chitosan core shell nanoparticles for the purpose of oral insulin delivery

Accepted Manuscript Preparation of polyurethane –alginate/chitosan core shell nanoparticles for the purpose of oral insulin delivery Aditi Bhattacharyya, Debarati Mukherjee, Roshnara Mishra, P.P. Kundu PII: DOI: Reference:

S0014-3057(17)30362-2 http://dx.doi.org/10.1016/j.eurpolymj.2017.05.015 EPJ 7871

To appear in:

European Polymer Journal

Received Date: Revised Date: Accepted Date:

27 February 2017 8 May 2017 10 May 2017

Please cite this article as: Bhattacharyya, A., Mukherjee, D., Mishra, R., Kundu, P.P., Preparation of polyurethane –alginate/chitosan core shell nanoparticles for the purpose of oral insulin delivery, European Polymer Journal (2017), doi: http://dx.doi.org/10.1016/j.eurpolymj.2017.05.015

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Preparation of polyurethane –alginate/chitosan core shell nanoparticles for the purpose of oral insulin delivery Aditi Bhattacharyyaa, Debarati Mukherjeeb, Roshnara Mishra b, P.P. Kundua, c* a

Department of Polymer Science and Technology, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, India b

c

Department of Physiology, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, India

Department of Chemical Engineering, Indian Institute of Technology, Roorkee-247667, Uttarakhand, India Corresponding Author: Prof. Patit P. Kundu (E-mail: [email protected]; [email protected])

Abstract: Waste polyethylene terephthalate (PET) is depolymerized through glycolysis and the glycolyzed product, bis (2-hydroxyethylene) terephthalate (BHET) is utilized in the synthesis of polyurethane as diol.

Polyurethane (PU) is incorporated in a core- shell nanoparticle

formulation along with alginate (ALG) and chitosan (CS) to develop an efficient oral insulin delivery vehicle.

Fourier transform infrared (FT-IR) spectrums of the polyurethane-

alginate/chitosan (PU-ALG/CS) nanoparticles confirm the presence of all elements distinctly. Nanoparticles of average particle size 90-110 nm are clearly visible from the images of scanning electron microscope (SEM) and transmission electron microscope (TEM). Unique characteristics of insulin loaded PU-ALG/CS nanoparticles are noticed in both in-vitro and invivo studies. More than 90% insulin encapsulation efficiency, sustained swelling, controlled insulin release from mucoadhesive nanoparticle formulation are the major causes of long term hypoglycaemic effects in diabetic mice and improved insulin bioavailability (10.36%). PUALG/CS nanoparticles are also found to be safe, according to the acute toxicity studies. Key Words: Polyurethane, chitosan, core-shell, nanoparticles, insulin bioavailability.

1.

INTRODUCTION

Hyperglycaemia caused due to the deficit of insulin hormones secreted from pancreatic beta cells or by the resistance of cells to the insulin secreted by the body is well-known as diabetes mellitus [1]. Exogenous insulin administration through subcutaneous injections of insulin is the most familiar way to control diabetes mellitus, although it causes sufferings resulted from pain, sensitivity, and drug scheduling complexities [2]. Suffering leads to negligence which results in major long term health care complicacies such as micro-vascular and macro-vascular problems [3]. Various alternative pathways of insulin administration were launched in the recent past and researchers are still working dedicatedly to find out other substitutes to reduce patient hassles. Insulin delivery through nasal, buccal, transdermal, rectal, ocular and oral routes using nanotechnology, gene delivery, implantable insulin pumps, pen devices, inhalers are the novel research advancements [4]. Although, oral delivery route is the most convenient method for everyone, but the oral delivery of insulin refers to low bio-availability. Rapid enzymatic degradation of insulin in the stomach and intestinal lumen, poor permeability across the intestinal epithelium and the absence of lipophilicity are the limitations of insulin ingestion through the oral route [2]. Therefore, a carrier system is essential to protect insulin from the insensitive environment of the stomach and small intestine when delivering orally [5]. Researches on oral insulin delivery are trying to deliver a fruitful outcome till date even though there is enough scope to bring out newer delivery systems. pH sensitive, biocompatible and biodegradable natural as well as synthetic polymeric micro or nano-particles were often used as drug delivery systems. Self-assembled [6], Cross-linked [7], core shell [8] polymeric nano-particles are the recent trends for oral insulin delivery systems. Nano-particles are particles ranging from 10 to 1000 nm in size and also of various shapes. Advantages of polymeric nano-particles are higher surface area to volume ratio, which provides faster dissolution and higher adsorption capacity for surface loading, protection from

proteolytic enzymes, enhanced mucoadhesion and increased retention in the GI tract [9]. Increased bioavailability, dose proportionality, smaller forms of drug dose having less toxicity, and finally reduced fed/ fasted variability are noted as few other significant benefits of nanoparticles [10]. Polymeric nanoparticles consisting of natural polymers such as chitosan [11], alginate [12], guar gum [13], gelatine [14] and synthetic polymers like polyethylene glycol [15], polyesters [16], polyurethanes [17,18] and combination of both natural and synthetic polymers in the form of polymer blends or polymer grafts or interpenetrating network are accepted materials of nanoparticles. Chitosan, a natural polysaccharide is the fully or partially N-deacetylated derivative of chitin [19]. Both chitin and chitosan are produced from crab or shrimp shells [20]. Chitosan has been extensively used in biomedical and pharmaceutical applications by reason of its remarkable properties like biocompatibility, biodegradability, anti-microbial, non-toxicity, mucoadhesion and non-immunogenicity [21,22].

Sodium alginate is another natural

biopolymer acquired from brown seaweeds, has been found to be an enormously potential polymer in drug delivery applications because of its hydrophilicity, biocompatibility and easy gelation [23]. Polyurethane is a biodegradable and biocompatible synthetic polymer widely used for biomedical applications, starting from medical devices like catheters, pacemakers to drug caring hydrogels, nanoparticles etc. [24,25]. Synthesis of polyurethane using depolymerised product of waste polyethylene terephthalate and utilising it for the betterment of oral insulin delivery vehicle with incorporation of natural polymers like alginate and chitosan is the main focus of this research work. Polyurethanealginate core and chitosan shell structure of nanoparticles were synthesized in our present research with the intention of obtaining higher encapsulation efficiency, more sustained insulin release as well as greater insulin bioavailability. Efficacy and safety of these core shell

nanoparticles were also investigated in an animal model for proper verification of in-vitro examinations.

2.

MATERIALS & METHOD

2.1.

Materials

PET flakes from waste PET bottles, ethylene glycol (EG), zinc acetate as catalyst and polyethylene glycol (PEG) with molecular weight of 600 were purchased from Merck, India. Hexamethylene diisocyanate (HDI) was purchased from Sigma Aldrich and nitrogen was purged through it before use. Sodium chloride, copper sulphate, sodium carbonate, potassium hydroxide, sodium hydroxide, Tris (hydroxymethyl) aminomethane, calcium chloride (CaCl 2) and hydrochloric acid (HCl) were purchased from Merck, India. Sodium potassium tartrate and Folin and Ciocalteu’s phenol reagent were supplied by Sisco Research Laboratory (SRL), India.

Low-viscosity, low-G (α-L-guluronic acid) ALG (β-D-mannuronic acid (M)/α-L-

guluronic acid (G) content 64.5%/35.5%) was purchased from Loba Chemie, India. Chitosan, alloxan monohydrate and insulin (Bovine insulin, 27 USP units per mg) was obtained from Sigma-Aldrich, India.

Lysozyme was purchased from Hi-media.

Sodium azide, sodium

carbonate and potassium ferricyanide were taken from Merck, India. Bovine Insulin ELISA kit from LILAC Medicare Pvt. Ltd., serum glutamate pyruvate transaminase (SGPT) ALAT (GPT)-LS kit and serum glutamate oxaloacetate transaminase (SGOT), ASAT (GOT)-LS kit from Piramal Health Care Limited, Mumbai, India, and lactate dehydrogenase LDH (P-L) kit, micro protein kit (Crest Biosystems, Goa, India), creatinine, Merckotest kit (Merck Limited, Mumbai, India) were purchased for the study. Multistix reagent strips (Siemens, Baroda, India) were purchased for qualitative analysis of different biochemical parameters of urine samples.

