Preparation, characterization and evaluation of antibacterial properties of epirubicin loaded PHB and PHBV nanoparticles

Journal Pre-proof Preparation, characterization and evaluation of antibacterial properties of epirubicin loaded PHB and PHBV nanoparticles

Kousar Perveen, Farha Masood, Abdul Hameed PII:

S0141-8130(19)36775-3

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.049

Reference:

BIOMAC 14076

To appear in:

International Journal of Biological Macromolecules

Received date:

23 August 2019

Revised date:

30 November 2019

Accepted date:

5 December 2019

Please cite this article as: K. Perveen, F. Masood and A. Hameed, Preparation, characterization and evaluation of antibacterial properties of epirubicin loaded PHB and PHBV nanoparticles, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2019.12.049

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© 2018 Published by Elsevier.

Journal Pre-proof Title: Preparation, characterization and evaluation of antibacterial properties of epirubicin loaded PHB and PHBV nanoparticles Kousar Perveen a, *, Farha Masood a, # 1, *, Abdul Hameed b a

Department of Biosciences, COMSATS University, Islamabad, Pakistan

b

SA Centre for Interdisciplinary Research in Basic Sciences, International Islamic University,

Islamabad, Pakistan

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* Both authors have made an equal contribution in this manuscript.

#1 Corresponding author address, tel. number (with country and area code): [email protected]; Department of Biosciences, COMSATS University, Islamabad, Pakistan, 00925190496122

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Journal Pre-proof Preparation, characterization and evaluation of antibacterial properties of epirubicin loaded PHB-PEG and PHBV-PEG nanoparticles Kousar Perveen, Farha Masood, Abdul Hameed ABSTRACT Poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) are considered as ideal drug carriers due to their non-toxic, biodegradable and biocompatible nature.

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In this study, the epirubcin (EPI) was used as a model drug. The blank (PHBo, PHBVo) and drug

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loaded (EPI-PHB-PEG, EPI-PHBV-PEG) nanoparticles were prepared by nanoprecipitation method. The average particle size, polydispersity index and zeta potential of blank and drug

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loaded nanoparticles were determined. While, the morphology of blank and drug loaded nanoparticles was evaluated by scanning electron microscopy. The drug loading efficiency of

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EPI-PHB-PEG nanoparticles was higher in comparison to EPI-PHBV-PEG nanoparticles. A

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sustained release of EPI was found over a period of 8 days at pH 4 from EPI-PHB-PEG and EPIPHBV-PEG nanoparticles in comparison to faster drug release at pH 7. The assessment of

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antibacterial properties of the drug loaded nanoparticles showed significant antibacterial properties against methicillin resistant Staphylococcus aureus, Escherichia coli and

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Key words:

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Pseudomonas aeruginosa bacterial strains as compared to an equivalent amount of free drug.

Antibacterial properties; epirubicin; poly-3-hydroxybutyrate; poly-3-hydroxybutyrate-co-3hydroxyvalerate; nanoparticles

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Journal Pre-proof INTRODUCTION An alarming increase in the emergence of new and existing diseases has motivated the researchers to introduce new methods of disease treatment for improving the patient compliance. Traditional disease treatment systems suffer several serious drawbacks such as shorter half-life of drug as a result of its degradation in human body, toxicity associated with its repetitive intake, failure to reach to the target site and non-specific tissue distribution. In this regard, use of biomaterials for drug delivery is a good choice. A biomaterial is a non-viable material that is

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designed or tailored to interact with biological systems for therapeutic and diagnostic purpose [1] [2] [3]. The key features of biomaterial-based drug delivery systems (DDS) include tunable

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stability and enhanced drug efficacy [4][5][6].

