Facile preparation of acrylamide grafted locust bean gum-poly(vinyl alcohol) interpenetrating polymer network microspheres for controlled oral drug delivery

Accepted Manuscript Facile preparation of acrylamide grafted locust bean gum-poly(vinyl alcohol) interpenetrating polymer network microspheres for controlled oral drug delivery Santanu Kaity, Animesh Ghosh PII:

S1773-2247(16)30045-4

DOI:

10.1016/j.jddst.2016.02.005

Reference:

JDDST 169

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 18 December 2015 Revised Date:

2 February 2016

Accepted Date: 19 February 2016

Please cite this article as: S. Kaity, A. Ghosh, Facile preparation of acrylamide grafted locust bean gumpoly(vinyl alcohol) interpenetrating polymer network microspheres for controlled oral drug delivery, Journal of Drug Delivery Science and Technology (2016), doi: 10.1016/j.jddst.2016.02.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

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Am-g-LBG

Am-g-LBG-PVA blend

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PVA

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Emulsion crosslinking

IPN microspheres

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For graphical abstract only

Controlled release of BH

ACCEPTED MANUSCRIPT ORIGINAL RESEARCH ARTICLE

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Facile preparation of acrylamide grafted locust bean gum-poly(vinyl alcohol)

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interpenetrating polymer network microspheres for controlled oral drug delivery

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Santanu Kaity and Animesh Ghosh*

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Department of Pharmaceutical Science and Technology, Birla Institute of Technology,

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Mesra, Ranchi- 835215, Jharkhand, India.

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* Corresponding author:

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Department of Pharmaceutical Sciences and Technology

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Birla Institute of Technology, Mesra

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Ranchi – 835215, Jhakhand, India.

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Tel.:+91-9470339587, Fax-+91-6512275290

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Email address: [email protected] (Animesh Ghosh)

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ACCEPTED MANUSCRIPT Abstract:

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Hybridization of natural and synthetic polymers is used to improve the physicochemical

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stability, swelling and drug release pattern of the respective materials in biological condition.

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In present study, a biodegradable, biocompatible and stable interpenetrating polymer network

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(IPN) of acrylamide grafted locust bean gum (Am-g-LBG) and poly(vinyl alcohol) (PVA)

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was developed by emulsion crosslinking method for spatial and temporal drug delivery. The

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IPN microspheres were prepared with the help of glutaraldehyde as a crosslinker for

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controlled oral delivery of buflomedil hydrochloride. The formulation parameters were

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optimized in terms of different gum:PVA ratio, crosslinker amount and drug loading. The

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microspheres were evaluated for their drug entrapment efficiency, particle size, swelling,

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SEM, FTIR, NMR, DSC, XRD and in vitro drug release profile. The particles showed well

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controlled release characteristics and continued to drug release following diffusion controlled

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release pattern. The drug release was for prolong time without collapsing the particle matrix.

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Thus, IPN microspheres based delivery system can be a better approach for controlled

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delivery of highly water soluble drugs like buflomedil hydrochloride.

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Key words: Controlled release; Acrylamide grafted locust bean gum; Poly(vinyl alcohol);

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Buflomedil hydrochloride; Interpenetrating polymer network.

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ACCEPTED MANUSCRIPT 1. Introduction:

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The prime aim of controlled release drug delivery is “spatial placement” and “temporal

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delivery”. Spatial placement refers to drug targeting to specific organs, tissue cells or even

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sub-cellular compartments; whereas, temporal delivery refers to control the rate of drug

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delivery to the target site.

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system can be a major advance towards delivering the drug molecules to their intended

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destination. Multi-particulate delivery systems have gained special interest because of their

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capability to meet the needs of spatial and temporal delivery of drug molecules with

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minimum fluctuation of plasma drug concentration. [2]

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An interpenetrating polymer network (IPN) is a composite of two polymers, which obtained

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when at least one polymer network is synthesized or cross-linked independently in the

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immediate presence of the other.

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improve mechanical properties and thermal stability.

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polymer based network systems has extensively studied for drug delivery purpose as they are

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biodegradable, biocompatible and can be tailored as intended.

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polymer combination of natural and synthetic origin has been found to be useful to control

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the release of short half-lived drugs under physiological conditions. [7]

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Recently, a large number of IPN microspheres have been developed using different polymer

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combinations for drug delivery purpose. Among them, Poly(vinyl alcohol) (PVA) based IPN

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systems are extensively studied

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biocompatibility and desirable physical properties such as sufficient mechanical strength and

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high degree of swelling in aqueous solution. [12] A combination of PVA with natural polymer

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may provide the system with better stability and improved mechanical strength to meet the

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major objectives of controlled release drug delivery.

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A strategically developed controlled release drug delivery

Chemical crosslinking between these polymers leads to [4]

In majority of the cases, the natural

[5, 6]

A judicially selected

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[1]

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[8-11]

due to its inherent non-toxicity, non-carcinogenicity,

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Various natural polymers have been explored for controlled-release microspheres

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development. Among them, guar gum, [10, 11] xanthan gum, [3] gellan gum, [9] locust bean gum,

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[13]

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acrylamide grafted locust bean gum and PVA was selected as matrix for IPN based

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microspheres development. To make a multi-particulate delivery system which will be able to

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deliver the API for prolong time, the delivery device should have sufficient integrity during

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its GI residence. This hybridization was selected to make the matrix crosslinkable and to

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provide sufficient integrity to the particulate delivery device during its GI residence.

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Locust bean gum (LBG) is a high molecular weight branch polysaccharide and is extracted

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from the seeds of carob tree Ceratonia siliqua. It is a non-starch polysaccharides consisting

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of galactose and mannose in the ratio of 1:4 and hence they are known as galactomannan. [15]

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LBG is consists of (1,4)-linked β-D mannopyranose backbone with branch points from their

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6-positions linked to α-D-galactose. The molecular weight of LBG ranges between 300,000

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and 1,200,000 Da. It is not easily soluble in water due to relatively hydrophobic nature of

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mannan unit and requires heating to affect solvation. [16]

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Recently, we have explored the efficiency of native LBG and carboxymethylated LBG

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(CMLBG) with PVA as IPN controlled release drug delivery device.

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observed poor entrapment efficiency of the particles. Moreover, in case of native LBG we

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observed miscibility problem with aqueous PVA solution. Grafting of polyacrylamide onto

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LBG can increase the aqueous solubility of resultant polymer.

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grafted LBG and PVA may be able to overcome the problems observed in case of LBG-PVA

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and CMLBG-PVA IPNs. So, in present study, IPN microspheres of acrylamide grafted locust

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bean gum (Am-g-LBG) and PVA was prepared for controlled oral delivery of a highly water-

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soluble drug, buflomedil hydrochloride (BH). BH is readily absorbed in the gastrointestinal

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tract and has a plasma half-life of approximately 2 to 3 h. The usual oral dose of BH is 300 to

[14]

has been reported till date. In present study, a novel combination of

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chitosan

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[13, 17]

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IPN with acrylamide

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600 mg/day. Long-term use of BH in high doses can produce drug accumulation and drug

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related toxicity.

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BH, can reduce the dose and dose related side effects.

