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Multilayered polysaccharide nanofilms for controlled delivery of pentoxifylline and possible treatment of chronic venous ulceration Jan Stana, Janja Stergar, Lidija Gradišnik, Vojko Flis, Rupert Johann Kargl, eleonore froehlich, Karin Stana Kleinschek, Tamilselvan Mohan, and Uros Maver Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00523 • Publication Date (Web): 04 Aug 2017 Downloaded from http://pubs.acs.org on August 5, 2017
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Biomacromolecules
Multilayered polysaccharide nanofilms for controlled delivery of pentoxifylline and possible treatment of chronic venous ulceration Jan Stanaa, Janja Stergarb, Lidija Gradišnikb, Vojko Flisc, Rupert Kargld, Eleonore Fröhliche, Karin Stana Kleinschekd, Tamilselvan Mohanf,*, Uroš Maverb,g,* a
Schön Klinik Vogtareuth, Department of Vascular and Endovascular Surgery, Krankenhausstraße 20, 83569 Vogtareuth, Germany b University of Maribor, Faculty of Medicine, Institute of Biomedical Sciences, Taborska ulica 8, SI-2000 Maribor, Slovenia c University Medical Centre Maribor, Division of Surgery, Department of Vascular Surgery, Ljubljanka ulica 5, SI-2000 Maribor, Slovenia d University of Maribor, Faculty of Mechanical Engineering, Laboratory for Characterisation and Processing of Polymers, Smetanova 17, SI-2000 Maribor, Slovenia e University of Graz, Institute of Chemistry, Heinrichstrasse 28, 8010 Graz, Austria f Medical University of Graz, Center for Medical Research, Core Facility Microscopy, Stiftingtalstraße 24, 8010 Graz, Austria g University of Maribor, Faculty of Medicine, Department of Pharmacology, Taborska ulica 8, SI-2000 Maribor, Slovenia
*Corresponding authors Assist. prof. dr. Uroš Maver, head of the Institute of Biomedical Sciences University of Maribor, Faculty of Medicine Institute of Biomedical Sciences Taborska ulica 8, SI-2000 Maribor, Slovenia
[email protected] Phone: +386 2 234 5823
Dr. Tamilselvan Mohan University of Graz Institute of Chemistry Heinrichstrasse 28, 8010 Graz, Austria
[email protected] Phone: +43 316 380 5413
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ABSTRACT
Local drug delivery systems made from non-toxic polysaccharide nanofilms have an enormous potential in wound care. A detailed understanding of the structural, surface, physicochemical and cytotoxic properties of such systems is crucial to design clinically efficacious materials. Herein, we fabricated polysaccharide-based nanofilms onto either a 2D model (SiO2 and Au-sensors) or on non-woven alginate 3D substrates using an alternating assembly of N,N,N-trimethylchitosan (TMC) and alginic acid (ALG) prepared by a spinassisted layer-by-layer (LbL) technique. These TMC/ALG multi-layered nanofilms are used for a uniform encapsulation and controlled release of pentoxifylline (PTX), a potent antiinflammatory drug for treatment of the chronic venous ulceration. We show a tailorable film growth and mass, morphology, as well as surface properties (charge, hydrophilicity, porosity) of the assembled nanofilms through control of the coating during the spin-assisted assembly. The uniform distribution of the encapsulated PTX in the TMC/ALG nanofilms is preserved even with when the amount of the incorporated PTX increases. The PTX release mechanism from the model and real systems is studied in detail and is very comparable for both systems. Finally, different cell based assays illustrated the potential of the TMC/ALG multilayer system in wound care (e.g., treatment chronic venous ulceration) applications, including a decrease of TNF-α secretion, a common indicator of inflammation.
Keywords: Trimethylchitosan, alginate, pentoxifylline, chronic venous ulceration, multilayered films, wound dressings
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Wound healing generally follows a well-defined yet complex cascade of processes commonly
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divided into four main stages; coagulation, inflammation, cell proliferation with repair of the
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matrix and epithelialization with remodeling of the scarred tissue1. Wounds are often
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classified as being either acute or chronic. In general, acute wounds are healed within an
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expected time frame, and if not, they in essence become chronic wounds. Wounds labeled
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“chronic” are often merely symptoms of other long-standing or overwhelming problems2, 3.