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hydrophilicity, chemical modifications, biocompatibility, non-toxicity, suitable hydrolytic

The nanotechnology has brought a revolution in the biomaterial-based DDS. Polymer

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nanoparticles are colloidal particles that lie between 1-1000 nm in diameter. Polymer

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nanoparticles have an integral role in the fabrication of biomaterial-based DDS due to their unique advantages, including controlled drug release, enhanced encapsulation efficiency,

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decreased side effects, reduced dose and dosing frequency [7][8][9][10]. Moreover, polymeric nanoparticles can be tailored to overcome the bioavailability and permeability issues of

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hydrophilic drug [11] [12].

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The Epirubicin (EPI) is an anthracycline. The EPI induces DNA damage by inhibiting the topoisomerase II activity, thus inducing apoptosis [13] [14]. The EPI is available for clinical trials in the form of hydrochloride. However, this formulation has inconsistency in circulation, ineffective selectivity and/or insufficient distribution in tissues [15] and poor bioavailability [16]. The poly-3-hydroxyalkanotes (PHA) are natural, non-toxic, biodegradable and aliphatic microbial polyester. The poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3hydroxyvalerate (PHBV) are two important members of PHA family, which are widely used as drug carriers, scaffolds and bio-implants[17][18] [19] [20] [21] [22] [23]. The D-3hydroxybutyric acid (D-3HB) monomers are obtained during hydrolytic degradation of PHB, which are present in the human blood under normal conditions. The rate of degradation of PHA is dependent on their molecular mass and monomeric composition [24][25]. The PHA is 3

Journal Pre-proof considered as a suitable candidate to encapsulate hydrophobic drugs due to its lipophilicity. However, the PHA nanoparticles could be tailored to ensure better encapsulation efficiency of hydrophilic drugs. Previously, PHB nanoparticles were used to encapsulate doxycycline hyclate (DOXY), which is a hydrophilic broad-spectrum antibiotic [5]. The resulting DOXY loaded PHB nanoparticles demonstrated potent antibacterial activity against E. coli and S. aureus as a result of

higher

drug

loading

efficiency.

In

another

study,

poly(3-hydroxybutyrate-co-3-

hydroxyhexanoate) (PHBHHx) nanoparticles were used to improve the

bioavailability of

insulin, (a water-soluble hormone) [4]. For this purpose, the insulin phospholipid complex was

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prepared first and then this complex was loaded into biodegradable PHBHHx nanoparticles.

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Approximately 20% of encapsulated insulin were released from the insulin phospholipid complex loaded PHBHHx nanoparticles over a period of 31 days with an initial burst release of

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5.42% in the first 8 h. Moreover, pharmacological bioavailability of insulin was also significantly enhanced. The polyethylene glycol (PEG) is a hydrophilic, non-toxic and blood

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compatible polymer and is considered to be safe for use by the U.S. food and drug administration

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[26]. The PEGylated PNPs has a long circulation time inside the body, thus enhancing the bioavailability of drugs. The PHB/PEG composite films exhibited better cell compatibility and

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faster degradation rate in comparison to pure PHB [27]. Therefore, in this study, Bacillus cereus FA11 was used to produce PHBV. While, the PHB was

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obtained from Sigma. The EPI was used as a model hydrophilic drug. Blank and EPI loaded

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PHB and PHBV nanoparticles were prepared by the nanoprecipitation method. During nanoparticle preparation, different amount (0.5, 1 and 2 mg) of the drug were added to obtain the maximum drug encapsulation efficiency and drug loading content. The particle size and zeta potential of blank and drug loaded nanoparticles were examined. The scanning electron microscopy, energy dispersive x-ray spectroscopy, Fourier transform infrared spectroscopy and x-ray diffraction analysis were used for characterization of blank and drug loaded PHB and PHBV nanoparticles. The release of EPI from PHB and PHBV nanoparticles were studied at pH 7 and pH 4 under in-vitro conditions. Furthermore, antibacterial properties of drug loaded PHB and PHBV nanoparticles were determined against different Methicillin resistant Staphylococcus aureus (Gram-positive), Escherichia coli and P. aeruginosa (Gram-negative) bacterial strains. MATERIALS AND METHODS 4