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The IPN particles of Am-g-LBG and PVA were developed by emulsion crosslinking method

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and the microspheres were evaluated for their drug entrapment efficiency, particle size,

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swelling, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy

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(FTIR), nuclear magnetic resonance (NMR) spectroscopy, differential scanning colorimetry

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(DSC) and X-ray diffraction (XRD) profile. An in vitro drug release study [in both acidic

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media (pH 1.2) and phosphate buffer (pH 6.8)] and kinetic modelling was performed to

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understand the drug release mechanism.

Therefore, a controlled-release approach, such as IPN microspheres of

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2. Materials:

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Buflomedil hydrochloride was obtained as gift sample from Fresenius Kabi Oncology

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Limited (Kalyani, West Bengal, India). Locust bean gum and poly(vinyl alcohol) (PVA;

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average molecular weight 125000 Da) were purchased from HiMedia Laboratories Pvt. Ltd.

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(Mumbai, India). Light liquid paraffin (LLP; viscosity 25−80 mPa at 20°C), hydrochloric

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acid (HCl; 30%, ultra-pure) and acetone (Density = 0.789 to 0.791 g/mL) were obtained from

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Rankem India Pvt. Ltd. (Mumbai, India). Span 80 was procured from Pioneer In-Organics

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(Delhi, India). Glycine and glutaraldehyde (GA; 25%, v/v) were supplied by Merck India

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Ltd. (Mumbai, India). All the reagents were used without further purification. Water used

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was of Milli-Q grade.

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3. Methods:

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3.1. Synthesis of acrylamide grafted locust bean gum (Am-g-LBG)

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ACCEPTED MANUSCRIPT Recently, we have reported the detailed method of synthesis and characterization of

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acrylamide grafted LBG. [18] In brief, 10 g of acrylamide (Am) was dissolved in 30 mL water

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and mixed with 120 mL aqueous dispersion of LBG (0.0083g/mL). 300 mg ceric ammonium

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nitrate (CAN) was dissolved in 30 mL of water and mixed with the Am-LBG mixture. The

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dispersion was irradiated by microwave (Laboratory scientific microwave system, Catalyst

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systems, India) at 480 W for 2.5 min. using alternate one minute heating and cooling cycle.

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The grafted gum was precipitated using acetone and washed with absolute and 30% aqueous

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ethanol. The grafted gum thus prepared was vacuum dried at 40⁰C to a constant weight and

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converted to fines for further use.

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3.2. Preparation of Am-g-LBG-PVA IPN microspheres

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Buflomedil hydrochloride (BH) entrapped IPN microspheres of PVA and Am-g-LBG were

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developed by water-in-oil (w/o) emulsion-crosslinking method (Figure 1). [14] Briefly, 20 mL

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of 2% (w/v) aqueous polymeric solution (total polymer amount was kept constant) was

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prepared by dispersing varying amounts of Am-g-LBG in aqueous PVA solution. The

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required amount of BH was added in the polymeric dispersion. The drug- polymer blend was

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slowly emulsified with light liquid paraffin (100 g) containing 1% (w/w) Tween-80 under

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constant mechanical stirring (IKA Labortechnik, Staufen, Germany) at 500 rpm. A milk

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white emulsion (w/o) was obtained. To this emulsion, GA (2.5 and 5 mL) containing 0.5 mL

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of 1 N HCl was added slowly and stirring was continued for 3 h. The crosslinked

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microspheres were then filtered and washed with acetone, 0.1 M glycine solution and water to

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remove excess amount of liquid paraffin, unreacted GA and surfactant, respectively.

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Complete removal of unreacted GA was confirmed by treating the filtrate with Fehling’s

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reagent. A negative result assured the absence of unreacted GA. Hardened microspheres were

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vacuum-dried at 40°C for 24 h and stored in desiccator until further use. The absence of

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ACCEPTED MANUSCRIPT unreacted GA was confirmed in dried particle matrix by making an aqueous dispersion of

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crushed dried particles and treating it in similar way as said earlier.

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For the preparation of blank microspheres similar method was adopted except the addition of

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drug into the system. Detailed formulation variables are given in Table 1.

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Insert Table 1 here.

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Insert Figure 1 here.

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3.3. Full factorial design

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In the present study, two-level [high level (+1) and low level (−1)], three-factor, full factorial

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design (8 batches) was used for the optimization of gum:PVA ratio, crosslinker and drug

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loads (independent variables). The responses (dependent variables) selected for optimization

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were drug entrapment efficiency, particle size, swelling and cumulative percentage drug

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release in dissolution study. The results (Tables 1) of response generated using the

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experimental designs were analysed by factorial models using Design Expert software

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(Version 7.0.0, Stat-Ease, Inc., Minneapolis). The 3D response plots are presented in Figure

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2.

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Insert Figure 2 here.

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3.4. Fourier transform infrared spectroscopy (FTIR)

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The FTIR spectrums of Am-g-LBG, PVA, BH, drug-polymer physical mixture, blank

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microspheres, and drug loaded microspheres were carried out by FTIR-8400S (Shimadzu,

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Japan) to confirm the formation of Am-g-LBG and compatibility of different ingredients of

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the IPN formulations. A small amount of each material was mixed with potassium bromide

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(KBr) (1 %w/w sample content), taken into sample holder and scanned in the range of 600 to

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4000 cm-1.

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ACCEPTED MANUSCRIPT 3.5. Solid state 13C NMR spectroscopy

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Solid state 13C NMR of Am-g-LBG, PVA and glutaraldehyde crosslinked blank formulation

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was studied to confirm the formation of Am-g-LBG and glutaraldehyde crosslinked IPN

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network. For solid state 13C NMR, approximately 300 mg of samples were inserted into the

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ceramic rotor on a Bruker AMX 400 spectrometer. The spectrum was recorded at 75.5 MHz.

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3.6. Differential scanning calorimetry (DSC)

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DSC thermograms of Am-g-LBG, PVA, BH, blank formulation and drug loaded formulations

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were obtained by using DSC-60 (Shimadzu, Japan). Each sample (3–7 mg) was accurately

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weighed into a 40 µL aluminium pan in a hermetically sealed condition. The measurements

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were conducted in nitrogen atmosphere between 30°C and 350°C at the heating rate of

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10°C/min. The Am-g-LBG and the drug loaded formulations were heated in-situ up to 110⁰C

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to make them moisture free. Then the samples were cooled to room temperature and reheated

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up to 350⁰C.

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3.7. Qualitative X-Ray diffractometry (XRD)

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Grinded samples of blank IPN microspheres, drug loaded IPN microspheres, BH were

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scanned from angular range of 10° to 60° 2θ, using an X-ray diffractometer (Bruker AXS D8

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Advance, Germany, Configuration: Vertical, Theta/2 Theta geometry: X-ray Cu, wavelength

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1.5406 A°, Detector: Si (Li) PSD. The diffractometer was run at a scanning speed of 2°/min

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and a chart speed of 2°/2 cm per 2θ.

