Molecular Mechanism of Poly(vinyl alcohol) Mediated Prevention of

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Molecular mechanism of polyvinyl alcohol mediated prevention of aggregation and stabilization of insulin in nanoparticles Sanjay Rawat, Pawan Gupta, Anil Kumar, Prabha Garg, C Raman Suri, and Debendra K. Sahoo Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5003653 • Publication Date (Web): 02 Feb 2015 Downloaded from http://pubs.acs.org on February 6, 2015

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Molecular Pharmaceutics

Molecular mechanism of polyvinyl alcohol mediated prevention of aggregation and stabilization of insulin in nanoparticles Sanjay Rawat1, Pawan Gupta2, Anil Kumar1, Prabha Garg2, C. Raman Suri1 and Debendra K Sahoo1,* 1 CSIR - Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India 2 Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Sector 67, Mohali, 160062, India.

Running title: Role of PVA in insulin loaded nanoparticles

__________________ * Corresponding author Debendra K. Sahoo CSIR-Institute of Microbial Technology Sector 39-A, Chandigarh 160036, India Phone: +91-172-6665324 Fax: +91-172-2690632 E-mail: [email protected]

ACS Paragon Plus Environment


Scanning electron micrograph of a nanoparticle

Docking of PVA on hexameric insulin

Polyvinyl alcohol (PVA)

Insulin 18

Poly (lactide-co-glycolide) (PLGA)

Nanoparticle

Control

F2

16

F1 F2

14 K J u le / K -m o le

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F3

12

F3 Control

10 8 6

F1

4 2 0 30

40

50 Temperature (˚C)


60

70

Differential scanning calorimetry of released insulin

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Molecular mechanism of polyvinyl alcohol mediated prevention of aggregation and stabilization of insulin in nanoparticles Sanjay Rawat1, Pawan Gupta2, Anil Kumar1, Prabha Garg2, C. Raman Suri1 and Debendra K Sahoo1,* 1

CSIR - Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India

2

Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and

Research, Sector 67, Mohali, 160062, India. ABSTRACT It is a challenge to formulate polymer based nanoparticles of therapeutic proteins as excipients and process conditions affect stability and structural integrity of the protein. Hence, understanding the protein stability and complex aggregation phenomena is an important area of research in therapeutic protein delivery. Herein we investigated the comparative role of three kinds of surfactant systems (Tween 20:Tween 80), small molecular weight polyvinyl alcohol (SMW-PVA) and high molecular weight PVA (HMWPVA) in prevention of aggregation and stabilization of hexameric insulin in poly(lactide-coglycolide) (PLGA) based nanoparticle formulation. The nanoparticles were prepared using solid-in-oil-in water (S/O/W) emulsification method with one of the said surfactant system in inner aqueous phase. The thermal unfolding analysis of released insulin using circular dichroism (CD) indicated thermal stability of the hexameric form. Insulin aggregation monitored by differential scanning calorimetry (DSC) suggested the importance of nuclei formation for aggregation and its prevention by HMW-PVA. Additional guanidinium hydrochloride based equilibrium unfolding and in silico (molecular docking) studies suggested maximum stability of released insulin from formulation containing HMW-PVA



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(F3).

Furthermore, in vivo studies of insulin loaded nanoparticle formulation (F3) in

diabetic rats showed its bioactivity. In conclusion, our studies highlight the importance of C-terminal residues of insulin in structural integrity and suggest that the released insulin from formulation containing HMW-PVA in inner aqueous phase was conformationally and thermodynamically stable and bioactive in vivo. Keywords: bovine insulin, poly(lactide-co-glycolide), polyvinyl alcohol, stability, aggregation, nanoparticles. __________________ *

Corresponding author. Phone: ++91-172-6665324; Fax: +91-172-2690632; E-mail: [email protected]


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1. INTRODUCTION The administration of structurally integrated form of insulin is one of the prerequisites for the treatment of diabetes (insulin dependent diabetes mellitus–IDDM), a disease caused by inability of the body to control blood glucose concentration. However, most protein drugs have short half lives, are unstable in biological fluids, and not fully absorbed from their route of administration because of their relatively high molecular weights.1-3 So formulation of biological macromolecules like insulin, that has an inherent tendency to undergo degradation processes, such as aggregation, oxidation, hydrolysis, and deamidation, for its sustained release as stable and structurally integrated active protein constitutes a challenge.46

There are a number of reports on preparation of biodegradable polymer (chitosan, PLGA,

alginate etc.) based nanoparticle formulations of insulin for administration through oral and intravenous routes, however, none of these could have proved to give completely stable form of insulin in the final formulation.7-9 Although the preparation of nanoparticles using PLGA as a polymer for controlled release of insulin has advantage but achieving it without aggregation and in structurally integrated form is still critical to formulation development. Among various methodology used for the preparation of nanoparticles, solid-in-oil-in-water (S/O/W) emulsion is reported to be preferred for better protein stability over other methods while water-in-oil-in-water (W/O/W) has advantage in terms of protein loading efficiency.10, 11 In formulating therapeutic proteins, both the type and concentration of the excipients matter. As the interaction of excipients with protein depends on their nature (charged, polar, hydrophobic or, surface active), excipients along with polymers used for the preparation of nanoparticles should be compatible with the structural integrity of the insulin. The common excipients used for providing stability to protein in nanoparticles as well as for



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its controlled release include polyethylene glycol (PEG), polyvinyl alcohol (PVA) and various sugar moieties. Among these excipients, PEG is known for stabilizing the protein by inhibiting adsorption of protein on PLGA and by protecting the hydrophobic core residues.12, 13 As the stability of protein during nanoparticle preparation can be affected by various harsh conditions like organic solvent and process parameters,14,

15

the use of

excipients that can protect certain important residues of insulin from external environment could be one of the strategies to stabilize the insulin towards aggregation and denaturation during formulation development. The insulin molecule consists of two polypeptide chains, an A chain of 21 amino acids and a B chain of 30 amino acids. Although circulating insulin is active as a monomer, it is synthesized and stored as hexamer, and in insulin crystal structure, three ‘insulin dimers’ arrange around two zinc ions to form a globular, hexameric structure.16 The B-chain of monomeric insulin contains a loop region (B1-B8), α-helix (B9B19) which is followed by a type I turn (B20-B23) and the C-terminal end of the B-chain that is an extended strand (B24-B29), making close nonbonding contacts to B-chain helix (B23 residue).17 When insulin monomers associate to form dimer, the extended C-terminal ends of the two molecules are brought together, forming a two-stranded antiparallel β-sheet, which is stabilized by hydrophobic contacts and intermolecular hydrogen bonds.18, 19 It has been reported that the C-terminal end of the B-chain (B25-B30) is involved in the shielding of A-chain residues in monomeric insulin.20 B26 tyrosine and B25 phenylalanine residues are important in the contact between the two monomers formed on dimerization.21 The available literatures also suggest that the B-chain residue which may be taking part in the protection of dimer-dimer interface (antiparallel β-sheet) in hexameric insulin is proline (B28) as mutation of this Pro (28) → Thr led to more flexible structure and the flexibility in