Journal Pre-proof Materials Poly-3-hydroxybutyrate (PHB, Mw= 150.00 kDa), polyethylene glycol (PEG, Mw=1450 Da), polyvinyl alcohol (PVA) (Mw = 2.2×104, 88% hydrolyzed), acetone and epirubincin hydrochloride (EPI. HCl) were purchased from Sigma. The PHBV (viscosimetric Mw=1.85x105 kDa) containing 15% molar of 3-hydroxyvalerate units was fermented from Bacillus cereus FA11 in our laboratory. The molecular weight of PHBV was determined by viscometer using intrinsic viscosity method. All reagents and solvents were of analytical or HPLC grade and used

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Preparation of blank and drug loaded nanoparticles

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without further purification. Deionized water (stakpure, Germany) was used in all experiments.

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The nanoprecipitation method was used for preparation of blank and drug loaded nanoparticles with some necessary modifications [28]. Briefly, for preparation of drug loaded EPI-PHB-PEG

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nanoparticles, the PHB (50 mg) and PEG (50 mg) were dissolved in 3 mL acetone as an organic

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phase. An aqueous phase was prepared by dissolving the polyvinyl alcohol (0.07%). The EPI. HCl was dissolved in an aqueous phase by adjusting it pH at 3. The organic phase was added

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into the aqueous phase in a dropwise manner during stirring at room temperature. Then, the mixture was gently stirred for overnight. The drug loaded nanoparticles were collected by centrifugation using a centrifuge (H16, Shimadzu) at 10000 rpm followed by three washes with

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deionized water and dried in vacuum heating oven (Memmert GmbH, Germany). The blank

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PHBo nanoparticles were prepared by the same method as described above without taking drug in aqueous phase. The purified PHBV was used for preparation of blank PHBVo and dug loaded EPI-PHBV-PEG nanoparticles. The composition and yield of different delivery systems prepared in this study are given in the Table I. Table I: Composition and identification code of nanoparticles. Polymer

PHB

EPI (mg)

Identification code

Yield of nanoparticles (%)

0

PHBo

26.49±0.33

0.5

EPI-PHB-PEG0

26.83±1.25

1

EPI-PHB-PEG1

26.25±0.65

2

EPI-PHB-PEG2

23.13±0.98 5

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PHBV

0

PHBVo

28.23±0.98

0.5

EPI-PHBV-PEG0

24.51±0.78

1

EPI-PHBV-PEG1

21.99±1.05

2

EPI-PHBV-PEG2

19.06±0.88

Characterization studies

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Scanning electron microscope (SEM) analysis

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SEM analysis was carried by using the JEOL (Japan) scanning electron microscope operated at an accelerating voltage of 1.0 kV. The nanoparticle suspension was prepared by dissolving 1 mg

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dried nanoparticles in 1 mL water. This suspension was dried over glass cover slip and put over

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aluminum stubs using both side adhesive carbon tape and viewed under the microscope after

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gold coating.

Energy dispersive X-ray spectroscopy (EDX)

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EDX was also performed to investigate the elemental composition of blank and drug loaded

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Particle size analysis

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nanoparticles under normal conditions in specific position.

The particle size, size distribution (polydispersity index, PDI) and zeta potential of blank and drug loaded nanoparticles was determined using Zeta Sizer Nano ZS 90 instrument (Malvern Instruments, Ltd., UK) at 25°C. The nanoparticles (1 mg) were dissolved in 1 mL water and the average size was evaluated by dynamic light scattering (DLS) technique at a room temperature. X-ray diffraction (XRD) analysis XRD analysis was performed using XRD equipment (X’Pert Pro PANalytical) with CuKα radiation operating at 40 kV and 30 mA. The scanning was performed from 5° and 30° at a scanning rate of 2θ/min. The average d-spacing value 𝑑̅ was calculated using Bragg’s law:

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Journal Pre-proof ̅̅̅̅ 𝑠𝑖𝑛𝜃̅ 𝑛𝜆 = 2𝑑 Fourier transform infrared (FTIR) spectroscopy The chemical structures of blank and drug loaded nanoparticles were characterized by using a Nicolet 6700 FTIR spectrophotometer (Thermo Electron Corp, Marietta, OH). The spectrum of the sample was obtained using an attenuated total reflectance mode with a diamond crystal in the

Drug loading (DL) and encapsulation efficiency (EE)

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range from 4000 to 500 cm-1.

