Synthesis of CSK-DEX-PLGA Nanoparticles for the Oral Delivery of

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Synthesis of CSK-DEX-PLGA nanoparticles for oral delivery of exenatide to improve its mucus penetration and intestinal absorption Yina Song, Yanan Shi, Liping Zhang, Haiyan Hu, Chunyan Zhang, Miaomiao Yin, Liuxiang Chu, Xiuju Yan, Mingyu Zhao, Xuemei Zhang, Hongjie Mu, and Kaoxiang Sun Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00809 • Publication Date (Web): 02 Jan 2019 Downloaded from http://pubs.acs.org on January 3, 2019

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Molecular Pharmaceutics

Yantai, 264005, China Sun, Kaoxiang; School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, 264005, China,

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Synthesis of CSK-DEX-PLGA nanoparticles for oral delivery of exenatide to improve its mucus penetration and intestinal absorption Yina Songa#, Yanan Shib#, Liping Zhanga, Haiyan Hua, Chunyan Zhanga, Miaomiao Yina, Liuxiang Chua, Xiuju Yana, Mingyu Zhaoa, Xuemei Zhangc, Hongjie Mua, Kaoxiang Suna,c

aSchool

of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug

Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China bSchool cState

of Pharmacy, Binzhou Medical University, Yantai 264005, China

Key Laboratory of Long-acting and Targeting Drug Delivery System, Luye

Pharmaceutical Co. Ltd., Yantai 264005, China #These

authors contributed equally to this work.

Corresponding Authors Yanan Shi, School of Pharmacy, Binzhou Medical University, Guanhai Road No. 346, Yantai 264005, China. E-mail: [email protected]; Tel: +86-18766453922; Fax: +86-535-3946187 Kaoxiang Sun, School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation


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Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Qingquan Road No. 32, Yantai 264005, China. E-mail: [email protected]; Tel: +86-535-3808266; Fax: +86-535-6706066



Abstract Oral absorption of exenatide, a drug for type 2 diabetes treatment, can be improved by using nanoparticles (NPs) for its delivery. To improve the mucus penetration and intestinal absorption of exenatide, we designed a block copolymer, CSKSSDYQC-dextran-poly (lactic-co-glycolic acid) (CSK-DEX-PLGA), and used it for preparation of exenatide-loaded NPs. The functionalized exenatide-loaded NPs composed of CSK-DEX-PLGA were able to target intestinal epithelial cells and reduce the mucus-blocking effect of the intestine. Moreover, the CSK modification of DEX-PLGA was found to significantly promote the absorption efficiency of NPs in the small intestine, based on in vitro ligation of intestinal rings and examination of different intestinal absorption sites. Compared with DEX-PLGA-NPs (DPs), the absorption of CSK-DEX-PLGA-NPs (CDPs) was increased in the villi, allowing the drug to act on goblet-like Caco-2 cells through clathrin-, caveolin-, and gap-mediated endocytosis. Furthermore, enhanced transport ability of CDPs was observed in a study on Caco-2/HT-29-MTX co-cultured cells. CDPs exhibited a prolonged hypoglycemic response with a relative bioavailability of 9.2% in diabetic rats after oral administration. In conclusion, CDPs can target small intestinal goblet cells and have a beneficial effect on oral administration of macromolecular peptides as a nanometer-sized carrier.

Keywords: targeted nanoparticle, oral delivery, mucus penetration, intestinal absorption


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1. Introduction Protein and peptide drugs have distinct biological activities and few side effects, and these factors are important for their use in clinical care. As a drug passes through the gastrointestinal tract, it encounters a series of obstacles, including the stomach and intestinal environment, mucus barrier, and tight connections between the cellular bypass pathways, before reaching the epithelial capillaries. As an example, insulin, a commonly used peptide drug for diabetes, reaches the bloodstream in its intact form at 6,000 Da) and PLGA (Mn > 20,000 Da). As shown in Table 1, the weight-average molecular weight (Mw) of DEX-PLGA was 36,061, coinciding with the sum of the Mn values of DEX (Mn > 8,000 Da) and PLGA (Mn > 28,000 Da). Table 1. GPC characterization of the DEX-PLGA block copolymer

DEX-PLGA

Mn (Da)

Mw (Da)

PDI

26239

36061

1.374342

Mn: number-average molecular weight; Mw: weight-average molecular weight.