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turn gave less stable structure of insulin.22, 23 The phenolic ligands used as preservative act by protecting this residue and favor the hexameric form indicating the role of this residue in stability of hexameric insulin.17, 24 So, protection of proline and residues of B-chain forming antiparallel β-sheet during nanoparticle preparation may be helpful in maintaining the structural integrity of hexameric insulin. One of the most important challenges in the development of protein formulations is to understand the structure of the protein (e.g., insulin) and its complex aggregation phenomena when it is present along with other excipients. In this study the basic understanding of protein stability and nuclei formation for aggregation and its prevention by surfactants such HMW-PVA has been addressed by changing the composition of inner aqueous phase with three different surfactant systems (F1 with Tween20:Tween 80 mixture, F2 with SMW-PVA and F3 with HMW-PVA) and varying their concentrations during nanoparticle preparations. The stability of the released insulin from these formulations with respect to secondary structure, tertiary structure and thermal behavior was analyzed by CD-spectroscopy, DSC and thermal-CD, respectively. The aggregation profile was evaluated by DSC, native-PAGE, and dynamic light scattering (DLS). In addition, molecular docking studies were performed to address the question of protection of important residues by surfactants used in inner aqueous phase during nanoparticle preparation. Furthermore, the effectiveness of in vivo delivery of insulin loaded nanoparticle formulation, F3, in lowering blood glucose level supported our biophysical characterization results. 2. MATERIALS AND METHODS 2.1. Materials. Insulin from bovine pancreas (MW ~5.8 kDa), polyethylene glycol (PEG) MW 8000, poly (D, L-lactide-co-glycolide) (PLGA) with a copolymer ratio of 50:50



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(lactic : glycolic(%)) MW of 40,000-75,000), polyvinyl alcohol MW of 30,000-50,000, polyvinyl alcohol MW 35,000-75,000, Tween 20, Tween 80, sodium azide, bicinchonic acid (BCA), guanidinium hydrochloride, acrylamide, streptozocin and ethyl acetate were obtained from Sigma–Aldrich (St. Louis, USA), and Pierce BCA 660 nm protein assay reagent was obtained from Thermo Scientific, USA (Pierce 660 nm Protein Assay reagent: Product code-22660, Pierce, USA). All other chemicals used were of analytical grade or highest grade available. 2.2. Preparation of nanoparticles. The S/O/W emulsification method2,

3, 25, 26

with

modification was employed to prepare the nanoparticles of insulin. The preparation of solid protein particles with PEG was carried out by mixing insulin (1 mg/mL, 2 mL) and PEG at molar ratio of 1:0.75 in 10 mM PBS (phosphate buffer saline, pH 7.4) and lyophilizing the samples in a freeze dryer (Martin Christ Alpha 1-2 LD, Osterode, Germany) at -54˚C for 12 h. Following sonication of the lyophilizates for 3 min in ethyl acetate (in 30 s on and 30 s off mode), the sonicated samples were centrifuged for 7 min at 4,000 rpm and the precipitates were collected after discarding supernatants. Primary emulsification in the organic phase containing polymer (PLGA) was done by redispersing PEG coated insulin nanoparticles in the organic phase (ethyl acetate containing PLGA) followed by sonication for 3 min (in 30 s on and 30 s off mode). Secondary emulsification in aqueous phase was accomplished by pouring 1mL of 1 % surfactant system (Tween 20:Tween 80 :: 3:1/ SMWPVA/ HMW-PVA with 0.5% ethyl acetate) into the redispersed particles (2 mL) suspended in organic phase. This system was then homogenized (ULTRA TURRAX T25, JANKE & KUNKEL, IKA Labortechnik, Germany) for 80 s and the homogenized secondary emulsion was immediately added to 50 mL of 0.5% PVA (MW 35,000- 75,000) (with 4% ethyl


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acetate) under stirring condition for 12 h. This led to solidification and evaporation of the organic phase. The precipitate was collected by centrifugation (at 10,000 rpm). The washing of prepared nanoparticles was done under centrifugation at 14,000 rpm and finally, lyophilization (under conditions as described before)2 resulted in solidified nanoparticles. The protein loading (%) and encapsulation efficiency (%) was calculated by measuring the protein content of the supernatant, collected after the centrifugation of the final step of nanoparticles preparation and measured by BCA micro-assay. The difference between the initial protein taken for the preparation of nanoparticles and the protein content of the supernatant after final centrifugation step (i.e., released protein) was used to calculate the loading (%) and encapsulation efficiency (%) of insulin as follows: Loading capacity (%) = (TI – FI)/N X100 Encapsulation efficiency = (TI-FI)/TI X100 TI: Weight of total insulin taken for nanoparticle preparation FI: Weight of free amount of insulin (left in supernatant after nanoparticle preparation) N: Weight of nanoparticles 2.3. Determination of size distribution of insulin loaded nanoparticles. For the size distribution analysis of nanoparticles, the dynamic light scatterer (DLS) (Delsa-Nano, BECKMAN COULTER) was used with light falling at an angle of 60˚ and scattering intensity of 10084 cps.1, 2 The experiments were repeated three times. 2.4. Scanning electron microscopy. Samples, in triplicates, were attached on a metal stub using a double sided adhesive and exposed to gold-spray under argon atmosphere for 15 min. Scanning electron microscopic (SEM) images were taken using SEM-JSM6100 (JEOL, USA) at 8- 15 KeV sputtering energy.2



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2.5. In-vitro release study of insulin from insulin loaded PLGA nanoparticles. Lyophilized nanoparticles (10 mg), in triplicate, were placed in round bottom microtubes with 1 mL phosphate buffer saline (PBS-10 mM, pH 7.4) containing 0.02 % sodium azide as a bacteriostatic agent. The suspended samples then kept in a rotary shaker at 37˚C and 100 rpm. The samples were centrifuged (at 10,000 rpm) periodically at 24 h interval and the separated supernatants containing released insulin in PBS (10 mM) were collected and analyzed in triplicates for protein concentration by micro BCA assay using Pierce 660 nm Protein Assay reagent (Product code-22660, Pierce, USA). 150 µl of micro BCA reagent were added to 10 µl of released insulin. Following 5 min incubation at room temperature, the absorbance of the samples was measured at 660 nm using a plate reader (Power Wave 340, BioTek, USA).27-28 The stability studies on 24 h released insulin were performed to investigate the structural integrity. 2.6. SDS-PAGE of released insulin. The intactness of released insulin was monitored by SDS-PAGE29 analysis using protein gel electrophoresis apparatus (Sigma-Aldrich, St Louis, USA). The homogeneity and molecular weight of the released insulin was determined by using 15 % polyacrylamide gels under reducing conditions (with βmercaptoethanol and boiling) and using a low molecular weight marker (Biorad, USA). 2.7. Far UV- circular dichroism (CD) spectra of released insulin. Retention of secondary structure was monitored using circular dichroism (CD) spectroscopy. The CD spectra were recorded in the far UV range from 200 to 250 nm in a spectropolarimeter (JASCO J-180) with an optical cell of 0.1 cm path length at 25˚C. The concentration of released insulin solution was adjusted to100 µg/mL in 5 mM PBS (pH 7.4). The results were expressed as molar ellipticities [θ] using the equation,