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About 5 mg drug loaded NPs were dissolved in 1 mL DMSO. This solution was centrifuged at

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14000 rpm for 15 min. The quantity of the EPI loaded in the drug loaded nanoparticles was determined by taking the optical density of supernatant at 480 nm using double-beam UV–vis

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spectrophotometer (Shimadzu). A standard curve of EPI.HCl (2-20 ug) (R2 = 0.9588) was made.

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The drug loading content (%) and encapsulation efficiency (%) were calculated as follows: Weight of drug in nanopaticles × 100 Weight of drug loaded nanoparticle

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Drug loading content (%) =

Total amount of drug added − Free drug × 100 Total amount of drug added

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In-vitro release studies

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Drug encapsulation efficiency (%) =

In-vitro release studies of EPI from EPI-PHB-PEG and EPI-PHBV-PEG nanoparticles were studied by a dialysis method at pH 4 and pH 7. An aliquot of sample was put within a hydrated dialysis membrane (molecular weight cut-off size of 8-14 kDa; Sigma). Then it was dialyzed against 35 mL of PBS at 37°C in a shaking incubator (JSSI-300T, Korea) at 150 rpm. An equivalent amount of free drug EPI.HCl was also dialyzed for comparison. The sample (3 mL) was taken out and replaced with same volume of fresh PBS at regular intervals. The concentration of drug released in the sample was determined by taking optical density at 480 nm. The in-vitro drug release data of EPI-PHB-PEG1 and EPI-PHBV-PEG1 was fitted into zero order, first order, Higuchi, Hixson-crowell and Korsmeyer-peppas’ s model kinetic models. The 7

Journal Pre-proof optimum model was subsequently selected, based on comparison of the relevant correlation coefficients [29]. Antibacterial studies The antibacterial activity of free drug, EPI-PHB-PEG1 and EPI-PHBV-PEG1 nanoparticles was determined according to the broth dilution assay [30]. The selected bacterial strains include methicillin resistant Staphylococcus aureus (Gram-positive bacteria), Escherichia coli ATCC 11775 and Pseudomonas aeruginosa ATCC 27853 (Gram-negative bacteria). The inoculum preparation of each bacterial strain was done in a sterilized nutrient broth. The optical density of

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inoculum was adjusted to 108 CFU/mL. Different dilutions of nanoparticles, i.e. 1000, 750, 500, 250 µg/mL and equivalent quantity of epirubicin were added into each test tube along with

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inoculum. The test tubes were then incubated at 37°C and 150 rpm for 18 hours. Agar spotting

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method was used to determine CFU/mL [31]. The fold change of treatments as compared to

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epirubicin (free drug) was calculated according to the equation [32].

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RESULTS AND DISCUSSION

Treated − Control Control

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Fold change =

Characterization of nanoparticles

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Scanning electron microscopy

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Fig. 1 (a-b) shows the morphologies of PHBo, EPI-PHB-PEG, PHBVo and EPI-PHBV-PEG loaded nanoparticles. The PHBo, EPI-PHB-PEG, PHBVo and EPI-PHBV-PEG nanoparticles demonstrated the spherical morphology. The ellipticine loaded PHBV nanoparticles showed the spherical morphology [33]. However, few agglomerates of nanoparticles were observed in scanning electron micrographs of PHBVo and EPI-PHBV-PEG nanoparticles. This behavior revealed that surface energies of both systems were not completely stabilized by PVA. EDX analysis The EDX analysis of PHBo, EPI-PHB-PEG, PHBVo and EPI-PHBV-PEG loaded nanoparticles are given in Fig. 2. Carbon, hydrogen and oxygen atoms were found in EDX analysis of blank 8

Journal Pre-proof and drug loaded nanoparticles. Additionally, the nitrogen atom was found in case of drug loaded EPI-PHB-PEG and EPI-PHBV-PEG nanoparticles due to presence of the epirubicin within the

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polymer matrix.