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3.8. Scanning electron microscopy (SEM)

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The surface morphology and topography of Am-g-LBG-PVA IPN microspheres were

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evaluated by scanning electron microscope (JSM-6390LV, Jeol, Japan). Before examination,

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ACCEPTED MANUSCRIPT the samples were mounted onto stubs using double-sided dried adhesive carbon tape and

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vacuum coated with gold palladium film (thickness 2 nm) by sputter coater (Edward S-150,

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U.K.) to make them electrically conductive. Representative sections were photographed for

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evaluation.

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3.9. Drug encapsulation efficiency (DEE)

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IPN particles were crushed in mortar and pestle and a specified amount (10 mg) was taken

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into 50 mL of phosphate buffer solution (pH 6.8), heated at 50°C for effective drug

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extraction. After 24 h, the suspension was subjected for filtration and centrifugation for the

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removal of polymeric debris. The supernatant was analysed with a spectrophotometer (UV-

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1800, Shimadzu, Japan) at λmax of 282 nm. All samples were analysed in triplicate. The drug

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entrapment efficiency (%) was calculated by using the following equation: [20]

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Encapsulation efficiency (%) = (Actual drug content / Theoretical drug content) × 100

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(1)

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3.10. Swelling study

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Equilibrium swelling study of IPN microspheres was done in different media. An accurately

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weighed amount of microspheres (W1) was immersed in 50 mL buffer (pH 1.2 and pH 6.8)

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and allowed to swell for 24 h at 37°C.The swollen microspheres was collected and the

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adhered liquid droplets on the surface of the particles was removed carefully with tissue

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paper and reweighed (W2) to an accuracy of ±0.01 mg on an electronic microbalance

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(Mettler, model AT120, Greifensee, Switzerland). The swelling index was calculated by the

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formula given below: [11]

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Swelling index = [(W2-W1) / W1]× 100

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Where, W2 and W1 are the swollen and dry weights of the gum/microspheres, respectively.

Eq.

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Eq. (2)

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ACCEPTED MANUSCRIPT 3.11. Particle size analysis

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Particle size of IPN microspheres were determined by using a Mastersizer 2000 Ver.5.40

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(Malvern Instruments Ltd., UK) which allows sample measurement in the range of 20–2000

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µm. The micro particles were dispersed in water and the size was measured using the laser

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light scattering technique. Polydispersity index (PDI) was determined according to the

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equation:

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Polydispersity index = [d(0.9) – d(0.1)]/d(0.5)

Eq. (3)

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where, d(0.9), d(0.5) and d(0.1) are the particle diameters determined respectively at the 90th,

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50th and 10th percentile of undersized particles. [21]

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3.12. In vitro drug release and release kinetic study

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In vitro drug release from the IPN microspheres were investigated in pH 1.2 for the initial 2

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h, followed by phosphate buffer of pH 6.8. All the experiment was performed in triplicate in

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a dissolution tester (Electro Lab, TDT-08L, India) equipped with eight baskets (glass jars) at

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the stirring speed of 50 rpm. An accurately weighed quantity of each sample (equivalent to

219

100 mg BH) was placed in 900 mL of dissolution medium maintained at 37.5⁰C. At regular

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intervals of time, sample aliquots were withdrawn and analysed using UV spectrophotometer

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(UV-1800, Shimadzu, Japan) at the fixed λmax of 282 nm.

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The release data were fitted in various empirical equations like Zero order (Qt = Kt), First

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order (ln Qt= ln Q0-Kt ), Higuchi kinetic (Qt= Ktn), Hixson-Crowell (Q01/3- Qt1/3 = Kt) and the

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power-law equation: Mt/M∞ = ktn, where Mt/M∞ is the fraction of drug release at time t, k is

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the release rate constant, and n is the diffusion exponent that denotes the drug-release

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mechanism. [22] A least squares regression method was used to determine the values of n and

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k. Values of n = 0.43 or less indicate Fickian transport, whereas values of n of 0.43–0.85

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ACCEPTED MANUSCRIPT indicate anomalous or non-Fickian drug transport. Exponent values of n greater than 0.85

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signifies the super case II transport mechanism.Q0 and Qt are amount of drug released at zero

230

and t time. K represents release rate constant.

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4. Results and discussion:

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The microwave assisted grafting method was used in present study to induce rapid energy to

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the reaction system and to reduce the reaction time. The ceric ion from CAN generates free

234

radical sites on LBG backbone (Figure 3). LBG free radicals used to couple with acrylamide

235

by covalent linkage. The reaction is terminated by coupling of two free radicals.

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details of synthetic parameters of IPN microspheres are given in Table 1. Acrylamide is an

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excellent monomer which is freely soluble in water and can be mixed with hydrophilic gum

238

solution to make a homogenous blend. Thus it can easily attach with the adjacent gum

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backbone by free radical polymerization thus reduce the chance of high amount of

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homopolymer formation. Moreover acrylamide in low dose is non-toxic, easily excreted from

241

body and plasma half life is very less (2-2.5 h). The method of IPN particle preparation and

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proposed reaction mechanism of IPN formation is shown in Figure 1 and Figure 3,

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respectively. The general mechanism of matrix crosslinking with GA, a bi-functional

244

crosslinker, was through developing an acetal ring between the hydroxyl groups of Am-g-

245

LBG and PVA with the aldehyde groups of GA. This aided the formation of the IPN

246

microspheres in the emulsion phase. [14]

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Insert Figure 3 here.

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4.1. Fourier transform infrared spectroscopy (FTIR)

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The FTIR spectral study of Am-g-LBG, buflomedil hydrochloride, PVA, physical mixture,

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blank microspheres and optimized drug loaded microspheres (Figure 4) was performed to

[18]

The

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ACCEPTED MANUSCRIPT evaluate the compatibility of different formulation ingredients and to confirm the formation

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of Am-g-LBG and GA crosslinked IPN matrix structure.

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As presented in Figure 4, the Am-g-LBG showed the bands at 1683 and 1651 cm-1 were

254

attributed to amide-I (C–O stretching) and amide-II (N–H bending) conferred by Am. [23] The

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peak at 2800–3566 cm−1 in Am-g-LBG was due to overlap of N–H stretching band of amide

256

group and O–H stretching band. A band at about 1454 cm−1 was due to the C–N stretching.

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Peak at 1010 cm−1 was attributed to CH-O-CH2 group which occurs during grafting reaction

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between OH group of C2 and π bond of acrylamide.

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evidenced that LBG was successfully modified to Am-g-LBG.

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For buflomedil hydrochloride major peaks were observed at about 2968 cm−1 for methoxy

261

CH stretching (CH3-O-). A small peak at about 1339 cm−1 was observed for aromatic C-N

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stretching. Absorption band at 1701 cm-1 was of ketonic carbonyl group (Figure 4).

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In case of PVA major peaks related to hydroxyl and acetate groups were present in FTIR

264

spectra. The large band observed between 3600 and 3200 cm−1 are linked to the O–H

265

stretching of the intermolecular and intramolecular hydrogen bonds. The vibrational band

266

observed between 2854 and 2944 cm−1 referred to the C–H stretching from alkyl groups and

267

the peaks between 1740–1716 cm−1 were due to the stretching C=O and C–O from acetate

268

group residues from PVA (Figure 4). A sharp peak obtained near 1456 cm-1 indicated the

269

bending vibration of CH2 groups. The intensity of the 1740– 1716 cm−1 strongly suggested

270

that it was less hydrolysed. [24]

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The physical mixture showed the peaks of all components (BH, PVA and Am-g-LBG) in its

272

spectra without significant changes, indicating compatibility of the ingredients. The peak

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intensities were observed to be reduced for all components due to the dilution effect.

