellent emulsifier in the food industry, the application of its combination with OCMC in the formulation of LbL capsules was also investigated. In vitro studies revealed that coating of GA-stabilized oil droplets with OCMC followed by genipin crosslinking conferred enhanced stability against disintegration in the simulated gastric fluid and sustainable release in the
Corresponding author. E-mail address: [email protected] (J.-X. Xiao).
https://doi.org/10.1016/j.msec.2019.110129 Received 11 August 2018; Received in revised form 18 August 2019; Accepted 23 August 2019 Available online 24 August 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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simulated intestinal fluid [21]. Hence, in vivo data are necessary to further validate the efficacy of this delivery system. Omeprazole is a lipophilic drug that is widely used for the prophylaxis and treatment of gastro-duodenal ulcers. This compound degrades rapidly in the acidic environment of stomach and undergoes hepatic first-pass metabolism [22]. For these reasons, its bioavailability following oral administration is very low, making it an ideal model for evaluating the intestine targeting performance of oral delivery systems. The purpose of this work is to evaluate the intestine-targeted delivery potency of the GA – OCMC LbL microcapsules through in vitro and in vivo assays by using omeprazole as a model. The result could provide valuable evidence for the application of OCMC and the LbL technique in the food and pharmaceutical industries for oral delivery system construction.
aluminum stubs with double-sided tape, coated for 250 s with 15 nm gold, then examined and photographed with a scanning electron microscope (JSM-840 A, Jeol, Ltd., Tokyo, Japan) at an accelerating voltage of 15 kV. 2.5. Omeprazole content determination
2. Materials and methods
The content of omeprazole was determined using a high-performance liquid chromatography (HPLC) method. The sample to be analyzed was filtered through a 0.45 μm membrane and 0.5 μL of the filtrate was injected to a HPLC1200 system equipped with an ZORBAX SBC18 column (Agilent, 250 × 4.6 mm, 5 μm). Elution was carried out isocratically using a mixture of 0.2 mol/L aqueous Na2HPO4 solution and acetonitrile (3:1, v/v) in 1 mL/min and 35 °C with wavelength of 302 nm [23]. The content of omeprazole was calculated by comparing the peak area with the standard curve.
2.1. Materials
2.6. Microencapsulation yield and drug load determination
OCMC with degree of substitution 0.35 was prepared in the same method as mentioned in a previous work [19]. GA and SPAN60 were gifts from Changsha Rongxing Biotechnology Co., Ltd. (Changsha, China). Omeprazole, rhodamine B, and fluorescein isothiocyanate were purchased from Sigma (St. Louis, MO, USA). Genipin was bought from Linchuan Biotechnology Co., Ltd. (Fuzhou, China). Soybean oil was obtained from a local supermarket in Qingdao, China. All other reagents were of analytical or higher grade.
The yield and drug load of the microcapsules were calculated as follows:
Microencapsulation yield (%) Weight of omeprazole in LbL microcapsules = × 100 Total weight of omeprazole added to system
Drug load (mg/g) =
2.2. Preparation of omeprazole-loaded GA – OCMC LbL microcapsules
Weight of omeprazole in LbL microcapsules Weight of LbL microcapsules
The weight of omeprazole in LbL microcapsules was determined indirectly by measuring its residual in the supernatant. After the GA – OCMC LbL microcapsules were separated by centrifugation, the supernatant was collected and subjected to omeprazole content determination in the HPLC method mentioned in section 2.5.
Our previous study revealed that the electrostatic reaction between OCMC and GA could occur in a wide pH range from 2.5 to 7.0 [19]. Since omeprazole is susceptible to destruction by gastric acid, the LbL microcapsules were assembled in pH 6.0 in this work. An amount of 10 g omeprazole was dissolved in 100 mL 0.5% (v/v) aqueous solution of dimethyl sulfoxide to yield a 10% (w/v) solution, which was mixed with soybean oil in volume ratio 1:4 and emulsified at 25 °C and 9000 rpm for 5 min in the presence of 2% SPAN60 (v/v). The resultant primary water-in-oil (W/O) emulsion was poured to four-fold volumes of 5% (w/v) GA solution and emulsified again at 3000 rpm for 3 min. The generated secondary water-in-oil-in-water (W/O/W) emulsion was blended with the same volume of 1% (w/v) OCMC solution to get an OCMC to GA mass ratio 1:4 [19]. The mixture was then adjusted to pH 6.0 with 0.1 mol/L HCl and maintained at 35 °C and 300 rpm magnetic agitation to allow OCMC deposition. Fifteen minutes later, the suspension was centrifuged at 4000 rpm for 10 min and the precipitate was crosslinked by dipping in 0.1 mg/mL, 0.3 mg/mL, or 0.5 mg/mL genipin solution at 30 °C for 6 h. Finally, the crosslinked microcapsules were collected and lyophilized for analysis. Control microcapsules were prepared in the same procedure, except that no crosslinking was applied.