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The venous ulceration is a common chronic wound type that presents a major socio-economic
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problem in the western society. In Europe 5% of the population suffers from chronic venous
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insufficiency, from which 1% develops chronic venous ulceration4. Histopathological and
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immunological findings have shown that chronic inflammation plays a pivotal role in the
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development of venous ulcerations5, 6. One of the possible hypotheses for the up-regulation of
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leukocyte activation that accompanies the chronic inflammation, could be in the rheological
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changes due to chronic venous insufficiency 7. Namely, the changed sheer stress on the
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endothelium of the vascular wall is responsible for the higher expression of ICAM-1
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(intracellular adhesion molecule 1), VCAM-1 (vascular intracellular adhesion molecule 1),
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PECAM-1 (platelet endothelial cell adhesion molecule 1), as well as E-, L-selectins, which
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contribute to an increased leucopedesis 6. In the next step the activation of the leukocytes,
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especially neutrophils and macrophages, takes place. This results in a higher concentration of
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their products (e.g., leukotrienes, free oxygen radicals and cytokines IL-1 (interleukin 1), IL-6
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(interleukin 6) and TNF-α (tumour necrosis factor)) in the wound fluid of chronic venous
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ulceration
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non-healing wounds, demonstrating its importance in chronic wound care 11, 12.
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Considering the mentioned, our study focused on the controlled release of pentoxifylline
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(PTX), an already clinically proven effective drug in the systemic treatment (oral
INTRODUCTION
8-10
. TNF-α is found in much higher concentrations in the wound fluid of chronic
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administration) of venous ulceration, where it was found to improve its healing rate 13, 14. PTX
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is a xanthine derivative, whose anti-inflammatory action can be related to a non-selective
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phosphodiesterase inhibition, raising intracellular cAMP (cyclic adenosine monophosphate)
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concentrations, inhibition of TNF-α and leukotriene synthesis, as well as the initiation of
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immunity 15, 16. Namely, the higher intracellular cAMP concentration decreases the activation
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of macrophages, concentration of superoxide anions and inhibits the release of lysosomal
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enzymes from polymorphonuclear cells
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dependent manner
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mechanisms, especially to the increase in prostaglandin E2 production, inhibition of natural
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killing cells18, reduction of the TNF- α expression and increased production of TGF-β
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(transforming growth factor beta), which leads to a reduced action of Th1 cells 15.
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Different fiber forming polymers are often used as drug carriers, absorbents or as moisturizers
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in wound dressings19, 20. Natural polysaccharides, such as regenerated cellulose (viscose),
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cellulose derivatives and alginate, are as part of various formulations (e.g., foams, hydrogels,
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non-woven materials) among the most common functional parts of different modern wound
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dressings
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sustained drug release, make alginate and chitosan (and its derivatives) highly interesting for
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the development of functional wound dressings
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derivative of the chitosan, is permanently charged and water soluble, and has been previously
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shown as suitable for preparation of controlled delivery formulations 25. Alginic acid (ALG) is
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a water soluble anionic polysaccharide isolated from seaweed and various microbial sources,
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and is mostly used in the form of a calcium salt. In contact with the wound exudate (with an
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excess of sodium ions) an exchange of Na+ and Ca2+ ions takes place, which leads to the
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formation of a viscous gel 23. The gel enhances the autolytic cleansing activity in the wound
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. The inhibition of TNF-α takes place in a dose
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. The anti-inflammatory effect of PTX is also related to other
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. Their already observed positive effect on wound healing and suitability for
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. N, N, N-trimethyl chitosan (TMC), a
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. The increase of Ca2+ ions in the wound leads to an
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and decreases the bacterial count
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improved hemocompatibility and to an increased rate of fibroblast proliferation 27, 28.
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Although multilayered and multifunctional wound dressings, especially the ones, created
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through the layer-by-layer (LbL) technique, are not a novelty in wound care
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products do not address the challenging issues in wound treatment, such as controlled
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therapeutic action or wound type specific healing 30, 31. The LbL technique has been perfected
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in the last decade and already successfully applied in different fields of research
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hard to overlook the potential of LbL in development of novel drug delivery systems that go
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beyond a simple passive diffusion based delivery, but provide different levels of control over
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the release
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multilayers for the controlled delivery of different anti-inflammatory drugs
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from previous LbL approaches to prepare nanofilms for controlled drug delivery, as well as
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considering the still many unexploited potentials in development of novel drug/material
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combinations as multilayered multifunctional wound dressings, served both as the foundation
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for this study.
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Based on the above mentioned, the aim of this study was the development of a platform for
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the design and testing of the potential of novel wound dressings, in the form of multilayers
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with an incorporated drug. Using a combination of a biocompatible cationic TMC and anionic
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ALG polysaccharides, and a potent anti-inflammatory drug PTX, we prepared and
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characterized an advanced wound dressing for the treatment of chronic venous ulceration
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based on controlled delivery of PTX. Among others, its potential for future use in wound care
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can be deduced from the different in vitro cell studies on human skin and immune cells,
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showing an improved cell viability and diminished inflammation after exposure to the
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developed system, respectively.