Other chemicals were of analytical grade and used directly without further

modification.

2.2.

Animals

Male Swiss albino mice (28 ± 2 g) from M/s Chakraborty Enterprise, Calcutta, India were housed under a controlled environment of room temperature (23 ± 2 oC), relative humidity (60 ± 5%, 12 h day/night cycle) along with a proper balanced diet and water ad libitum. The animal ethical committee, Department of Physiology, University of Calcutta has approved all the animal experiments according to the guidelines of the ethical committee, CPCSEA Ref. No. IAEC/IV/Proposal/RM-02/2015 dated 13.10.2014.

2.3.

Polyurethane synthesis using the depolymerised product of PET

PET waste was depolymerised using glycolysis process and the monomer BHET was separated from the reaction product as described in the previous reported research [23,26]. The reaction product was the mixture of monomer bis (2-hydroxyethylene) terephthalate (BHET), dimers and oligomers along with reactant residues. BHET was separated from the mixture by hot water treatment followed by filtration. Dimers as well as oligomers do not dissolve in hot water whereas monomer does.

Therefore filtrate part containing BHET

monomer was kept in 4 oC temperature to get crystalline BHET. It was further filtered and washed several times with distilled water to eliminate reactant residues like ethylene glycol and the catalyst zinc acetate. The elemental analysis of BHET and catalyst zinc acetate was performed using Scanning electron microscope (SEM) fitted with Energy-dispersive X-ray spectroscopy (EDX) to confirm the complete removal of the catalyst (Provided in the supplementary document).

Polyurethane was synthesized with BHET as one of the diols along with polyethylene glycol and hexamethylene diisocyanate [26]. 0.013 mol BHET was melted at 110oC under nitrogen atmosphere followed by incorporation of HDI (1.682 g, 0.02 mol) in to the reaction flask.

0.007 mol of PEG 600 was further added as a chain extender to the synthesized isocyanate group (NCO) terminated prepolymer. Almost 3-4 hour reaction time was required at 180oC to get a homogeneous dense yellow solution of polyurethane. Finally, synthesized polyurethane was cooled to room temperature and dissolved it in chloroform to prepare polyurethane films.

2.4.

Preparation of polyurethane –alginate blend

Freeze dried polyurethane was finely dispersed in water by overnight agitation. A particular amount of sodium alginate was mixed within the polyurethane dispersion and blended homogeneously at a ratio of 7:3 (PU: ALG) to incorporate the pH sensitive characteristics.

2.5.

Preparation of blank and insulin loaded core shell PU-ALG/CS nanoparticles

5mg/ml of CaCl2 solution was drop-wise added within polyurethane-alginate (PU-ALG) blend solution while the whole mixture was continuously sonicated for 15-20 minutes using a probe sonicator at room temperature (23 ± 2oC). pH of the PU-ALG blend was maintained at 5.1 before the addition of crosslinker of aqueous calcium chloride solution. Sonication was continued for another 15 minutes to allow the crosslinking of alginate for the formation of the core consisting of semi-interpenetrating network of alginate and polyurethane. After 15 minutes of the addition of crosslinker, 1% chitosan solution was added into PU-ALG core nanoparticle and sonicated for another 15 minutes to prepare PU-ALG core and CS shell nanoparticles of various compositions. 1mg/ ml of insulin stock solution was prepared by dissolving insulin powder in 0.1 M HCl solution. Insulin is hydrophobic molecule, and easily dissolves in a buffer at relatively low pH compared to the isoelectric point of insulin (pI = 5.8). Finally, 0.1 N of Tris(hydroxymethyl) aminomethane solution was used to adjust the pH of insulin solution to 8.0–8.4 and added to the calcium chloride solution for the crosslinking of the

chitosan shell and thereby lead to the preparation of insulin loaded fully crosslinked (core and shell) PU-ALG/CS nanoparticles. According to a an earlier report [27], dissolution of the insulin by addition of base at the iso-electric point, was performed to assure full solubility of the drug. As mentioned by the researchers, pH value of an aqueous solution of insulin should be adjusted above neutral, preferably (pH > 8) to provide advanced physical stability to the insulin formulations. Other processes were exactly the same as that for blank nanoparticle preparation. Blank and insulin loaded nanoparticles were freeze dried at -80oC (Optizen, Germany) before using it for different in-vitro, ex-vivo and in-vivo characterizations.

2.6.

FT-IR Spectroscopy

Attenuated total reflectance- Fourier transform infrared (ATR-FTIR) (Model-Alpha E, Bruker, Germany) spectrometer was used to perform FT-IR spectroscopic analysis with a scanning range from 4000 to 500 cm−1 for 42 consecutive scans at room temperature (23 ± 2oC). Polyurethane, alginate, PU-ALG nanoparticles, chitosan, insulin and PU-ALG/CS core shell nanoparticles were analysed using ATR mode. Wave-numbers of FT-IR Spectrums were expressed as a mean ± SD, n = 6.

2.7.

X-ray diffraction

An wide-angle X-ray diffractometer (Panalytical X-ray diffractometer, model X’pert powder) with a graphite monochromator and CuKα source was used to perform X-ray diffraction study of PU, calcium alginate and chitosan films, crosslinked PU-ALG nanoparticles, core shell ALG/CS, PU-ALG/CS nanoparticles, insulin powder and insulin loaded PU-ALG/CS nanoparticles. Samples were placed on a quartz sample holder at room temperature (23 ± 2 oC)

and were scanned at a diffraction angle of 2θ from 1 to 60 degree at the scanning rate of 1o per minute. The X-ray generator was operated at 40 kV and at 30 mA.

2.8. In-vitro enzymatic degradation of chitosan: chitosan matrices were initially weighed (Wo) and taken in 50 ml vials and filled with 25 ml of 0.1mol/L phosphate buffered saline (PBS) (pH 7.4) containing 0.5mg/mL Lysozyme and 0.5 mg/ml sodium azide. The vials were incubated at 37 oC in an incubator with continuous agitation at 120 RPM. Samples were withdrawn from media at fixed time intervals (1. 5, 10, 20 and 30 days) and washed thoroughly with distilled water to remove remaining PBS or lysozyme on the polymer surface. Polymer matrices were freeze dried for 24h and weighed to calculate weight after enzymatic degradation (Wd). All studies were carried out in triplicates. The percentage of remaining weight of polymer matrices was calculated using the following formula [28]. (1) The supernatant was also collected from each vial at similar time intervals to determine the content of reducing sugar. Modified Schale’s method [29] was followed to determine the content of reducing sugar in the supernatant collected in different time durations. 1.5 ml of centrifuged supernatant was taken in a test tube. A solution was prepared by dissolving 0.5 g of potassium ferricyanide in 0.5 mol/L sodium carbonate to 1 L of total solution. 2ml of potassium ferricyanide solution was mixed with the previously taken supernatant.

Test tubes were immediately sealed with

aluminium foil and kept in a water bath of 100 oC for 15 minutes. Test tubes were further cooled to room temperature and the absorbance of the solution was measured using a UV-vis spectrophotometer at 420 nm. All tests were performed in triplicates.

2.9.

DLS and Zeta

The particle sizes of PU-ALG, ALG/CS and PU-ALG/CS nanoparticles and zeta potential of polyurethane, alginate, chitosan, insulin, PU-ALG nanoparticles, alginate/chitosan (ALG/CS) nanoparticles and PU-ALG/CS nanoparticles with and without insulin were determined using dynamic light scattering (DLS) with Zetasizer Nano ZS (Malvern Instrument, UK). The size and zeta tests were performed in distilled water. All studies were carried out in triplicates.

2.10. SEM A scanning electron microscope (Hitachi, Japan, Model: 3400 N) was used to determine the size and surface morphology of insulin loaded PU-ALG/CS core shell nanoparticles. 10 μl of the nanoparticle suspension was placed on a glass slide and dried at room temperature (23 ± 2oC). The samples were fixed to the stub and gold sputtered to neutralize the charging effects before scanning in SEM with an acceleration voltage of 20 KV.

