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Interactions between chloramphenicol, carrier polymers and bacteria – implications for designing electrospun drug delivery systems countering wound infection Liis Preem, Mohammad Mahmoudzadeh, Marta Putrins, Andres Meos, Ivo Laidmäe, Tavo Romann, Jaan Aruväli, Riinu Härmas, Artturi Koivuniemi, Alex Bunker, Tanel Tenson, and Karin Kogermann Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00524 • Publication Date (Web): 03 Nov 2017 Downloaded from http://pubs.acs.org on November 4, 2017
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Molecular Pharmaceutics
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Interactions between chloramphenicol, carrier polymers and bacteria – implications for
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designing electrospun drug delivery systems countering wound infection
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Liis Preema, Mohammad Mahmoudzadehb, Marta Putrinšc, Andres Meosa, Ivo Laidmäea,
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Tavo Romannd, Jaan Aruvälie, Riinu Härmasd, Artturi Koivuniemib, Alex Bunkerb, Tanel
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Tensonc, Karin Kogermanna,*
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a
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b
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University of Helsinki, Viikinkaari 5E, P.O. Box 56, FI-00014 University of Helsinki, Finland
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c
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d
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e
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Estonia
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*Correspondence to: Karin Kogermann (Telephone: +372 737 5281; Fax: +372 737 5289).
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E-mail address:
[email protected] (K. Kogermann).
Institute of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, Estonia Drug research program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy,
Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia Institute of Chemistry, University of Tartu, Ravila 14a, 50411, Tartu, Estonia
Institute of Ecology and Earth Sciences, University of Tartu, Ravila 14a, 50411, Tartu,
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Abstract
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Antibacterial drug-loaded electrospun nano- and microfibrous dressings are of major interest
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as novel topical drug delivery systems in wound care. In this study, chloramphenicol (CAM)
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loaded polycaprolactone (PCL) and PCL/polyethylene oxide (PEO) fiber mats were
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electrospun and characterized in terms of morphology, drug distribution, physicochemical
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properties, drug release, swelling, cytotoxicity and antibacterial activity. Computational
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modelling together with physicochemical analysis helped to elucidate possible interactions
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between the drug and carrier polymers. Strong interactions between PCL and CAM together
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with hydrophobicity of the system resulted in much slower drug release compared to the
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hydrophilic ternary system of PCL/PEO/CAM. Cytotoxicity studies confirmed safety of the
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fiber mats to murine NIH 3T3 cells. Disc diffusion assay demonstrated that both fast and slow
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release fiber mats reached effective concentrations and had similar antibacterial activity. A
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biofilm formation assay revealed that both blank matrices are good substrates for the bacterial
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attachment and formation of biofilm. Importantly, prolonged release of CAM from drug-
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loaded fibers helps to avoid biofilm formation onto the dressing and hence avoids the
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treatment failure.
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Keywords: electrospinning; polymeric carrier; intermolecular interactions; biofilm; wound
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infection; molecular dynamics simulation
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1. Introduction
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Biofilm and infection are increasingly associated with delayed wound healing.1,2 This is
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devastating to both patients and healthcare resources, for example, in the UK, the cost of
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managing wounds in 2012/2013 was retrospectively estimated to be between £4.5 to £5.1
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billion.3 As novel treatment strategies are needed, antibacterial drug-loaded electrospun nano-
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and microfibrous dressings are of major interest as topical drug delivery systems (DDSs) for
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managing wound infections.
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Polymeric DDSs have the capability to deliver drugs to the site of action at a controlled rate
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and establish localized, clinically relevant drug concentrations for extended periods of time,
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hence have the potential to improve the therapeutic efficacy, reduce toxicity and enhance
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patient compliance.6,7 Prolonged drug release is beneficial in wound infection treatment as the
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frequent need to change the dressings could harm the regenerating tissue and also be painful
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and inconvenient to the patient. Local delivery of antibiotics by topical delivery device
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enables maintenance of high local antibiotic concentration for an extended period of time
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without systemic toxicity.8 Also, the antibiotic concentrations need to be carefully maintained
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to avoid the development of persistent and resistant microorganisms.9
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Electrospinning is a simple and versatile technique for the production of polymeric nano- and
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microfibers using an electrostatically driven jet of polymer solution or polymer melt.