3.1.2. Synthesis of CSK-DEX-PLGA To prepare the CSK-conjugated DEX-PLGA copolymer (CSK-DEX-PLGA), CDI was added to activate the hydroxyl groups of the DEX blocks. The aminated CSK was then coupled to the hydroxyl group of DEX by an amide bond. The composition mechanism of CSK-DEX-PLGA is shown in supplementary data Figure S3 (B). Conjugation of CSK to the DEX-PLGA copolymer was evaluated by HPLC and the 1H-NMR spectrum [30]. As shown in supplementary data Figure S3 (C) and (D), there were obvious peaks related to the CSK peptide before the coupling



reaction, while the polypeptide was only minimally detected after the coupling reaction, indicating that the basic reaction of CSK was complete. HPLC was carried out to monitor the polypeptide-coupled polymer materials. The solvent used in the reaction was DMSO, which was employed during the polypeptide synthesis to ensure that the polypeptides were not degraded during the preparation process. The disappearance of the CSK peak in the HPLC peaks after the CSK coupling reaction was not caused by degradation of CSK, but by attachment of CSK to the polymer. The 1H-NMR spectrum results showed that the peaks at δ 7.0 and 6.7 ppm were the peaks of the tyrosine group in the CSK peptide, meaning that the surface CSK peptide was successfully coupled with PLGA-DEX. We chose D2O as the solvent to specifically observe the nuclear magnetic peaks of the hydrophilic chain and avoid interference from the PLGA peaks with the CSK polypeptide peaks. In this study, we precisely synthesized and characterized a block copolymer composed of DEX and PLGA by reductive amination of DEX. The aminated CSK was then coupled to the hydroxyl group of DEX by an amide bond. Finally, CSK-DEX-PLGA block copolymers were successfully prepared. 3.2. Preparation and characterization of DPs and CDPs DEX-PLGA was used as a carrier material to prepare exenatide-loaded NPs (termed DPs) and CSK-DEX-PLGA was used to prepare exenatide-loaded NPs (termed CDPs). The main steps in the CDP preparation are illustrated in supplementary data Figure S4 (A). Both types of NPs were prepared by the double emulsion method. The morphologies of DPs and CDPs were characterized by TEM


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(supplementary data Figure S4 (B)). The results demonstrated that DPs and CDPs were homogeneously dispersed as individual spherical particles. The particle sizes of NPs were in the range of 100–200 nm, as measured by a zeta potential and particle sizer. As shown in Table 2, the particle sizes of DPs and CDPs were 104.4 ± 5.3 nm and 136.3 ± 8.9 nm, respectively. The diameters of DPs and CDPs are shown in supplementary data Figure S5. The particle size of CDPs was larger than that of DPs, indicating that CSK was successfully bound to DEX-PLGA. The size of NPs influences their absorption in the gastrointestinal tract. The sizes of the prepared DPs and CDPs were around 100 nm, which was suitable for absorption by intestinal epithelial cells. Particles larger than 500 nm cannot be absorbed by absorption bubbles, meaning that only particles less than 500 nm can reach the circulation system [41]. A small particle size leads to increased surface area and biofilm adhesion. A large number of NPs entering the intestinal tract will be localized at the Peyer node, where biomolecules pass through the biological mucosa in complete form, thereby improving the bioavailability of oral drugs [4, 42]. The zeta potentials of DPs and CDPs were 0.48 ± 0.025 mV and 0.15 ± 0.019 mV, respectively (Table 2, supplementary data Figure S6). Drug absorption in the intestine occurs through the transcellular route or paracellular route as shown in Figure 1. The cell pathway is primarily dependent on the size and charge of NPs. The surface of the cell membrane (mucus layer) has a negative charge, meaning that positively charged and electrically neutral NPs can easily pass through the mucus layer [43]. The zeta potentials of DPs and CDPs met this electrical requirement.



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Table 2. Size, polydispersity index (PDI), zeta potential (mV), encapsulation efficiency (%), and drug loading (%) of exenatide-loaded DPs and CDPs

Formations

Size(nm)

PDI

Zetapotential

Encapsulation

Drug loading

(mV)

efficiency (%)

(%)