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PEG (d) nanoparticles.

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Fig. 1: Scanning electron micrographs of PHBo (a), EPI-PHB-PEG (b), PHBVo (c) and EPI-PHBV-

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Journal Pre-proof Fig. 2: EDX analysis of PHBo (a), EPI-PHB-PEG (b), PHBVo (c) and EPI-PHBV-PEG (d) nanoparticles.

Particle size and polydispersity index The kinetics of drug release from polymeric nanoparticle are dependent on average particle size and polydispersity index of nanoparticles [34]. The average diameter and PDI of PHBo, EPIPHB-PEG, PHBVo and EPI-PHBV-PEG nanoparticles is given in Table II. The diameters of drug loaded nanoparticles were increased as compared to blank nanoparticles. This increase in

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diameters of drug loaded nanoparticles was due to an adsorption and encapsulation of epirubicin

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within the polymeric matrix. The diameter of roxithromycin loaded PLGA nanoparticles was increased as a result of roxithromycin encapsulation within the PLGA nanoparticles [35]. The

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higher values of PDI observed in case of drug loaded EPI-PHB-PEG and EPI-PHBV-PEG

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nanoparticles were a result of drug encapsulation within the polymeric matrix in comparison to blank PHBo and PHBVo nanoparticles. Similarly, a high value of PDI was found in the case of

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ellipticine loaded PHBV-15 nanoparticles as compared to blank PHBV-15 nanoparticles [33].

Zeta potential

Diameter (nm) 150.3 ±0.7 160.6±1.4 140.5±1.5 152.3±0.6

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Samples PHBo EPI-PHB-PEG PHBVo EPI-PHBV-PEG

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nanoparticles.

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Table II: The diameter, polydispersity index and zeta potential of blank and drug loaded

PDI 0.9±1.2 0.9±1.4 0.7±1.9 0.8±0.6

Zeta potential (mV) -16 -18 -21 -23

The zeta potential values of PHBo, EPI-PHB-PEG, PHBVo and EPI-PHBV-PEG nanoparticles is given in Table II. The higher negative zeta potential of nanoparticles is demonstrating their stability in the suspension [36][37]. Both blank and drug loaded nanoparticles exhibited negative value of zeta potential due to terminal -COOH group present in PHA. The value of zeta potential ranged from -16.1 to -23 mV. There was adsorption of cationic drugs (such as EPI) on the surface of nanoparticles due to electrostatic interactions.

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Journal Pre-proof XRD analysis Fig. 3 (a-b) shows the X-ray diffractograms of the blank and drug loaded nanoparticles. Two characteristic reflections appeared at 2θ = 13.9° and 16.8° in the diffractrogram of blank PHBo and PHBVo nanoparticles were attributed to the (020), (110) lattice planes of the orthorhombic unit cell of PHB, respectively [38] [39]. On the other hand, a significant decrease in intensities of all characteristic reflections was observed in the diffractogram of EPI-PHB-PEG and EPIPHBV-PEG drug loaded nanoparticles. Moreover, no additional reflections related to EPI were

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observed in the diffractogram of EPI-PHB-PEG and EPI-PHBV-PEG nanoparticles, which indicated the phase transition of drug molecules during the formation of nanoparticles. This

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behavior was possibly due to the development of strong electrostatic intermolecular interactions between the polymer matrix and drug molecules. Similarly, the disappearance of characteristic

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reflection of roxithromycin was evident in XRD diffractrogram of HPβCD-ROX/PLGA NPs and

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HPβCD-ROX/PLGA NPs [35]. The crystallite size of blank and drug loaded nanoparticles is given in Table III. The size of crystallite at (020) plane was increased in case of drug loaded

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PHB and PHBV nanoparticle as a result of drug entrapment.