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[θ]= 3300 ∆Abs/ cl. Where ∆Abs is the observed difference in absorbance for left and right circular components of the incidence light, c is the concentration in mol L-1 and l is the path length in cm.13 2.8. Near UV- circular dichroism (CD) spectra of released insulin. The tertiary structure of the insulin was analyzed by spectral scan in near UV range (250-300 nm) using spectropolarimeter (JASCO J-180) with an optical cell of 0.1 cm path length at 25 ˚C. The concentration of control and released insulin at 100 µg/mL (in 5 mM PBS, pH 7.4) was chosen for this study.5 2.9. Fluorescence spectra of released insulin. To confirm the retention of the tertiary structure of insulin containing tyrosine residues, the released insulin was analyzed by fluorescence spectroscopy (Cary Elipse, Victoria, Australia). Excitation was done at 260 nm and intrinsic fluorescence emission was measured in range of 280 to 350 nm wavelengths. The concentration of control and released insulin at 100 µg/mL (in 5 mM PBS, pH 7.4) was chosen for this study.13 2.10.

Thermal stability analysis

2.10.1. Thermal circular dichroism (CD) spectroscopy of released insulin. The CD spectra were recorded with a (JASCO J-180) spectropolarimeter. For thermal denaturation studies, the ellipticity of solution of native insulin (control) was followed at 216, 220 and 276 nm as a function of temperature. Released insulin from all three formulations and control were heated between 25˚C and 95˚C with a constant heating ramp of 1˚C /min. On reaching the endpoint of 95˚C, the transition temperature was immediately reversed to cool the sample down at a constant rate of 1˚C or 2˚C per min. A cuvette of 1.0 cm path length was employed for thermal stability studies with proteins in solution. The pre-transitional



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baseline, describing the ellipticity of native state, θN and the post-transitional baseline, θD, with normalized ellipticity as a function of temperature were used to obtain thermal unfolding temperature (Tm).24 2.10.2. Differential scanning calorimetry (DSC) of released insulin. Thermal denaturation of insulin was monitored with a high sensitivity differential scanning calorimeter (Model NANO-DSC, TA-instrument, USA). Thermogram of released insulin from all three formulations, each taken in three sets (with differing concentrations of surfactant mixtures), was obtained between 25 and 100˚C, at a scan rate of 10, 20 30, 60 and 90˚C/h. All results were averages of at least three independent measurements. The calorimetric data was analyzed by using NANOANALYZER (TA-instrument, USA) software to obtain the melting temperature (Tm).4, 6 2.11.

Equilibrium unfolding of insulin in presence of guanidinium hydrochloride

(Gdn-HCl). Released and control (insulin) at a final concentration of 100 µg/mL was incubated in Gdn-HCL solution at 37˚C for 12 h to attain equilibrium. CD measurements were done on spectropolarimeter (JASCO J-180) using 0.1 cm path length cuvette at 25 ˚C. Measurements were recorded at 222 and 276 nm over 30 seconds for secondary and tertiary structure analysis respectively.30 2.12. Analysis of insulin aggregation by native-PAGE. The aggregation profile of released insulin was monitored by native-PAGE using protein gel electrophoresis apparatus (Sigma-Aldrich, St Louis, USA). The homogeneity and molecular weight of the released insulin was determined by using 10 % polyacrylamide gel under non reducing conditions29 and using a standard protein gel marker (Sigma-Aldrich, St Louis, USA)


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2.13. Analysis of insulin aggregation by Dynamic light scattering (DLS). The released insulin from different formulations was analyzed by DLS using Zetasizer (Nano ZS, Malvern Instrument) at room temperature. Monitoring of decay constant (Autocorrelation function) was done in zeta sizing cuvette by collecting scan of released insulin from each formulation and control in triplicates.31 2.14. Fluorescence quenching studies of released insulin using acrylamide. Intrinsic fluorescence emission of tyrosine residues of control and released insulin was monitored in presence of different concentrations of acrylamide (10-100 mM) keeping insulin concentration constant at 100 µg/mL. The excitation and emission wavelengths chosen were 260 nm and 305 nm respectively. Fluorescence intensities were corrected for dilution effects and fluorescence quenching data were analyzed by using Stern-Volmer plot.24 2.15. Computational studies. The molecular structure of polyvinyl alcohol (PVA) was built using SYBYL7.1 molecular modeling package installed on a Silicon Graphics Fuel Work station running IRIX 6.5. Tripos force field, Gasteiger Huckel partial atomic charges32 and Powell’s conjugate gradient method33 were used for minimization of all molecules with 0.05 Kcal/mol energy gradient convergence criterion. The insulin protein (PDB ID: 1OLY) was used for docking studies of PVA. This protein is hexa-homodimer protein. AutoDock tool (ADT) was used to prepare the PVA and protein (deleting all water molecules, adding polar hydrogen’s and loading Kollman United Atoms charges) and perform docking calculations. A grid box with spacing 0.375 and dimensions 60 × 60 × 60 points around the anti-parallel β-sheet of protein was constructed. There are six anti-parallel β-sheets present in this protein, so docking was repeated six times for binding of PVA at anti-parallel β-sheet using the same grid size, but grid centers were different in each anti-



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parallel β-sheet. The Lamarckian genetic algorithm (LGA) was employed for docking study.34-36 2.16. In vivo release and In-vivo evaluation of insulin nanoparticles formulation with HMW-PVA (F3) The in vitro release profile analysis of insulin from F3 was carried out by placing lyophilized nanoparticles (containing 996 µg of insulin), in triplicates, in round bottom microtubes and suspending it in 1 mL of 10 mM PBS at pH 7.4 containing 0.02 % sodium azide as a bacteriostatic agent. The sample tubes were incubated in a rotary shaker at 37˚C and 100 rpm. The nanoparticle samples were centrifuged (10,000 rpm, 5 min) at different time intervals and the supernatant containing released insulin in PBS were collected and analyzed by BCA protein assay (Pierce 660 nm Protein Assay reagent) in triplicate. Fresh PBS (1 mL) was added to the tubes containing nanoparticles at each sampling point and the incubation continued. The concentrations of released insulin from insulin-loaded nanoparticles was estimated by taking 10 µL of protein sample in a 96-well plate and adding 150 µL micro BCA working solution into each well followed by incubation of the plate at 37˚C for 5 min. The absorbance of the samples was measured at 660 nm using a plate reader (Power Wave 340, BioTek, USA).2 For in vivo evaluation of insulin loaded nanoparticle formulation F3, female Sprague Dawley rats, weighing between 220 and 280 g, were randomly distributed in to four groups (n=6 for each group) and subjected to overnight fasting. Diabetes was induced by the intraperitoneal administration of optimized dose of Streptozocin (STZ) (75 mg/kg of body weight) dissolved in 10 mM of citrate buffer, pH= 4.1. The blood glucose concentration was measured using glucometer (Glucocard 01-mini from Piramel Healthcare, India) and