DPs

104.4±5.3

0.201±0.035

0.48± 0.025

76.23±1.57

5.37±0.85

CDPs

136.3±8.9

0.189±0.073

0.15±0.019

71.64±3.21

4.62±1.37

The encapsulation efficiency and drug loading of the prepared NPs were determined and calculated (Table 2). The encapsulation efficiency of DPs was 76.23 ± 1.57% and the drug loading was 5.37 ± 0.85%. The encapsulation efficiency of CDPs was 71.64 ± 1.37% and the drug loading was 4.62 ± 1.37%. The values for CDPs were slightly lower than those for DPs, possibly because the molecular weight of the carrier material for CDPs was larger than that for DPs. 3.3 Mucus aggregation and mucus penetration studies NPs need to avoid strong entanglement with mucin fibers for rapid mucus penetration. Therefore, we evaluated the interactions between mucin and CDPs or DPs by investigating the retention of NPs after CDP and DP dispersion into mucus and measuring the amount of unbound NPs at a fixed time point. As shown in Figure 2 (A), there was no significant difference in mucus retention between the CDP and DP groups. However, the retention of the free FITC-exenatide solution was lower than that of the CDP and DP groups, which may be explained by the strong


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electrostatic repulsion between free FITC-exenatide and the negatively charged mucus. Compared with the free FITC-exenatide groups, the electrostatic repulsion between NPs and mucus was lower because the interactions between the electrically neutral NPs and the mucus layer were weak, and the mucus layer had a strong electrostatic repulsion toward negative charges. A small amount of NPs was dispersed in a large amount of mucus, regardless of whether a small amount of positive charge was present on the CDPs or DPs. These small differences were insignificant in the presence of the large amount of mucus, thus explaining the similar retention of the two NP groups.

Figure 2. (A) Percentages of mucin-particle aggregation in rat intestinal mucus for DPs and CDPs at 37 °C. (B) Papp values of DPs and CDPs during incubation with mucus for 2 h in a mucus penetration study. Data represent means ± SD (n = 3). *p < 0.05, versus DP groups; ##p < 0.05, versus FITC-exenatide groups.

As shown in Figure 2 (B), the mucus permeation rate of CDPs was significantly increased, being 1.55-fold higher than that of DPs and 2.1-fold higher than that of the free solution groups. This arose because CDPs were mostly electrically neutral and had localized negative electrical properties related to the small amount of CSK



attached to their outermost layer [26]. A previous study showed that CSK rapidly crosses the overlying mucus and enters the epithelium via receptor-mediated pathways [8]. Furthermore, the mucus was electrically negative and the DPs were electrically neutral, whereas DEX was positively charged and thus had a stronger attraction to the mucus and affected the mucus permeation of DPs. Thus, CDPs exhibited desirable mucus permeability. 3.4 In vivo transport of NPs after oral administration The biodistribution of NPs was visually investigated using an in vivo imaging system. After oral administration, the fluorescence intensity of NPs in the stomach decreased over time and the accumulation in other major organs (heart, liver, spleen, lungs, and kidneys) increased (Figure 3 (A)). At 2 h post-administration, the fluorescence intensity of CDPs in the small intestine (especially the jejunum) was significantly higher than that of DPs, indicating that CDPs had a high targeting efficiency for the small intestine. In contrast, the DP group showed no significant difference in fluorescence intensity in all parts of the small intestine, indicating that the absorption of DPs in the small intestine was not specific. These results also demonstrated that CSK was targeted to the small intestinal goblet cells [36]. In rats, the transit times for the jejunum, small intestine, and total gastrointestinal tract were reported to be 80 min, 3 h, and 11 h, respectively [44, 45]. At the next time point, there was no significant difference between the two groups because of the effect of gastrointestinal motility on NPs. At 24 h post-administration, the fluorescence intensity was weak in both groups, indicating that the drug was either digested or


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excreted in vitro.

Figure 3. Distribution of DIR in different intestinal segments (A) and heart, liver, spleen, lung and kidney (B) of rats was monitored by in vivo imaging at different time points after oral administration of DPs or CDPs in rats.

The accumulation of DiR in other major organs (heart, liver, spleen, lung, and kidney) was also increased. As shown in Figure 3 (B), the CDP group showed obvious fluorescence intensity in the liver at 2 h after administration, but no



fluorescence was observed for the DP group. At 4–12 h after administration, the accumulation of DiR in the liver, spleen, and kidney was increased. Compared with DPs, the fluorescence intensity of CDPs in the main organs was stronger, indicating that the intestinal absorption rate of the targeted groups was faster. The fluorescence intensity was significantly decreased after 24 h, indicating that the drug was either digested or absorbed [36]. 3.5. Absorption studies using a ligated ileum loop model Recently, epithelium targeting has been considered an efficient approach to further enhance the oral absorption of NPs [46]. Modification of NPs with a specific ligand, especially a peptide ligand, is expected to lead to greater binding with the epithelium and subsequently better uptake. The CSK peptide was identified to specifically target goblet cells, the second largest cell population in the epithelium [5]. Previously, TMC-based epithelium-targeted NPs were prepared by conjugating TMC with the CSK peptide [5, 47]. The relative bioavailability of CSK-modified TMC NPs in diabetic rats was 1.5 times higher than that of unmodified TMC NPs. Other studies showed that target recognition was partly affected by the presence of mucus, possibly through partial disintegration of NPs in the mucus [5, 47]. To determine where and how rapidly CDPs were absorbed in the gut, we investigated different absorption sites in the intestine (Figure 4 (A)). The fluorescence intensity was strongest in the duodenum and gradually became weaker in the posterior intestine, possibly because NPs passed through the duodenum first and had a very high concentration in this region. Consequently, there was no significant difference in