2.11. TEM Micrographs taken with transmission electron microscope (JEOL JEM 2100 HR) were used to analyse the morphology of insulin loaded PU-ALG/CS nanoparticles. A drop of PU-ALG/CS core shell nanoparticle suspension was placed on a carbon coated copper grid and the water was allowed to get evaporated in a vacuum drier. The grid containing insulin loaded PU-ALG nanoparticles was scanned under the TEM.

2.12. Swelling study of nanoparticles using DLS

Swelling studies of ALG/CS and PU–ALG/CS core shell nanoparticles and PU-ALG nanoparticles were performed in three buffer solutions with different pH corresponding to the changing pH of the gastrointestinal tract (pH 1.2, pH 6.8 and pH 7.4) concurrently at fixed intervals of time. Nanoparticles were initially allowed to swell at pH 1.2 and then transferred to the buffer solutions of pH 6.8 and 7.4 simultaneously. Nanoparticles were centrifuged each time before transferring it to another media. All swelling studies were performed in triplicates. Particle sizes of the nanoparticles at different pH were determined by using dynamic light scattering with Zetasizer Nano ZS (Malvern Instrument, UK).

2.13.

Drug loading content and drug encapsulation efficiency (EE) of the

nanoparticles Insulin-encapsulated ALG/CS and PU-ALG/CS nano-formulations were centrifuged at 14,000 RPM for 30 min at room temperature (23 ± 2oC) to separate the clear supernatant for the analysis of insulin content in the solution. Lowry’s assay method was used for the estimation of residual insulin in the supernatant obtained after filtration of the solution. The absorbance of insulin solution was determined using a UV spectrophotometer (Optizen Korea, Japan) at 660 nm. All experiments were done in triplicate to calculate drug loading content [30] and drug encapsulation efficiency [8] by the following formula: (2)

(3)

2.14.

In-vitro insulin release profile

Insulin release profiles of insulin-loaded ALG/CS and PU-ALG/CS core shell nanoparticles and PU-ALG nanoparticles were executed following the earlier mentioned protocol [7]. Freeze dried nanoparticles were immersed into buffer solutions at different pH corresponding to GI tract (i.e., pH 1.2, 6.8 and pH 7.4) with mild agitation using drug dissolution apparatus (Glass Agencies, India). Samples were centrifuged at specific time intervals to get the supernatant and an aliquot from each sample was taken out. The same amount of the supernatants was replaced with the fresh dissolution media each time after withdrawal of the sample. The concentration of the released insulin in the aliquot of each sample was determined by Lowry’s assay using a UV spectrophotometer at 660 nm. All of the dissolution runs were performed in triplicate. The cumulative amount of insulin released at different time intervals was calculated using the following formula and plotted against time to obtain the insulin release pattern. (4) Where C, D refers to the concentration of insulin in the dissolution medium and volume of the dissolution medium, respectively. In vitro insulin release data were fitted to zero-order, first-order, Higuchi, Hixon-Crowell and Korsmeyer-Peppas models to establish the insulin release mechanism of PU-ALG, ALG/CS and PU-ALG/CS nanoparticles. Korsmeyer-Peppas model is widely used to confirm the release mechanisms. (5) where, Mt / M∞ denotes the fraction of drug released at time (t), K is a constant showing structural and geometric characteristics of the particles, n is mentioned as the release exponent indicating the diffusion mechanism.

Values of release exponent (n) = 0.45, 0.45 < n < 0.89 and 0.89 indicates Fickian (Case I) diffusion, non-Fickian (anomalous) diffusion and zero-order (Case II) transport respectively [31].

2.15. Ex-vivo mucoadhesion study Freshly excised tissue from the small intestine of mice was used for the mucoadhesion studies of PU-ALG/CS nanoparticles. A part of the small intestine was taken out from the sacrificed male Swiss albino mice of 28±2 g weight and cleaned carefully by flashing saline solution to remove luminal contents. Intestinal part was incised, opened and then attached in a glass support using an adhesive. PU-ALG/CS core shell nanoparticles were spread evenly on the intestinal surface and left on the intestinal mucosa for 15 minutes. Glass slide was mounted at an angle of 45o on a petri plate under a constant flow of phosphate buffer (pH 7.4) at 10 ml/minute.

Percentage of adherence of PU-ALG/CS nanoparticles on the intestine was

calculated by deducting the amount of washed out nanoparticles from the weight of applied nanoparticles [32]. (6) Where, Wa =Weight of nanoparticles applied; Ww = weight of nanoparticles washed out.

2.16. Pharmacological response of insulin loaded PU-ALG/CS core shell nanoparticles in mice Inter-peritoneal alloxan injection was administrated to the male Swiss albino mice to develop diabetic animal model. The diabetic mice were fasted overnight before treatment and fasting continued till the completion of the experiment, only allowing water ad libitum. Insulin encapsulated PU-ALG (100 IU/kg body weight (b.w.), ALG/CS (100 IU/kg body weight) and

PU-ALG/CS nanoparticles with different doses (50 and 100 IU/kg body weight) were fed orally with a feeding needle to the diabetic animals (n = 6 each group). Another set of animals was fed orally with 50 IU/kg b.w. insulin solution, mice treated with subcutaneous insulin injection (5 IU/kg b.w.) were used as control and another set of diabetic mice without any treatment was also investigated as negative control. ACCU-CHEK’s glucose meter was used to check the blood glucose level of treated mice at regular intervals of time.

2.17.

Relative insulin bioavailability after oral administration of insulin loaded

PU-ALG/CS core shell nanoparticles. Diabetic mice divided into seven groups (n = 6) were treated with (Group I) oral insulin solution of 50 IU kg−1 b.w., (Groups II & III) oral insulin-loaded PU-ALG/CS core shell nanoparticle of 50 and 100 IU kg−1b.w., (Group VI) oral insulin-loaded PU-ALG nanoparticle of 100 IU kg−1b.w. , (Group V) oral insulin-loaded ALG/CS core shell nanoparticle of 100 IU kg−1b.w, (Group VI) subcutaneous injection of insulin solution (5 IU kg−1 b.w.) and (Group VII) control diabetic mice without any treatment. Blood samples were collected from a retro orbital vein and centrifuged at 5000 RPM, for 10 min at 4 oC to separate blood serum. Serum insulin levels were determined using enzyme-linked immunosorbent assay (ELISA). Area under the curves (AUC) calculated from the concentration versus time plot. The AUC of the orally fed insulin-loaded PU-ALG/CS nanoparticles and subcutaneous (SC) injection of insulin along with their respective doses were used to calculate the relative bioavailability. Following formula was used to calculate the relative bioavailability of insulin [7]. (7)

2.18. Toxicity studies of the ALG/CS and PU-ALG/CS nanoparticles in animal models.

Toxicity study of PU-ALG/CS nanoparticles was carried out to determine LD50 value according to OECD (2001) guidelines 425. Mice were fasted overnight before the treatment, and the PU-ALG/CS core shell nanoparticle solution was orally fed with a single dose of 5000 mg/kg body weight to perform limit test. Acute toxicity studies were performed for oral treatment of ALG/CS and PU-ALG/CS core shell nanoparticles. Three sets of animals (n=6) were separated as Group I: animals received only 1 ml of 0.9% saline perorally, considered as the control, group II is animals orally fed with ALG/CS nanoparticles of 150 mg kg −1b.w. dose, group III and group IV are the animals fed with PU-ALG/CS core shell nanoparticles of 150 and 5000 mg kg−1b.w. dose orally. After 24 hours, stored urine was collected and blood was taken from retro-orbital veins of the animals of all three groups.

2.18.1. Liver function test Serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and lactate dehydrogenase activity (LDH) were tested using blood serum to determine the liver function of the mice treated with ALG/CS and PU-ALG/CS core shell nanoparticles.

2.18.2. Nephrotoxicity test Serum creatinine, urine creatinine and urine microprotein and urine urea estimations were performed to detect the nephro-toxicity of ALG/CS as well as PU-ALG/CS core shell nanoparticles. Qualitative analysis of leukocytes, nitrite, urobilinogen, protein, blood, ketone, bilirubin, glucose, pH and specific gravity was performed with urine samples of ALG/CS and PUALG/CS nanoparticle fed mice and control mice using Orinasys reagent strips.

Liver and kidney were initially attached in phosphate-buffered formalin (10%) for Pathohistological diagnosis.