2.7. Particle size and zeta potential determination Lyophilized microcapsules were dispersed in 25% (v/v) isopropyl alcohol and vortexed for 3 min in the presence of 0.05% (w/v) Tween 80 [24]. Then, the particle size and zeta potential were measured at 25 °C using a Zetasizer Nano ZS (Malvern, Worcestershire, England). 2.8. Swelling behavior in simulated gastrointestinal fluids Lyophilized microcapsules were weighed (W0) and immersed in the simulated gastric (pH 1.2 HCl solution), intestinal (pH 6.8 PBS solution), and colonic (pH 7.4 PBS solution) fluids at 37 °C in sequence. The incubation in each simulated fluid lasted 3 h and the swollen microcapsules were transferred to the next solution at the end of each incubation. At selected intervals, the swollen microcapsules were taken out, blotted on filter paper, and weighed (Wt). The swelling ratio was then calculated using the following equation:
2.3. Confocal laser scanning microscopy (CLSM)
Swelling ratio =
OCMC and GA were labeled with fluorescein isothiocyanate and rhodamine B respectively in exactly the same methods as mentioned in a previous report [21]. After the remaining dyes were washed away with ethanol, the labeled polyelectrolytes were lyophilized and subjected to omeprazole-loaded microcapsule preparation in the same procedure described above. CLSM images of the samples were then taken by a LEICA TCS SP5 II system (Leica Microsystems, Solms, Germany).
Wt
W0 W0
2.9. In vivo delivery performance evaluation A total of 210 SPF-grade Kunming mice weighed 18–22 g were purchased from Qingdao Drug Inspection Institute, China. Laboratory animal handling and experimental procedures were performed in strict compliance with the requirements of Provisions and General Recommendation of Chinese Experimental Animals Administration Legislation. After acclimatization to standard laboratory conditions for 1 week, the animals were fasted for 24 h, but allowed free access to water during this period. The mice were then randomly divided into 5 groups, including one control group and four experimental groups, with
2.4. Scanning electron microscopy (SEM) The freeze-dried microcapsule powders were mounted on circular 2
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42 mice in each group. Animals in the control group were intragastrically administered with omeprazole in dosage 10 mg/kg·body weight as the primary W/O emulsion mentioned in section 2.2 and those in the other four experimental groups with omeprazole-loaded LbL microcapsules that were not crosslinked or crosslinked by 0.1 mg/mL, 0.3 mg/mL, or 0.5 mg/mL genipin for 6 h in the same equivalent dosage as the control group. At 0 h, 0.5 h, 1.0 h, 1.5 h, 2.0 h, 3.0 h, and 8.0 h post drug administration, 6 mice were randomly selected from each group and 0.5 mL blood was collected from eye ground vein to heparinized tubes. The blood samples were centrifuged at 9000 rpm and 4 °C for 10 min and the plasma was collected and stored in −80 °C until use. Upon omeprazole content determination, the plasma was thawed and mixed with the same volume of acetonitrile – water mixture (9:1, v/v). The suspension was vortexed for 1 min, then allowed to settle while refrigerated at 4 °C. Twenty minutes later, the samples were vortexed for another 20 s and centrifuged at 9000 rpm and 4 °C [25]. The supernatant was then collected for omeprazole content determination in the HPLC method mentioned in section 2.5. The plasma concentration of omeprazole was plotted versus time and three pharmacokinetic parameters, including maximal plasma concentration (Cmax), time taken to reach Cmax (tmax), and area under the concentration-time curve from time 0 to 8 h (AUC), were estimated by using the DAS 2.0 program (Drug and Statistics, Anhui, China). Furthermore, the relative bioavailability of omeprazole was calculated by comparing the AUC of the LbL formulations with that of control.