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fluorescence intensity in the duodenum between the two groups. However, the fluorescence intensity of CDPs in the jejunum and ileum was stronger than that of DPs, suggesting that CDPs had higher uptake than DPs. In the presence of intestinal peristalsis and NP destruction by the gastrointestinal environment, the fluorescence intensity in the posterior part of the intestinal segment was decreased. Because of the larger surface area of the microfolds and the slower transport rate of substances in the colon, the DP groups showed better absorption at this location compared with the CDP groups. DEX was previously shown to be degraded in the colon by colonic enzymes, and is thus considered to be an important material for colonic drug targeting [38]. Another reason why the DP group showed better absorption in the colon was that CSK targeted goblet cells in the small intestine, resulting in a large amount of local absorption of CDPs in that region. Consequently, only a small fraction of the remaining CDPs entered the large intestine via peristalsis compared with the amount of DPs entering the large intestine, and thus the DP group showed stronger fluorescence intensity in the colon and rectum than the CDP group. In addition, the gastrointestinal environment damaged the NPs, meaning that the tail of the intestinal fluorescence intensity decreased.



Figure 4. (A) Different intestinal segments were taken out and then the different parts of the intestine were photographed by CYTATION imaging reader after intragastric administration of DPs or CDPs labeled with Nile red. (B) Ex vivo ligated intestinal loop assay. The relative numbers of transported CDPs and DPs after 150 min are shown. Data represent means ± SD (n = 3). *p < 0.05, CDPs versus DPs.

The purpose of NPs is to transport their embedded exenatide across the intestinal epithelium into the blood circulation. Therefore, a NP permeability study was conducted using an ex vivo ligated intestine loop assay [48]. First, the absorption of the fluorescent NPs was examined in different intestinal segments. We found that the fluorescence intensities in the duodenum, jejunum, ileum, and colon were the strongest. Thus, we carried out an in vitro ligation experiment using these four segments of the intestine, and investigated the transport of CDPs and DPs in each of the four segments. As shown in Figure 4 (B), the unit area transport values for CDPs


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in the duodenum, jejunum, and ileum after 150 min were approximately 1.5-fold higher than those for DPs. However, in the colon, the transport volumes of the two types of NPs were similar. These results were consistent with our previous experiments, and indicated that CDPs were specifically and efficiently absorbed in the small intestine through the existence of receptors that interact with CSK, thereby mediating specific binding of NPs to small intestinal goblet cells and promoting the absorption of NPs in the small intestine. 3.6 Cell experiments 3.6.1 Cytotoxicity evaluation of polymers To assess the cytotoxicity of DPs and CDPs after administration, the cellular viability of Caco-2 cells was evaluated by MTT assays. DPs and CDPs were incubated with Caco-2 cells for 24 or 48 h, and the cell viability was assessed. The percentage cell survival was 80%–120% after incubation with 50–1000 μg/mL DPs or CDPs, indicating no significant impact on Caco-2 cell viability at both time points and consistent with a previous report [49]. The effects of exenatide on the viability of Caco-2 cells are shown in supplementary data Figure S7. The results confirmed that DPs and CDPs were not significantly cytotoxic. 3.6.3 Investigation of the cellular internalization and uptake mechanism The results for in vitro intestinal ligation and in vivo transport of NPs after oral administration showed that the CSK peptide had a potential targeting effect toward goblet cells. However, as previously reported by Kang et al. [27], its specific targeting pathway was unclear. Therefore, the possible mechanisms for cellular uptake of CSK