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Electrospun fibers have many useful properties for wound care applications, including oxygen
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permeability, high effective surface area and porosity promoting hemostasis and absorbing
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wound exudate. Also, electrospinning allows relatively easy incorporation of different drugs
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or growth factors into the dressing matrix, making it a promising DDS. The morphology of
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electrospun nanofibers is similar to a natural skin extracellular matrix that promotes cell
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adhesion, migration and proliferation and reduces the scar formation.4,5
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Chloramphenicol (CAM) is a broad-spectrum antibiotic first isolated from Streptomyces
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venezuelae.10 By diffusing through the bacterial cell wall and reversibly binding to the
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bacterial 50S ribosomal subunit, it interferes with peptidyltransferase activity and prevents
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peptide bond formation, hence impairing bacterial protein synthesis and bacterial cell
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proliferation. Systemic therapeutic use is mostly limited to severe infections for which the
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benefits of the drug outweigh the risks of the potential toxicities, most importantly affecting
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the hematopoietic system.11,12 More commonly it is used in the local treatment of
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conjunctivitis and keratitis13, but could also be used in the local wound infection management.
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Polycaprolactone (PCL) is a synthetic polyester widely used in tissue engineering and drug
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delivery due to its biocompatibility, biodegradability, low cost and non-toxicity.14,15 It has
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been used alone or in combination with other polymers as a carrier for electrospinning of
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several antibiotics to achieve controlled release.16–20 Nevertheless, its hydrophobicity may
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limit the absorption of wound exudate and interactions with cells to promote wound healing.
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Polyethylene oxide (PEO) is a nonionic and highly hydrophilic polymer widely used in
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DDSs.21,22 It is known to suppress protein adsorption and thus is used in anti-inflammatory
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polymeric coatings for implantable biomaterials and devices.23 Due to its hydrophilic nature it
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can balance the hydrophobicity of PCL.15
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The compatibility of solvent(s), carrier polymer(s) and drug molecules determines the success
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of stable electrospinning process as well as physicochemical and biopharmaceutical properties
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of resulting DDSs.24 A considerable amount of research has been performed in the use of
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electrospinning to produce controlled release nanofibrous DDSs. However, insufficient
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emphasis has been put to investigating how interactions between carrier polymers,
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electrospinning solvents and drug molecules translate into structural and physicochemical
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properties, which shape the biopharmaceutical properties of the DDS and bring about new
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interactions with both bacteria and eukaryotic cells of the regenerating tissue. 4 ACS Paragon Plus Environment
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Our aim was to develop antibacterial drug-loaded electrospun matrices for the prevention and
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local treatment of wound infections. It was our aim to understand the effect of different carrier
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polymers on the drug release, drug-polymer interactions and evaluate the antibacterial
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capacity of the developed matrices and understand how this is influenced by both bulk matrix
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properties and interactions at the molecular level. In a novel approach, we have
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complemented our experimental analysis with molecular dynamics simulation (MD), to
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provide additional mechanistic insight into the molecular interactions between PCL and PEO
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with CAM. This represents the first such instance of the use of MD to study the interaction of
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this set of molecules.
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2. Materials and Methods
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2.1 Materials
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Polycaprolactone (PCL) (Mn 80,000), polyethylene oxide (PEO) (Mw ≈ 900,000),
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chloramphenicol (CAM) (PubChem CID: 5959), methanol (gradient grade) and chloroform
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(puriss p.a.) were purchased from Sigma-Aldrich. All other materials were of reagent grade or
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better and were used as received without any further purification.
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2.2 Preparation of electrospinning solutions and electrospinning of the fiber mats
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Four different solutions for electrospinning were prepared. Solution of PCL was prepared by
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dissolving PCL in a solvent mixture of chloroform and methanol (3:1 V/V) to obtain 12.5%
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(w/V) solution. Solution of PCL/PEO was prepared similarly, only the concentration of PCL
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was 10% and PEO 2% (w/V). Drug-loaded solutions were prepared by adding CAM to both
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solutions. The amount of CAM was 4% (w/w) based on the dry weight of the polymer(s) in
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both cases. Solutions were allowed to mix overnight by the aid of magnetic stirrer.
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Electrospinning was conducted using NanoNC electrospinning robot (South Korea). For the
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case of PCL drug-loaded and blank solution, the optimized solution feed rate was 1 ml/h and 5 ACS Paragon Plus Environment
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the voltage applied was 9 kV. The distance between the spinneret and collector plate was 14
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cm. For the case of PCL/PEO drug-loaded and blank solution, the chosen feed rate was 2.5
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ml/h, voltage 12 kV and distance between the spinneret and collector plate 17 cm. The
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needles used were 25 and 23 G, respectively. Approximately 3 ml of solution was electrospun
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to obtain fiber mats, which were stored in the airtight plastic bags at ambient conditions
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(temperature of 22 ± 1 °C and RH of 20 ± 2%) until further analyses. For the solid state
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characterization of drug within fibers, physical mixtures and amorphous CAM were prepared
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(Supporting Information).