The tissues were embedded in paraffin and sectioned

subsequently. Haematoxylin and eosin (H&E) were used to stain the tissue sections.

2.19. Statistical analysis All the results were expressed as a mean ± SE, n=3 for in vitro studies and n=6 for in vivo studies. The significance level was determined by one-way ANOVA following Tukey’s post hoc test. P < 0.05 was considered as significant.

3.

RESULTS AND DISCUSSION

Polyurethane-Alginate/ Chitosan (PU-ALG/CS) core shell nanoparticles has been investigated in terms of its competency as well as safety for the purpose of oral delivery of insulin. PUALG core was formed by partial ionotropic pre-gelation of PU-ALG blend due to the presence of alginate and subsequently contouring shell was constructed through polyelectrolyte complexation of chitosan. Insulin was added in the crosslinker solution to encapsulate it within the core during gelation. Most of the excess unbounded insulin was present either in the surface or in the solution, which was finally encapsulated within the shell. As reported in our previous research [7], PU-ALG nanoparticles showed lesser encapsulation efficiency for the same amount of insulin compared to the PU-ALG/CS nanoparticles. Encapsulation efficiency significantly increased due to the presence of the outer shell incorporated over PU-ALG core. Secondly, it was also reported, the initial bulk release of insulin from PU-ALG nanoparticles at acidic pH in spite of negligible swelling might be due to weakly interacted insulin molecules

on nanoparticle surface.

Those weakly bounded insulin molecules were supposed to be

covered with chitosan shell. Spectroscopic and morphological characteristics of the polymeric nanoparticles were analysed using FT-IR spectroscopy, SEM and TEM. Size and charge distribution of the nanoparticles were examined using DLS and Zeta potential. The hypoglycaemic effect of insulin loaded PUALG/CS nanoparticles as well as pharmacological bioavailability of insulin were studied in animal models. Toxicity assay involving different parameters was performed to determine whether the nanoparticles are toxic or not.

3.1.

FT-IR Spectroscopic analysis:

FT-IR spectrums of polyurethane, sodium alginate, polyurethane-alginate blend nanoparticles, chitosan,

alginate/chitosan

nanoparticles,

insulin

and

nanoparticles, insulin

loaded

polyurethane-alginate/chitosan polyurethane-alginate/chitosan

core

shell

core

shell

nanoparticles were illustrated in Figure 1A, 1B, 1C, 1D, 1E and 1F correspondingly. FT-IR spectrum of polyurethane (Figure 1A) demonstrated the presence of OH group, N-H linkages, ester-carbonyl groups (C=O), and aromatic C-H group linkages at 3400-3500 cm-1, 1715 cm-1 and 725 cm-1 respectively. Absorption bands at 2861 cm-1 to 2932 cm-1, 1536 cm-1 and 1250 cm-1 were indicated the stretching vibrations of the C-H group, N-H groups and C-O-C group [23]. Figure 1B demonstrated the FT-IR spectrum of sodium alginate nanoparticles. Peaks at 3000-3600 cm-1, 1593 cm-1and 1415 cm-1 signified the existence of OH groups, C=O groups and carboxylate salt in the nanoparticles. Peaks at 2927 cm-1 and 2859 cm-1 were attributed to the aliphatic C-H group and presence of C-O-C linkages was confirmed due to the peaks at1000-1200 cm-1 [8]. FT-IR spectrum of PU-ALG nanoparticles in Figure 1C showed peaks at 3000-3600 cm-1 due to OH groups, 2850 cm-1 and 2917 cm-1 attributed to the stretching of

C-H group stretching. Characteristic peaks of polyurethane such as 1716 cm-1 and 1605 cm-1 due to the presence of C=O group, 1537 cm-1 for stretching vibration of NH, 1250 cm-1 for CO-C group and finally 728 cm-1 due to the presence of C-H linkages in the aromatic system was observed. Carboxylate salt anion peak at 1434 cm-1, C=O stretching vibrations peaks at 1084 cm-1 and 1017 cm-1 confirmed the presence of alginate in the nanoparticle system. Figure 1D demonstrated the FT-IR spectrum of chitosan where peaks at 3000 cm-1 -3500 cm-1 indicated the OH and NH stretch overlap, peaks at 2920 cm-1 and 2873 cm-1 were due to C-H stretch. Peaks at 1633 cm-1, 1536 cm-1, 1148 cm-1 and 1062 cm-1were due to C=O stretching, N-H bending, bridge-O stretch and C-O stretching respectively [8].

IR spectrum of ALG/CS

nanoparticles (Figure 1E), shows that the asymmetrical stretching of C=O groups at 1587 cm−1 and the peak for carboxylic salt ion shifted to 1405 cm−1 after mixing with ALG. Presence of OH and NH groups were confirmed by the broad peak at 3000 cm−1 to 3500 cm−1 [8]. Figure 1F, illustrated the FT-IR spectrum of PU-ALG/CS nanoparticles. Broad peak at 3000-3600 cm-1for OH and NH group overlap and peak at 2933 cm-1 for C-H stretching, 1714 cm-1 and 725 cm-1 indicated C=O group and aromatic C-H linkages present in PU. Peaks at 1620 cm-1 and 1543 cm-1 were present for C=O stretching and NH bending of chitosan, 1420 cm-1 appeared due to the presence of carboxylate ion in alginate. FT-IR spectrum of insulin in Figure 1G illustrates peaks at 3285 cm-1and 1641 cm-1 for NH stretching and C=O stretching of amide I.

The presence of N-H bend and C-N stretching corresponding to amide II was

confirmed by the peak at 1512 cm-1 [7]. Finally, FT-IR spectrum of insulin loaded PU-ALG/CS core shell nanoparticles (Figure 1H) shows peaks at 3000 cm-1 to 3600 cm-1 and 2880 cm-1 to 2938 cm-1, indicating OH as well as NH stretch overlap and C-H stretching respectively, however, appearance of peak at 3318 cm-1 confirmed the presence of insulin. Peaks at 1715 cm-1 and 727 cm-1 attributed to C=O group and aromatic C-H linkages, peaks at 1684 cm-1 with a shift from 1641 cm-1 indicated the C=O

stretching due to the presence of insulin [8]. Peaks at 1605 cm-1, 1538 cm-1 were present for C=O stretching and NH bending of chitosan with little shift; peak of carboxylate ion also shifted to 1414 cm-1. Characteristic peaks of polyurethane-alginate, chitosan and insulin were clearly evident in this FT-IR spectrum; this indicates towards well prepared insulin encapsulated core shell nanoparticles.

Figure 1. FT-IR spectrum of A. Polyurethane film, B. Alginate film, C. Polyurethanealginate nanoparticles, D. Chitosan film, E. Alginate/chitosan nanoparticles, F. Polyurethane-alginate/chitosan nanoparticles, G. Insulin powder and H. Insulin loaded Polyurethane-alginate/chitosan nanoparticles

3.2.

X-ray diffraction analysis:

X-ray diffraction of PU-ALG/CS/Insulin NPs, insulin powder, PU-ALG/CS NPs, ALG/CS NPs, chitosan film, PU-ALG NPs, alginate and polyurethane films are shown in Figure 2. XRD spectra of polyurethane showed broad hump near 2θ=24 o. As mentioned in our previous work [23] presence of peak near 24 o indicated amorphous nature of polyurethane film. Alginate film and PU-ALG nanoparticles illustrated a broad hump between (2θ) 20 to 30 degrees also indicated the amorphous nature of the nanoparticles in accordance to our previously reported results [7]. XRD spectrum of chitosan film illustrated hump near 11 and 23 degrees indicating the crystalline nature of chitosan. Strong intermolecular hydrogen bonds between hydroxyl as well as amino groups and the regularity of polymer chain structure formed due to these bonds might be the cause for the appearance of these peaks [33]. Presence of small hump near 13 degrees in the XRD spectrum of ALG/CS nanoparticles along with a broad peak near 20 to 25 degrees accurately point towards the incorporation of chitosan over alginate nanoparticles. PU-ALG/CS nanoparticles showed the dominance of incorporating polyurethane along with alginate and chitosan as the intensity of the hump at 20 to 25 degrees increased compared to ALG/CS nanoparticles. Again, XRD spectrum of insulin illustrated peaks near 10 degrees and 21 degrees. Finally, the presence of a similar hump near 9 to 10 degrees and existence of broad hump at (2θ) 20 to 25 degrees confirmed presence of insulin in PU-ALG/CS nanoparticles.