peptide-modified CDPs and unmodified DPs were investigated in Caco-2 cells, which comprise approximately 80% of the mature goblet cells with tight junctions in a monolayer [11]. The intracellular uptakes of coumarin-6 labeled CDPs and DPs were also measured in Caco-2 cells. Using a high-resolution live cell imaging system, the fluorescence intensity of CDPs was higher than that of DPs at different time points, based on the green fluorescence of the coumarin-6 label. By labeling the nucleus with a blue fluorescent probe (Hoechst 33342), we determined that NPs were absorbed into the cytoplasm rather than into the nucleus (Figure 5 (A)). The uptakes of CDPs and DPs were time-dependent, indicating that both uptakes occurred by active transport. The fluorescence intensity in the CDP group was higher than that in the DP group at different time points, indicating that CSK promoted the uptake of CDPs [50]. The goblet cell-targeting ability of the CSK peptide-modified CDPs likely facilitated the targeted absorption [5]. A quantitative investigation of NP uptake into Caco-2 cells was performed by flow cytometry. At different time points, the fluorescence of CDPs was significantly higher than that of DPs (p < 0.05; Figure 5 (B)). This phenomenon was attributed to CSK-mediated transcytosis into the small intestinal epithelium simulated by Caco-2 cells. CSK enabled more CDPs to enter the cells by binding to the small intestinal goblet cells. Overall, CDPs showed augmented cellular uptake of coumarin-6 with simultaneous help from NPs and the CSK peptide compared with DPs.


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Figure 5. Cellular uptake study. (A) High-resolution live cell imaging system images of Caco-2 cells after incubation with CDPs and DPs. (B) Relative fluorescence intensity of coumarin-6 in Caco-2 cells. Data represent means ± SD (n = 3). *p < 0.05, CDPs versus DPs. (C) Relative amounts of uptake of CDPs and DPs by Caco-2 cells in the presence of different endocytosis inhibitors. Data represent means ± SD (n = 3). **p < 0.01, *p < 0.05, #p < 0.05, CDPs versus DPs. (D) Relative numbers of NPs transported across the cell monolayer for 4 h. Data represent means ± SD (n = 3). *p < 0.05, #p < 0.01.

To confirm the influence of CSK on cellular uptake, the mechanisms were investigated under different conditions. NPs were incubated with Caco-2 cells in the



presence of different specific inhibitors including chlorpromazine, formalin, EIPA, and M-β-CD to examine clathrin-, caveolin-, macropinocytosis-, and lipid raft-mediated endocytosis pathways, respectively [50, 51]. As shown in Figure 5 (C), EIPA treatment significantly decreased the cell uptake of DPs (p < 0.01), while that of CDPs remained unchanged, indicating that uptake of DPs was closely related to macropinocytosis [50, 51]. Chlorpromazine is a cationic amphiphilic agent that inhibits clathrin-mediated endocytosis by disrupting the assembly of clathrin adaptor proteins at the cell surface. Chlorpromazine treatment significantly reduced the uptake of CDPs by about 50% (p < 0.05), but there was no significant change in DP uptake [32]. Formalin was used as an inhibitor of caveolin-mediated endocytosis. The uptake of CDPs was reduced by about 35% with formalin (p < 0.05), while DP uptake was unaffected. The presence of M-β-CD significantly reduced the uptakes of CDPs and DPs by 60% (p < 0.05) and 20% (p < 0.01), respectively, indicating that lipid rafts were involved in their energy-dependent endocytosis. These results demonstrated that active transport processes involving adsorptive endocytosis may play an important role in the uptakes of both types of NPs [26], and that CSK enhanced the epithelial internalization of NPs by changing the uptake pathway through specific ligand-receptor interactions. 3.6.4 In vitro transepithelium transport studies The absorption of NPs into the circulatory system suggests their transport across intestinal epithelial cells into the bloodstream. Thus, once exenatide permeates the apical side of the monolayers, either transcellularly or paracellularly, it should be


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released and delivered to the basolateral side. Therefore, we used a co-cultured cell model consisting of absorptive enterocyte-like Caco-2 cells and mucus-producing HT29-MTX cells to evaluate the effect of NPs in overcoming the intestinal epithelial cell barrier. This co-cultured cell model simulates the intestinal epithelium based on the presence of a mucus layer and has a constant transepithelial electrical resistance (TEER) value close to that of the intestinal epithelium [52]. Thus, it should be a suitable in vitro model to evaluate the permeability of orally delivered vehicles as well as the influence of mucus on absorption. Cell monolayers with TEER values within the range of 300–450 Ωcm2 were used [5]. The cumulative amounts of FITC-exenatide transported under different conditions are shown in Figure 5 (D). In all cases, FITC-exenatide was transported in a time-dependent manner. The amounts of FITC-exenatide permeating the Caco-2/HT29-MTX cell monolayers were greatly increased by modification with the CSK peptide at all time points. To further validate the effectiveness of the CSK peptide modification, we performed a competitive transport experiment. The permeation of FITC-exenatide from the Free CSK+FITC-exenatide-CDP groups was significantly lower than that in the FITC-exenatide-CDP groups, which may have arisen by the excessive free CSK competing with FITC-exenatide-CDPs to interact with receptors on the Caco-2 cell surface. The amounts of transported FITC-exenatide in the Free CSK+FITC-exenatide-DP groups and FITC-exenatide-DP groups were similar, indicating that the transport of DPs was independent of CSK. These results



