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Preparation of exenatide-loaded linear poly(ethylene glycol)-brush poly(l-lysine) block copolymer: potential implications on diabetic nephropathy

The poly(ethylene glycol)-b-brush poly(l-lysine) polymer (PEG-b-(PELG50-g-PLL3)) was synthesized and evaluated as a nanocarrier for prolonging delivery of exenatide through the abdominal subcutaneous injection route. The isoelectric point of exenatide was 4.86, and exenatide could combine with PEG-b-(PELG50-g-PLL3) polymers via electrostatic interactions at pH 7.4. This polymer was a good candidate for achieving prolonged drug delivery for exenatide, considering its high molecular weight. Besides the physicochemical characterization of the polymer, in vitro and in vivo applications were researched as a sustained exenatide delivery system. In the in vitro release research, 20.16%–76.88% of total exenatide was released from the PEG-b-(PELG50-g-PLL3) polymer within 7 days. The synthesized block-brush polymers and exenatide–block-brush polymers were analyzed by nuclear magnetic resonance spectroscopy, gel permeation chromatography, transmission electron microscopy, nanoparticle size instrument, and scanning electron microscopy. The best formulation was selected for in vivo experimentation to achieve blood glucose control in diabetic rat models using free exenatide as the control. The hypoglycemic action of the formulation following subcutaneous injection in diabetic rats lasted 7 days, and the results indicated that exenatide–block-brush polymers demonstrate enhanced long-acting hypoglycemic action. Besides the hypoglycemic action, exenatide–block-brush polymers significantly alleviated diabetic nephropathy via improving renal function, decreasing oxidative stress injury, decreasing urinary albumin excretion rate, mitigating albumin/creatinine ratio, reducing blood lipids, abating kidney index, weakening apoptosis, and downregulating expression of connective tissue growth factor. All of the results suggested that PEG-b-(PELG50-g-PLL3) polymers could be used as potential exenatide nanocarriers, with efficient encapsulation and sustained release.


Introduction
Diabetic nephropathy (DN) is a life-threatening complication in patients with long-standing diabetes. 1 Exenatide therapy is essential in the treatment of DN. 2 However, exenatide therapy is limited due to its short plasma half-life of 2.4 h and, thus, dosing is very difficult and frequent injections are required, which are inconvenient for patients and result in low compliance. 3 Considerable interest has been generated over the past few decades in the development of effective protein-delivery systems, such as hydrogels, [4][5][6][7][8][9] polymeric nanoparticles, [10][11][12][13][14][15][16] nano-or microcapsules, [17][18][19] and 4664 Tong liposomes for peptides and proteins. Many researchers in this field are striving to identify appropriate delivery systems to load peptides and proteins such as exenatide. However, there is still no commercially applicable delivery system that is available for efficient delivery of exenatide. Therefore, development of appropriate delivery systems for exenatide still faces challenges.
Exenatide administered by injection has activity that is sustained for a very short time due to faster degradation/ consumption. 3 Moreover, frequent exenatide injections are needed for sustained pharmacological action and, as a result, are painful for patients. 3 To avoid frequent injections for patients, many investigators are developing potential exenatide delivery systems. Some reports in the literature showed that loading exenatide onto nanoparticles prolongs the plasma half-life of exenatide. 20,21 In this study, we first introduce brush poly(l-lysine) (PLL)-based block-brush polymers as potential exenatide carriers. The synthesis of the block-brush poly(ethylene glycol)-b-brush poly(llysine) (PEG-b-(PELG 50 -g-PLL 3 )) polymers refers to our previous work. 22 The negatively charged exenatide combines with positively charged PLL brush block through electrostatic interactions at pH 7.4 (structure shown in Figure 1). The loaded exenatide ensures sustained release, which can improve the protective effect and bioactivity of exenatide. The loading capacity of exenatide was decided via vitro release. In this in vivo research study, we continue to evaluate the sustained pharmacological effect of the exenatide-loaded block-brush polymer PEG-b-(PELG 50g-PLL 3 ) in prolonging blood glucose control and significantly alleviating DN in comparison with free exenatide as the control.

