pH sensitive insulin-loaded nanohydrogel increases the effect of oral insulin in diabetic rats

Abstract

Background: There are different methods for insulin administration in diabetic patient. Nano-hydrogel is one of the most talented drug carrier for its sensitivity to environmental stimulus.

Methods and results: NIPAAm–MAA–HEM copolymers were synthesized by radical chain reaction. The copolymers were characterized with Scanning electron microscopy (SEM) and Transient electron microscopy (TEM). Copolymers were loaded with regular insulin by modified double emulsion method. Diabetic rats are used for feeding insulin-loaded nanohydrogel. Analysis of the results from the measurement of the amount of blood insulin from the rats blood that received insulin in nanohydrogel loaded form compared with rats that received pure insulin is significantly high, which confirm that insulin has been able to pass from the stomach acid barrier by nanohydrogel and is absorbed from the intestine. Blood sugar levels from tested rats indicate that with increasing amount of insulin, blood sugar levels fall down.

Conclusion: Our study confirms that insulin has been able to pass from the stomach acid barrier by nanohydrogel and be absorbed from the intestine.

Keywords:

• PH-sensitive nanohydrogels
• diabetes mellitus
• drug delivery
• insulin

Introduction

Diabetes mellitus (DM) is a disorder caused by decreased production of insulin or by decreased ability to use insulin and that is considered by relative insulin deficiency in response to increase in insulin request induced by insulin resistance, leading to increased blood glucose levels (Alejandro et al. 2015, Karnoosh-Yamchi et al. 2014). Type-1 diabetes comprises those forms of diabetes that are principally due to insulin deficiency. Type-2 diabetes contains those forms that result from a primary defect in insulin resistance (Akhter and Nijhu 2012, Davaran et al. 2011, Jafari et al. 2011). The diabetic foot syndrome includes a number of pathologies, including diabetic neuropathy, Charcot’s neuroarthropathy, peripheral vascular disease, foot ulceration, osteomyelitis, are responsible for extra morbidity and mortality, reduced quality of life, loss of independence, and the potentially avoidable end point, amputation. Subjects with diabetic foot problems are also probable to harbor other associated complications of diabetes such as nephropathy, retinopathy, ischemic heart disease, and cerebrovascular disease (Akhter and Nijhu 2012, Assaad-Khalil et al. 2015).

Insulin is used to treat diabetes since 1922. There are different methods for insulin administration (Karnoosh-Yamchi et al. 2014).

First, Lowman et al. loaded insulin on microspheres of poly methacrylic-ethylene glycol and gave it orally to normal and diabetic pests (Lowman 1999). In another study, polymers of acrylic acid–N-isopropyl acrylamide and meta-butyl methacrylic acid with molar ratio of 10:5:85 was used (Peppas 2004, Ramkisson-Ganorkar 1999). Alginate microparticles produced by emulsification method was used as a carrier for the drug delivery of insulin by Reis et al. (2007). Used nanoparticles protect insulin from stomach acid, but had little biosafety and showed side effects (Karnoosh-Yamchi et al. 2014).

Smart polymers are soluble, surface-coated or cross-linked polymers exhibiting comparatively large and sharp physical or chemical changes in response to small physical or chemical stimuli, such as the temperature, pH, solvent composition, addition of special ions, and electric fields (Gupta et al. 2006, Sahiner et al. 2006). Hydrogels are cross-linked, macromolecular polymer networks occupied in a solvent, synthesized to exhibit large volumetric swelling in response to a variety of environmental stimuli. They are three-dimensional cross-linked polymeric structures that are able to swell in the aqueous environment (Üzüm and Karadağ 2007).

Nano-hydrogel is one of the most talented drug carrier for its sensitivity to environmental stimulus (Zhang and Gu 2006). Nanogels have recently attracted significant attention in the controlled release technology for diagnostic and therapeutic applications (Argentiere et al. 2010).

The main goal of the present research was to provide pH sensitive nanohydrogels containing human insulin and examination the nanohydrogel behavior for oral administration of insulin in diabetic rat.

Materials

Methacrylic acid (MAA) (Merck chemical Co., Germany), Hydroxy ethyl methacrylate (HEMA) (Merck chemical Co., Germany), N-iso propyl acryl amide (NIPAAm) obtained from Acros Organics (NJ), Pure human insulin was purchased from EXIR, Iran Hormone, Alloxan monohydrate (Acros), NaCl (Merck chemical Co., Germany), BPO (Merck chemical Co., Germany), 1,4-Dioxan (Di gung chemical Co., Korea), n-Hexan (Merck chemical Co., Germany), Trichloromethane (Merck chemical Co., Germany), Dichloromethane (Merck chemical Co., Germany), Centrifuge sigma (5000RPM), Centrifuge HEROLAB (13000RPM), Vacuum rotary (er100-pro, China), Sunicator (vp200h, Hiescher, Germany), FT–IR spectrometer instrument (FT–IR, Tensor 27 Bruker), Scanning electron microscopy (SEM, Mira3 tescan 5.0 KV), Transmission electron microscopy (TEM, Zeiss EM10C 80 KV), Rat (Razi Institute, Iran), Ketamine hydrochloride (Germany), Heparinated Capillary (Fisher scientific).