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FTIR analysis of Am-g- LBG

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In the placebo Am-g-LBG-PVA IPN formulation, a considerable intensity reduction of the

275

O–H peaks from the PVA was noted, giving a possible indication of acetal bridge formation.

276

[25]

277

to ether linkage (C-O-C stretching) formed between the –OH group of PVA and Am-g-LBG

278

by the help of glutaraldehyde. [10] Peaks near about 1735 and 1650 cm −1 may be due to C=O

279

group of PVA acetate and amide-II (N–H bending) conferred by Am-g-LBG, respectively

280

(Figure 4). Thus, it can be said that Am-g-LBG and PVA was successfully crosslinked by GA

281

to form the network structure.

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The drug loaded microspheres showed the additional peaks of BH in FTIR spectra than that

283

of placebo microspheres. Peaks at about 2925 cm−1 (methoxy CH stretching), 1338 cm−1

284

(aromatic C-N stretching) and 1702 cm-1 (ketonic carbonyl group) were due to BH (Figure 4).

285

No additional peaks were observed in the drug incorporated formulation than that of BH and

286

formulation components indicating that the ingredients itself and the process was compatible

287

for the preparation of BH loaded Am-g-LBG-PVA IPN microspheres.

288

Insert Figure 4 here.

289

4.2. Solid state 13C NMR spectroscopy

290

As per the earlier reports, the major peaks in the 13C NMR spectrum of LBG observed at δ =

291

62.0 ppm for the C-6 carbon of mannan unit, δ = 70-80 ppm for the signals of C-2, C-3 and

292

C-5 carbon atom, δ = 81.0 ppm for the C-4 mannan carbon atom and a sharp peak at δ =

293

102.7 ppm for C-1 mannan carbon atom. [13]

294

In the

295

CH2-CH-)n groups, formed due to Am polymerization, peak at δ=180.8 ppm for carbon atoms

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A small absorption peak at about 1250 cm −1 was observed in the spectrum which was due

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C NMR spectrum of Am-g-LBG (Figure 5), the peak at δ=42.5 ppm was for (-CH-

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ACCEPTED MANUSCRIPT of –CONH2 group. The NMR data strongly suggests that LBG was successfully converted to

297

Am-g-LBG.

298

The NMR spectrum of PVA consists of one broad peak at 45.8 ppm due to the methylene

299

carbon and a group of three peaks at δ = 62.8, 72.9, and 82.6 ppm from the oxymethine

300

carbon (Figure 5).

301

The

302

42.2 ppm due to the (-CH-CH2-CH-)n groups of acrylamide polymerization. The absorption

303

peak at δ = 61.3 ppm and 72.8 ppm are for PVA methine carbon. Peak at δ = 101.9 ppm is

304

due to the acetal carbon atom

305

group due to cross linking by glutaraldehyde proves the formation of IPN (Figure 5). The

306

broadened peak at δ = 177.4 ppm is for carbon atoms of –CONH2 group. Hence, the solid

307

state NMR spectra of the IPN particles showed a strong evidence of formation of

308

glutaraldehyde crosslinked IPN matrix of Am-g-LBG and PVA.

309

Insert Figure 5 here.

310

4.3. Differential scanning calorimetry (DSC)

311

From the DSC study it was observed that the Am-g-LBG showed its transition mid point

312

temperature (Tm) at about 238⁰C (Figure 6). It was almost similar as reported earlier. [18] PVA

313

showed its characteristic melting peak at about 192⁰C and a degradation peak was observed

314

at about 322⁰C (Figure 6). In case of pure BH, a sharp melting peak was observed at about

315

197⁰C which was matching with previous reports. [13] In case of placebo microspheres, peaks

316

due to individual components were hard to detect. However, a broad endothermic peak was

317

observed at about 288⁰C. This may be an indication of formation of a polymer composite

318

which degrades at a temperature other than the degradation points of individual basic

[17]

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C NMR spectrum of placebo Am-g-LBG-PVA IPN microspheres showed peak at δ=

which formed between the Am-g-LBG and PVA hydroxyl

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ACCEPTED MANUSCRIPT components. In drug loaded formulation, the endothermic peak at about 196⁰C was due to the

320

melting of entrapped BH (Figure 6). A broad endothermic peak was observed at about 278⁰C

321

which may be due to the degradation of the Am-g-LBG-PVA composite network structure.

322

Thus, from DSC study it can be said that a complex network structure was formed between

323

Am-g-LBG and PVA crosslinked by GA. Moreover, BH was successfully encapsulated in the

324

IPN polymer matrix.

325

Insert Figure 6 here.

326

4.4. Qualitative X-Ray diffractometry (XRD)

327

From the diffractogram of BH (Figure 7), it can be said that, it is highly crystalline in nature.

328

BH showed its characteristic peaks at 2θ of 6-50⁰. Sharp intense peak at about 2θ of 6⁰, 15⁰

329

and 21⁰ proves its crystalline nature. Drug loaded microspheres showed a broad peak at about

330

2θ of 20⁰. However, other peaks have disappeared in BH loaded microspheres assuring that

331

drug is molecularly dispersed in the polymer matrix. The peak intensity at 2θ of 20⁰ is higher

332

in drug loaded formulation than that of placebo confirms the presence of BH in IPN matrix.

333

Insert Figure 7 here.

334

4.5. Scanning electron microscopy (SEM)

335

The SEM image reveals surface morphology of the IPN particles and represented in Figure 8.

336

The particles which were prepared by PVA only (A9, Figure 8a) were having smooth surface

337

and well defined spherical structure. The particles prepared by Am-g-LBG and PVA in

338

combination with less amount of GA (Figure 8b) showed some pores on the surface.

339

However, the particles prepared with high amount of GA were appeared to be solid and there

340

was no pore on the surface (Figure 8c). This happened because of the higher crosslinking of

341

the polymer matrix. The surface appeared to be tough and showed evidence of network

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15

ACCEPTED MANUSCRIPT structure (Figure 8d). The cross sectional view of microspheres (Figure 8e and 8f) also

343

showed a strong evidence of the formation of well-defined polymer network structure.

344

Insert Figure 8 here.

345

4.6. Drug encapsulation efficiency (DEE)

346

The drug entrapment efficiency of Am-g-LBG-PVA IPN microspheres (Table 1) was

347

observed in the range of 51.32±1.94 (A2) to 73.51±2.78% (A1).This improvement in DEE

348

was due to the optimum miscibility of two phases and the highly branched structure of the

349

grafted gum which prevented the leaching of drug during the crosslinking stage. [18] The drug

350

entrapment capacity was observed to be dependent on the gum:PVA composition, crosslinker

351

and drug loads. A higher amount of Am-g-LBG (formulation code A2 - A5) led to reduced

352

DEE due to increased hydrophilicity of the system and inability of crosslinker to crosslink the

353

total system (Table 1). Similar findings were reported by Sullad et al. (2010).