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glucose sensor strips (Arkray healthcare Pvt. Ltd., Japan). When glucose level reached to 450-550 mg/ dl of blood (after 5 days of STZ treatment) the rats were considered to be diabetic. The first group (Group I) was kept as diabetic control and treatments of groups IIIV were performed with subcutaneous (S.C.) injection of Insulin aspart solution (5 IU/ kg of body weight) (Novorapid from Novo Nordisk) to Group II and the third (Group III) and fourth (Group IV) groups were subjected to treatment with subcutaneous (S.C.) administration of bovine insulin loaded nanoparticles prepared with HMW-PVA as a surfactant (F3) (at 15 IU/Kg body weight and 25 IU/Kg body weight, respectively). The blood samples at diﬀerent time points (0, 1, 2, 4, 6 h) were analyzed for plasma glucose level by tail prick method.1, 37-39 The experiments were performed using protocols approved by the institutional animal ethics committee of CSIR-Institute of Microbial Technology, Chandigarh. 3. RESULTS AND DISCUSSION 3.1. Preparation of insulin loaded PLGA nanoparticles. The size of the nanoparticles is one of the major factors for the successful delivery of any protein.14, 15 In our study, three kinds of nanoparticles (F1, F2 and F3) were prepared using three different surfactant systems in inner aqueous phase while outer aqueous phase contained HMW-PVA in all formulations and the loading and encapsulation efficiency of insulin in these three formulations are presented in Table 1. These preparations showed different particle size distribution when analyzed by DLS and SEM (Figure 1A, 1B). Formulation, F1, containing Tween 20:Tween 80 mixture showed 800 nm mean particle diameter with PDI=0.248 while F2 (containing SMW-PVA) and F3 (containing HMW-PVA) were having particles with 4.5 µ mean diameter and PDI= 0.272 and 400 nm mean diameter and PDI=0.122, respectively.



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The SEM micrograph (Figure 1B) analysis of nanoparticles of all three formulations (F1, F2 and F3) showed particle size distribution similar to those obtained by DLS analysis (Figure 1A). 3.2. Thermal stability analysis 3.2.1. Thermal circular dichroism (CD) spectroscopy of released insulin. Circular dichroism spectroscopy can be used to monitor changes in secondary and tertiary structure of a protein with respect to change in temperature.12,

24

When thermal scan of released

insulin along with control was carried out at 216 nm, which is characteristic wavelength of β-sheet (Figure 2), the ellipticity for control hexameric insulin was found to be more negative before transition midpoint temperature but less negative after that. The first increase in ellipticity could be due to the dissociation of hexameric unit into dimeric units and finally due to loss of secondary structure it was forming aggregates (decrease in ellipticity).4 The released insulin from all three prepared formulations showed more negative ellipticity than control and the transition midpoint temperature of released insulin from formulation prepared with HMW-PVA (F3) was highest among all formulations. This suggested a possible role of HMW-PVA in the protection of residues which could have taken part in the formation of aggregates, especially B chain residues that formed the antiparallel β-sheet at dimer-dimer interfaces and C-terminal proline residue (B28) in hexameric form of insulin. This was evident from the results of fluorescence quenching studies where acrylamide was used as a neutral quencher to study the accessibility of tyrosine residues of dimer-dimer interface. This interface contains three tyrosine residues namely TyrA14, TyrB16 and TyrB26 which are shielded in hexameric form and hence, not accessible for acrylamide.24, 40 The change in linearity of fluorescence intensity of released


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insulin on gradual addition of acrylamide is presented as Stern Volmer plot (Figure 3). In Stern Volmer plot, the control and released insulin from formulation F3 showed more linearity than insulin released from other two formulations which indicated the protection of tyrosine residues and dimer-dimer interface of insulin in F3. This further supports the role of HMW-PVA in protection the above said residues. On the other hand proline is reported to have a role in both short and long range effects on the structural stability of residues involved in the architecture of insulin.41 Hence, it could be stated that this residue might have protected the antiparallel β-sheet at the dimer-dimer interface of hexameric form of insulin.17,

42

The molecular docking studies (Figure 4) were performed to see polymer

binding to the proline site or antiparallel β-sheets of hexameric form (chains B, D, F, H, J and L). It was found that PVA binds near to antiparallel β-sheet and proline residue, resulting in the prevention of nuclei formation for protein aggregation. In HMW-PVA (Sigma–Aldrich Product code: P8136), apart from 87% alcoholic component, 13% polyvinyl acetate is also present as compared to 98% alcoholic component and 2% polyvinyl acetate in SMW-PVA (Sigma–Aldrich Product code: 363138). As evident, charged residues like lysine present in C-terminal end and glutamine in nearby chains (chain F and H) of hexameric form of insulin may also impart stability to the protein. There could also be a chance of binding of polyvinyl acetate to these charge residues. Therefore, molecular docking studies were again performed for polyvinyl acetate. It was observed that polyvinyl acetate binds near to lysine and glutamine through polar interactions (electrostatic and H-bonding) and provides additional protection to hexameric insulin in F3 in comparison to the same in other two formulations, F1 and F2 (Figure 4 and Table 2).40



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When ellipticity of released insulin at 222 nm wavelength was monitored (characteristic of α-helix of protein) the thermal denaturation graph showed similar pattern of denaturation profile as that observed at 216 nm. This showed that the α-helical structure of insulin also denatured in same manner with transition midpoint temperature near 53-55˚ C. The insulin released from all three formulations showed similar unfolding pattern. The far UV CD scan from 200-250 nm also suggested that the stability of secondary structure was highest in insulin released from formulation F3 prepared using HMW-PVA (Figure 5A) and also, released insulin from the same formulation (F3) was found to have the highest thermal transition midpoint among all samples including control. To confirm whether the hexameric form of insulin was retained in the released form, the near UV CD scan and thermal unfolding analysis of released insulin from all formulations were carried out and compared with native insulin (taken as control). The wavelength of 276 nm was chosen for the thermal unfolding analysis because insulin hexamer shows the characteristic tyrosyl CD signal at 276 nm (Figure 2C). Main contributor to this signal was the interaction at the dimer-dimer interface of hexamers.4 The near UV CD scan from 250300 nm showed a marked increase in ellipticity (at characteristic 276 nm for tyrosyl residue) in case of insulin released from formulation F3 prepared with HMW-PVA (Figure 5B). Also, the integrity of insulin released from all formulations was maintained as evident from the SDS-PAGE analysis (Figure 6). Furthermore, in all cases, the thermal unfolding profile of released insulin indicated an increase in thermal stability and CD spectral scan in near and far UV range indicated structural and conformational integrity of insulin when compared with control. However, insulin released from nanoparticles prepared with HMWPVA (F3) showed maximum stability among all samples.