Methods

Nanohydrogel synthesizing

NIPAAm–MAA–HEMA copolymer was synthesized by radical chain reaction with 80:8:12 ratios, respectively. Reaction was carried out in 1, 4-dioxan and under Nitrogen gas-flow. Benzoyl peroxide (0.3 mol % with respect to the monomers) was added as initiator of polymerization. The mixture was magnetically stirred and degassed with nitrogen gas for 30 min. The polymerization was fulfilled at 70 °C for 24 h under nitrogen atmosphere. The synthesized polymers were precipitated using n-Hexane in liquid nitrogen bath. The polymerization yield of the experiment was 86.5%. Figure 1 shows whole scheme protocol of nanohydrogel synthesized.

Figure 1. Whole scheme of nanohydrogel synthesizing protocol.

Insulin loading

Modified double emulsion method was used for loading insulin in the polymers. Poly vinyl alcohol (PVA) was used as stabilizer with 0.1% concentration. First, 100 mg of the synthesized and freeze dried copolymer was dissolved in 5 mL of chloroform. Then, 50 IU (international unit) of insulin as aqueous solution was added in solution and sonicated for 60 s. Then, 50 mL of PVA was added to the mixture and sonicated for 50 s. The mixture was located in vacuum rotary for 30 min for further evaporation of chloroform and loading of insulin and then centrifuged in 13 000 rpm at 25 °C. Loaded polymers were lyophilized and stored in 2–8 °C for further use. Figure 2 shows whole scheme protocol of insulin loading to nanohydrogel (Abbasi et al. 2014, 2016b, Daraee et al. 2016a, 2016b, Ebrahimi et al. 2016, Mirakabad et al. 2016, Nasrabadi et al. 2016).

Figure 2. Whole scheme of insulin loading to nanohydrogel protocol.

TEM and SEM microscopy and FT-IR spectroscopy

Scanning electron microscopy (SEM) is used for evaluating size and size distribution, and Transient electron microscopy (TEM) is used for evaluating shape and size of the spherical nanohydrogels. The structure of insulin loaded on poly (NIPAAm–MAA– HEMA) nanohydrogels was showed using FT-IR spectroscopy (Aval et al. 2016, Chung et al. 2016, Ebrahimnezhad et al. 2013, Mollazade et al. 2013, Nejati-Koshki et al. 2013, Pourhassan-Moghaddam et al. 2013, 2014).

Studies on diabetic rats

Type 1 diabetes mellitus (IDDM) in rats initiated with a single intraperitoneal injection of alloxan 120 mg per kg of body weight and the physiologic serum (NaCl 0.9%) is used as the alloxan solvent (Demerdash et al. 2005, Kumar et al. 2006, Ragavan and Rishnakumari 2006). Increasing blood glucose level between 200 and 300 mg/dl is the criteria of diabetes (Demerdash et al. 2005, Kulkarni et al. 2002, Ragavan and Rishnakumari 2006, Viana et al. 2004).

A total of 20 male rats with weigh ∼260–280 g were selected and were made diabetic by injecting intraperitoneally alloxan (120 mg/kg). Three days after injection, diabetics was confirmed by using a glucometer and blood levels above 400 confirmed it (Abbasi et al. 2016a, Ahmadi et al. 2014, Alimirzalu et al. 2014, Alizadeh et al. 2014, 2016, Davoudi et al. 2014, Eatemadi et al. 2014, Ghasemali et al. 2013, Hosseininasab et al. 2014, Kouhi et al. 2014).

The rats were randomly divided into three groups. Rats by intraperitoneal injection of ketamine (44 mg/kg) were anesthetized and to control diabetics, a blood sample was taken from the eye by capillary heparinated and then in the order:

1. Control group were given distilled water by gavage.

2. Group 1 were fed with nanoparticles containing insulin dissolved in distilled water by gavage equivalent 20 IU/kg (5 IU for each rat with weight average 250 gr). (3 mg of insulin-loaded nanohydrogel containing 5 IU insulin)

3. Group 2 were fed with pure insulin (20 IU/kg) dissolved in distilled water by gavage.

After gavage, rats were anesthetized by ketamine and the blood sampling was done in each 90 min for 6 h (for four times), blood samples were collected in the 1.5 ml ependorfs and were centrifuged in 4000 rpm for 10 min to separate serum, serum samples were collected in 0.5 ml ependorfs and the amount of insulin and blood glucose levels are measured.