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, existing
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. It is
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. Recently, several studies have also focused on the use of LbL-based
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. Learning
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2.1
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N,N,N-trimethylchitosan chloride (TMC 66, Mw: 90 kDa, degree of acetylation (DA): 32%,
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degree of cationization (DSNMe3+Cl-: 66%)) with medical grade quality was purchased from
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Kitozyme S.A. (Herstal, Belgium). Sodium salt of alginic acid (ALG) and PBS buffer tablets
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were purchased from Sigma-Aldrich (Maribor, Slovenia) and used as received. A so called
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Soft Alignattamponade (SeaSorb, Coloplast, Denmark) was used as the substrate for the
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preparation of the multilayers on a “real” wound dressing. Milli-Q (18.2 MΩ cm at 25 °C)
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water from a Millipore water purification system (Billerica, USA) was used for all sample
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preparations. Quartz crystal microbalance (QCM) crystals coated with SiO2 (QSX303) were
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purchased from LOT-Oriel, Germany. All chemicals were used without further modification.
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Different cell lines were used for the safety and efficiency testing, namely the human skin
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derived fibroblast cell cultures (ATCC-CCL-110, Detroit 551, and ATCC-CCL-171, MRC-5,
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both from LGC Standards, UK) and THP-1 human monocytes obtained from Cell Line
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Services were cultured in Roswell Park Memorial Institute (RPMI, Gibco, USA).
EXPERIMENTAL Materials and methods
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2.2
Substrate cleaning and multi-layered polysaccharide nanofilm preparation with pentoxifylline (PTX) drug
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2.2.1 Substrate cleaning
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Slicon wafers (Si-wafers, Topsil, Germany) with (100) surface orientation were cut into
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pieces of 1 x 1 cm2 and used as base-substrates for the multilayer preparation. In brief, the Si-
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wafers were cleaned with ethanol, rinsed with Milli-Q water and immersed into a “piranha”
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solution of 1:3 (v/v) H2O2 (35%)/H2SO4 for 1 h. After that, they were soaked in Milli-Q water
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for another hour, washed with Milli-Q water and blow-dried with N2 gas. The cleaned wafers
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were then used for the subsequent deposition of TMC and ALG.
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2.2.2 Multilayer preparation
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Briefly, 50 µL of TMC (0.1%, w/v, dissolved in 150 mM NaCl electrolyte) was deposited on
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the static Si-wafers, which was then rotated for 30 s with a spinning speed of 3000 rpm and
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acceleration of 2500 rpm/s. Subsequently, 50 µL of ALG (0.1%, w/v, dissolved in 150 mM
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NaCl electrolyte) was spin coated on the TMC layer using the same preparation parameters as
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described above. Likewise, two more bilayers of TMC/ALG were created. Each coated layer
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(TMC or ALG) was designated with the numbers 1, 2, or 3, for the number of layers in
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respective samples (i.e., 3TMC, 2ALG etc.).
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2.2.3 Drug loading
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Pentoxifylline (PTX) drug was incorporated into each bilayer by a simple dropping approach.
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50 µL of PTX, dissolved in Milli-Q water at different concentrations (1, 5, 10, 15, 20
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mg/mL), was deposited on the TMC layer in each bilayer and dried at 40 °C for 2 h. As soon
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as the PTX drug solution was dried, the next layer of ALG was created by spin coating as
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described in section 2.2.2. Afterwards, 50 µL of Milli-Q water was deposited on the final
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ALG layer and spin coated using the same parameters as mentioned in section 2.2.2. To create
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multilayers with PTX drug on ‘real’ alginate wound dressing (sterile SeaSorb of 1 x 1 cm2
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size), instead of Si-wafers, the same procedure as stated before was used.
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The resulting multi-layered film structure is shown in Figure 1. The final sample names
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represent the number of respective layers and the number of PTX depositions (xTMC, yALG,
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zPTX), as well as the used PTX concentration for the respective sample (i.e., 3TMC-3ALG-
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3PTX_15 for the sample composed of three bilayers, whereas PTX with the starting
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concentration of 15 mg/ml, was added thrice). The sample prepared on the alginate substrate,
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was denoted as ALG_3TMC-3ALG-3PTX_20. As seen from the name, in this sample, PTX
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was added thrice using a concentration of 20 mg/ml.
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Figure 1: Schematic depiction of the prepared multi-layered films.