354

increased amount of GA helped in higher drug entrapment due to the formation of rigid

355

polymer matrix (A1 and A6). Interestingly an increased drug loading showed least effect on

356

the DEE of the IPN microspheres (A1 and A8). This may be due to the saturation of the

357

matrix by the optimum drug concentration.

358

The three dimensional plot of the effects of different formulation variables on %DEE is

359

shown in Figure 2a. The mathematical relationship of DEE with the independent variables

360

was generated by MLRA and expressed as:

361

[11]

An

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DEE (%) = 63.58+6.29A+3.48B+1.35C

Eq. (4)

362

Where, A is gum:PVA ratio, B is glutaraldehyde amount and C is % drug loading. Here we

363

observed that all the independent variables were having positive impact on %DEE as was

364

observed experimentally. ANOVA analysis indicated that the model was significant (P < 16

ACCEPTED MANUSCRIPT 0.05) with R2 value 0.99. The adjusted (0.98) and predicted (0.96) R2 were reasonably in

366

good agreement.

367

4.7. Swelling study

368

The swelling of Am-g-LBG-PVA IPN microspheres (Table 1) was observed to be moderately

369

pH dependent. The extent of crosslinking played a major role in swelling of the microspheres.

370

Highly crosslinked particles (prepared with high amount of GA) showed less swelling (A1,

371

A3, A4 and A8) due to the absence of free hydrodynamic volume in their matrix. The

372

presence of higher amount of grafted gum in microspheres matrix resulted in higher swelling

373

(A2 in pH 1.2 and 6.8, respectively) (Table 1). This happened because of the hydrophilic

374

nature of the grafted gum. This postulation was further confirmed by the swelling

375

characteristics of microspheres having only PVA (A9) which showed least swelling in both

376

the media since it was devoid of grafted gum. The swelling of particles in acidic media (pH

377

1.2) was less in all cases than that of phosphate buffer (pH 6.8). Under pH 6.8, the presence

378

of ionisable groups in the component polymers which deprotonated and exhibited higher

379

swelling ratio due to the collective electrostatic repulsion forces between the ionized groups.

380

[26]

381

A slight increase in swelling was observed in case of formulation having higher drug loading

382

(A3 and A4). This was due to the increased hydrophilicity of the matrix in presence of highly

383

hydrophilic BH.

384

The effects of formulation variables on swelling as observed from MLRA are shown in

385

Figure 2c, 2d and expressed as:

386

Swelling% (pH 1.2) = 255.36 - 13.34A - 14.33B

387

Swelling % (pH 6.8) = 332.93 - 11.27A - 9.91B

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17

Eq. (5) Eq. (6)

ACCEPTED MANUSCRIPT Where, A is gum: PVA ratio and B is GA amount. ANOVA analysis indicated that the

389

factorial model was significant (P < 0.05) having R2 value of 0.91 and 0.92 for pH 1.2 and

390

pH 6.8, respectively. The adjusted (0.88 for both pH 1.2 and 6.8) and the predicted (0.78 and

391

0.79 for pH 1.2 and pH 6.8, respectively) R2 values were in reasonably good agreement. Here

392

both the gum: polymer ratio and GA showed negative effect and drug loading (%) showed

393

negligible influence on swelling as observed experimentally.

394

4.8. Particle size analysis

395

The arithmetic mean diameter of IPN particles varied from 371.06 µm to 766.06 µm (Figure

396

9, Table 1). In a fixed IPN blend composition, higher crosslinker amount resulted in

397

reduction of particle size (For A1 particle size is 449.46 µm, for A6 size is 519.36 µm). This

398

was due to the fact that as the amount of crosslinker was increased, the density of the polymer

399

matrix increased to a higher extent and reduced the internal void space.

400

The particle size was observed to be increased with the amount of Am-g-LBG (For A1

401

particle size was 449.46 µm, for A3 size was 608.76 µm) (Table 1). This can be explained on

402

the basis of hydrodynamic viscosity concept, i.e., with increased Am-g-LBG amount, the

403

interfacial viscosity of the polymer droplets in the emulsion was increased and resulted in the

404

formation of larger particles.

405

An increased drug loading (For A1 and A8 particle size was 429.70 µm and 449.46 µm

406

respectively) also resulted in the formation of bigger particles (Figure 9). The reason behind

407

this was, drug molecules might have hindered the inward shrinkage of the polymer matrix by

408

occupying the free volume spaces within the matrix. The polydispersity index of the Am-g-

409

LBG particles was less and it was in the range of 0.82 and 1.32 (Table 1). This result

410

indicates that the particles formed are of narrow size distribution range.

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18

ACCEPTED MANUSCRIPT 411

The effects of independent variables on the particle size are shown in Figure 2b. The

412

mathematical relationship is expressed as:

413

Particle size = 557.60 – 88.94A – 47.64B

414

Where, A is gum:PVA ratio and B is GA amount. ANOVA analysis indicated that the model

415

was significant (P < 0.05) with R2 value 0.87. The adjusted (0.83) and predicted (0.69) R2

416

were almost in good agreement. Here also we observed the negative effect of gum: PVA

417

ratio and GA on the size of IPN particles. It was in good agreement with experimental

418

observation.

419

Insert Figure 9 here.

420

4.9. In vitro drug release and release kinetic study

421

The cumulative percentage drug release vs. time plot of different batches of Am-g-LBG-PVA

422

IPN microspheres are presented in Figure 10. It was observed that the drug release from the

423

IPN microspheres were dependent upon major factors like crosslinker amount, polymeric

424

blend ratio and to less extent on the amount of drug loading.

425

Effect of crosslinker: In fixed formulating parameters, when the amount of crosslinker was

426

increased from 2.5 mL to 5 mL, the amount of drug release was decreased. In formulation A1

427

and A6 polymer composition (1:2) and drug loading (50%) was same but amount of

428

crosslinker was different. A1 showed less drug release (64%) than A6 (68%) (Figure 10).

429

This may be due to the higher crosslinking of the matrix which prevent solvent imbibition

430

and network erosion leading to high release retardant property.

431

Effect of gum:PVA blend ratio: When the blend ratio of IPN particles were changed from 1:2

432

to 1:1 in fixed crosslinker and drug loading percentage, the drug release increased from 64%

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Eq. (7)

19

ACCEPTED MANUSCRIPT (A1) to 79% (A3) and 68% (A6) to 87% (A2). This was due to the hydrophilic nature of the

434

grafted gum which interacted with media to swell and erode in a faster rate and helped in

435

faster drug release. Particles composed of only PVA (A9) showed 74% drug release in 12 h

436

(Figure 10).

437

Effect of drug loading: Formulations, having different drug loading in a fixed gum: PVA

438

ratio and crosslinker amount, showed different extent of drug release. For example, A2 (25%

439

drug loading, 87% drug release) showed retarded drug release property than A5 (50% drug

440

loading, 93% drug release) (Figure 10) though other parameters for both the formulations

441

were same. It may be due to the reason that, concentration gradient, the driving force, will be

442

high in high drug load formulations and promoted faster drug release. Moreover, low drug

443

load matrix would have a greater gum and polymer fraction to act as the barrier to drug

444

release.