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2.3. Scanning electron microscopy (SEM)
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The morphology and diameter of electrospun fibers was observed under SEM (Zeiss EVO 15
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MA, Germany). Randomly selected areas of the fiber mats were mounted on aluminum stubs
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and magnetron-sputter coated with a 3-nm gold layer in an argon atmosphere prior to
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microscopy.
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2.4 Chloramphenicol (CAM) content and distribution within fibers and fiber mats
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To determine the content of drug loading and its distribution within electrospun matrices, high
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performance liquid chromatography (HPLC) and Raman scattering microspectroscopy (RSM)
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analyses were performed. HPLC analyses were performed using Shimadzu Prominence LC20
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with PDA detector (wavelength at 275 nm) and according to the official European
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Pharmacopoeia method for a related substance CAM sodium succinate. HPLC was equipped
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with a column Phenomenex Luna C18(2), 250 x 4.6 mm, 5 mm, and the mobile phase used
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was 20 g/l solution of phosphoric acid R, methanol R, and water R (5:40:55 V/V/V). The flow
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rate was 1.0 ml/min, and injection volume was 20 µl. The CAM-loaded mats were cut into 1
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and 3 cm2 pieces, weighed and dissolved in dichloromethane and methanol (3:1 V/V). Pieces
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were taken from both center and edges of the matrix to see if differences occurred in drug
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distribution.
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In addition to HPLC analysis, RSM mapping was used in order to visualize the CAM
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distribution within the fiber samples. RSM was performed using Reinshaw InVia micro-
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Raman spectrometer (Reinshaw, England) with CCD Camera (1040x256) and 785 nm diode
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laser excitation. Exposure time of 150 s and 50x objective was used for the measurements.
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Raman mapping data were collected on approximately 92 (height) 119 (width) µm area of the
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fibers in the spectral range of 700 to 1800 cm-1 with 1 cm-1 resolution. The maps were
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collected with 1.2 µm step size in both direction and consisted of 5000-10000 points. Bright
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field images of the samples were taken before each Raman mapping.
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2.5 Mercury Intrusion Porosimetry (MIP) and Brunauer-Emmett-Teller (BET) analyses
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The pore size distribution and density of electrospun fiber mats were determined by MIP
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using the POREMASTER-60-17 porosimeter (Quantachrome Instruments, USA). An MIP
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analysis was used to measure pores with diameters in the range 0.032–10 µm. The porosity of
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the fiber mats, describing the percentage of void volume of the sample, was calculated from
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the Equation 1: % = 1 −
∙ 100% 1
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where ρf is the density of fiber mat and ρm is the bulk density of corresponding materials,
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more precisely PCL 1.145 g/cm3, PEO 1.21 g/cm3 and CAM 1.547 g/cm3.
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Krypton adsorption isotherms were measured on degassed (>58 h, 25 °C, vacuum) samples
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using an ASAP 2020 Accelerated Surface Area and Porosimetry System (Micromeritics,
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USA) at -195.75 C°. The specific surface areas (SBET) were calculated from sorption data
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2.6 X-ray diffraction (XRD)
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The XRD patterns of the starting materials, physical mixtures and electrospun fibers were
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obtained with X-ray diffractometer (D8 Advance, Bruker AXS GmbH, Germany). The XRD
4
experiments were carried out in a symmetrical reflection mode (Bragg–Brentano geometry)
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with CuKa radiation (1.54 Å). The scattered intensities were measured with the LynxEye one-
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dimensional detector including 165 channels. The angular range was from 5° to 40° 2-theta
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with the step size of 0.0198° 2-theta. The degree of crystallinity (Xc) was calculated with
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Bruker AXS software Topas 4-1 (Eq. 2): =
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∙ 100% 2
+
where Ic is the diffracted intensity of the crystalline phase and Ia is the diffracted intensity of
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amorphous phase.