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2.3
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The wettability of TMC and ALG coated layers was measured by using Dataphysics contact
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angle measurement system OCA15+ (Dataphysics, Germany) with the sessile drop method
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and a drop volume of 3 µl. All measurements were carried out at room temperature.
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Determination of the SCA was based on the analysis of the drop shape and was performed
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with the software provided by the manufacturer (software version SCA 20). All the
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measurements were performed on at least three independent substrates with a minimum of
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three drops per surface and. The results are reported as average values with standard errors.
Contact angle (CA) measurement
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2.4
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ATR-IR spectra were recorded using an Agilent Cary 630 FTIR spectrometer with the
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diamond ATR module at a scan range of 4000–650 cm-1 with a step of 1 cm-1. The scans were
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performed on three different places in 8 repetitions on either for each sample surface, as well
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as after preparation of each respective layer.
Attenuated total reflectance infrared (ATR-IR) spectroscopy
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2.5
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Layer thickness of TMC and ALG coated films was determined by profilometry using a
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DEKTAK 150 Stylus Profiler from Veeco (Plainview, NY, USA). The scan length was set to
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1000 µm over the time duration of 3 s. The diamond stylus had a radius of 12.5 µm and the
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force was 3 mg with a resolution of 0.333 µm/sample and a measurement range of 6.5 µm.
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The profile was set to hills and valleys. Prior to surface scanning, the coated layers were
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scratched to remove the TMC/ALG films in order to determine the thickness of the coating
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using a step-height profile. The thickness was determined at 3 independent positions. The
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samples were measured after each coating of either TMC or ALG.
Profilometry
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2.6
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A QCM-D instrument (model E4) from Q-Sense, Gothenburg, Sweden was used to calculate
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the mass of each deposited layer. The instrument simultaneously measures changes in the
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resonance frequency (∆f) and energy dissipation (∆D) when the mass of an oscillating
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piezoelectric crystal changes upon increase/decrease in the mass of the crystal surface due to
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the added/deduced sample mass. Dissipation refers to the frictional losses that lead to
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damping of the oscillation depending on the viscoelastic properties of the material. For a rigid
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adsorbed layer that is fully coupled to the oscillation of the crystal, ∆fn is given by the
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Sauerbrey equation40 (1):
Quartz crystal microbalance with dissipation measurements (QCM-D)
∆m = C
∆f n n
(1)
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where ∆fn is the observed frequency shift, C is the Sauerbrey constant (-0.177 mg·Hz-1·m-2 for
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a 5 MHz crystal), n is the overtone number (n = 1, 3, 5, etc.), and ∆m is the change in mass of
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the crystal due to the adsorbed layer.
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2.7
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The surface morphology of the samples was characterized by atomic force microscopy (AFM)
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in tapping mode with an Agilent 7500 AFM multimode scanning probe microscope (Keysight
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Technologies, Santa Barbara, USA). The images were acquired after drying the samples with
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N2 gas. The images were scanned using silicon cantilevers (ATEC-NC-20, Nanosensors,
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Germany) with a resonance frequency of 210–490 kHz and a force constant of 12–110 N m-1.
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All measurements were performed at room temperature. All images were recorded with a
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resolution of 2048 x 2048 pixels and were processed using the freeware Gwyddion software
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package.
Atomic force microscopy
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2.8
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In vitro drug release studies were performed using an Automated Transdermal Diffusion Cells
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Sampling System (Logan System 912-6, Somerset, USA). The drug-loaded samples were
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placed heads up into the Franz diffusion cell. The receptor compartment was filled with PBS
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solution (pH 7.4) and its temperature was maintained at 37 °C. During the dissolution testing,
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the medium was stirred continuously with a magnetic bar at 50 rpm. Samples were collected
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over a period of six days at different time intervals (1, 5, 10, 20, 30, 60, 120, 180, 240, 360,
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1440, 2880, 4320, 5760, 7200 and 8640 min), while the released/dissolved PTX concentration
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in the receptor medium was determined using UV-Vis spectrophotometry (Cary 60 UV-
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Visible Spectrophotometer, Agilent, Germany) by quantification of the absorption band at
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276 nm. The withdrawn sample volumes were replaced by fresh PBS solution. Due to sample
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withdrawal, followed by sample dilution through media replacement, sink conditions were
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ensured. In the calculation of concentrations using the Beer-Lambert law, this dilution was
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accounted for. All release studies were performed in three parallels. For all obtained release
In vitro drug release
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results, the confidence interval was determined as ±/√ , where t is a Student's t-
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distribution, s is the standard deviation, and x is the number of measurements.