445

The effect of formulation variables on CPR is shown in figure 2e and the mathematical

446

relation is expressed as:

447

CPR = 75.90 - 9.08A – 4.18B

448

Where, A is gum:PVA ratio and B is amount of GA. ANOVA analysis indicated that the

449

model was significant (P < 0.05) with R2 value 0.95. The adjusted (0.93) and predicted (0.88)

450

R2 were in good agreement. The effect of gum: polymer ratio and GA was similar to that of

451

experimental evidence.

452

After generating the model polynomial equations to relate the dependent and independent

453

variables, the best optimized amount of independent variables was finalized in terms of

454

maximum gum: PVA ratio, minimum amount of crosslinker and maximum drug loading to

455

achieve the dependent variables in terms of maximize DEE%, particle size in range,

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Eq. (8)

20

ACCEPTED MANUSCRIPT maximize swelling and minimum CPR. The software generated solution was in good

457

agreement with batch A6.The optimized Am-g-LBG-PVA IPN particles showed 68.08% drug

458

release at 12 h.

459

Release kinetic: The invitro release data were fitted in different empirical equations and

460

presented in Table 2. It was observed that the best fit was with Higuchi release kinetic,

461

suggesting that the release of drug molecules from IPN matrix was diffusion controlled. The

462

drug release data were also fitted with the Power law equation and the values of n varied

463

from 0.845 to 1.078. For A4 drug release was non Fickian and for the rest the release pattern

464

followed super case II transport mechanism.

465

Insert Figure 10 here.

466

Insert Table 2 here.

467

5. Conclusion:

468

In present study Am-g-LBG-PVA IPN microspheres were successfully prepared by emulsion

469

crosslinking method by using glutaraldehyde as a crosslinker. Among various formulations

470

the A6 batch was the optimized formulation. The particles were spherical with narrow size

471

distribution. The particle showed promising controlled release property with moderate pH

472

sensitivity. This type of polymeric composites may be fruitful as a promising biomaterial to

473

solve the major loop holes of controlled release of highly water soluble drugs with short half

474

life.

475

Conflict of interest:

476

The authors report no conflicts of interest. The authors alone are responsible for the content

477

and writing of the article.

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21

ACCEPTED MANUSCRIPT Acknowledgements:

479

Santanu Kaity is thankful to CSIR, New Delhi for financial support as CSIR-SRF [grant no.:

480

09/554(0029)/2011. EMR-I]. The authors are also thankful to CIF and the authority of BIT-

481

Mesra, Ranchi for providing necessary facilities for this work.

482

References:

483

[1] H. Kojima, K. Yoshihara, T. Sawad, H. Kondo, K. Sako, Extended release of large

484

amount of highly water soluble diltiazem hydrochloride by utilizing counter polymer in

485

polyethylene oxides (PEO)/polyethylene glycol (PEG) matrix tablets, Eur. J. Pharm.

486

Biopharm. 70 (2008) 556-562.

487

[2] A. Dashevsky, A. Mohamad, Development of pulsatile multiparticulate drug delivery

488

systemcoated with aqueous dispersion Aquacoat® ECD, Int. J Pharm. 318 (2006) 124-131.

489

[3] S. Ray, S. Banerjee, S. Maiti, B. Laha, S. Barik, B. Sa, U. K. Bhattacharyya, Novel

490

interpenetrating network microspheres of xanthan gum–poly(vinyl alcohol) for the delivery

491

of diclofenac sodium to the intestine- in vitro and in vivo evaluation, Drug Deliv. 17 (2010)

492

508-519.

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[4] S. Mishra, R. Bajpai, R. Katare, A. K. Bajpai, Preparation, characterization and

494

microhardness study of semi interpenetrating polymer networks of poly(vinyl alcohol) and

495

crosslinked poly(acrylamide), J. Mater. Sci. - Mater. Med. 17 (2006) 1305-1313.

496

[5] T. R. Bhardwaj, M. Kanwar, R. Lal, A. Gupta, A. Natural gums and modified natural

497

gums as sustained-release carriers, Drug Dev. Ind. Pharm. 26 (2000) 1025-1038.

498

[6] V. Vijan, S. Kaity, S. Biswas, J. Isaac, A. Ghosh, Microwave assisted synthesis and

499

characterization of acrylamide grafted gellan, application in drug delivery, Carbohydr.

500

Polym. 90 (2012) 496-506.

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ACCEPTED MANUSCRIPT [7] A. Lohani, G. Singh, S. S. Bhattacharya, A. Verma, Interpenetrating polymer networks as

502

innovative drug delivery systems, J. of Drug Del. 1 (2014) 1-11.

503

[8] A. G. Sullad, L. S. Manjeshwar, T. M. Aminabhavi, Novel semi-interpenetrating

504

microspheres of dextran-grafted-acrylamide and poly(vinyl alcohol) for controlled release of

505

abacavir sulfate, Ind. Eng. Chem. Res. 50 (2011) 11778-11784.

506

[9] S. A. Agnihotri, T. M. Aminabhavi, Development of novel interpenetrating network

507

gellan gum-poly(vinyl alcohol) hydrogel microspheres for the controlled release of

508

carvedilol, Drug Dev. Ind. Pharm. 31 (2005) 491-503.

509

[10] K. S. Soppimath, A. R. Kulkarni, T. M. Aminabhavi, Controlled release of

510

antihypertensive drug from the interpenetrating network poly(vinyl alcohol) – guar gum

511

hydrogel microspheres, J. Biomat. Sci- Polym. E. 11 (2000) 27-43.

512

[11] A. G. Sullad, L. S. Manjeshwar, T. M. Aminabhavi, Novel pH sensitive hydrogels

513

prepared from the blends of poly(vinyl alcohol) with acrylic acid-graft-guar gum matrixes for

514

isoniazid delivery, Ind. Eng. Chem. Res. 49 (2010) 7323-7329.

515

[12] M. C. Hassan, N. A. Peppas, Structure and applications of poly(vinyl alcohol) hydrogel

516

produced by conventional crosslinking or by freezing/thawing methods, Adv. Polym. Sci.

517

153 (2000) 37-62.

518

[13] S. Kaity, J. Isaac, A. Ghosh, Interpenetrating polymer network of locust bean gum-poly

519

(vinyl alcohol) for controlled release drug delivery, Carbohydr. Polym. 94 (2013) 456-467.

520

[14] P. B. Kajjari, L. S. Manjeshwar, T. M. Aminabhavi, Novel interpenetrating polymer

521

network hydrogel microspheres of chitosan and poly(acrylamide)-grafted-guar gum for

522

controlled release of ciprofloxacin, Ind. Eng. Chem. Res. 50 (2011) 13280-13287.

523

[15] K. S. Parvathy, N. S. Susheelamma, R. N. Tharanathan, A. K. Gaonkar, A simple non-

524

aqueous method for carboxymethylation of galactomanans, Carbohydr. Polym. 62 (2005)

525

137-141.