Figure 2: X-ray diffraction graphs of PU-ALG/CS/Insulin NPs, insulin powder, PUALG/CS NPs, ALG/CS NPs, chitosan film, PU- ALG NPs, alginate and polyurethane films.

3.3.

Enzymatic degradation of chitosan:

Chitosan is mainly degraded by the enzymes which can hydrolyse the glucosamine linkages present in chitosan. Bacterial enzymes present in the colon such as lysozyme mainly takes part in the degradation of chitosan [34],[29]. The weight of chitosan matrices decreased gradually up to 56.92% after 30 days degradation time (Figure 3A).

After the mentioned time of

degradation chitosan films became very fragile and relatively thinner than initially taken chitosan film.

Content of reducing sugar present in the supernatant collected from degradation media of chitosan in different intervals of time are plotted in figure 3B. Concentration of reducing sugar was found to be increased very slowly in the initial time durations. However, it was elevated after 10 days interval and resulted gradual rise in reducing sugar content. 0.058mg/ml reducing sugar was calculated after initial 5 day whereas concentrations of reducing sugar were 0.21 mg/ml, 0.55 mg/ml and 0.74 mg/ml after 10, 20 and 30 days interval. β-(1→4) glycosidic bond cleavage occurs due to enzymatic degradation of chitosan by lysozyme resulting, increased level of reducing sugar in the degradation media [29]. Percentage of weight loss and concentration of reducing sugar clearly indicated successful degradation of chitosan films.

Figure 3(A): Degradation percentage of chitosan matrices undergone in-vitro degradation by lysozyme. (B) Reducing sugar content of degradation media.

3.4.

Particle size and zeta potential:

Nano-particles have comparatively larger surface areas than similar masses of larger particles. A greater sum of the material can come into contact with surrounding when the surface area per mass of material increases. Therefore, smaller sizes directly affect the reactivity of the material. Particle sizes of PU-ALG nanoparticles were reduced to 40-60 nm; this particle size is smaller than our previous research (70- 90 nm) [7]. Particle sizes of PU-ALG/CS core shell nanoparticles were found to be within a range of 90-110 nm. The average size of PU-ALG nanoparticles at weight ratio of 7:3:2 (PU: Alginate: Insulin) ALG/CS nanoparticles at weight ratio 3:3:2 (CS: ALG: Insulin) and PU-ALG/CS nanoparticles at weight ratio of 3:7:3:2 (CS: PU: ALG: Insulin) were 56.85 nm 156.12 nm and 105.7 nm respectively (Figure 4A). Particle sizes of the PU-ALG/CS core shell nanoparticles are smaller than other core shell nanoformulations [8],[35]. We have also performed size analysis of ALG/CS and PU-CS/ALG nanoparticles for different ratios of chitosan as 1:3:2, 2:3:2 and 3:3:2 (CS: ALG: Insulin). The size of the nanoparticles increased with increasing amounts of chitosan. The repulsion between the excess positive charges accumulated in the presence of higher amount of chitosan might be the reason behind the simultaneous size enlargement [8]. As represented in Figure 4B, Zeta potential values of polyurethane, alginate, chitosan, PUALG/CS NPs, ALG-CS NPs, insulin, insulin loaded PU-ALG NPs and insulin loaded PUALG/CS NPs are found to be as -11.6 mV, -13.1 mV, 49.5 mV, 43.5 mV, 21 mV, 9.34 mV,27.3 mV and 38.5 mV respectively. ALG/CS core shell nanoparticles showed a noticeable change from ALG nanoparticles as zeta potentials become positive (21 mV) from -13.1 mV for ALG nanoparticles. Accumulation of surplus positive charges due to chitosan shell around ALG core.is the probable reason of this phenomenon.

The zeta potential of PU-ALG

nanoparticles was -15.3 mV whereas PU-ALG/CS nanoparticles showed a sharp increase to 43.5 mV similarly due to the presence chitosan shell. Zeta potential of insulin encapsulated

PU-ALG/CS nanoparticles showed a considerable change of zeta potential as 38.5 mV, which is certainly due to the incorporation of negatively charged insulin molecules having zeta potential value of -9.34 mV. -25 mV ˃ Zeta potential ˃ +25 mV

indicate great electrostatic stability of polymer

nanoparticles [7]. It is extremely important to have high zeta potentials especially for the micro/nano-carriers of drugs or proteins [36]. Both blank and insulin loaded PU-ALG/CS NPs showed zeta potential values of 43.5 mV and 38.5 mV respectively indicating the electrostatic stabilization of colloidal system of the nanoparticles.

Figure 4(A) Particle sizes of PU-ALG nanoparticles, ALG/CS and PU-ALG/CS core/shell nanoparticles. (B) Zeta potentials of Polyurethane, Alginate, Chitosan, PU-ALG nanoparticles, PUALG/CS nanoparticles, ALG/CS nanoparticles, insulin and insulin loaded PU-ALG/CS.

3.5.

Dynamic swelling study of ALG/CS, PU-ALG and PU-ALG/CS nanoparticles.

Figure 5 demonstrated the changes in the size of ALG/CS, PU-ALG/CS and PU-ALG/CS core shell nanoparticles due to swelling in different solutions of pH buffers (pH 1.2, pH 6.8 and pH 7.4) simultaneously according to the pH of the gastrointestinal tract of animal body. Hydrodynamic size of the nanoparticles in pH 1.2 buffer solutions showed no change in the size of the nanoparticles.

Hydrodynamic swelling characteristics showed a continuous

variation of the size (d, nm) of the nanoparticles when immersed in pH 6.8 buffer. At pH 6.8, the size of the nanoparticles increased at a lower rate at the beginning (up to 120 minutes) and then increased at a much higher rate. Though nanoparticles immersed in pH 7.4 buffer also showed similar characteristics as in the case of pH 6.8, however, overall enhancement of the diameter of nanoparticles in buffer solution pH 6.8 was found to be less than pH 7.4. This result explains poor swelling of nanoparticles in the acidic pH of the stomach and excellent sustained swelling of PU-ALG/CS nanoparticles in intestinal pH.

Unique swelling

characteristic (i.e change in higher rate of swelling) at intestinal pH during the 5 th to 7th hours for PU-ALG/CS nanoparticles were thought to be due to the core shell structure of the nanoparticles. The shell prepared with chitosan started swelling primarily and probably the core began to swell relatively later than the shell. PU-ALG initiates swelling slowly and increases in a sustained manner which might be the reason behind these swelling features.

Figure 5. Swelling study of PU-ALG/CS, ALG/CS and PU-ALG nanoparticles using dynamic light scattering.

3.6.

Morphological analysis:

Figure 6 shows the micrographs of scanning electron microscope and transmission electron microscope captured to analyse the morphological features of the PU-ALG/CS core shell nanoparticles.

Most of the core-shell nanoparticles were found to be spherical in shape

according to both SEM and TEM micrographs. As per the images were shown, the outer surface of the nanoparticles was smooth and even. TEM images (Figure 6 (B & C)) are the clearest evidence for the core shell structure where the PU-ALG core evenly covered with chitosan shell. TEM images of PU-ALG nanoparticles were reported in our previous research [7]. Opaque, mostly spherical nanoparticles were clearly visible from the images. There were no such shell observed in case of PU-ALG nanoparticles. On the other hand, TEM images of PU-ALG/CS nanoparticles showed the clear transparent cover over dark opaque nucleus. We concluded that cover lining as the chitosan shell from the comparative observation with previous research. Thickness of the chitosan shell depends upon a few factors such as the

concentration of the polymer [37], electrostatic interaction of the polymeric shell with the core polymer etc. [38]. Particle sizes of the core shell nano-formulation were varied within a range of 80-100 nm surrounding the PU-ALG core of 40-60 nm sizes. Particle sizes of PU-ALG/CS core shell nanoparticles were found to be slightly larger during DLS analysis because of the hydrodynamic diameter measurement through this process.

Figure 6. (A) SEM and (B&C) TEM images of polyurethane-alginate/chitosan core shell nanoparticles.

