Fabrication of Progesterone-Loaded Nanofibers for the Drug Delivery Applications in Bovine
© The Author(s). 2017
Received: 8 August 2016
Accepted: 9 December 2016
Published: 14 February 2017
Progesterone is a potent drug for synchronization of the estrus and ovulation cycles in bovine. At present, the estrus cycle of bovine is controlled by the insertion of progesterone-embedded silicone bands. The disadvantage of nondegradable polymer inserts is to require for disposal of these bands after their use. The study currently focuses on preparation of biodegradable progesterone-incorporated nanofiber for estrus synchronization. Three different concentrations (1.2, 1.9, and 2.5 g) of progesterone-impregnated nanofibers were fabricated using electrospinning. The spun membrane were characterized by scanning electron microscopy, X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis, and Fourier transform infrared spectroscopy. Uniform surface morphology, narrow size distribution, and interaction between progesterone and zein were confirmed by SEM. FTIR spectroscopy indicated miscibility and interaction between zein and progesterone. X-ray analysis indicated that the size of zein crystallites increased with progesterone content in nanofibers. Significant differences in thermal behavior of progesterone-impregnated nanofiber were observed by DSC. Cell viability studies of progesterone-loaded nanofiber were examined using MTT assay. In vitro release experiment is to identify the suitable progesterone concentration for estrus synchronization. This study confirms that progesterone-impregnated nanofibers are an ideal vehicle for progesterone delivery for estrus synchronization of bovines.
Electrospinning is a technique used to form nanoscale fibers. It is quite versatile for fabricating nanofibers from various synthetic or natural polymers . In literature , reported functional electrospun nanofibrous composite structures can also be produced by incorporating functional additives in the fiber matrix or on the fiber surface. The development of nanostructured systems for the delivery and sustained release of molecules towards specific targets represents a frontier area of nanoscience and nanotechnology, with the possibility of contributing substantially to advances in animal reproduction . Improving delivery techniques that minimize toxicity of drug has a significant effect on its efficacy. Overall, nanosized delivery systems enhance the therapeutic efficacy of several bioactive molecules, including reproductive hormones, by simply improving their pharmacokinetic and pharmacodynamic properties . These systems are able to carry a wide variety of molecules enhancing their sustained release, showing low systemic toxicity, allowing targeted treatment, and avoiding premature inactivation [5, 6]. Electrospun polymer-based fibers have been investigated for providing different types of controlled drug release profiles, such as immediate, delayed, sustained, and biphasic releases [7, 8]. Among them, sustained drug release is gaining considerable attention as a method of administering and maintaining desired drug concentrations in the blood within a specified therapeutic window [9–11].
Zein is a mixture of proteins with different molecular weights in corn gluten. Apart from biodegradability and biocompatibility, zein has low hydrophilicity, high elasticity, and film-forming capabilities, and it is considered a potential raw material for bioengineering application .
The artificial induction and synchronization of estrus in production animals is critical to ensure a positive balance of the cost-benefit equation of the artificial insemination related activities. The usual administration of hormones must be very precise. The controlled hormone release is a current technological challenge. One interesting agent to be tested in such delivery system is the progesterone, a steroid hormone naturally produced by the corpus luteum of the ovaries of mammals and involved in their pregnancy. In veterinary medicine, exogenous progesterone is used as a potent drug for suppression of estrus and ovulation, making possible the synchronization of the estrus and ovulation cycles in livestock animals . In this sense, the present study aims to investigate the release characteristics of progesterone-impregnated zein nanofiber obtained by electrospinning process. In addition, the ability of progesterone-loaded zein nanofibers to provide sustained drug release was studied.
Zein from corn and progesterone were purchased from Sigma-Aldrich (USA). Ethanol 99.7% purity was supplied by Merck
Progesterone-Loaded Nanofiber Fabrication
Zein was dissolved in ethanol and kept under vigorous stirring overnight at room temperature. Various concentrations (1.2, 1.9, and 2.5 g) of progesterone were dissolved in ethanol for an hour at room temperature. Both solutions were mixed for an hour. Progesterone-loaded zein fibers prepared by electrospinning were spun using a voltage of 24 kV, working distance of 12 cm, and feed rate of 2 μL min−1. Electrospinning processes were carried out under ambient conditions (24 ± 3 °C with relative humidity 57 ± 4%) .
Characterization of Progesterone Loaded Zein Nanofiber
Scanning Electron Microscopy
Scanning electron microscopy is used to check the surface morphology of three different concentrations (1.2, 1.9, and 2.5 g) of progesterone-incorporated nanofiber. The SEM characterization of electrospun nanofiber was performed using JEOL JSM-6480 V (accelerative voltage 20 kV) scanning electron microscopy at the Nanotechnology Department of SRM University, Chennai. The nanofiber samples collected on the aluminum foil was peeled out and then mounted on SEM sample holder using graphite-impregnated adhesive conductive black carbon tape, coated with platinum, and visualized under SEM at various magnifications.