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In order to fit the release results and consequently evaluate the release kinetics, two well-
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known models to describe the release from different pharmaceutical formulations, were
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applied. Namely, the zero-order kinetics as shown in equation (2) and the modified
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Korsmayer-Peppas model, equation (3) 41.
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Zero order kinetics = +
(2)
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where Mt is the amount of drug dissolved in time t, M0 is the initial amount of drug in the
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solution (most time M0 = 0) and k0 is the zero-order release constant expressed in unit’s
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concentration/time.
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Modified Korsmayer-Peppas model
= ∙ +
(3)
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where Mt is the fraction of drug released at time t, M∞ is the total amount of drug in the
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system, k is the constant of apparent release, n the diffusion exponent and b the amount that
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can be associated with the burst release.
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2.9
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2.9.1 Preparation of extracts
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The extraction was carried out in compliance with ISO10993-5 and ISO 10993-12 regulations
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(10993-5 AAI. Biological evaluation of medical devices - Part 5: Tests for in vitro
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cytotoxicity. 2009.10993-12 I. Biological evaluation of medical devices – Part 12: Sample
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preparation and reference materials. 2007) by incubation of 3cm2 material per ml cell culture
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medium for 24h at 37°C.
Cell based assays
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2.9.2 Cells and exposures
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MRC-5 human fibroblasts (ATCC-CCL-171) were cultured in in Minimal Essential Medium
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+ Earle’s salts (MEM, Gibco, USA), 10 wt.% fetal bovine serum (FBS, GE Health Sciences,
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UK), 2mM L-glutamine, 1 wt.% penicillin/streptomycin at 37°C and 5 wt.% CO2. THP-1
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human monocytes obtained from Cell Line Services were cultured in Roswell Park Memorial
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Institute (RPMI, Gibco, USA) 1640 medium,10 wt.% FBS, 2mM L-glutamine, 1 wt.%
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penicillin/streptomycin.
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MRC-5 cells were exposed to the pure eluates, 1:2, 1:5, 1:10 and 1:20 dilutions with MEM +
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10 wt. % FBS. THP-1 cells were exposed to eluates in 1:2, 1:4, 1:10 and 1:20 dilution with
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RPMI 1640 + 10 wt.% FBS.
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2.9.3 Viability/cytotoxicity screening in contact with in eluates
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MRC-5 cells/well of a 96 well were seeded 24h prior to the exposures to the eluates. Cell
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density was 16,000/well for 24h, 13,000 for 48h and 11,000 for the 72h exposures. The
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different densities were needed to obtain the subconfluent growth conditions suggested for
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cytotoxicity testing. Treatment of cells with 2µl 70 wt. % EtOH + TritonX100 (1+1) for 10
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min prior to the measurement of viability was used as positive control. After exposure to the
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eluates for 24h, viability was determined using the measurement of dehydrogenase activity
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(CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay, Promega, USA) and ATP
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content (CellTiter-Glo Luminescent Cell Viability Assay, Promega, USA).
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2.9.4 CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay/MTS
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The assay was used according to the instruction in the user manual. In short, MTS solution
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and the PMS solution were thawed, 100 µl of the PMS solution was mixed with 2 ml of MTS
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solution and 20 µl of the combined MTS/PMS solution was added to 100 µl of each well.
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Plates were incubated for 2 h at 37°C and 5 wt.% CO2 in a cell incubator. Absorbance was
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read at 490 nm on a plate reader (SPECTRA MAX plus 384, Molecular Devices, USA). In
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parallel, cells were viewed by bright-field microscopy to confirm the MTS data. Cell viability
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was calculated according to the following formula (4):
ℎ % = 100 ×
$%&'()* − $%&'()* 490 3 $,-.-) − $/)&0
(4)
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CellTiter-Glo Luminescent Cell Viability Assay for ATP content was used according to the
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manufacturer’s instruction. Plates were equilibrated to room temperature for approx. 30
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minutes and reconstituted CellTiter-Glo Reagent was added 1:1 to the amount of cell culture
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medium present in each well. Plates were shaken for 2 min and incubated for additional 10
244
min at RT before well contents were transferred to a luminescence compatible 96 well plate
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and luminescence was read on a Lumistar (BMG LabTech, USA).
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2.9.5 Viability/cytotoxicity screening in direct contact
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MRC-5 cells (400,000 cells for 24h; 332,500 cells for 48h, and 266,000 cells for 72h
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exposure) were seeded on glass coverslips (control for optimal cell growth) or on PTX loaded
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multilayers and not loaded multilayers placed in a conventional plastic well. After 24h, 48h,
251
and 72h of incubation cells were viewed by bright field microscopy. After 72h growth
252
substrates with the cells were transferred to another well for determination of viability using
253
dehydrogenase activity, cells in the glass bottom plate were analyzed in the well. Cells were
254
removed from the substrates and cells counted using CASY TT cell counter (Innovatis Roche,
255
Switzerland).