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3.2.2. Differential scanning calorimetry (DSC) of released insulin. Differential scanning calorimetry is one of the important tools to monitor the thermodynamic transitions associated with protein stability (unfolding and aggregation).41 Thermal unfolding is recognized by a sharp endothermic peak while exothermic peak indicates the aggregation of any protein.6 The thermal scanning of hexameric insulin (control) and insulin released from different formulations was performed at pH 7.4 using DSC (Figure 7). It was evident that the thermal unfolding of insulin followed the biphasic denaturation and that resulted in two distinct transitions with Tm’s of ~45 and ~100˚C. This biphasic denaturation might be due to the presence of zinc in hexameric form of insulin4 and could also be related to the aggregation of insulin.6 This was further supported by our study on native insulin (control) which showed first endotherm with a transition midpoint temperature (Tm) of ~45˚C while the second endotherm at ~100oC was followed by aggregation. The released insulin from F1 and F2 formulations showed similar biphasic denaturation with first endothermic transition at ~45˚C and second endothermic peak, Tm at 99 and 95oC, respectively, while F3 showed first endothermic transition with a Tm value of 53.0oC and the second endotherm above 100˚C. This increase in Tm value (thermal stability) of endotherm, from 45˚C to 53˚C in case of F3 could be related to the protection of residues which were easily prone for unfolding and subsequently aggregation. The effect of the concentration of surfactants on thermal stability of released insulin from nanoparticles was studied by varying the concentration of surfactant in inner aqueous phase of respective formulations (F1, F2 and F3) from 0.5 % - 4 %. Though Tm values of released insulin was highest at 1 % surfactant concentration in all cases, it was maximum in case of formulation F3 prepared with HMWPVA in inner aqueous phase (Table 3). This observation suggests that at 1 % optimum



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concentration the surfactant is easily accessible to the residues which form the antiparallel β-sheet (tyrosine and proline). This was further supported by our earlier observations of acrylamide quenching studies and fluorescence spectra of released insulin and also previous reports on the same.13,

24

It may also be said that B chain residues while forming the

antiparallel β-sheet at dimer-dimer interface in hexameric insulin, if get separated from hexamer, might be acting as a nuclei for the further addition of dimeric units and propagated to amyloid fibrils.43-45 To prove that the second transition was following the aggregation DSC studies of the released insulin were carried out at different scan rates. When insulin (control) was heated at a scan rate of 10, 20, 30, 60 and 90˚C/ h, with increase in the scan rate the protein showed a shift in position of the exothermic peak to even higher temperature (Table 4), i.e., higher the scan rate is, larger the shift in the position of exothermic peak to higher temperature.5 Likewise when the released insulin from formulation F1 (containing Tween 20:Tween 80) and F2 (containing SMW-PVA) were heated at different scan rate, similar patterns of Tm values of exotherm (as in case of control insulin) were observed. However, when insulin released from third formulation (F3) containing HMW-PVA was scanned at different rates, the exothermic transition appeared above 100˚C at all scan rates. These observations clearly suggested the formation of nuclei for aggregation of insulin46 in control and formulations F1 and F2 as increase in scan rate caused a gradual shift of exothermic peak to higher temperature.

However, in case of third formulation (F3)

containing HMW-PVA, it maintained the tertiary structure of the hexameric insulin and an increase in Tm value of endothermic peak (did not show any exothermic peak till 100˚C at scan rate of 60˚C/h) indicated increase in thermal stability of protein without aggregation. Furthermore, the autocorrelation function analysis of released insulin in terms of cumulant


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fit of scattered light indicated slow decay of the insulin released from F3 when compared with other two formulations. In summary, Native-PAGE and dynamic light scattering (DLS) studies of insulin released from F3 (Figure 8 and Figure 9, respectively) suggested no aggregation. 3.3. Effect of guanidinium hydrochloride (Gdn-HCl) on the released insulin 3.3.1.Effect on tertiary structure. Denaturant guanidine hydrochloride (Gdn-HCl) was chosen to alter the interactions governing the tertiary structure of hexameric insulin, as it acts by interacting with solvent exposed charge residues and peptide backbone carbonyl groups of protein, and by disruption of non-covalent interactions.47 The equilibrium unfolding experiment30 in which sequential addition of Gdn-HCl (1-8 M) was reported to be sufficient to denature tertiary structure48 was chosen for this study. It is reported that the anti-parallel β-sheet between two different dimeric units, van der Wall interactions and coordination of zinc metal with histidine residues of B-chain of monomeric unit, and B28 proline residues contributed towards establishment of the tertiary structure of hexameric form.18,

21, 23

In our study, the denaturation of insulin using Gdn-HCl was followed by

monitoring CD spectra of native hexameric insulin (control) as well as those of insulin released from three formulations (F1, F2 and F3) at 276 nm (characteristics of tyrosyl residues of insulin)4. The comparison of native insulin CD spectra with those of released insulin from three prepared formulations emphasized the importance of protection of certain residues forming the overall hexameric structure. It is evident from Figure 10A that the insulin released from F2 showed loss of tertiary structure compared to native hexameric insulin and insulin released from other two formulations. This could be due to the inability of SMW-PVA in F2 to protect certain amino acid residues (charged residues like lysine and



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glutamine) which are also important for tertiary structure of insulin. The formulation, F1 (containing Tween 20:Tween 80) retained the tertiary structure as evident from its more negative ellipticity as compared to control and this could be due to binding and protection of hydrophobic surface residues of protein by these nonionic surfactants.49 In case of F3, it could be strongly suggested that this formulation containing HMW-PVA (with both alcoholic (87 %) and acetate (13 %) components), by protecting proline residues, antiparallel β-sheet and charged residues at the dimer-dimer interface, retained the whole tertiary structure of hexameric insulin. This result was supported by a previous observation that stated when proline residue was mutated to other residue like alanine the transition midpoint of Gdn-HCl decreased and this in turn decreased the protein stability.50 This proline residue if not protected will be destabilized by Gdn-HCl due to the shift of proline in the hydrophobic core.49 These observations were further supported by the fluorescence spectra of released insulin (Figure 11) which indicated that the maintenance of tertiary structure in formulation F3 containing HMW-PVA is more than that in F1 and F2. 3.3.2.Effect on secondary structure. The hexameric insulin is a globular α-helical containing protein. The CD-spectra at 222 nm in presence of Gdn-HCl can be used to monitor the secondary structural changes. We performed equilibrium unfolding experiment by sequential addition of 1-8 M Gdn-HCl to control (native hexameric insulin) and released insulin solutions which caused the secondary structural perturbation. The monomeric insulin contain four tyrosine residues and various solvent exposed polar residues which are acted upon by Gdn-HCl causing the charge delocalization and stacking of side chain of aromatic amino acids (tyrosine).40,