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2.7 Attenuated total reflection Fourier transformed infrared (ATR-FTIR) spectroscopy
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ATR-FTIR spectroscopy was performed on pure substances, physical mixtures and
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electrospun matrices using an IRPrestige-21 spectrophotometer (Shimadzu Corp., Kyoto,
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Japan) and Specac Golden Gate Single Reflection ATR crystal (Specac Ltd., Orpington, UK).
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The spectra were collected between 600 and 4000 cm-1, each spectrum was the average of 60
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scans.
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2.8 Differential Scanning Calorimetry (DSC)
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Two different DSC equipment were used for thermal analysis (Mettler Toledo and
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PekinElmer DSC4000). The measurement conditions were kept the same. Pure substances,
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physical mixtures and electrospun fibers were analyzed under 50 ml/min dry nitrogen purge in
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crimped aluminum pans without pinholes at a heating/cooling rate of 10 °C/min from 0 to 180
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2.9 Contact angle and in vitro drug release from CAM-loaded fiber mats
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The contact angle between the electrospun fibers and deionized water was measured with the
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sessile drop method (CAM 200, Attension/Biolin Scientific Oy, Espoo, Finland). A drop of
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deionized water was applied to the fibers deposited as a thin layer on a gold surface. The
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contact angle was analyzed using the Attension software (Attension/Biolin Scientific Oy,
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Espoo, Finland). Each sample was measured in triplicate. The in vitro drug release of CAM
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from drug-loaded electrospun fiber mats was carried out using 1 cm2 samples (n=3) cut from
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the matrix. These were weighed, placed into 10 ml of PBS (pH 7.4) at 37°C in 50 ml plastic
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tubes. The tubes were put into dissolution apparatus vessel (Dissolution system 2100, Distek
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Inc., NJ, USA) containing water and maintained at 37°C. The tubes were rotated by the
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paddles at the speed of 100 rpm. Aliquots of 2 ml were removed and replaced with the same
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amount of PBS at set time points. The aliquots were analyzed using UV-spectroscopy
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(Shimadzu UV-1800). Wavelength of maximum absorption (λ=278 nm) was chosen for drug
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release analysis.
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2.10 Swelling and weight loss of electrospun fiber mats
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A set of 4 cm2 samples (n=3) were cut from the matrices and weighed, then immersed into 10
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ml of deionized water at 37 °C for 24 h. After that, the samples were tapped dry with a filter
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paper to remove free surface water and weighed. Swelling index and weight loss were
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calculated as reported previously.26
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2.11 Computational methods
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A set of MD simulations, all 100 ns in length, were carried out in order to obtain insight into
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the mechanisms behind the interactions between CAM molecules and both PCL and PEO. All
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simulations were performed using the GroMACS 5.0.4 MD simulation package.27 For each of
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the control systems without CAM molecule, a single simulation was performed. For the 9 ACS Paragon Plus Environment
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systems containing both the drug and polymer (PCL with CAM and PEO with CAM), five
2
different simulations were performed starting from five different initial configurations and
3
then the results obtained were averaged together in order to achieve improved statistics and to
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exclude any possible effect of the selection of the initial configuration on the final results. For
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both polymers, one of the initial configurations was selected through ligand docking using the
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Autodock package28 and the other four through randomly placing one CAM molecule in a
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simulation box containing one PCL or PEO chain. Thus, 12 systems were simulated in total:
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one solvated PCL system, one solvated PEO system, five solvated PCL/CAM systems and
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five solvated PEO/CAM systems (Table 1).
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Table 1. Number of molecules/ions in each of the simulated systems System
PCLa PEOb CAMc Water chains chains molecules molecules
Pure PEO
-
1
-
2805
Ions (Na+ and Cl-) 16
Simulation box volume [nm3] 85.18
PEO/CAM autodocked
-
1
1
989
6
30.66
PEO/CAM random1
-
1
1
986
6
30.66
PEO/CAM random2
-
1
1
988
6
30.66
PEO/CAM random3
-
1
1
991
6
30.66
PEO/CAM random4
-
1
1
987
6
30.66
Pure PCL
1
-
-
843
4
25.41
PCL/CAM autodocked
1
-
1
1131
6
34.96
PCL /CAM random1
1
-
1
1134
6
34.96
PCL/CAM random2
1
-
1
1136
6
34.96
PCL/CAM random3
1
-
1
1137
6
34.96
PCL/CAM random4
1
-
1
1133
6
34.96
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a
PCL – polycaprolactone; bPEO – polyethylene oxide; cCAM - chloramphenicol
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In order to make the comparison of the results gained for the PCL/CAM and the PEO/CAM
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systems possible, polymer lengths were chosen such that both systems had similar molecular
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weight and identical number of polar/nonpolar atoms; for all cases a PCL chain containing 10 ACS Paragon Plus Environment
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two monomers and a PEO chain containing five monomers were used. All details regarding
2
parametrization of all molecules, including our new topology for CAM, are included in
3
Supporting Information.