256 257
2.9.6 Measurement of TNF-α secretion
258
1x10E6 THP-1 cells were seeded in 12-wells and treated with eluates in different
259
concentrations alone and in combination with the inflammatory stimulus 500 ng/ml
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lipopolysaccharide (LPS, Escherichia coli 055:B5, Sigma, Germany) for 24h. The release of
261
the cytokines was measured using the TNF-α ELISA set from BD Biosciences (BD
262
OptEIA™, USA) according to the protocol given by the producers. In short, plates were
263
coated with the respective anti- TNF-α (capture) antibody, exposed to standards and samples
264
(in the appropriate dilution), incubated with the biotinylated detection antibody plus
265
streptavidin-coupled horseradish peroxidase and finally with the peroxidase substrate
266
tetramethylbenzidine. After termination of the reaction by addition of 2N sulfuric acid
267
absorbance was read at 450 nm with correction at 570 nm on a SPECTRA MAX plus 384
268
photometer (BMG LabTech, USA).
269 270
2.9.7 Exposure to samples from the in vitro release testing
271
To perform biocompatibility (according to ISO 10993-5 standard) testing with the human skin
272
derived fibroblast cell culture (ATCC-CCL-110, Detroit 551, LGC Standards, UK), solutions
273
of respective samples, withdrawn at the desired time intervals during the release testing, were
274
incubated with the fibroblast cell culture in P96 microtiter plates. Each well was filled with
275
cell suspension containing 60,000 cells and after 24 h, when the cells attached, it was
276
supplemented with the dissolved sample solutions, as described above. The solutions used
277
were diluted in a ratio of 1:2 in the cell culturing media (Advanced Dulbecco’s modified
278
Eagle’s medium (ADMEM, Gibco, Grand Island, NY, USA)), supplemented with 5 wt.%
279
FBS. Cytotoxic effects on the cell culture were observed after 24 h of incubation at 37 °C and
280
5 wt.% CO2. Cell viability was determined via the reduction reaction of the tetrazolium salt
281
MTT (3(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)42.
282 283
3
RESULTS AND DISCUSSION
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3.1
Multilayer characterization: layer thickness, roughness, wettability, composition and morphology
286
Understanding the growth, the surface and the physicochemical properties of multi-layered
287
systems built on 2D substrates is a highly important base knowledge for design and
288
development of ‘real-3D’ materials such as wound-dressings and materials for tissue
289
engineering applications. Therefore, we carefully analysed the layer thickness (t), roughness
290
(r), mass (m), wettability and morphology of the multilayer polysaccharide nanofilms created
291
from the alternating deposition of TMC and ALG. Figure 2 shows the results of the measured
292
layer thicknesses, the respective sample roughness’s (determined by profilometry
293
measurements) and the Sauerbrey (dry)-mass for each spin coated layer and also for the rinsed
294
multilayer. The total thickness increases linearly with the number of layers deposited; which
295
is also reflected in the roughness (Figure 2a). The layer thickness is increased from ca. 90 nm
296
(1TMC) to ca. 280 nm after three bilayers. The obtained layer thickness for the second and
297
third TMC deposition is almost the same (2TMC: 43 nm, 3TMC: 42 nm). In the case of ALG,
298
the layer thickness decreases with increasing deposition step (1ALG: 40 nm > 2ALG: 34 nm
299
> 3ALG: 29 nm). The roughness values slightly increase as the number of deposition steps
300
increases for both TMC and ALG. Upon rinsing with water, the reduction of film thickness
301
(24%, t: 220 nm) and roughness (r: 13.5 nm) is clearly visible, indicating that all loosely
302
bound materials are removed and a smooth film is formed. The mass of each deposited layer
303
was also estimated using QCM-D (Figure 2b). The latter is a highly sensitive and suitable
304
technique, often used to determine the mass of dry and rigid adsorbed layers directly from the
305
change in frequency using the Sauerbrey equation (1), in particular, when the change in
306
dissipation is below 2 x 10-6
307
0.22 ± 0.003 x 10-6 for each layer. It has to be noted that the spin coated layers were also
308
always dried with the stream of nitrogen gas before they were assembled into QCM-D
43, 44
. In our case, the observed change in dissipation is less than
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309
chambers for the measurements of frequency and dissipation change. The results of Sauerbrey
310
mass is shown in Figure 2b. These depict that the mass (µg/cm2) also increases linearly with
311
respect to deposition steps as in the case of layer thickness and roughness measurements. The
312
Sauerbrey mass for the 1TMC layer is 932 ± 12 µg/cm2. For the 2TMC and 3TMC layer the
313
deposited mass remained constant 400 ± 15 µg/cm2, which is 55% less than the mass
314
estimated for the first layer. In the case of ALG deposition, the mass is decreased with the
315
increasing number of deposition steps. The mass of the multilayer is also reduced up to 29%
316
after rinsing with water. The above results are in excellent correlation with the thickness
317
values obtained from profilometry measurements, indicating that QCM-D is a reliable and
318
direct technique for the measurements of deposited dry layers.