suggest that the increased transportation of CDPs was mediated by the specific affinity of the CSK peptide, because the free CSK peptide inhibited the uptake of CDPs in a concentration-dependent manner but had no effect on the unmodified DPs. 3.7 In vitro drug release and enzymatic stability of NPs For oral delivery of protein drugs, the enzymatic stability of nanocarriers is important. Drugs must overcome the gastrointestinal environment, resist degradation, and then be absorbed by the intestinal epithelium to carry out their therapeutic role. Thus, it is necessary to investigate the capability of NPs to protect exenatide against gastrointestinal enzymes [36]. Among the various proteases present in the gastrointestinal tract, exenatide is mainly degraded by pepsin and trypsin in the intestinal lumen and mucus layer [53]. The ability of the prepared NPs to protect their loaded exenatide against digestive enzymes was investigated with pepsin and trypsin. The percentages of intact exenatide remaining in enzymatic environments are shown in Figure 6 (A) and (B). Compared with free exenatide, the enzymatic stability of exenatide delivered by DPs was significantly improved for all samples examined, indicating that DPs protect exenatide against enzymatic degradation. In the simulated gastrointestinal fluid containing enzymes, both the exenatide solution and NPs were extremely unstable. Therefore, we performed a supplemental study on the stability of exenatide in the absence of enzymes. As clearly shown in Figure 6 (C) and (D), the stability of exenatide in the NP and solution groups was significantly improved in the absence of enzymes, confirming that enzymes in the gastrointestinal tract are a major factor in the destruction of exenatide. Of note, the


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exenatide-DP groups had higher levels of residual exenatide than the exenatide solution groups. Considering the complex pH environment in the gastrointestinal tract, we examined the release behavior of the NPs in SGFsp for 2 h, and then in SIFsp for the following 30 h. Because peptides can be degraded rapidly in simulated gastrointestinal fluid containing enzymes, we used released gastrointestinal fluid lacking enzymes [37]. The in vitro release profile of exenatide was examined at pH 1.2 and 7.4, which simulated the pH environment of the fasting stomach and small intestine, respectively (Figure 6 (E)). During the first 0.5 h in gastric fluid, the release in the NP groups was slow compared with that in the exenatide solution groups. After 2 h at pH 1.2, the cumulative release in all groups was about 60%, but the release profile in the NP groups was flatter than that in the solution groups. This indicated that when the drug was encapsulated in DPs, a sustained release effect was observed. The cumulative release in the exenatide-Zn2+ DP groups eventually reached about 70%, while that in the exenatide-Zn2+ solution groups was 80%, indicating that the encapsulation of polymer materials had a protective effect on exenatide [26]. Both the Zn2+-containing DP groups and Zn2+-containing solution groups had higher cumulative release than the Zn2+-free groups, suggesting that the presence of Zn2+ improved the stability of exenatide [32]. These results were also consistent with the pH stability results. Similar release profiles were observed for other PLGA-based nanocarriers. The phospholipid complex used in our nanoplatform also delayed the release process, as reported previously [54].



Figure 6. Residual exenatide-Zn2+-DPs and exenatide solutions after incubation in SGFsp (A), SIFsp (B), SGF (containing pepsase) (C), and SIF (containing trypsase) (D). (E) Exenatide released from exenatide-DPs, exenatide-Zn2+-DPs, exenatide solution, and exenatide-Zn2+-solution in vitro. Data represent means ± SD (n = 3). **p < 0.01, *p < 0.05, versus the exenatide-Zn2+- solution group.

3.8 In vivo pharmacokinetic studies


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Figure 7. Plasma levels of exenatide at different times. Comparisons of the plasma levels of exenatide in Sprague-Dawley rats following oral administration of exenatide Zn2+-DPs, exenatide-Zn2+-CDPs, or exenatide-Zn2+ solution (100 µg/kg) are shown, with subcutaneous injection of exenatide solution (10 µg/kg) as a positive control. Data represent means ± SD (n = 5).