3.7.

Insulin loading content and encapsulation efficiency:

Figure 7A and 7B illustrated the insulin loading content and insulin encapsulation efficiencies of ALG/CS and PU-ALG/CS nanoparticles where ratios of chitosan, polyurethane-alginate blend and insulin were taken at assorted weight ratios. ALG/CS nanoparticles of different

ratios of chitosan, alginate and insulin such as 1:3:1, 2:3:1, 3:3:1, 3:3:0.5 and 3:3:2 showed 12.6%, 11.83%, 11%, 6.07% and 17.25% of insulin loading content respectively. However, insulin loading content was found to be low. PU-ALG/CS nanoparticles illustrated 7.25%, 7.08%, 6.71%, 3.59% and 12% for different chitosan: polyurethane-alginate: insulin ratios as 1:7:3:1, 2:7:3:1, 3:7:3:1, 3:7:3:0.5 and 3:7:3:2. Alginate/Chitosan nanoparticles showed 63% encapsulation efficiency for ratio 1:3:1 (Chitosan: Alginate: Insulin).

Nanoparticles with

different ratios such as 2:3:1, 3:3: 3:3:0.5 and 3:3:2 were 71%, 77%, 79% and 64% as illustrated in the figure 4ii. Polyurethane-Alginate/Chitosan nanoparticles of ratio 1:7:3:1 (Chitosan: Polyurethane: Alginate: Insulin) resulted with 87% encapsulation efficiency. Subsequently, the amount of chitosan was increased keeping the ratio of insulin and PU-ALG blend constant. Percentage of insulin encapsulation efficiency, increased gradually with the increasing quantity of chitosan in the core shell nanoparticles. 92% and 94% encapsulation efficiencies were calculated for the CS: PU: ALG: Insulin ratio of, 2:7:3:1 and 3:7:3:1 respectively. The ratio of polyurethane to alginate was kept same (7:3) because this ratio was found to be more proficient than 9:1 and 8:2 ratios in our previous works [26]. As per mentioned result, 3:7:3 ratios for chitosan to polyurethane-alginate showed maximum insulin encapsulation. Again, the quantity of insulin was varied in further experiments maintaining the ratio of polymers fixed in 3:7:3 (chitosan: polyurethane: alginate). Encapsulation efficacies in the case of polymer to insulin ratios, 13:0.5 and 13:2 of PU-ALG/CS nanoparticles were estimated as 97% and 90%. Insulin encapsulation efficiency of chitosan/ alginate core shell nanoparticles with weight ratios 3:1:1 (alginate: chitosan: insulin) was reported to be 78.3% [8] whereas incorporation of polyurethane showed a much better percentage of encapsulation (87%). Encapsulation efficiency of alginate nanoparticles for ratio 0.6:0.03 (alginate: insulin) was 22%, whereas 78% encapsulation efficiency was reported in our previous work for PUALG nanoparticles with PU: ALG: insulin ratio of 1.4:0.6:0.03 [7]. So, incorporation of PU

definitely improved the encapsulation efficiencies.

In our present work, encapsulation

percentages reduced with increasing quantity of insulin; however, when we have worked with the ratio of 3:7:3:2 (CS: PU: ALG: INSULIN), it offers 90% encapsulation.

Core shell

nanoparticle preparation with the polymer to insulin ratio of 13:2 elaborately included 3 mg chitosan, 7 mg polyurethane, 3 mg alginate and 2 mg insulin, although, practically 1.8 mg (90% of 2 mg) insulin was encapsulated. Actual insulin loading for other ratios (13:1 and 13: 0.5) was extremely low such as 0.94 mg and 0.485 mg respectively; therefore, our obvious choice was the polymer to insulin ratio 13:2.

Insulin loading contents of PU-ALG/CS

nanoparticles were unsatisfactory compared to ALG/CS nanoparticles due to higher polyurethane content, however, improvement in the encapsulation efficiencies of PU-ALG/CS nanoparticles compared to previously reported research was a great encouragement for our present work.

Figure 7(A). Loading content and (B). Encapsulation efficiency of insulin loaded ALG/CS and PU-ALG/CS nanoparticles. 3.8.

In-vitro insulin release profile:

Figure 8 illustrated the insulin release characteristics of PU-ALG/CS and ALG/CS core shell nanoparticles as well as PU-ALG nanoparticles performed at different pH conditions such as pH 1.2, pH 6.8 and pH 7.4 simultaneously at 37oC. Initially, there is a slight insulin release (13.7 %, 15.77% and 14.17% ) from the PU-ALG/CS, ALG/CS and PU-ALG nanoparticles at pH 1.2 up to one hour, followed a decrease in cumulative value of insulin release, indicating no release of insulin at pH 1.2. Few weakly interacted insulin with the polyelectrolyte on the nanoparticle surface might be the cause of that initial release and least swelling of the nanoparticles in that pH protected insulin from the acidic environment of gastric fluid [7]. Insulin encapsulated in PU-ALG/CS nanoparticles released moderately into pH 6.8 buffer solution up to 50% till 10th hour whereas sustained release of insulin was noticed at pH 7.4. The continuous increase of insulin release was noticed from 11 th hour till the end of the study. The release characteristics showed sustained and continuous enhancement in the percentage of insulin release and the maximum insulin release till 20 th hour was 98.32%.

PU-ALG

nanoparticles and ALG/CS core/shell nanoparticles showed similar characteristics in the acidic environment. However, about 60% and 81% of the encapsulated insulin released from PUALG and ALG/CS nanoparticles at 10th hour while immersed in pH 6.8 buffer. Maximum (93.33%) encapsulated insulin released at pH 7.4 within 15th hour for ALG/CS nanoparticles whereas PU-ALG nanoparticles taken 17 hours to release 97.5% insulin.

In a previous

research, chitosan/alginate core shell nanoparticles showed maximum 84% release of the encapsulated insulin [8]. The augmented interaction between the cationic chitosan shells with alkaline pH 7.4 buffer solution was believed to be the reason of solvent diffusion into the alginate core followed by the chitosan shell. Alginate nanoparticles show burst release of

insulin in the alkaline solutions which might be another cause of uninterrupted continuous release of insulin from chitosan/alginate nanoparticles whereas release characteristics of PUALG is prolonged and continuous as concluded from our previous research [7]. The release pattern of PU-ALG/CS nanoparticles during the initial hours at pH 6.8 might be due to the initiation time of swelling of the PU-ALG core of the nanoparticles. Different insulin release patterns observed at changing pH conditions could be explained by the electrostatic interactions between the polymers like alginate and chitosan and their pKa values. At pH 3-4, carboxylic groups of alginate are half protonated, therefore partially uncharged because the pKa value of alginate is around 3.5 [39]. Electrostatic interactions of alginate and chitosan also become weaker in this pH. pKa value of chitosan is about 7 therefore, similar characteristics of electrostatic interactions results when immersed at pH near to its pKa value [40].

Figure 8. In vitro insulin release study from PU-ALG/CS, ALG/CS and PU-ALG nanoparticles at different pH.

Mathematical analysis of the in-vitro insulin release pattern revealed the diffusion controlled and swelling controlled insulin release. Correlation coefficient (R ≥ 0.99) values of different models illustrated in table 1 which confirmed that the release data fit well into the empirical equation. The “n” release exponents indicated a non-Fickian (anomalous) transport (0.45 < n < 0.89) of the nanoparticles for simultaneous drug release system at pH 1.2, pH 6.8 and pH 7.4 respectively.

Table 1: Drug release mechanisms of PU-ALG/CS nanoparticles

Drug release mechanism of PU-ALG/CS core shell nanoparticles (Values are shown as mean ± SE (n=3) P ˂ 0.05, Significant) Nanoparticles

Models Zero order

First order

(r2)

(r2)

ALG/CS

0.8632

0.7567

PU-ALG

0.9651

PU-ALG/CS

0.9872

3.9.