smoother and the matrix became more intensified (Fig. 2b–d), indicating that genipin crosslinking increased the compactness of the microcapsules. 3.3. Microcapsule properties Zeta potential measurement helps to confirm the polyelectrolyte deposition through LbL assembly. As shown in Table 1, all the microcapsules were positively charged, indicating successful coating of the GA-emulsified oil droplets by OCMC. This result was consistent with the CLSM observation in Fig. 1 and an earlier report that coating with carboxymethyl chitosan reversed the zeta potential of hyaluronic acidcoated aminated mesoporous silica nanoparticles from negative to positive [28]. Besides, the net charge of the microcapsules decreased with genipin concentration rise. This was because that the crosslinking reaction occurred in the –NH2 groups of OCMC [29]. Higher genipin concentration meant more –NH2 consumption by crosslinking and consequently fewer positive charges of the microcapsules. The microencapsulation yield of uncrosslinked microcapsules was around 50%. Genipin crosslinking increased this parameter, but the differences were not significant. The variation of drug load exhibited the similar pattern. Such results indicated that genipin crosslinking slightly decreased the permeability of the OCMC layer and increased the retention of omeprazole in the microcapsules during the entrapment process. Genipin crosslinking significantly decreased the particle size of the microcapsules and 0.5 mg/mL genipin caused a particle size of 89 μm compared with 155 μm of the uncrosslinked counterpart. This result was consistent with the SEM observation (Fig. 2) that crosslinking increased the compactness of the microcapsules and similar works that crosslinking by tea polyphenol decreased the size of OCMC-coated zein nanoparticles [16] and crosslinking by formaldehyde reduced the size of chitosan microspheres in a dose-dependent manner [26]. The particle size of microcapsules is extremely important for the release rates of carried bioactive compounds and ultimately how much is absorbed into the body and hence the overall efficacy of the compounds [30]. Therefore, genipin crosslinking might be an effective variable to tailor the delivery potency of the GA - OCMC LbL microcapsules.
2.10. Statistical analysis All the experiments except otherwise specified were performed on triplicate samples and values were expressed as means ± SD. Differences between mean values were compared using the one-way analysis of variance (ANOVA) by SPSS 16.0 software. Differences were statistically significant at p < 0.05. 3. Results and discussion Crosslinking is an effectively way to fabricate the properties of coatings [1,26] and genipin crosslinking degree has been found greatly affect the in vitro delivery potency of OCMC-coated LbL microcapsules [21]. Hence, the characteristics and delivery potency of the GA – OCMC LbL microcapsules were evaluated as a function of genipin concentration in this work.
3.4. In vitro swelling behavior As can be seen in Fig. 3, the swelling ratio of the uncrosslinked microcapsules approached zero immediately after contact with the simulated gastric fluid, indicating that the microcapsules disintegrated rapidly in the medium due to the nature of electrostatic interaction. The same phenomenon was also seen in the native chitosan – GA polyelectrolyte complex [31] and the possible reasons had been detailed by Abbaszad Rafi and Mahkam [32]. Genipin crosslinking obviously increased the stability of the microcapsules against disintegration in the simulated gastric fluid as evidenced by their measurable swelling ratios, which was quite beneficial for the delivery of acid-susceptible compounds. It should be noted that crosslinking by higher genipin concentrations led to greater swelling and the ratios decreased gradually as the incubation in the simulated gastrointestinal fluids proceeded. This was because that disintegration and water adsorption occurred simultaneously to the microcapsules during the incubations. For the microcapsules with a lower crosslinking degree, though they were easier in water adsorption due to their looser network structure, they suffered greater disintegration and hence exhibited lower swelling ratios; while for the microcapsules with a higher crosslinking degree, though they were more resistant to water absorption, they possessed better stability against disintegration and consequently displayed higher swelling ratios. As the incubation in the simulated fluids continued, disintegration dominated and the swelling ratios of the microcapsules decreased gradually as a result. The gradual disintegration of the microcapsules in
3.1. CLSM The CLSM images of the LbL microcapsules that were not crosslinked or crosslinked by 0.5 mg/mL genipin were taken as representatives to see if the desired core-shell structure was formed. As shown in Fig. 1, obvious spheres could be seen in both the micrographs and the red-labeled droplets (corresponding to GA) were surrounded by green fluorescence (corresponding to OCMC), indicating that OCMC had deposited onto the surface of GA-emulsified omeprazole droplets and microcapsules of the core-shell structure were produced successfully. 3.2. SEM The microstructures of the omeprazole-loaded LbL microcapsules were illustrated in Fig. 2. The freeze-dried microcapsules existed as clusters and only very few individual microcapsules could be identified. The same problem also observed in the vanilla oil-loaded chitosan – GA microcapsules and had been ascribed to the high viscosity of chitosan that hindered the separation of microcapsules from the matrix [27]. The surface of the uncrosslinked microcapsules (Fig. 2a) was rough with many wrinkles. After crosslinking by genipin, the surfaces became 3
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Fig. 1. CLSM images of the omeprazole-loaded GA – OCMC LbL microcapsules that were not crosslinked (a) or crosslinked by 0.5 mg/mL genipin for 6 h. The green and red fluorescence in the images is emitted by OCMC and GA respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
the simulated intestinal and colonic fluids might favor the sustainable release of omeprazole during transit in the gastrointestinal tract. The different swelling and disintegration behavior of the formulations depicted in Fig. 3 implied that the microcapsules might possess diverse delivery performances.