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2.12 In vitro indirect cytotoxicity of electrospun fiber mats
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The CellTiter-Glo® Luminescent Cell Viability Assay (Promega) was used as described by
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the manufacturers to investigate the cytotoxic effects of the fluid extracts of the fibers on
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murine fibroblastic NIH 3T3 cells. The cells were cultured in Dulbecco’s Modified Eagle
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Medium (DMEM) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and
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1% penicillin-streptomycin. Cells were maintained at 37 °C in 5% CO2 incubator. The fluid
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extracts were prepared by immersing the drug-loaded and blank fibers in the same growth
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medium and kept in the incubator for 24 h. The cells were cultivated on the 96 well-plates to
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near confluency and then the medium was replaced with the extracts (12.5, 25, 50, 100 or 200
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µg/ml) or fresh medium and incubated for 24 h. Both treated and untreated cells were then
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transferred to opaque-walled 96-well plate in fresh culture medium (100 µl per well),
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alongside with control wells containing medium without cells to obtain a value for
16
background luminescence. The plate was equilibrated at room temperature for 30 minutes.
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After that 100 µl of CellTiter-Glo® Reagent was added to each well and mixed for 2 minutes
18
on an orbital shaker to induce cell lysis. The plate was further incubated at room temperature
19
for 10 minutes and then luminescence recorded with VarioskanFlash 4.00.53 luminometer to
20
quantify cell viability by the amount of ATP present, which correlates to the presence of
21
metabolically active cells. Each test was run in duplicate.
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2.13. Microbial strains
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Gram-negative bacteria used in this study were laboratory strain Escherichia coli (E. coli)
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MG1655 and uropathogenic E. coli CFT073.29 The latter was kindly provided by Prof Harry
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Mobley. Preparation of DMSO stocks is described in Supporting Information.
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2.14 Disc diffusion assay
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Antibacterial activity of electrospun fibers was determined with disc diffusion assay.
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Overnight liquid cultures of both E. coli strains were grown from DMSO stocks and the cell
7
number was adjusted with fresh LB to about 3x107 colony-forming units (CFU)/ml. 100 µl of
8
these dilutions were spread onto the surface of LB agar plates. Discs cut from fiber matrices
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with a diameter of 6 mm were applied to these plates. Positive controls were prepared by
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immersing 6 mm filter paper discs with 20 µl of CAM solution, so each disc contained 20 µg
11
of the drug. Untreated filter paper was used as a negative control. The concentration of CAM
12
in positive control was similar to that in drug-loaded fibers. The plates were incubated at 37
13
°C for 24 h. The inhibition zones free of bacterial growth were determined. Tests were run in
14
triplicate.
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2.15 Biofilm assay
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Biofilm formation protocol was established based on the work of Brackman et al.30 Overnight
17
liquid culture of E. coli CFT073 in LB was grown from DMSO stocks. The culture was
18
diluted to about 5x107 CFU/ml with Dulbecco’s Modified Eagle medium (DMEM)
19
supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS). 1 ml of the bacterial
20
dispersion was added to 1 cm2 samples in 24 well-plates. The well-plates were incubated at 37
21
°C for 24, 48 or 72 h. After that the samples were rinsed twice with PBS and put into 10 ml of
22
fresh PBS in 50 ml plastic tube. For disrupting the biofilm alternating vortexing (Vortex-
23
Genie 2, Scientific Industries) and sonication (Bandelin Sonorex digital 10 P, operating at
24
20% of maximum power) was performed in 30 s cycles. Each cycle was repeated 6 times, as 12 ACS Paragon Plus Environment
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this was seen to provide best compromise between biofilm disruption and bacterial viability.
2
The CFUs were determined by making 10-times dilutions of the dispersion, plating these as 5
3
µl drops on LB agar plates and counting the CFUs at optimal dilutions after 18 h of
4
incubation.
5
2.16 Statistical analyses
6
Results are expressed as a mean ± standard deviation (SD). Statistical analysis was performed
7
by applying one-way ANOVA and post-hoc pairwise t-tests with MS Excel 2013 software
8
(p