Materials and methods Materials
The FITC-PEG-NH 2 (M w =5 kDa), exenatide, streptozocin (STZ), and other reagents used in this research were from Sigma-Aldrich (St Louis, MO, USA). EC9706 cells were purchased from Shanghai Fu Xiang Biotechnology Co., Ltd., Shanghai, People's Republic of China. synthesis and cytotoxicity of Peg-b-(Pelg 50 -g-Pll 3

Pharmacodynamic research of exenatide
The SD rats were randomly divided into two groups (n=5 per group). Exenatide group: exenatide 5 μg/kg was administered as a single subcutaneous injection; in the exenatide-PEG-b-(PELG 50 -g-PLL 3 ) group, exenatide-PEG-b-(PELG 50 -g-PLL 3 ) 5 μg/kg was given as a single subcutaneous injection. Blood samples were collected from the tail vein of rats at predetermined time points, and plasma exenatide concentration was assayed by ELISA.

Diabetic animal model
The SD rats were fed a high-fat diet (fat content 40%) for ten weeks and then STZ (dissolved in citrate buffer, pH 4.5) was administered at a dose of 35 mg/kg as a single intraperitoneal injection. A total of 72 h later, diabetes was confirmed by measuring blood glucose levels using the glucose oxidase-peroxidase (GOD-POD) methods. 23 Animals which had blood glucose levels 16.7 mmol/L were used in the experiments.
Treatment with exenatide-Peg-b-(Pelg 50 -g-Pll 3 ) complex in diabetic animal model Fifteen diabetic male rats were randomly assigned to one of three groups (n=5, per group): 1) control group; 2) exenatide group: 5 μg/kg as a single abdominal subcutaneous injection; and 3) exenatide-PEG-b-(PELG 50 -g-PLL 3 ) group: 5 μg/kg as a single abdominal subcutaneous injection. At the predetermined time points, blood glucose levels were measured using the GOD-POD methods. 23 Treatment with exenatide-Peg-b-(Pelg 50 -g-Pll 3 ) complex in DN Ten normal SD rats and 30 diabetic male rats were assigned to four groups (n=10, per group): 1) sham group: normal SD rats were fed a standard diet; 2) DN group: saline buffer of equal quantities was subcutaneously administered for eight weeks in diabetic male rats; 3) exenatide group: 5 μg/kg as a single abdominal subcutaneous injection daily for eight weeks continuously in diabetic male rats; and 4) exenatide-PEG-b-(PELG 50 -g-PLL 3 ) group: 5 μg/kg as a single abdominal subcutaneous injection every 7 days, continuously administered for eight weeks to diabetic rats. At the predetermined time points, blood samples and one of the kidneys were collected. Fasting blood glucose (FBG), body weight, serum lipid, fasting plasma insulin (FINS), homeostasis model insulin resistance index (HOMA-IR), insulin sensitivity index (ISI), blood urea nitrogen (BUN), serum creatinine (Scr), urinary albumin excretion rate (UAER), mitigating albumin/creatinine ratio (ACR), superoxide dismutase (SOD), malonyldialdehyde (MDA), apoptotic cell ratio, kidney weight/body weight, and expression of connective tissue growth factor (CTGF) were measured.

statistical measurement
All of the data were analyzed by mean values ± standard deviation (SD). Between-group analyses were conducted by ANOVA. A value of less than 0.01 (P0.01) was regarded as indicating statistical significance.

exenatide release in vitro
The release of exenatide from PEG-b-(PELG 50 -g-PLL 3 ) and FITC-PEG-b-(PELG 50 -g-PLL 3 ) was measured using a dialysis method (MWCO, 100 kDa) at normal temperature, with 5 mL of exenatide-loaded polymer. The cumulative release ratio of exenatide from exenatide-block-brush polymers is shown in Figure 5. After 2 h, approximately 22.89% of the exenatide is released from exenatide-blockbrush copolymers, indicative of an initial burst release of exenatide. Approximately 55% of the exenatide is released after 3 days and approximately 76.88% after 7 days, indicative of biphase release profiles in the exenatide-block-brush polymers (Table S1).

Plasma exenatide concentration
Pharmacodynamic study confirms that, in rats treated with exenatide solution, plasma exenatide concentrations increase rapidly, reaching the peak within 2 h (678 ng/mL), followed by a remarkable decline after 24 h (0 ng/mL; Figure 6). In contrast, the concentration of the exenatide-PEG-b-(PELG 50 -g-PLL 3 ) complex gradually peaks within 3 h (87.65 ng/mL) and remains at a comparatively low level by 7 days (2.14 ng/mL on day 7; Table S2).