Higuchi

Drug transport mechanism

Hixson Crowell

Korsmeyer peppas

Korsmeyer peppas

(r2)

(r2)

(n)

0.9416

0.9388

0.9506

0.6634

Nondiffusion

0.8799

0.9638

0.943

0.9538

0.8023

Non-fickian diffusion

0.9478

0.9371

0.9215

0.9259

0.6017

Non-fickian diffusion

2

(r )

fickian

Ex-vivo mucoadhesion studies:

Freshly excised mouse intestine, PU-ALG/CS nanoparticle incorporated intestine before washing and during the time of washing with pH 7.4 buffer solution are demonstrated in Figure

9A, 9B and 9C respectively. Adherence of the PU-ALG/CS nanoparticles to the mucosal layer of the intestine was found to be quite excellent, even after continuous gush of buffer solution onto it throughout 30 minutes of washing. The amount of nanoparticles washed out during this study was less than 10% and the mucoadhesive nature of these core shell nanoparticles is also evident from the negligible visible difference of Figure 9B and 9C.

Mucoadhesive and

superior adsorption qualities of sodium alginate, polyurethane as well as chitosan were supposed to be the compulsion for the attachment of PU-ALG/CS nanoparticle in the mouse intestine. Alginate is mentioned as an anionic mucoadhesive polymer due to the presence of carboxyl terminal groups [7],[41] whereas, the presence of positively charged chitosan in the PU-ALG/CS nanoparticles is the reason of strong electrostatic interaction with negatively charged intestinal mucosa. The interaction between nanoparticles and mucosal surface causes redistribution of F-actin and the tight junctions protein, allowing increased paracellular permeability [8],[42]. Excellent prolonged attachment of the nanoparticles to the mucosal surface is one of the major causes for a sustained and controlled release of the encapsulated insulin as evidenced from the following discussion.

Figure 9. Ex-vivo mucoadhesion study of PU-ALG nanoparticles (A. Freshly excised mouse intestine, B. before and C. after washing with pH 7.4 buffer).

3.10. Pharmacological response of insulin loaded PU-ALG/CS nanoparticles: Hypoglycaemic effects of insulin encapsulated PU-ALG/CS core shell nanoparticles on the alloxan treated diabetic mice models were investigated. Insulin loaded nanoparticles were fed orally to the overnight fasted swiss albino mice and remained fasted throughout the experiment, however, water is allowed ad libitum. Insulin loaded ALG/CS nanoparticles at 100 IU/kg body weight dose, PU-ALG/CS nanoparticles at different doses such as 50 IU/kg body weight and 100 IU/kg body weight, PU-ALG nanoparticles at 100 IU/kg body weight dose and insulin solution at 50 IU/kg body weight dose were administrated orally whereas, subcutaneous injection of insulin at 5 IU/kg body weight dose was given for relative study. A baseline of average blood glucose levels was estimated before the administration of oral formulations. Variations in the average blood glucose levels after insulin administration either by the oral route or by subcutaneous injection were plotted against time in Figure 10A. Changes in blood glucose levels of saline treated diabetic mice (Control) with time were also investigated. PU-ALG/CS nanoparticles showed improvement in terms of sustained hypoglycaemic response than PU-ALG nanoparticles. Insulin loaded PU-ALG/CS nanoparticles efficiently decreased the blood glucose level in a gradual manner except during 5 th to 7th hours where the hypoglycaemic effect was noticed to be almost unchanged or hardly augmented.

This

phenomena is thought to be due to slow insulin release during swelling initiation of the core i.e. PU-ALG. Insulin loaded PU-ALG/CS nanoparticles reduced the level of blood glucose up to 98 mg/dl and 131 mg/dl for the insulin doses of 100 IU/kg body weight and 50 IU/kg body weight respectively at the 10th hour. The better hypoglycaemic effect in the case of higher dose indicated the greater absorption as well as the higher concentration gradient of insulin in the intestine. The blood glucose level was reduced to 105 mg/dl at the 8 th hour in case of PU-ALG

nanoparticles with insulin dose of 100 IU/kg body weight. ALG/CS nanoparticles of 100 mg/kg body weight dose reduced the blood glucose level to 123 mg/dl at the 6th hour and returned to the basal level within 10 hours. Insulin loaded PU-ALG/CS nanoparticles was found to be effective for several hours because it took more than 18 hours to reach the basal blood glucose level. As mentioned in the earlier report [8], sustainability of the insulin loaded CS/ALG core shell nanoparticles was till 9 hours. So, it can be concluded that incorporation of polyurethane served a vital role in the enduring release of insulin from the prepared nanoparticles. Orally fed 50 IU/kg body weight dose of insulin solution was incompetent to show noticeable effects in comparison with these nanoparticles. On the contrary, subcutaneous injection of insulin at 5 IU/kg body weight dose showed an instantaneous hypoglycaemic effect in the 1st hour and ultimate minimization of the blood glucose level at 115 mg/dl within the next hour. Blood glucose level of the insulin injected mice started increasing soon after that and maximum time elapsed to return to its initial level was 6 to 7 hours. Saline treated diabetic mice did not show any significant change in blood glucose level during the long fasting time. It also eliminated the probabilities of any influence in the reduction of blood glucose level.

Figure 10 (A). In-vivo pharmacological response (B) Serum insulin concentration after peroral treatment of insulin loaded ALG/CS, PU-ALG and PU-ALG/CS nanoparticles.

3.11. Relative insulin bioavailability of insulin loaded PU-ALG/CS nanoparticles: Serum insulin levels of the insulin treated diabetic animals were investigated at the simultaneous time intervals along with the blood glucose measurements. The area under the curves (AUC) were calculated from the graphs plotted (Figure 10B) for subcutaneous and various oral treatments of insulin. The relative bioavailability of insulin for insulin loaded PUALG/CS nanoparticles was calculated from the calculated AUC values using the previously mentioned formula. Mice treated with subcutaneous injections of insulin reached at the peak of serum insulin concentration at the 2nd hour after insulin administration whereas, serum insulin concentrations of insulin solution fed mice was found to be insignificantly low. Orally fed

insulin loaded PU-ALG nanoparticle showed maximum serum insulin concentration at the 8 th hour, however, PU-ALG/CS nanoparticles encapsulated insulin of both 50 mg and 100 mg per kg body weight dose demonstrated the highest concentration of serum insulin at the 10th hour after the oral feeding. The relative bioavailability of insulin calculated from the AUC (0-18 hours) of orally administrated 50 IU and 100 IU/kg body weight, insulin dose of PU-ALG/CS nanoparticles were ∼9.57% and ∼10.36% respectively. 8.75% insulin bioavailability was reported for insulin (100 IU/kg body weight) loaded PU-ALG nanoparticles in our previous work [7] and similar result (∼8.73%) was also found when repeated for a relative study of the current research. Bioavailability of ALG/CS nanoparticles was calculated as ∼5.56%. It was reported that oral treatments of insulin loaded alginate/chitosan [43] and dextran sulphate /chitosan [44] (100 IU/kg body weight) nanoparticles showed 7% and 3.4% bioavailability. 100 IU/kg body weight dose of insulin loaded chitosan/alginate core shell nanoparticles demonstrated 8.11% oral insulin bioavailability [8].

Oral insulin bioavailability of PU-

ALG/CS was found to be improved in comparison with previous researches and it also signified the better intestinal absorption of insulin resulted from good mucoadhesion, longlasting protracted existence of insulin in the gastrointestinal tract [45]. 3.12. Acute toxicity studies of PU-ALG/CS nanoparticles: 3.12.1. Lethal dose: No mortality was perceived by the oral administration of 5000 mg/kg body weight dose of PUALG/CS nanoparticles, ensuring the safe utilization of these nanoparticles as an insulin carrying vehicle. 3.12.2. Hepato-toxicity tests: ASAT (GOT) and ALAT (GPT) levels of swiss albino mice were determined for control as well as different oral doses of PU-ALG/CS nanoparticles. A significant increase in the liver specific enzymes such as ASAT (aspartate aminotransferase) and ALAT (alanine

aminotransferase) are the signs of liver related diseases together with liver parenchyma damages. SGPT level of control mice, and the mice fed with ALG/CS nanoparticles of 150 mg/kg body weight, PU-ALG/CS nanoparticles of 150mg/kg body weight and 5000 mg/kg body weight dose were 36.9 U/L, 71.34 U/L, 79.98 U/L and 116.43 U/L respectively as mentioned it the Figure 11A. The reference range of SGPT level is 28-184 U/L and all accounted values fall within this range. SGOT reference range is 55-251 U/L and SGOT values estimated (Figure 11B) also resided within this range such as 71.93 U/L for saline treated control mice, 78.9 U/L for 150 mg/kg body weight dose of ALG/CS nanoparticle treated mice, 83.95 U/L for 150 mg/kg body weight and 173.55 U/L for 5000 mg/kg body weight dose of PU-ALG/CS nanoparticle treated mice.