Statistical analysis

Statistical data were performed by student’s t-test on SPSS version16 and charting was performed by Microsoft Excel software 2010 version (Redmond, WA).

Results

The product of synthesized nanohydrogel is in crystal form. This product for subsequent applications could be kept at room temperature or in refrigerator or in freezer. The SEM micrographs of pure PNIPAAm–MAA–HEMA nanohydrogels (Figure 3(A)) and insulin loaded on poly (NIPAAm– MAA–HEMA nanohydrogels (Figure 3(B)) are shown.

Figure 3. SEM micrographs.

The TEM micrographs of pure PNIPAAm–MAA–HEMA nanohydrogels (Figure 4(A)) and insulin loaded on poly (NIPAAm– MAA–HEMA nanohydrogels (Figure 4(B)) are shown.

Figure 4. TEM micrographs.

Also, FT-IR is done for pure PNIPAAm–MAA–HEMA nanohydrogels and insulin loaded on poly (NIPAAm– MAA–HEMA nanohydrogels) for evaluation of loading percent.

Analysis of the obtained results from the measurement of the amount of blood insulin is shown in Figure 5 in this diagram, it is known that insulin in the rats blood that received insulin in nanohydrogel-loaded form compared with rats that received pure insulin is significantly high, that confirm insulin has been able to pass from the stomach acid barrier by nanohydrogel and be absorbed from the intestine.

Figure 5. Column chart from the amount of blood insulin collected in different times (the zero time is related to before gavaging).

Blood sugar levels from tested rats is shown in Figure 6, which indicate that with increasing amount of insulin, blood sugar levels fall down that are compatible with the experimental findings and also mapped charts show that the extent of absorption of insulin in case connected to the nanohydrogel is increased briefly.

Figure 6. Column chart from the amount of blood glucose collected in different times (the zero time is related to before gavaging).

By comparing the graphs of Figure 5 and 6 is evident that blood sugar levels has come down with insulin increasing, the minor changes are probably the result of the stress caused by the shock of the comatose, repeated injections, blood sampling, and animal fasting during the test time.

Discussion

Our in vivo study shows that attached insulin is synthesized nanohydrogel in 1,4 dioxan solvent without cross linker has lowest release and absorption in the stomach’s acidic environment and most of liberation and absorption in the intestinal environment and this is consistent of Peppas and Ramkisson Ganorkar works that in study were used three acrylic acid polymers, n isopropyl acrylic amide, and butyl meta acrylic acid with 10: 5: 85 molar ratio, the result showed that insulin in the acidic environment of the stomach has the least releasing while in pH = 7/4 insulin releasing done well (Peppas 2004, Ramkisson-Ganorkar 1999).

The results obtained from the measurement of the amount of rat insulin and blood sugar levels shows that by increasing the amount of insulin or blood sugar levels come down that are consistent with the experimental findings. Because the groups classification was carried out completely random and blood glucose in rats between 200 and 600, and due to this that the average of blood glucose and insulin in rats of each group are calculated and statistical analysis was performed on average, so perhaps there are rats in the control group, which have higher blood sugar in control group and has the same reason.

Analysis of the results show as well as that the extent of absorption of insulin in case connected to the nanohydrogel increase, this work would alter with results of Lowman et al, that first they were loaded with insulin on poly-ethylene glycol and Methacrylic microspheres and were gave orally to diabetic and healthy rats, in this study it was observed that in the acidic environment of the stomach because of the formation of intermolecular polymer complex and trapping insulin inside the polymer molecular network, and trapped insulin was preserved from proteolytic analyses and in neutral and alkaline intestinal environments this complex breaks down and medicine can be released (Lowman 1999).

Conclusions

Our main goal in this study is to provide pH sensitive Nanohydrogel from N- iso propyl polyacrylamide + Meta-acrylic acid + Hydroxyethyl methacrylate in 1, 4 Dioxane solvent without cross-linker addition and load the human pure insulin by modified dubbed emulsion and gavage it for diabetic rats and measuring plasma insulin and glucose levels at specified intervals.

Acknowledgements

The authors thank Department of Medical Nanotechnology, Faculty of Advanced Medical Science of Tabriz University for all supports provided. This work was funded by 2013 Drug Applied Research Center Grant.

Disclosure statement

The authors declare that they have no competing interests.

Funding

This work was funded by 2013 Drug Applied Research Center Grant.