levels beyond 2 h, indicating that the drug was quickly metabolized by mice. For the uncrosslinked LbL microcapsules, the omeprazole plasma level displayed similar profile, but showed significantly higher values, implying that coating the primary emulsion droplets with OCMC improved the adsorption of omeprazole. Crosslinking by the three genipin concentrations further boosted the plasma omeprazole level and the drug was still detectable 2 h post administration, suggesting the sustainable release potency of the microcapsules. Crosslinking by 0.1 mg/ mL genipin caused the highest peak omeprazole level of 14.1 μg/mL compared with 6.6 μg/mL and 5.6 μg/mL of 0.3 mg/mL and 0.5 mg/mL genipin respectively at 0.5 h; as the metabolism proceeded, its plasma omeprazole level showed a much sharper decrease and lower values
3.5. Pharmacokinetic studies Fig. 4 is the plasma concentration versus time profile after the intragastric administration of omeprazole at a single dose of 10 mg/ kg·body weight. As for the control formulation, the omeprazole level in plasma increased rapidly during the first 1 h, then declined to very low
Fig. 2. SEM micrographs of the omeprazole-loaded GA – OCMC microcapsules that were not crosslinked (a) or crosslinked by 0.1 mg/mL (b), 0.3 mg/mL (b), and 0.5 mg/mL genipin for 6 h (× 100). 4
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Table 1 Microencapsulation yield, drug load, particle size, and zeta potential of omeprazole-loaded GA – OCMC LbL microcapsules. Sample
Microencapsulation yield (%)
OCMC GA Uncrosslinked 0.1 mg/mL 0.3 mg/mL 0.5 mg/mL
49.53 54.69 56.05 56.60
± ± ± ±
Drug load (mg/g)
a
5.48 2.7a 5.35a 2.85a
495.26 546.94 564.98 566.02
± ± ± ±
a
54.82 27.02a 53.48a 58.55a
Particle size (μm)
Zeta potential (mV)
3936.33 ± 105.58 414.1 ± 45.77 154.76 ± 18.72a 115.47 ± 8.43b 102.65 ± 9.8b 89.15 ± 8.28c
16.37 ± 0.4 −10.12 ± 0.3 3.1 ± 0.51a 2.89 ± 0.09ab 2.24 ± 0.22bc 1.76 ± 0.18c
Note: Values of the same column without same letter(s) indicate significant difference at p < 0.05.
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Table 2 Pharmacokinetic parameters estimated after a single dose of 10 mg/kg·bw omeprazole formulated in emulsion or omeprazole-loaded GA - OCMC LbL microcapsules.