51

As B-chain residues protects A-chain that formed the

hydrophobic core of hexameric form,19 Gdn-HCl induced structural changes due to the


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above described actions led to the unfolding and exposure of hydrophobic core residues to outer environment. All secondary structures of a protein are held together by hydrophobic core residues and protection of hydrophobic core protects the secondary structure of the protein.13 On comparison of CD-spectra of control hexameric insulin with that of released insulin from all three formulations (Figure 10B), insulin released from F1 (formulation containing Tween 20:Tween 80) showed less denaturation than control hexameric insulin as nonionic surfactants are known to protect hydrophobic surface residues.49 The insulin released from formulation F2 containing SMW-PVA showed even less denaturation compared to F1 and this could be due to the protection of the hydrophobic core of the protein by alcoholic component of SMW-PVA. In case of F3, the formulation contained HMW-PVA with both alcoholic (87 %) and acetate (13 %) components. The alcoholic component of HMW-PVA protects both proline and the anti-parallel β-sheet of dimer-dimer interface and this interface in turn protects the above said hydrophobic core. In addition, the acetate component of HMW-PVA protects the charged residues like lysine and glutamine of anti-parallel β-sheet and nearby A-chain, respectively (Table 2). Hence, it is inferred that HMW-PVA in inner aqueous phase maintained the secondary structure of hexameric insulin in F3. This phenomenon is also supported by a previous study which states that any mutation in residues covering hydrophobic core of insulin could lead to the decrease in thermodynamic stability.52 Furthermore, stepwise unfolding (dimer formation) followed by aggregation because of aromatic stacking interaction between residues forming anti-parallel β-sheet could also be contributing to the above phenomenon.53 Hence, it may be articulated that HMW-PVA by protecting above said residues enables the protection of hydrophobic core and subsequently prevents insulin aggregation.



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3.4.

In vivo evaluation insulin from formulation containing HWA-PVA in inner

aqueous phase (F3) The biophysical characterization studies of insulin released from three different nanoparticle formulations (F1, F2 and F3) showed maximum stability of insulin from formulation containing HWA-PVA in inner aqueous phase (F1) and these results were complemented by in silico studies. To further evaluate the bioactivity of insulin in nanoparticle formulation F3, hypoglycemic activity in streptozocin induced diabetic Female Sprague Dawley rats following subcutaneous (S.C.) administration of insulin preparation was determined. The animal were divided into four groups (Group I, II, III and IV) and keeping Group I animal as diabetic control, insulin loaded nanoparticles were injected to Group III (15 IU insulin/Kg body weight) and Group IV (25 IU insulin/Kg body weight) where as standard insulin Aspart ((Novorapid- a fast acting analogue of recombinant human insulin) was injected to Group II. When the blood glucose level of Group III and IV rats was compared with that of Group I (diabetic control), insulin loaded nanoparticle formulations were found to reduce the blood glucose level (from base level) in both Group III and IV rats (Figure 12B)37-38, though greater reduction in blood glucose level was observed in the animals receiving higher dose of insulin. The initial sudden decrease in blood glucose levels in Group III and IV was similar to that observed in Group –II and could be due to the initial burst release of insulin (In vitro studies showed the initial burst release of insulin from F3 to be ~15.6 % in 8 h, presented in Figure 12A). However, unlike Group II, blood glucose level in Group III and Group IV was maintained in controlled manner up to 8 h. This study


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highlighted the rapid onset, controlled release and bioactivity of insulin from insulin loaded nanoparticle formulation F3.54-55 CONCLUSION The preparation of insulin nanoparticles with promising stability is still a challenge in formulation development. In this study, we report the basic understanding of using different surfactant systems in inner aqueous phase in PLGA based nanoparticles preparation and their effect on the stability and aggregation of the protein. In formulation F3, HMW-PVA was found to be an excellent protectant of C-terminal proline residue, charged residues and residues of antiparallel β-sheet of hexameric form as indicated by various spectroscopic and calorimetric studies of released insulin from different nanoparticles formulations. This was supported by in silico (molecular docking) studies. DSC studies of released insulin further indicated the importance of nuclei formation for the aggregation. Finally, the in vivo evaluation of insulin in F3 formulation prepared with HMW-PVA showed the formulation (F3) retaining bioactivity of insulin and proved to be effective in reducing the blood glucose level with rapid onset of action. In addition this study also highlights the importance of understanding and selecting surfactant system in optimum concentration for formulation development and how this knowledge can be applied in the development of other biopharmaceuticals delivery system. ACKNOWLEDGEMENT This work was supported by Council of Scientific and Industrial Research, Government of India. SR acknowledges his fellowship from Department of Biotechnology, Government of India. The authors acknowledge the help of Dr. N. Khatri for animal experiments and Dr. S.



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Mukhopadhya of Indian Institute of Science Education and Research, Mohali, India for Raman Spectroscopy.

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(49) P. Garidel, C. Hoffmann, A. Blume, A thermodynamic analysis of binding interaction between polysorbate 20 and 80 with human serum albumins and immunoglobulins: a contribution to understanding colloidal protein stabilization. Biophys. Chem. 2009, 143, 70- 78. (50) G. Pappenberger, H. Aygun, J. W. Engels, U. Reimer, G. Fischer, T. Kiefhaber, Nonprolyl cis peptide bonds in unfolded proteins cause complex folding kinetics. Nat. Struct. Biol. 2001, 8, 452-458. (51) J. Heyda, M. Kozisek, L. Bednarova, G. Thompson, J. Konvalinka, J. Vondrasek, P. Jungwirth, Urea and guanidinium induced denaturation of a Trp-Cage miniprotein. J. Phys. Chem. B 2011, 115, 8910-8924. (52) B. Xu, Q. X. Hua, S. H. Nakagawa, W. Jia; Y. C. Chu, P. G. Katsoyannis, M. A. Weiss, A cavity-forming mutation in insulin induces segmental unfolding of a surrounding α-helix. Protein Sci. 2002, 11, 104–116. (53) L. A. Woods, G. W. Platt, A. L. Hellewell, E. W. Hewitt, S. W. Homans, A. E. Ashcroft, S. E. Radford, Ligand binding to distinct states diverts aggregation of an amyloid-forming protein. Nat. Chem. Biol. 2011, 7, 730-739. (54) A. Arora, I. Hakim, J. Baxter, R. Rathnasingham, R. Srinivasan, D. A. Fletcher, Samir Mitragotri, Needle-free delivery of macromolecules across the skin by nanoliter-volume pulsed microjets. Proc. Natl. Acad. Sci. USA 2007, 104, 42554260. (55) Z. Gu, A. A. Aimetti, Q. Wang, T. T. Dang, Y. Zhang, O. Veiseh, H. Cheng, R. S. Langer, D. G. Anderson, Injectable nano-network for glucose-mediated insulin delivery. ACS Nano 2013, 7, 4194-4201.