XRD patterns were generated from nonwoven fibrous mat using a Rigaku D/Max ULTIMA 11 X-ray diffractometer (Japan). The X-rays are generated by a cathode ray tube filtered to produce monochromatic radiation directed towards the sample. The interaction of the incident rays with the sample produces constructive interference (and diffracted rays). The diffracted intensity were recorded from 0 to 1400 at 2θ angle and the pattern was recorded by Cu K radiation with 1.5418 Å and graphite monochromatic filtering wave at a tube voltage of 40 kV and tube current of 30 mA, and scanning in the region of 0 to 70 at 6 min−1 with incident beam.
Differential Scanning Calorimetry
Differential scanning calorimetry (DSC) measurements (Mettler Toledo DSC 821e, Schwerzenbach, Switzerland) were performed on samples of 5 mg in the range of −100 to 200 °C at a heating rate of 10 °C/min (N2 atmosphere 80 L/min). The glass transition temperature (Tg) was evaluated with the Stare-software version 6.01 (Mettler Toledo, Schwerzenbach, Switzerland; calibration with indium and zinc). Zein films were stored over silica gel or at different relative humidities for 5 days prior to measurement to achieve different water contents. The relative humidity (r.h.) was controlled by saturated salt solutions (KCH3COO 22% r.h.; NaCl 75% r.h.; ZnSO4 85% r.h.; pure water 100% r.h.) The predicted Tg values were calculated with the Gordon-Taylor-equation.
Fourier Transform Infrared Spectroscopy
Nanofiber functional groups were analyzed using FTIR spectroscopy. A pinch of the sample was placed into the sample holder and FT-IR spectra (Spectrum Rx1, Perkin Elmer) were recorded in the range 4000–400 cm−1a to a resolution of 4 cm−1.
MTT Viability Assay
Vero cells from ATCC are used for the MTT assay. One hundred-microliter Vero cells at the concentration of 3 × 103 cells/well were seeded in 96-well plates containing DMEM and incubated in 5% CO2 at 37 °C for 24 h. The medium was changed after 1 h and 100 μL of different concentrations (20,000, 10,000, 1000, 500, 250, and 100 μg/ml) of the 1.2 g progesterone-loaded nanofiber dissolved with PBS was added to the wells and incubated for 24 h at 37 °C in the CO2 incubator. One hundred microliter of MTT (5 mg/mL) was added to the wells containing cells and nanofibers of different concentrations. It was incubated at 37 °C for 4 h. The medium was then removed and 20 μL of DMSO was added to the wells. It was then shaken and incubated at 37 °C for 15 min and the absorbance was measured at 575 nm.
In Vitro Release of Progesterone from Nanofiber
The in vitro release studies were performed at three different progesterone concentrations (1.2, 1.9, and 2.5 g) of nanofibers in a shaker at 37 °C. A weighed quantity of the fibers (20 mg) was suspended in PBS of pH 7.4. Then, it was kept in a shaker for seven days at 37 °C. The sample was withdrawn at regular one day intervals up to 7 days and replaced with the same volume of freshly prepared PBS pH 7.4. The withdrawn samples were used for OD measurement at 237 nm by a UV-visible spectrophotometer (Shimadzu, model UV-2601).
Results and Discussion
In this, the electrospinning of zein nanofibers was mostly carried out by using ethanol system which resulted in ribbon-like fiber morphology due to the rapid mat formation of the fiber core because of the very fast evaporation of the solvent [15, 16].
Characterization of Progesterone Loaded Zein Nanofiber
Scanning Electron Microscopy
X-ray Diffraction Method
Differential Scanning Calorimetry
In Vitro Release of Progesterone from Nanofiber
The results of the current study confirm some miscibility of progesterone on hydrophobic biopolymers according to SEM, XRD, DSC, TGA, and FTIR. The electrospinning can be appropriately used to encapsulate active agents in biodegradable and biocompatible polymers, providing a hormone release sustainably. The increases in the concentration of progesterone affect the nanofiber size and morphology was confirmed by SEM. Progesterone at various concentrations did not affect the structural integrity of the nanofibers. Progesterone was found to have the effect of plasticizer when added to zein polymer. The electrospinning can be appropriated used to encapsulate active agents in biodegradable and biocompatible polymers, providing a hormone release sustainably. This study clearly indicated that 1.2 g progesterone-loaded zein nanofibers can be potentially used in controlled delivering of progesterone, in livestock animals for estrus synchronization.
We thank TANUVAS and TRPVB for providing facility and Department of Biotechnology, Government of India, for funding this project.
Department of Biotechnology, Government of India, for funding this work through Translational Research Platform for Veterinary Biologicals grant number 102/IFD/DBT/SAN 2681/2011-2012 dated 29.09.2011.
CK carried out the experiments. MS participated in the sequence alignment and drafted the manuscript. GKJ performed the characterization work. RS and GJ conceived of the study and helped to draft the manuscript. GDR participated in the design of the study. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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