Table 3. Pharmacokinetic parameters of exenatide following administration of different exenatide formulations Exenatide-Zn2+solution((i.g.)

Exenatide-Zn2+DPs(i.g.)

Exenatide-Zn2+-CDPs

Exenatide solution

(i.g.)

(s.c.)

Dose(µg/ml)

100

100

100

10

Cmax(pg/ml)

585.47± 141.15

1078.15± 101.38

1720.44±180.68

3827.8± 579.28

Tmax(h)

6

6

6

0.25

AUC(pgh/ml)

7382.34±2527.81

16221.21±4347.15

26068.49±1339.60

28253.82±3006.60

BR(%)

2.6

5.7

9.2

100



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Cmax: maximum plasma concentration; Tmax: time at which Cmax is attained; BR: relative bioavailability; i.g.: intragastric administration; s.c.: subcutaneous administration (n = 5 for all groups).

Bioavailability is an important parameter that determines the pharmacodynamic effects of peptide drugs. We investigated the bioavailability of exenatide after oral administration of exenatide-Zn2+-CDPs, exenatide-Zn2+-DPs, and exenatide-Zn2+ solution, and subcutaneous administration of exenatide solution. The plasma exenatide concentration-time profiles and related pharmacokinetics parameters are shown in Figure 7 and Table 3, respectively. The plasma concentrations of exenatide in the exenatide-Zn2+ oral administration groups were very low. Subcutaneous administration elicited a maximum plasma concentration at 0.25 h. However, oral administration of exenatide-Zn2+-CDPs and exenatide-Zn2+-DPs resulted in a maximum plasma concentration at 6 h post-administration. exenatide-Zn2+-CDPs

Furthermore, was

greater

the

maximum

than

that

for

plasma

concentration

exenatide-Zn2+-DPs.

for The

bioavailability of oral exenatide-Zn2+-CDPs was 9.2% compared with 100% after subcutaneous administration. The bioavailability for exenatide-Zn2+-CDPs was 1.61-fold higher than that for non-targeted exenatide-Zn2+-DPs. These data suggest that CSK promoted interactions with the small bowel goblet cells and permitted transport of more exenatide into the small intestine. 3.9 Hypoglycemic activity in vivo The hypoglycemic effects of orally encapsulated exenatide-DPs, exenatide-Zn2+


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DPs, exenatide-Zn2+-CDPs, and exenatide-Zn2+ solution were evaluated in type 2 diabetic mice. As expected, the free exenatide-Zn2+ solution and saline did not induce hypoglycemic effects, because the protein was easily digested by proteases in the gastrointestinal tract. In contrast, hypodermic injection of exenatide (10 μg/kg) significantly decreased the blood glucose level, with the blood glucose value reaching 50% of the initial value at 1 h. Because of the degradation of NPs in the gastrointestinal environment and the absorption barrier in the intestinal epithelium, the dosage of oral NPs was set at 100 μg/kg. Of note, the two dosing regimens based on the different NPs resulted in various therapeutic effects. As shown in Figure 7 (A), exenatide-Zn2+-DPs induced a maximal blood glucose reduction of 85.2% after 4 h, while exenatide-CDPs achieved a better hypoglycemic effect (75%) at the same time point. From 6–12 h, the glucose level after exenatide-Zn2+-CDP administration increased from 76% to 80%, while that after exenatide-Zn2+-DP administration increased from 76% to 91%. According to the above in vitro results, encapsulation in CDPs was an effective way to protect exenatide against degradation and improve its permeability. After oral delivery of exenatide-Zn2+-CDPs, a significant hypoglycemic effect was observed with a significantly improved pharmacological availability compared with orally administered exenatide-Zn2+ solution. Furthermore, the hypoglycemic effects after exenatide-Zn2+-CDP administration were maintained for a longer time, because exenatide stimulates pancreatic β-cells to release insulin under hypoglycemic conditions.



Figure 8. (A, B) Blood glucose levels in diabetic rats following single oral administration (A) and multiple oral administrations (B) of saline, exenatide-Zn2+-DPs, exenatide-Zn2+-CDPs, or exenatide-Zn2+ solution (100 µg/kg), with subcutaneous injection of exenatide solution (10 µg/kg) as a positive control. Data represent means ± SD (n = 6). *p < 0.05, **p < 0.01, versus the oral exenatide-Zn2+ solution group. #p < 0.05, versus the exenatide-Zn2+-DP group.