319 320
Figure 2: Layer thickness and roughness (a), and QCM-D dry mass (b) of multilayer
321
polysaccharide nanofilms assembled from the oppositely charged TMC and ALG.
322 323
The successful alternating coating of TMC and ALG was verified by static water contact
324
angle SCA(H2O) measurements (Figure 3); the latter gives meaningful information to show
325
the hydrophobic/hydrophilic characteristics of the coating. The hydrophilic film surface
326
(ALG) represents a lower contact angle value whereas the hydrophobic film surface (TMC)
327
exhibits a greater contact angle value. In general, the contact angle value (i.e., surface
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wettability) is sensitive to chemical functionalities of the outermost layer. The measured
329
SCA(H2O) exhibits a zigzag feature with the number of deposited layers, implying the
330
alternating coating of TMC and ALG on the surface. With the first TMC coating the
331
SCA(H2O) increased to 23.2 ± 0.7° in comparison to SiO2 surface (SCA(H2O): 10 ± 0.2°, data
332
not shown). This result is in accordance with the previous work where the TMC layer
333
adsorbed on cellulose substrate exhibited a lower contact angle (SCA(H2O): 24 ± 1°)45. The
334
subsequent ALG layer results in a decreased contact angle. These trends of increase and
335
decrease in SCA(H2O) is also repeated for next two bilayers, indicating that ALG is more
336
hydrophilic compared to TMC. The increased hydrophobicity (i.e., contact angle) for the
337
second and third TMC layers can be attributed also to the higher surface roughness as shown
338
in Figure 2a. After rinsing with water, the multilayer is becomes more hydrophilic with an
339
SCA(H2O) of 20.8 ± 0.1. An explanation can be the combination of change in surface
340
composition, reduced layer thickness and roughness (Figure 2). As a result of rinsing, various
341
functional groups such as -OH, -COOH, -NH2, and -N(CH3)3 originating from both TMC and
342
ALG, which are hydrophilic, can be exposed on the surface.
343 344
Figure 3: Water contact angle of multilayer polysaccharide nanofilms assembled from the
345
oppositely charged TMC and ALG.
346
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347
Results from ATR-IR measurements of multilayer built up on QCM-D Au-sensors (without
348
PTX incorporation) are shown in Figure 4a. The spectra show three main peaks, two of them
349
characteristic for TMC (1~1564 cm-1 for N-H, and 2~1479 cm-1 for C-H bending vibrations)
350
46
351
of the mentioned peaks indicate an increase in respective polymer content, indirectly
352
confirming the successful alternating coating of TMC and ALG layers. The latter is also in
353
agreement with the results of CA, QCM-D and profilometry measurements, where increasing
354
mass, thickness and hydrophilicity/hydrophobicity were observed as the result of multilayer
355
formation. The spectrum of PTX incorporated films exhibits several characteristic peaks,
356
which can be used for identification of its presence in the samples (Figure 4b).
, and one for ALG (3~1024 cm-1 for C-O stretching vibrations)47. The increasing intensities
357
358
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359
Figure 4: ATR-IR spectra of multilayer polysaccharide nanofilms prepared (a) without and
360
(b) with PTX incorporation at different concentrations. The spectra of multilayers built on
361
alginate dressing and pure PTX drug is shown in (b).
362 363
As seen from Figure 4b, the characteristic peaks for PTX (1701 and 1658 cm−1 for –CO, and
364
amide –CO stretching modes) 48 are present in all deposited layers in addition to the peaks of
365
polysaccharides, indicating successful PTX incorporation. Increasing peak intensities are an
366
expected result of an increasing amount of PTX in respective samples. Figure 4b also shows
367
the spectra for the sample, where an alginate dressing was used as a substrate instead of the
368
Si-wafer. In this case, also the characteristic peaks PTX peaks are observed again confirming
369
the successful PTX incorporation.