Blood glucose levels
We measured the average blood glucose levels over time for rats administered exenatide solution and exenatide-loaded polymers and, compared with the Control group, we found that diabetic male rats injected with the exenatide solution maintained blood glucose levels within the normal range for approximately 3 h, after which glycemia slowly increases, with initial hyperglycemic values achieved within 4.5 h.

assessment of sOD activity and MDa level of renal tissues in DN
Both SOD activity and MDA level were examined with the xanthine oxidase and thiobarbituric acid method. 25,26 SOD activity was measured after eight weeks ( Figure 10A). SOD activity in the DN group was less than in the sham group (P0.01; SOD activity: sham 76.32±2.39 U, DN group 31.25±1.45 U). Administration of exenatide increased SOD activity (48.69±1.19 U), compared with that in the DN group (P0.01). Administration of exenatide-PEG-b-(PELG 50 -g-PLL 3 ) remarkably increased SOD activity (61.27±2.08 U), which was markedly higher than in the DN group (P0.01). The MDA level was measured after eight weeks ( Figure 10B), and was higher in the DN group than in the ) group is denoted by *P0.01, **P0.01, and ***P0.01, respectively. (B) A significant increase from the sham group, a significant decrease from the DN group, and a significant decrease from the DN group is denoted by *P0.01, **P0.01, and ***P0.01, respectively. (C) A significant increase from the sham group, a significant decrease from the DN group, and a significant decrease from the DN group is denoted by *P0.01, **P0.01, and ***P0.01, respectively. (D) A significant increase from the sham group, a significant decrease from the DN group, and a significant decrease from the DN group is denoted by *P0.01, **P0.01, and ***P0.01, respectively (TC, TG, and LDL); and a significant decrease from the sham group, a significant increase from the DN group, and a significant increase from the DN group is denoted by *P0.01, **P0.01, and ***P0.01, respectively (hDl). (E) A significant increase from the sham group, a significant decrease from the DN group, and a significant decrease from the DN group is denoted by *P0.01, **P0.01, and ***P0.01, respectively. (F) A significant increase from the sham group, a significant decrease from the DN group, and a significant decrease from the DN group is denoted by *P0.01, **P0.01, and ***P0.01, respectively.

assessment of kidney weight/body weight
The kidney weight/body weight was measured after eight weeks (Figure 11), and was lower in the DN group than in the sham group (P0.01; kidney weight/body weight: sham group 4.71±0.69, DN group reached 14.27±0.86).

assessment of the Uaer on DN
The UAER was measured after eight weeks (Figure 12), and was higher in the DN group than in the sham group (P0.01; UAER: sham group 2.03±0.39 mg, DN group 56.75±1.13 mg). Administration of exenatide decreased UAER (30.56±3.29 mg), compared with that in the DN group (P0.01).

assessment of mitigating acr on DN
The mitigating ACR was measured after eight weeks (Figure 13), and was higher in the DN group than in the sham group (P0.01; mitigating ACR: sham group 0.03± 0.003 mg/μmol, DN group 0.19±0.003). Administration of exenatide decreased the mitigating ACR (0.16±0.007), compared with the mitigating ACR in the DN group (P0.01).

assessment of apoptotic cell ratio
The apoptotic cell ratio was detected using flow cytometry, using techniques that were previously reported, 27 and was measured after eight weeks ( Figure 14). The apoptotic cell ratio in the DN group was higher than in the sham group (P0.01). Administration of exenatide decreased the apoptotic cell ratio, compared with that in the DN group (P0.01). Administration of exenatide-PEG-b-(PELG 50 -g-PLL 3 ) remarkably decreased the apoptotic cell ratio, which was markedly lower than in the DN group (P0.01).

Measurement of cTgF expression
The methodology to evaluate CTGF expression has been described previously. 28 Briefly, renal tissue was homogenized in protein lysate buffer and resolved on polyacrylamide SDS gels, and then electrophoretically transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 3% BSA, incubated with primary antibodies against active CTGF and, subsequently, with alkaline phosphataseconjugated secondary antibodies that were developed using 5-b-romo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. The blot was stained with anti-β-actin antibody, and the level of protein was normalized with respect to β-actin and density; CTGF expression was measured after eight weeks (Figure 15), and was higher in the DN group than  in the sham group (P0.01). Administration of exenatide decreased CTGF expression, compared with CTGF expression in the DN group (P0.01). Administration of exenatide-PEG-b-(PELG 50 -g-PLL 3 ) remarkably decreased CTGF expression, which was markedly lower than that in the DN group (P0.01).