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ACCEPTED MANUSCRIPT [16] D. R. Picout, S. B. Ross-Murphy, K. Jumel, S. E. Harding, Pressure cell assisted solution

527

characterization of polysaccharides. 2. locust bean gum and tara gum, Biomacromolecules. 3

528

(2002) 761-767.

529

[17] S. Kaity, A. Ghosh, Carboxymethylation of locust bean gum: application in

530

interpenetrating polymer network microspheres for controlled drug delivery, Ind. Eng. Chem.

531

Res. 52 (2013) 10033-10045.

532

[18] S. Kaity, J. Isaac, P. Mahesh, A. Bose, T. W. Wong, A. Ghosh, Microwave assisted

533

synthesis of acrylamide grafted locust bean gum and its application in drug delivery,

534

Carbohydr. Polym. 98 (2013) 1083-1094.

535

[19] A. Dubourg, R. F. Scamuffa, An experimental overview of a new vasoactive drug:

536

buflomedil HCl, Angiology.32 (1981) 663-675.

537

[20] C.S. Angadi, S. L. Manjeshwar, T. M. Aminabhai, Stearic acid-coated chitosan-based

538

interpenetrating polymer network microspheres: controlled release characteristics, Ind. Eng.

539

Chem. Res. 50 (2011) 4504-4514.

540

[21] B. F. Oliveira, M. H. A. Santana, M. I. Ré, Spray-dried chitosan microspheres cross-

541

linked with d, l-glyceraldehyde as a potential drug delivery system: preparation and

542

characterization, Braz. J. Chem. Eng. 22 (2005) 353-360.

543

[22] R. W. Korsemeyer, R. Gunny, E. Doelker, P. Buri, N. A. Peppas, Mechanism of solute

544

release from hydrophilic polymers, Int. J. Pharm. 15 (1983) 25-35.

545

[23] R. C. Mundargi, A. S. Patil, T. M. Aminabhavi, Evaluation of acrylamide grafted-

546

xanthan gum copolymer matrix tablets for oral controlled delivery of antihypertensive drugs,

547

Carbohydr. Polym. 69 (2007) 130-141.

548

[24] G. Andrade, E. F. Barbosa-Stancioli, A. A. P. Mansur, W. L. Vasconcelos, H. S.

549

Mansur, Small-angle x-ray scattering and FTIR characterization of nanostructured poly(vinyl

550

alcohol)/silicate hybrids for immunoassay applications, J. Mater. Sci. 43 (2008) 450-463.

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ACCEPTED MANUSCRIPT [25] H. S. Mansur, C. M. Sadahira, A. N. Souza, A. A. P. Mansur, FTIR spectroscopy

552

characterization of poly(vinyl alcohol) hydrogel with different hydrolysis degree and

553

chemically crosslinked with glutaraldehyde, Mater. Sci. Eng- C. 28 (2008) 539-548.

554

[26] K. H. Leong, L. Y. Chung, M. I. Noordin, K. Mohamad, M. Nishikawa, Y. Onuki, M.

555

Morishita, K. Takayama, Carboxymethylation of kappa-carrageenan for intestinal-targeted

556

delivery of bioactive macromolecules, Carbohydr. Polym. 83 (2011) 1507-1515.

RI PT

551

557

SC

558 559

M AN U

560 561 562 563

EP AC C

565

TE D

564

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ACCEPTED MANUSCRIPT 1

Table 1. Representation of formulation code, different formulation variables, DEE, particle

2

size, PDI and equilibrium water uptake.

3

Gum:PVA

GA

Drug

DEE%

Particle

code

(Am-g-

(mL)

loading

(±SD, n=3)

size

LBG:PVA)

7 8 9

1:2

5.0

50

73.51±2.78

A2

1:1

2.5

25

51.32±1.94

A3

1:1

5.0

50

62.83±1.04

A4

1:1

5.0

25

A5

1:1

2.5

A6

1:2

A7

1:2

A8

1:2

A9

0:1

449.46

1.18

SC

A1

pH 6.8

235.13

317.28

1.03

281.55

354.17

608.76

1.12

252.49

332.10

59.74±1.82

551.91

1.32

247.36

328.97

50

55.29±1.43

766.06

1.00

293.41

361.58

2.5

50

68.11±2.15

519.36

1.09

255.27

328.85

2.5

25

65.68±1.55

476.12

0.82

248.53

326.77

TE D

M AN U

659.42

5.0

25

72.17±1.36

429.70

0.99

229.15

313.74

2.5

50

52.13±2.88

371.06

0.85

129.68

163.47

EP

6

pH 1.2

(µm)

AC C

5

Equilibrium water uptake (%)

[d(0.5)]

(%)

(w/w)

4

PDI

RI PT

Formulation

10 11

12 43

ACCEPTED MANUSCRIPT 13

Table 2.Representation of kinetic modelling of in vitro dissolution data Korsemeyer-Peppas Formulation

Zero

First

Higuchi

Hixson

code

order

order

kinetic

Crowell

A1

0.908

0.696

0.968

A2

0.906

0.699

A3

0.912

A4

R2

0.934

0.969

0.953

0.962

0.928

0.986

0.721

0.969

0.94

0.864

0.915

0.72

0.97

0.94

0.845

A5

0.929

0.742

0.972

0.947

0.86

A6

0.879

0.677

0.952

0.916

0.983

A7

0.864

0.664

0.938

0.902

1.032

A8

0.893

0.679

0.962

A9

0.863

0.651

0.938

15

T80%

0.391

0.219

0.353

0.959

0.195

0.282

0.956

0.151

0.263

0.961

0.151

0.263

0.942

0.239

0.385

0.931

0.262

0.413

SC

0.949

0.926

0.928

0.948

0.221

0.366

0.9

1.078

0.929

0.284

0.438

AC C

EP

TE D

16

M AN U

14

0.24

RI PT

n

T50%

44

ACCEPTED MANUSCRIPT Figure captions: Figure 1: Pictorial representation of microspheres formation by emulsion crosslinking method. Figure 2: Three dimensional response surface plots: showing the effects of synthetic

Cumulative percentage drug release in dissolution study.

RI PT

condition on (a) DEE%(b) Particle size (c) Swelling in pH 1.2 (d) Swelling in pH 6.8 (e)

Figure 3: Proposed reaction mechanism of formation of acrylamide grafted locust bean gum

SC

and glutaraldehyde crosslinked acrylamide grafted locust bean gum poly(vinyl alcohol) hybridized network.

M AN U

Figure 4: FTIR spectra of PVA, physical mixture (PM), placebo, drug loaded formulation (DLF), buflomedil hydrochloride (BH) and acrylamide grafted locust bean gum (Am-gLBG). Figure 5: Solid state

13

C NMR spectra of Am-g-LBG, Am-g-LBG-PVA microspheres and

TE D

PVA.

Figure 6: DSC spectra of buflomedil hydrochloride (BH), PVA, Am-g-LBG, placebo microspheres and drug loaded microspheres.