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Figure Legends Figure 1: Particle size analysis of PLGA based nanoparticles of different formulations (F1, F2, F3) prepared with three different surfactant systems (Tween20:Tween 80 mixture, SMW-PVA and HMW-PVA) in inner aqueous phase: (A) Size distribution analysis by DLS: F1: 800 nm mean diameter with PDI=0.248; F2: 4.5 µ mean diameter with PDI=272;, F3: 400 nm mean diameter with PDI=0.122. (B) Scanning electron microscope images of nanoparticles. Spherical larger particle in F1 and F2 while F3 showed smaller size spherical particles. Figure 2: Thermal circular dichroism (CD) of released insulin: (A) Thermal unfolding of released insulin at 216 nm, released insulin from F1, F2, F3 and native insulin. The released insulin from F3 retained maximum ellipticity when heated between 25˚C and 95˚C with a constant heating ramp of 1˚C /min. (B) Thermal unfolding of released insulin at 222 nm, released insulin from F1, F2, F3 and native insulin. The released insulin from F3 retained maximum ellipticity when heated between 25˚C and 95˚C with a constant heating ramp of 1˚C /min. (C) Thermal unfolding of released insulin at 276 nm, released insulin from F1, F2, F3 and native insulin. The released insulin from F3 retained maximum ellipticity when heated between 25˚C and 95˚C with a constant heating ramp of 1˚C /min. Figure 3: Stern-Volmer plot of released insulin (acrylamide quenching analysis). Fo and F correspond to fluorescence intensities in absence and presence of acrylamide, respectively. The insulin released from formulation F3 showed the maximum linearity of the graph close to native insulin.


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Figure 4: Docking of high molecular weight PVA (HMW-PVA) and anti parallel β-sheet on proline28 of insulin hexamer: (a) Docking pose of PVA with insulin Chain B (near Pro28): The docking studies found that PVA was placed near to Pro28 of anti parallel β sheet of Chain B and forming H-bonding interactions with important residues (listed in Table 3). b) Docking pose of PVA with insulin Chain J (near Pro28): The docking studies found that PVA was placed near to Pro28 of anti parallel β sheet of Chain J and forming H-bonding interactions with important residues (listed in Table 1). (c) Docking pose of PVA with insulin Chain D (near Pro28): The docking studies found that PVA was placed near to Pro28 of anti parallel β sheet of Chain D and forming H-bonding interactions with important residues (listed in Table 1). (d) Docking pose of PVA with insulin Chain H (near Pro28): The docking studies found that PVA was placed near to Pro28 of anti parallel β sheet of Chain H and forming Hbonding interactions with important residues (listed in Table 1). (e) Docking pose of PVA with insulin Chain F (near Pro28): The docking studies found that PVA was placed near to Pro28 of anti parallel β sheet of Chain F and forming Hbonding interactions with important residues (listed in Table 1). (f) Docking pose of PVA with insulin Chain L (near Pro28): The docking studies found that PVA was placed near to Pro28 of anti parallel β sheet of Chain L and forming H-bonding interactions with important residues (listed in Table 1). (g) Residues involve in interaction with poly vinyl acetate: Binding pose of Poly vinyl acetate in chain F of insulin. This showed the docking interactions of HMW-PVA with Lys and Gln residues of chain F which could have resulted in protection of insulin protein.



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Capital alphabet represents the chain name. Meshes are showing the close contact of poly vinyl acetate with nearby atoms of residues; Red for oxygen, Blue for nitrogen, Grey for carbon and green dots represent hydrogen bonding. (h) Residues involve in interaction with poly vinyl alcohol: Binding pose of Poly vinyl acetate in chain H of insulin. This showed the docking interactions of HMW-PVA with Lys and Gln residues of chain H which could have resulted in protection of insulin protein. Capital alphabet represents the chain name. Meshes are showing the close contact of poly vinyl acetate with nearby atoms of residues; Red for oxygen, Blue for nitrogen, Grey for carbon, green dots represent hydrogen bonding. (i) Model proposed for the protection of hexameric insulin: The hexameric insulin was represented along with six molecules of PVA in each anti parallel β sheet of each chain from B, J, D, H, F and L (near to Pro28). After the docking pose analysis, this complex (hexameric insulin-PVA) was found to be stable as compared to insulin without PVA. Docking pose of hexameric insulin (PDBID-(10LY) by Autodock 4.5 showing poly vinyl alcohol (PVA) binding to proline and antiparallel β-sheet of every chain (Chain D, J, H, F, L, B). Figure 5: Conformational circular dichroism (CD) of insulin released from nanoparticles of different formulations (F1, F2, F3) prepared with three different surfactant systems (Tween20:Tween 80 mixture, SMW-PVA and HMW-PVA) in inner aqueous phase: (A) Far UV CD-spectra of released insulin: The insulin released from formulation F3 showed the maximum retained secondary structural contents (mean residual ellipticity at θ values 208 and 222).


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(B) Near UV CD-spectra of released insulin: The insulin released from formulation F3 maximum retained the secondary structural contents (mean residual ellipticity at θ value 276: characteristic of tyrosine residues. Figure 6: SDS-PAGE of released insulin from nanoparticles of different formulations (F1, F2, F3) prepared with three different surfactant systems (Tween20: Tween 80 mixture, SMW-PVA and HMW-PVA) in inner aqueous phase. The intactness of released insulin was maintained in all the three formulations (F1, F2 and F3). Figure 7: Differential scanning calorimetric (DSC) thermogram of released insulin from nanoparticles of different formulations (F1, F2, F3) prepared with three different surfactant systems (Tween20:Tween 80 mixture, SMW-PVA and HMW-PVA) in inner aqueous phase. The native insulin was taken as control. The insulin released from F3 showed the maximum thermal stability (Tm 53°C) when heated between 10˚C and 100˚C with a constant heating ramp of 1˚C /min. Figure 8: Native-PAGE of insulin released from nanoparticles of different formulations (F1, F2, F3) prepared with three different surfactant systems (Tween20:Tween 80 mixture, SMW-PVA and HMW-PVA) in inner aqueous phase. The intactness of released insulin was maintained in insulin released from all three formulations (F1, F2 and F3). Figure 9: Analysis of rate of decay of released insulin by DLS. The insulin released from different nanoparticle formulations (F1, F2, F3) prepared with three different surfactant systems (Tween20:Tween 80 mixture, SMW-PVA and HMW-PVA) in inner aqueous phase showed least rate of decay in insulin released from formulation F3 in comparison to insulin released from F1 and F2 and native insulin taken as control.