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Fig. 3: Wide angle X-ray diffractrogram of PHBo, EPI-PHB-PEG (a), PHBVo and EPI-PHBV-PEG (b) nanoparticles at 2θ = 2-40°. 12

Journal Pre-proof Table III: Crystallite size and peak position determined from the X-Ray diffraction

EPI-PHB-PEG

PHBVo

EPI-PHBV-PEG

Peak position (°2θ)

FWHM (°2θ)

Crystallite size (nm)

020

13.46

0.3149

25.42

110

17.0424

0.2755

29.18

020

13.5143

0.2952

27.12

110

16.8919

0.4723

17.02

020

13.4178

0.2362

33.89

110

16.5602

0.9446

8.51

020

13.4728

0.2362

33.90

110

16.9406

0.2952

27.23

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PHBo

(hkl) planes

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Sample code

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patterns of blank and drug loaded nanoparticles.

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FTIR analysis

Fig. 4 shows the FTIR spectra of PHB, PHBV, PEG, and EPI. The spectrum of PEG

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demonstrated the absorption bands at 3448 cm-1 (OH stretching vibration), 2866 cm-1 (CH stretching vibration), 1457 and 1344 cm-1 (CH bending vibration), 1290 cm-1 (OH stretching

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vibration) and 1093 cm-1 (C-O-H stretching vibration), respectively[40]. While, the IR spectrum of PHB is characterized by absorption bands at 2932, 2975, 1375, 1456 cm-1 (C-H stretching

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vibrations form -CH2, -CH3 and -CH bonds), 1720 cm-1 (C=O stretching) and 1275 cm-1 (C-O stretching) [41] [42]. In case of the IR spectrum of PHBV, the absorption bands at 2956 and 2872

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cm-1 were corresponding to the anti-symmetric and symmetric vibrations of C-H bonds, respectively. The absorption bands at 1710 and 1270 cm-1 represented ester carbonyl group (C=O) and C-O stretching vibration of PHB [23]. The absorption bands at 1456 and 1390 cm-1 were indicating the presence of anti-symmetric and symmetric bending of C-H bonds, respectively [43]. The absorption bands present at 1142 and 1130-1100 cm-1 were corresponding to the anti-symmetric vibration of C-O-CO linkage between monomers and the anti-symmetric vibrations of -C-O-C- bonding in PHBV [40]. The IR spectrum of pure EPI showed characteristic bands at 3402 cm-1 (N-H stretching vibrations of the primary amines), 3250 cm-1 (O-H stretching vibrations), 1723 (C=O stretching), 1618 cm-1 (-C=N stretching vibrations of EPI aromatic ring) and 870 cm-1 (N-H wagging vibrations)[44].

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Fig. 4: FTIR spectra of poly-3-hydroxybutyrate (PHB), poly-3-hydroxybutyrate-co-3-

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hydroxyvalerate (PHBV), polyethylene glycol (PEG), and epirubicin drug (EPI) in the

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range of 4000-400 cm-1.

Fig 5 (a-b) shows FTIR spectra of PHBo, EPI-PHB-PEG, PHBVo and EPI-PHBV-PEG

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nanoparticles. The IR spectra of blank and drug loaded nanoparticles showed the characteristics absorption bands of raw components. There was no considerable change in intensity of the

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absorption bands of blank and drug loaded nanoparticles. A broad band appeared in a region from 3445-3164 cm-1 in the IR spectrum of PEG was disappeared after nanoparticle formation due to condensation reactions between PEG and PVA. A small absorption band at 1510 cm-1 was observed in the FTIR spectrum of drug loaded nanoparticles as a result of encapsulation of EPI. A sharp absorption band observed in the FTIR spectrum of pure EPI at 870 cm -1 was diminished in the FTIR spectra of drug loaded nanoparticles due to the conjugation of EPI molecules within the polymeric matrix of PHB, PVA and PEG. The hydrogen bonding, electrostatic and other non-covalent interactions are thought to be involved in loading of EPI on PHB-PEG and PHBVPEG nanoparticles. Similar mechanism of EPI loading in poly(butyl cyanoacrylate) nanoparticles was already reported [45]. In another study, the FTIR spectrum of EPI-loaded Fe@Si-PW