The blood glucose levels after multiple administrations are shown in Figure 8 (B). Five repeated administrations had no hypoglycemic effect in the exenatide-Zn2+ solution and saline groups. The blood glucose level in the exenatide-Zn2+-CDP groups remained at approximately 70% to 80%, while that in the subcutaneous groups ranged from 80% to 85%. However, the blood glucose levels in the exenatide-Zn2+-DP groups ranged from 80% to 95%. These results demonstrated that the presence of CSK enhanced the pharmacological effect of exenatide. The long-lasting hypoglycemic effect of orally administered exenatide-Zn2+-CDPs was partly derived from the slow release of exenatide from the NPs. In this experiment, there was no significant change in the blood glucose level in the subcutaneous groups, which may be explained by the rapid absorption after subcutaneous administration and the rapid


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onset of the hypoglycemic effect. The reduced blood glucose level had increased when the blood glucose was measured at 12 h after administration. Overall, the control of blood glucose levels by oral administration of exenatide-Zn2+-CDPs was stable. Exenatide improves the function of pancreatic β-cells and has a role in stimulating insulin secretion [32, 55]. As shown in supplementary data Figure S8, the staining intensity in the exenatide-Zn2+-CDP groups was higher than that in the exenatide-Zn2+-DP groups. These results were consistent with the plasma concentrations and blood glucose levels associated with exenatide. The area of insulin-stained pancreatic tissue in the exenatide-Zn2+-CDP groups was significantly larger than that in the exenatide-Zn2+-DP groups, suggesting that the presence of CSK caused more exenatide to be absorbed into the circulation. 4. Conclusions A CSK-conjugated DEX-PLGA copolymer was synthesized and used for preparation of exenatide-loaded CSK-targeted NPs by the double emulsion method. The results indicated that the mucus-blocking effect of the intestine was reduced for CDPs. Moreover, CDPs had an excellent affinity for epithelial cells in vitro, resulting in enhanced cellular internalization and transepithelial transport. In vivo experiments showed that oral administration of modified CDPs loaded with exenatide-Zn2+ had a good hypoglycemic effect in type 2 diabetic mice. Our study demonstrates the potential of the developed CDPs for oral delivery of exenatide, and suggests that this system should be further investigated for the delivery of other protein and peptide



Page 48 of 51

drugs. Supporting Information: Supplementary Figures: Figure.S1. (A) Synthesis scheme of DEX-HMDA diamine conjugate; (B) 1H-NMR spectra of DEX (a), hexamethylene diamine (b), and DEX-NHS (c) in D2O Figure.S2. (A) Synthesis scheme of DEX-PLGA blocked copolymer; (B) 1H spectra of DEX-NH2 in D2O (a), PLGA-NHS in DMSO-d6 (b), and DEX-PLGA blocked copolymer in DMSO-d6 (c) Figure.S3. (A) GPC chromatograms of DEX-PLGA block copolymer. (B) Synthesis scheme of CSK-DEX-PLGA. (C) HPLC chromatographic peaks before CSK coupling reaction (a), HPLC chromatographic peak after CSK coupling reaction (b). (D) 1H spectra of DEX-PLGA in D2O (a), CSK in D2O (b), and CSK-DEX-PLGA blocked copolymer in D2O (c) Figure.S4. (A) Schematic diagram of main steps in the preparation of CDPs. (B) TEM images of CDPs and DPs. Figure.S5. The particle size distribution of DPs and CDPs, which was measured by dynamic lighter scattering (DLS) method Figure.S6. Potential Diagram of DPs and CDPs Figure.S7. Survival rate of cells incubated with different concentrations of materials for 24 h (A) and48 h (B). Figure.S8. (A) Photomicrographs of pancreatic tissue following administration of (a) exenatide-Zn2+ solution (oral), (b) exenatide-Zn2+-DPs, (c) exenatide-Zn2+-CDPs, (d) saline and (e) exenatide solution (s.c.). (B)Area of beta cells in pancreatic tissue following

administration

of

exenatide-Zn2+-DPs,


exenatide-Zn2+-CDPs,

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exenatide-Zn2+ solution (oral), saline and exenatide solution (s.c.). Funding This work was supported by the Natural Science Foundation of Shandong (No. ZR2014HM062). Acknowledgments We thank Edanz Group China (www.liwenbianji.cn/ac) for editing the English text of a draft of this manuscript. Declaration of interest The authors report no conflicts of interest in this work. References 1.

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Synthesis of CSK-DEX-PLGA Nanoparticles for the Oral Delivery of

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