Serum LDH (Serum Lactate

Dehydrogenase) levels of control and treated (150mg per kg body weight dose of ALG/CS nanoparticles,150 mg and 5000 mg per kg body weight dose of PU-ALG/CS nanoparticles) animals are 231.76 U/L, 285.76 U/L, 282.13 U/L and 399.5 U/L respectively. LDH level plotted in Figure 11C was comparatively high for higher doses of the polymer; however, it was maintained within the reported reference range of 230-460 U/L. Acute liver toxicity studies did not show any difficulties in liver functions as a consequence of the nanoparticle treatment. 3.12.3. Nephro-toxicity tests: Levels of Serum creatinine, urine creatinine, urine micro-protein and urine urea were tested to investigate the effects of PU-ALG/CS nanoparticles on kidney functionality. Concentrations of serum creatinine were 0.81 mg/dl for saline fed mice, 0.85 mg/dl for 150 mg/kg body weight dose of ALG/CS nanoparticles, 0.89 mg/dl for 150mg/kg body weight dose feed of PUALG/CS nanoparticles and 1.01 mg/dl for 5000 mg/kg body weight dose of PU-ALG/CS nanoparticles.

All results within the range of 0.7-1.1 mg/dl signified no damage to the

functioning nephrons. Figure 11D showed visible differences in the serum creatinine values between control and nanoparticle treated mice; however, values did not exceed reference range.

Figure 11E illustrated the urine creatinine values of control animal, ALG/CS nanoparticles and PU-ALG/CS nanoparticle treated animals as 9.3 mg /kg body weight, 11.63 mg /kg body weight (150 mg /kg body weight), 13.01 mg /kg body weight (150 mg /kg body weight) and 21.05 mg /kg body weight (5000 mg /kg body weight) respectively. Again, these values were within the reference range (8.4-24.6 mg /kg body weight) and hence proved no nephrotoxicity. Normal distribution of water in the blood and tissues were maintained by the protein in the form of albumin and globulin functions. Enhanced glomerular permeability as well as reabsorption due to tubular malfunctioning, referred to be the major causes of proteinuria. Analysing urine microprotein is necessary for the detection of any abnormalities related to protein absorption. Reference range is reported as 28-140 mg/ 24 hour and the values of urine microprotein represented in Figure 11F were 77.33 mg/24 hour, 93.05 mg/24 hour, 86.59 mg/24 hour and 112.34 mg/24 hour for control animal, orally administrated 150mg/kg body weight ALG/CS nanoparticles, 150 mg and 5000 mg/kg body weight PU-ALG/CS nanoparticles, respectively. Urine microprotein amounts signified no renal toxicity as the values were within the normal range. Finally, urea concentration of urine was also found to be following the normal range (333-583 mmol/24 hour), ensuring no toxicity. As illustrated in the Figure 11G, oral feeding of 150 mg/kg body weight ALG/CS nanoparticles demonstrated urine urea concentration of 381.34 mmol/24 hour, 150 mg/kg body weight and 5000 mg/kg body weight PU-ALG/CS nanoparticles showed urine urea concentration of 378.89 mmol/24 hour and 511.5 mmol/24 hour, respectively; these values are comparatively higher than those for the control mice (340.34 mmol/24 hour). Since all parameters related to hepatotoxicity, as well as nephrotoxicity exhibited no toxic effects after oral administration of different doses of PU-ALG/CS core shell nanoparticles, hence these nanoparticles can be used safely for the purpose of oral insulin delivery.

All experimental data groups were estimated in comparison with control groups by the t-test for the significance level of *P˂ 0.05.

Figure 11. Acute toxicity study of PU-ALG/CS nanoparticles treated animals, (A. Serum SGPT, B. Serum SGOT, C. Serum LDH, D. Serum creatinine, E. Urine creatinine, F. Urine microprotein and G. Urine urea).

3.12.4. .Pathohistological tests: No visible changes were found in the ALG/CS and PU-ALG/CS nanoparticle treated mice’s liver and kidney tissue sections as illustrated in the Figure 12. Haematoxylin and eosin (H&E) were used to strain liver and kidney sections of ALG/CS as well as PU-ALG/CS nanoparticle

treated animals, which were found to be similar compared to the saline treated animals. Central vein along with the radiating hepatic cells was clearly visible in all experimental sets. Hepatotoxicity was not found in the case of polymer treated liver tissues according to the illustrated images. Kidney tissue sections of both ALG/CS and PU-ALG/CS nanoparticle treated mice also did not show any abnormalities. No trace of major toxic effect of the nanoparticles on the kidney was confirmed due to the presence of renal corpuscles along with the Bowman’s capsule, kidney tubules lined by cuboidal epithelium, urinary space in the form of clear space and the glomerulus in both control and nanoparticle treated animal kidney tissues.

Figure 12. Sections of liver and kidney (H&E staining, magnification 40X) of control and treated mice [.control, alginate/chitosan (150 mg/kg b.w.), polyurethane – alginate/chitosan (150 mg/ kg b.w.) and. polyurethane –alginate/chitosan (5000 mg/ kg b.w) nanoparticles).

Quantitative analyses of various biochemical parameters performed with PU-ALG/CS nanoparticle treated animals did not show adequate disparity compared to saline treated (control) and ALG/CS nanoparticle treated animals. Table 2 illustrated the quantitative values

to confirm the safe use of these polymer nanoparticles as an effective device for oral insulin delivery.

Table 2: Qualitative analysis of different biochemical parameters of urine in PU-ALG/CS nanoparticle treated animals. Parameters

Control animal (0.9% NaCl treated)

Treated animal

Treated animal

Treated animal

with ALG/CS NPs (150mg/Kg body weight)

with PU-ALG/CS NPs (150mg/Kg body weight)

with PU-ALG/CS NPs (5000mg/Kg body weight)

Leukocytes (LEU)

-ve

-ve

-ve

-ve

Nitrite (NIT)

-ve

-ve

-ve

-ve

Urobilinogen( URO)

0.2

0.2

0.2

0.2

Protein (PRO)

30+

30+

30+

100++

PH

8

8

8

9

Blood (BLO)

-ve

-ve

-ve

-ve

Specific Gravity (SG)

1.0

1.0

1.0

1.0

Ketone (KET)

-ve

-ve

-ve

-ve

Bilirubin (BIL)

-ve

-ve

-ve

-ve

Glucose (GLU)

100+

100+

100+

100±

4.

CONCLUSION:

Insulin loaded PU-ALG/CS core shell nanoparticles were prepared and characterized to evaluate its structural, physical, morphological properties, etc. Qualitative analyses such as

insulin encapsulation, insulin release studies demonstrated improved results compared to previous formulations. In vivo pharmacological response of the insulin loaded mucoadhesive PU-ALG/CS nanoparticles was found to be remarkable in terms of sustainable lowering the blood glucose in diabetic mice. Relative insulin bioavailability of insulin loaded PU-ALG/CS nanoparticles in the animal body was found to be improved (10.36%) compared to our previously reported work.

Acute toxicity studies including histopathological study also

provided evidence for the safe usage of the developed core shell PU-ALG/CS nanoparticles as oral insulin delivery medium.

5.

ACKNOWLEDGEMENT:

We are extremely grateful to Department of Science and Technology, Government of India for their financial support used for this work and the project entitled ‘Development of Polyurethane vehicle based on Polyethylene Terephthalate (PET) waste for controlled protein drug delivery’; Sanction No: DST/DISHA/SoRF-PM/045/2013.

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Highlights

 Synthesis of polyurethane using depolymerised polyethylene terephthalate.  Insulin loaded polyurethane-alginate/chitosan core-shell nanoparticles preparation.  Oral delivery of insulin loaded core-shell nanoparticles in animal model.  Toxicity assessment of these core-shell nanoparticles in animal model

Graphical abstract