Discussion
We evaluated the role of exenatide and exenatide-PEG-b-(PELG 50 -g-PLL 3 ) in the development and progression of DN. Exenatide and exenatide-PEG-b-(PELG 50 -g-PLL 3 ) treatments heighten renal function and ameliorate metabolic anomalies, including reduction of body weight, FBG, FINS, TC, TG, LDL, HOMA-IR, ISI, and kidney weight/body weight, as well as increase HDL and UAER, and mitigate ACR. Exenatide and exenatide-PEG-b-(PELG 50 -g-PLL 3 ) treatment, moreover, ameliorate oxidative stress injury and decrease renal cell apoptosis. Importantly, this research provides the first evidence that exenatide and exenatide-PEG-b-(PELG 50 -g-PLL 3 ) treatments ameliorate the severity of DN with decreased CTGF expression.
This study reports a technique to increase both in vitro release and in vivo safety and efficacy of exenatide, administered as a subcutaneous abdominal injection, using encapsulation of a PEG-b-(PELG 50 -g-PLL 3 )-exenatide complex. In our previous work, 22 we confirmed that PEG-b-(PELG 50 -g-PLL 3 ) has been intensively used as a potential insulin nanocarrier, and the 1 H-nuclear magnetic resonance (NMR) spectra and gel permeation chromatograms (GPCs) of PEG-b-(PELG 50 -g-PLL 3 ) are shown in Figure S2, confirming that the synthesis proceeded in a controlled manner with successful synthesization. In addition, PEG-b-(PELG 50 -g-PLL 3 ) and exenatide-PEG-b-(PELG 50 -g-PLL 3 ) on EC9706 cells exhibited high cell viability at concentrations as high as 500 mg/L ( Figure S2). In this regard, we choose PEG-b-(PELG 50 -g-PLL 3 ) to load Figure 15 cTgF expression. renal tissues of rats in the sham, DN, exenatide, and exenatide-Peg-b-(Pelg 50 -g-Pll 3 ) complex groups were collected after eight weeks, and results were expressed as mean ± SD. A significant increase from the sham group, a significant decrease from the DN groups, and a significant decrease from the DN groups is denoted by *P0.01, **P0.01, and ***P0.01, respectively. Abbreviations: cTgF, connective tissue growth factor; DN, diabetic nephropathy; Peg-b-(Pelg 50 -g-Pll 3 ), poly(ethylene glycol)-b-brush poly(l-lysine) polymer.

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Tong exenatide onto for sustained release of exenatide in order to maintain the effect of long-acting blood glucose suppression and alleviation of DN. Another PEG-b-(PELG 50 -g-PLL 3 ) polymer acts as a nanocarrier and protects exenatide from quick degradation in rats; it can enhance efficacy as well as decrease dosage and frequency of injections. On the other hand, PEG-b-(PELG 50 -g-PLL 3 ) can aggrandize the stability and bioactivity of exenatide in circulation.
Exenatide (a 39-amino acid polypeptide) is a natural dipeptidyl peptidase-IV resistant GLP-1 receptor agonist that shares 82% homology with human GLP-1 in the N-terminal segment. 29,30 Many studies have shown that exenatide can lower hyperglycemia, suppress glucagon secretion, reduce energy intake and gastric motility, increase β-cell mass in type 2 diabetes, and alleviate DN. 2,31-33 However, the plasma half-life of exenatide is very short and necessitates frequent injections that are a source of pain for patients. 3 In this study, we found PEG-b-(PELG 50 -g-PLL 3 ) to be a better sustained exenatide-release system. Furthermore, CTGF is an important factor that modulates DN. 34 CTGF is geared to the CCN protein family, which plays an important role in modulating synthesis of extracellular matrix. 35,36 In patients with type 2 diabetes mellitus, a high serum CTGF content is closely correlated with mortality due to end-stage kidney disease secondary to DN. 37 In addition, augmented CTGF expression on a biopsy specimen from patients at different stages of DN is associated with progression of DN. 38 In this study, exenatide and exenatide-PEG-b-(PELG 50 -g-PLL 3 ) treatment inhibit DN by decreasing CTGF expression.
To sum up, we find that PEG-b-(PELG 50 -g-PLL 3 ) polymers can prolong the plasma concentration of exenatide in SD rats and enhance the plasma half-life of exenatide. In addition, PEG-b-(PELG 50 -g-PLL 3 ) can sustain a comparatively low glucose level for up to 7 days, compared to free exenatide in type 2 diabetes, and significantly ameliorates DN.

Conclusion
We synthesized a PEG-b-(PELG 50 -g-PLL 3 ) polymer that significantly enhances the bioactivity of exenatide. Administration of exenatide-PEG-b-(PELG 50 -g-PLL 3 ) significantly promoted the plasma concentration of exenatide in SD rats, significantly prolonged blood glucose suppression, and alleviated DN.

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