EP

Figure 7: XRD patterns of BH, drug loaded microspheres and placebo microspheres. Figure 8: Scanning electron microscopy images of (a) Group of PVA microspheres (b) Am-

AC C

g-LBG-PVA network microspheres prepared by 2.5 mL GA (c) Am-g-LBG-PVA network microspheres prepared by 5.0 mL GA (d) Surface of Am-g-LBG-PVA network microspheres prepared by 5.0 mL GA (e-f) Cross sectional image of Am-g-LBG-PVA microspheres.. Figure 9: Particle size distribution of Am-g-LBG-PVA CR particles (A1-A9). Figure 10: In vitro dissolution characteristics of different batches of Am-g-LBG-PVA CR microspheres. 26

ACCEPTED MANUSCRIPT

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Am-g-LBG was added slowly and stirred for 3hr

0.5 mL 1 N Hcl was added and stirred for 30 min

Solution was cooled to room temperature

M AN U

SC

PVA dissolved in water (80⁰C)

Am-g-LBG-PVA blend was slowly emulsified with paraffin

Light liquid paraffin with Tween 80 mixed for 1 hr in a propeller stirrer

AC C

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Required amount of 25% GA was added and stirred for 3 h (500 RPM)

Particles vacuum dried in petridish and kept in desiccator until further use

Particles formed

Particles were filtered washed with acetone and 0.1 M glycine

SEM image of the particles

Figure 1: Pictorial representation of microspheres formation by emulsion crosslinking method. 27

ACCEPTED MANUSCRIPT

Design-Expert® Software Factor Coding: Actual DE% 73.51

(a)

51.32 X1 = A: Gum Polymer ratio X2 = B: Glutaraldehyde

75

Actual Factor C: % Drug loading = 0

70

RI PT

65

DE%

60 55 50

1

0.5 0

SC

1

0.5

0

-0.5

B: Glutaraldehyde

-0.5

A: Gum Polymer ratio

-1

M AN U

-1

Design-Expert® Software Factor Coding: Actual PS 766.06 429.7

700

(b)

600

PS

Actual Factor C: % Drug loading = 0

TE D

800

X1 = A: Gum Polymer ratio X2 = B: Glutaraldehyde

500

AC C

EP

400

1

0.5

0

B: Glutaraldehyde

1 0.5

-0.5 0 -0.5 -1

-1

A: Gum Polymer ratio

28

ACCEPTED MANUSCRIPT Design-Expert® Software Factor Coding: Actual Swelling(1.2) 293.41 229.15

(c)

X1 = A: Gum Polymer ratio X2 = B: Glutaraldehyde

300

Actual Factor C: % Drug loading = 0

260 240

RI PT

Swelling(1.2)

280

220

1

1

0.5

0.5

0

B: Glutaraldehyde

-0.5

SC

0

-0.5

A: Gum Polymer ratio

X1 = A: Gum Polymer ratio X2 = B: Glutaraldehyde

370

360

350

340

330

AC C

EP

Swelling(6.8)

Actual Factor C: % Drug loading = 0

(d)

TE D

Design-Expert® Software Factor Coding: Actual Swelling(6.8) 361.58 313.74

-1

M AN U

-1

320

310

1 1 0.5

0.5 0

0 -0.5

B: Glutaraldehyde

-0.5 -1

29

-1

A: Gum Polymer ratio

ACCEPTED MANUSCRIPT Design-Expert® Software Factor Coding: Actual CPR 93.16

(e)

59.99 X1 = A: Gum Polymer ratio X2 = B: Glutaraldehyde Actual Factor C: % Drug loading = 0

100 90

70

RI PT

CPR

80

60 50

1

1

0.5

0.5

0

0 -0.5

-0.5

SC

B: Glutaraldehyde

A: Gum Polymer ratio

-1

M AN U

-1

Figure 2: Three dimensional response surface plots: showing the effects of synthetic condition on (a) DEE%(b) Particle size (c) Swelling in pH 1.2 (d) Swelling in pH 6.8 (e)

AC C

EP

TE D

Cumulative percentage drug release in dissolution study.

30

ACCEPTED MANUSCRIPT CH2 OH O H

OHH H OH H

H O OH H2 C

H O

H H OH OH H

H H OH

O OH H

OH H

H

O

O CH2 OH

H

CH2 OH O H H OH OH H H H

H

O

H OH

OH H H

O CH2 OH

RI PT

Locu st bean gum

Ceric ammoninum nitrate as redox initiator Microwave irradiation

H OH

H OH

H

Acrylamide

O H

CH 2 O

H H OH OH H

OH H

H

CH2 H H OH

H H OH

O OH H

O

O CH2 O CH2

H

C

H

SC

OHH

OH O H

OH O

OH H H

H

H

CONH2

Initiation

H

O

OH H H

OH

OH

H

H

OH

n OH

H

H

CH2 O

H H OH

H

H

OH H

H

OH O

O

H

CH 2 O CH 2 C CH 2

CONH2

C

CONH2

C

CONH 2

CH C

OH

H H

O

CONH2

O

O

H

H

H

H

O

H OH

OH H H

O

H CH 2

CH O

CH2 CONH2

CH2 C

H

O

AC C

CH

H OH O

(CH2 )3

CH2

H

OH H

Elongation and termination

O

CH2

EP

H

CH2

H

O

OH

OH H

Gluteraldehyde Hydrochloric acid

Polyvinyl alcoh ol

O

H

OCH(CH2)3 CHO

TE D

CH2

H

H2 C

Acrylamide grafted locust bean gum CH2CH

H

O

M AN U

CH2

CONH 2

CH2 H

CH2 CH

C

CONH2

H 2C

n H

C

CONH2

CH H

C

CONH2

Hybridized network of acrylamide graf ted locu st bean gum and Poly(vinyl alcohol)

Figure 3: Proposed reaction mechanism of formation of acrylamide grafted locust bean gum and glutaraldehyde crosslinked acrylamide grafted locust bean gum-poly(vinyl alcohol) network. 31

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 4: FTIR spectra of PVA, physical mixture (PM), placebo, drug loaded formulation (DLF), buflomedil hydrochloride (BH) and acrylamide grafted locust bean gum (Am-g-

AC C

EP

LBG).

32

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Figure 5: Solid state 13C NMR spectra of Am-g-LBG, Am-g-LBG-PVA network and PVA.

33

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

Figure 6: DSC spectra of buflomedil hydrochloride (BH), PVA, Am-g-LBG, placebo

AC C

EP

microspheres and drug loaded microspheres.

34

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Figure 7: XRD patterns of BH, drug loaded microspheres and placebo microspheres.

35

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 8: Scanning electron microscopy images of (a) Group of PVA microspheres (A9) (b) Am-g-LBG-PVA CR microspheres prepared by 2.5 mL GA (c) Am-g-LBG-PVA CR microspheres prepared by 5.0 mL GA (d) Surface of Am-g-LBG-PVA CR microspheres prepared by 5.0 mL GA (e-f) Cross sectional image of Am-g-LBG-PVA microspheres.

36

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

A1

37

A2

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

A3

38

A4

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

A5

39

A6

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

A7

40

A8

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

A9

AC C

EP

TE D

Figure 9: Particle size distribution of Am-g-LBG-PVA hybridized particles (A1-A9).

41

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 10: In vitro dissolution characteristics of different batches of Am-g-LBG-PVA CR

AC C

EP

TE D

microspheres.

42