Uncrosslinked Crosslinked by 0.1 mg/mL genipin Crosslinked by 0.3 mg/mL genipin Crosslinked by 0.5 mg/mL genipin
13
Swelling ratio
11 9 7 5 3 1
1
2
3
4
5
6
7
8
tmax (h)
Cmax (μg/mL)
AUC (h·μg/mL)
Relative bioavailability
Control Uncrosslinked 0.1 mg/mL 0.3 mg/mL 0.5 mg/mL
0.83 ± 0.29 0.83 ± 0.27 0.5 ± 0 1 ± 0.5 0.5 ± 0
0.29 ± 0.09 0.94 ± 0.32 14.11 ± 4.14 6.14 ± 0.27 5.41 ± 2.60
1.56 ± 0.12 4.57 ± 3.32 13.67 ± 2.83 7.52 ± 0.35 7.10 ± 1.56
1 2.93 8.76 4.82 4.55
to 13.67 h·μg/mL was recorded in 0.1 mg/mL genipin. As the crosslinker concentration increased to 0.3 mg/mL and 0.5 mg/mL, the AUC decreased sharply to 7.52 and 7.10 h·μg/mL, but were still higher than the control formulation, indicating that excessive crosslinking retarded the release of omeprazole from the microcapsules due to their compact network structure. Consequently, the relative bioavailabilities of omeprazole in the four LbL formulations with crosslinking degree from 0 (no crosslinking) to the highest were 2.93, 8.76, 4.82, and 4.55 by comparing with the control formulation. Owning to the improved water solubility and more desirable pHsensitivity, OCMC has been regarded as an attractive alternative to chitosan for the development of oral delivery systems [15]. Nanoparticles prepared by crosslinking OCMC alone with Ca2+ showed certain pH-sensitivity [33]. The application of OCMC as a pH-sensitive coating has been reported by few researchers as well. It was reported that deposition of OCMC to zein nanoparticle followed by crosslinking with tea polyphenols [16] or Ca2+ [17] greatly reduced the release of loaded β-carotene or vitamin D3 and provided excellent stability in simulated gastrointestinal conditions. Compared with chitosan, OCMC coating created better controlled release for zein nanoparticles in gastric fluid, because OCMC could form a gel layer in contact with acidic medium that could function as barrier against the diffusion of loaded cargo, while chitosan is highly soluble under the pH of gastric fluid and might decompose quickly. For these reasons, the OCMC-coated zein nanoparticles have been proposed as a promising carrier for the intestine-targeted delivery of bioactive compounds [17,34]. The in vivo results of the current work further confirmed that coating with OCMC could create the intestine-targeted delivery potency for resultant microcapsules. Crosslinking by genipin further enhanced the delivery performance of the microcapsules, which depended greatly on the degree of crosslinking and a moderate crosslinking could yield a relative bioavailability of up to 8.76. Since GA is a common emulsifier and genipin is a safe and natural crosslinker [35], the GA – OCMC LbL system could be used for the oral delivery of nutraceuticals in the food and pharmaceutical industries.
-1 0
Sample
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Time (h) Fig. 3. Swelling behavior of the omeprazole-loaded GA - OCMC LbL microcapsules as a function of genipin concentration in simulated gastric fluid (0–3 h, pH 1.2), intestinal (3–6 h, pH 6.8), and colonic (6–9 h, pH 7.4) fluids.
Fig. 4. Plasma concentration of omeprazole versus time after a single oral administration of the omeprazole-loaded GA - OCMC LbL microcapsules crosslinked by different genipin concentrations.
than the other two genipin concentrations. This variation pattern indicated that a lower crosslinking degree could provide quicker omeprazole release due to the greater disintegration of the microcapsules in the gastrointestinal tract, but a higher crosslinking degree yielded better sustainable release owing to the compacter structure of the microcapsules. This trend was consistent with the swelling behavior of the microcapsules depicted in Fig. 3. Table 2 summarized the pharmacokinetic parameters deduced from the plasma level versus time profile of different omeprazole formulations. When omeprazole was fed as an emulsion (control formulation), the AUC was around 1.56 h·μg/mL. Upon entrapment by GA – OCMC assembly, the AUC increased markedly to 4.57 ± 3.32 h·μg/mL. Genipin crosslinking further increased the index and the highest value of up
4. Conclusion The intestine-targeted delivery performance of GA – OCMC LbL microcapsules was evaluated by using acid-susceptible omeprazole as a model. CLSM observation confirmed the successful preparation of LbL 5
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microcapsules with the core-shell structure. Genipin crosslinking did not affect the microencapsulation yield or drug load, but significantly decreased the particle size and positive charge of the microcapsules, and increased their stability against disintegration in the simulated gastric fluid. Entrapment by GA - OCMC LbL assembly greatly improved the oral bioavailability of omeprazole and its delivery performance could be tailored by varying the genipin crosslinking degree. Hence, the GA – OCMC LbL system could be a promising vehicle for the oral delivery of nutraceuticals.
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