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Figure 10 Equilibrium unfolding study of released insulin from nanoparticles of different formulations (F1, F2, F3) prepared with three different surfactant system (Tween20:Tween 80 mixture, SMW-PVA and HMW-PVA) in inner aqueous phase in presence of Gdn-HCl: (A) Circular dichroism at 276 nm: The change in ellipticity of released insulin and native insulin on incubation in Gdn-HCl solution at 37 ˚C for 12 h. (B) Circular dichroism at 222 nm: The change in ellipticity of released insulin and native insulin on incubation in Gdn-HCl solution at 37 ˚C for 12 h. Figure 11 Tertiary structure analysis of insulin released from nanaoparticles of different formulations (F1, F2, F3) prepared with three different surfactant system (Tween20: Tween 80 mixture, SMW-PVA and HMW-PVA) in inner aqueous phase by fluorescence spectroscopy. The fluorescence emission intensity (305 nm) of insulin released from different formulations (F1, F2, F3) and native insulin on excitation at 260 nm showed similar emission intensity profile. Figure 12 (A) In vitro release profile of insulin from nanoparticle formulation with HMWPVA in inner aqueous phase (F3). The profile of in vitro release of insulin from F3 was obtained by placing lyophilized nanoparticles (containing 996 µg of insulin), in triplicate, in round bottom microtubes and suspending it in 1 mL of 10 mM PBS at pH 7.4 containing 0.02% sodium azide as a bacteriostatic agent and incubating in a rotary shaker at 37˚C and 100 rpm. (B) In vivo evaluation of insulin nanoparticle formulation F3 (containing HMW-PVA in inner aqueous phase). The comparison of subcutaneously administered insulin loaded nanoparticle formulation, F3 (15 IU/Kg body weight or 25 IU/Kg body weight) and Insulin


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aspart solution (5.0 IU/Kg body weight) with diabetic control. Data represents the mean±SD, n=6 per group.



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Figure 1 (A) (A1) F1

(A2) F2

(A3) F3

(B) (B1) F1

(B2) F2

(B3) F3


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Figure 2 (A)

(B)

(C)



Figure 3 160

Control F1 F2 F3

140 120 100 0

F /F

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

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80 60 40 20 0 0

10

20

30 40 50 60 70 Acrylamide (mM)

80

90


100

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Figure 4 (a)

(b)

(d)

(c)

(e)

(g)

(f)

(h)

(i)



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Figure 5 (A)

(B)


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Figure 6 Marker

Control

F1

F2

Myosin (200 kDa) β-galactosidase (116 kDa) Bovine serum albumin (67 kDa) Carbonic anhydrase (29 kDa)

Soybean trypsin inhibitor (21.5 kDa)

Lysozyme (14.7 kDa) Aprotinin (6.5 kDa)


F3


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Figure 7


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Figure 8 Marker Control

F1

F2

F3

240 kDa 150 kDa 66 kDa 29 kDa

5.8 kDa



Figure 9 Cumulants Fit

1.0

F1

F2 F3

0.9

0.8

Control

0.7 G1 Correlation Function

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0.6

0.5

0.4

0.3

Buffer

0.2

0.1

0.0 1

10

100

1000

10000

Time (µs)


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Figure 10 (A)

(B)



Figure 11 120

Fluorescence intensity ( a. u.)

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

Control F1

100

F2 F3

80 60 40 20 0 280

294

308

322

336

350

Wavelength (nm)


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Figure 12 (A)

(B)



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Table 1 Loading and encapsulation efficiency of insulin in PLGA based nanoparticles with different surfactant system in inner aqueous phase Surfactant system in inner

Insulin Loading (% w/w)

aqueous phase

Insulin encapsulation efficiency (% w/w)

F (Tween 20: Tween80 mixture)

6.2 ± 0.38

52.5 ± 3.3

F2 (SMW-PVA)

6.9 ± 0.39

75.0 ± 3.9

F3 (HMW-PVA)

6.5 ± 0.31

73.5 ± 2.5


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Table 2 Docking scores for interaction between C-terminal residues of anti-parallel β-sheet and poly vinyl alcohol in terms of binding energy. Figure No.

Protein

Interactions

Chain

Autodock Score (kcal/mol)

Figure 1 (Magenta)

Chain B

A-Glu4; B-Thr27; J-Glu21

2.16

Figure 2 (Cyan)

Chain J

B-Tyr16; J-Gln4, Pro28

1.99

Figure 3 (Yellow)

Chain D

D-Thr27; H-Glu21, Tyr16

1.83

Figure 4 (Blue)

Chain H

H-Tyr26, Gln4; G-Val3,

-0.07

Glu4, Thr8 Figure 5 (Dark Green)

Chain F

F-Gln4, Thr27, Lys29

-1.54

Figure 6 (Orange)

Chain L

F-Tyr16; L-Thr27; K-Glu4

-1.74



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Table 3

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Thermal stability of released insulin at different surfactant concentration

Concentration of surfactant

F1, Tm (˚C)

F2, Tm (˚C)

F3, Tm (˚C)

0.25

48.0 ± 1.08

44.90 ± 0.69

47.00 ± 1.77

0.50

47.43 ± 1.91

46.45 ± 0.89

47.00 ± 0.76

1.00

48.90 ± 0.28

51.22 ± 0.52

52.78 ± 1.40

2.00

48.30 ± 1.13

47.76 ± 0.28

44.70 ± 1.07

3.00

47.00 ± 0.76

49.46 ± 1.15

45.00 ± 1.21

4.00

47.90 ± 1.98

47.46 ± 0.28

42.00 ± 0.91

in inner aqueous phase (%)


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Table 4

Thermal stability of released insulin at different scan rate

Scan rate Control (˚C/h)

F1, Tm (˚C)

F2, Tm (˚C)

F3, Tm (˚C)

10

80 ± 1.00

80 ± 2.60

76 ± 2.24

100 ± 2.44

20

88 ± 1.04

83 ± 1.53

82 ± 2.08

105 ± 4.63

30

94 ± 3.90

87 ± 1.15

85 ± 2.00

113 ± 2.08

60

100 ± 3.25

100 ± 2.53

94 ± 1.53

117 ± 1.24

90

100 ± 2.26

100 ± 0.46

100 ± 1.73

117 ± 2.52


Molecular Mechanism of Poly(vinyl alcohol) Mediated Prevention of

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