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Journal Pre-proof nanoparticles demonstrated the shift of absorption band from 1620 cm-1 (amide bond) to 1611

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cm-1 as a result of development of electrostatic interaction and/or hydrogen bonding [46].

Fig. 5: FTIR spectra PHBo, EPI-PHB-PEG1, PHBVo and EPI-PHBV-PEG1 nanoparticles in the range of 4000-400 cm-1 (a) and 2000-400 cm-1(b). 15

Journal Pre-proof Drug encapsulation efficiency The comparison of drug loading and drug encapsulation efficiencies of EPI loaded PHB and PHBV nanoparticles containing variable amounts of EPI is given in Fig. 6. An increase in loading efficiency of EPI in EPI-PHB-PEG and EPI-PHBV-PEG nanoparticles was observed with an increase in the amount of EPI. Further increase in the amount of EPI led to decrease in the loading efficiency. A high loading efficiency of EPI observed in case of drug loaded PHB nanoparticles was due to its high molecular weight. On the other hand, the higher valerate

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content was responsible for high drug encapsulation efficiency in case of EPI-PHBV-PEG nanoparticles. Epirubcin has a low tendency to bind to lipophilic polymers and organic phase,

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therefore, generally leaks into outer aqueous phase and led to low drug content. In this study, the

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pH of the aqueous phase was adjusted to 3 in order to improve loading efficiency as reported

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earlier in case of epirubicin loaded poly-lactic-co-glycolic acid nanoparticles [47].

Fig. 6: A comparison of drug encapsulation efficiency (%) and drug loading content (%) of different drug delivery systems (n=2). 16

Journal Pre-proof In-vitro drug release studies Drug release studies by the dialysis method showed relatively fast diffusion of free EPI·HCl through the dialysis membrane within the first 3 h at pH 4 and pH 7 (Fig. 7). Approximately 15% of the total drug content was released from the EPI-PHB-PEG1 nanoparticles within the first 3 h. Further release of the drug was very slow. This fast released 15% corresponded to the nonentrapped drug in the formulation. While, the entrapped EPI molecules developed a relatively strong association with the nanoparticles and only a small fraction of the loaded drug was

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released during the experiment. A sustained release of EPI was found over a period of 8 days at pH 4 from EPI-PHB-PEG1 and EPI-PHBV-PEG2 nanoparticles, indicating the pH sensitive

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release of EPI from a polymer matrix. On the other hand, the fast release of EPI was observed at

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pH 7 from EPI-PHB-PEG1 and EPI-PHBV-PEG2 nanoparticles over a period of 2 days.

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Different kinetic models, i.e., zero order, first order, Higuchi model, Korsmeyer–Peppas were used to analyze the drug release kinetics (Table III).

The analysis of relevant correlation

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coefficients of kinetic models suggested the Higuchi model as a best fit model to explain the release profile of EPI from EPI-PHB-PEG1, EPI-PHBV-PEG2, EPI-PHB-PEG1, EPI-PHBV-

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PEG2 at pH 7 and 4. The n values of both systems obtained from Korsmeyer-peppas’s model was less than 0.45 (quasi Fickian-diffusion)[48]. The observations indicated the drug release

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mechanisms predominantly followed by matrix diffusion with little involvement of erosion.

PHBV-PEG1.

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Table III: Mathematical kinetic models applied to in-vitro drug release of EPI-PHB-PEG1 and EPI-

Kinetic models System

EPI-PHBPEG1 EPI-PHBVPEG2

Parameter pH

Zero order R2

First order R2

7

0.8776

4

Korsmeyer-peppas’ s model 2 R n 0.306 0.9218

Higuchi

Hixon

R2

R2

0.9392

0.9439

0.661

0.6834

0.7551

0.7714

0.4598

0.2843

0.133

7

0.8776

0.9265

0.9516

0.7012

0.9116

0.313

4

0.4669

0.5126

0.6815

0.5305

0.4976

0.122

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Fig. 7: In vitro cumulative drug release (%) of EPI from EPI-PHB-PEG1, and EPI-PHBV-PEG2 nanoparticles at pH 4 (a, c) and pH 7 (b, d).

Antibacterial properties

Fig. 8 (a-c) shows antibacterial activity of free drug and different concentrations of EPI-PHBPEG1, and EPI-PHBV-PEG2 nanoparticles against E. coli, P. aeruginosa and Methicillin resistant Staphylococcus aureus. Approximately 6.5-fold increase in antibacterial activity of EPI-PHBPEG1 nanoparticles was found against MRSA in comparison to an equivalent amount of free drug. The encapsulation of drug into polymeric core prevents its recognition by efflux pumps and thus its escape from drug-resistance mechanisms [49]. The kanamycin sulphate loaded PHB nanofibers significantly inhibited the growth of drug resistant Staphylococcus aureus due to the 18

Journal Pre-proof sustained release profile of drug from nanofibers compared to the free drug [50]. Similarly, the antibacterial activity of EPI-PHBV-PEG2 nanoparticles was 3.6-fold increased against E. coli as compared to free drug. While, approximately, one-fold increase in antibacterial activities of EPIPHB-PEG1 and EPI-PHBV-PEG2 nanoparticles was observed against P. aeruginosa than free drug. On the other hand, bacterial growth was evident in the presence of blank PHB-PEG and PHBV-PEG nanoparticles, thus indicating their no toxicity to bacterial cells. Sobhani, et al., reported that the antibacterial activity of chitosan loaded ciprofloxacin nanoparticles was higher against S. aureus (a Gram-positive bacterium) as compared to the E. coli (a Gram negative

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bacterium) [51].

Figure 8: Antibacterial activity of free drug and different concentrations of EPI-PHB-PEG1, and EPI-PHBV-PEG2 nanoparticles against E. coli (a) P. aeruginosa (b) and Methicillin resistant Staphylococcus aureus (c).

Conclusions The PHBV was produced by Bacillus cereus FA11 using shake flask fermentation. The PHB was used as a standard in this study. Epirubicin was used as a model drug. The blank (PHB-PEG, PHBV-PEG) and drug loaded (EPI-PHB-PEG, EPI-PHBV-PEG) nanoparticles were produced by a nanoprecipitation method. The blank and drug loaded nanoparticles showed the spherical 19

Journal Pre-proof shape with narrow size distribution and negative zeta potential values. The maximum drug loading efficiency was observed in case of EPI-PHB-PEG nanoparticles. A sustained release of EPI from EPI-PHB-PEG1 and EPI-PHBV-PEG1 nanoparticles was evident at pH 4 over a period of 8 days in comparison to free drug. Moreover, it is hereby shown that the encapsulation of EPI into PHB-PEG and PHBV-PEG nanoparticles considerably improved the antibacterial properties of the drug. The results of the present study demonstrated a great potential of PHB and PHBV based drug loaded nanoparticles for sustained release of hydrophilic drug in the biomedical field.

work

was

supported

by

the

research

provided

by

(Project

No:

-p

5312/Federal/NRPU/R&D/HEC/2016) to the Dr. Mas

funding

ro

This

of

ACKNOWLEDGEMENT

re

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Journal Pre-proof Author statement Farha Masood: Conceptualization, Writing- Original draft preparation, Editing, Supervision. Kousar Parveen: Data curation, Visualization, Investigation. Software, Methodology,

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Abdul Hameed: Supervision. Reviewing

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