370 371
Figure 5: AFM height images of multilayer polysaccharide nanofilms, incorporated with
372
PTX at different concentrations. Top: 1 x 1 cm2 and bottom: 5 x 5 cm2.
373 374
The morphology of multilayers incorporated with PTX at different concentrations is shown in
375
Figure 5. It can be observed that regardless of the PTX content, at lower PTX concentration
376
(1 mg/mL), a rather smooth surface with a roughness in the range of 2 nm is observed in
377
contrast to multilayers prepared without PTX (not shown), which showed a rough surface
378
with a higher roughness (RMS: ~ 6 nm). These changes (morphology and roughness) clearly
379
indicate that the incorporated PTX has influenced the surface characteristics of the film
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380
already at the lowest concentration. Although the roughness differences are not significant
381
except for lowest PTX concentration, the observed morphology is different for each sample. It
382
is suggested that, at higher concentrations, the PTX molecules are packaged tightly and
383
organized differently in the multilayer built up leading to surfaces with differently formed
384
structures. The samples with the highest amount of incorporated PTX (20 mg/mL), exhibit
385
additional surface features, an indication that the increasing drug amount leads to formation of
386
bigger PTX aggregates. Since these seem to be still in the nanorange, this have no significant
387
influence on the overall sample roughness. Nevertheless, the latter indicates that a further
388
increase in the incorporated amount of PTX could result in even bigger agglomerates that
389
could significantly influence the overall multilayer film stability. Finally, it can be observed
390
that all samples (regardless of the PTX content) exhibit features that could present nano-holes
391
or pores in their structure. This could significantly influence the release performance of such
392
materials. This aspect is in more detail discussed below in section 3.2, describing the in vitro
393
drug release results.
394 395
3.2
396
Multi-layered dressings are commonly employed in clinical wound care for treating chronic
397
wounds49, 50. For this purpose, different commercial materials are combined on site in the
398
form of a “sandwich”. Such an approach enables combining different properties of respective
399
layers (ensuring appropriate moisture, drug delivery, air permeability etc.) to maximize
400
healing efficiency
401
composed of PTX containing bilayers based on TMC and ALG, two well-known
402
polysaccharides in wound related applications52, 53. Such a layered structure with multiple
403
functionalities and different physicochemical properties was chosen in order to suit the
404
treatment of the chronic venous ulceration for individual patients. This can be achieved
Controlled pentoxifylline delivery – possible clinical applicability?
31, 51
. In this study we prepared multi-layered model wound dressings
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405
through a possible adjustment of the PTX concentration (and hence the dose of the drug), its
406
effective and prolonged delivery up to five days without the need to change the dressing, as
407
well as exploiting the overall wound healing promoting effects of the used polysaccharides
408
TMC and ALG. Overall, such a dressing could significantly lower the necessary treatment
409
costs per patient for this type of wound.
410
In order to test the possible applicability of the chosen preparation methodology in a real
411
clinical setting, we prepared the same multilayers with PTX also on a non-woven alginate
412
dressing (SeaSorb from Coloplast, which is clinically used) and compared its release
413
performance with the model system built on Si-wafers. The obtained results from the in vitro
414
release testing are shown in Figure 6. Four important findings are presented in different
415
diagrams describing our results. These are explained in the following subchapters.
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Figure 6: Results from the in vitro release study: a) PTX mass as a function of time, b)
418
linearity testing of the dependence between the drug loading and released PTX mass, c) the %
419
of release PTX as a function of time, d) first derivatives of the release data for all samples, e)
420
fitting of the release profiles up to 360 min using the Zero order model, and f) fitting of the
421
release data from 360 min to 8640 min using the Korsmayer-Peppas model.
422
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3.2.1 The cumulative released DCF mass as a function of time
424
Figure 6a shows the results from the cumulative amount (mg/cm2) of released PTX from
425
respective samples as a function of time. The most likely general scenario to describe the
426
release profiles is that most of the PTX was relatively loosely packed inside the layers without
427
any significant interaction with the host materials. Here it has also to be stated that the final
428
step in the preparation of the multi-layered samples, namely the rinsing with water, followed
429
by spin coating, presumably led to a homogenous PTX distribution through the layers (see
430
Figure 7).
431 432
Figure 7: Schematic depiction of the influence of the final multilayer preparation step on
433
PTX distribution in the sample. Black arrows show the presumable burst like release, whereas
434
the red arrows present the slower release after 360 min.
435 436
This part of the incorporated drug is released in a “burst” like fashion (evident for the first
437
couple of minutes for all samples). The amount of released PTX due to this, and hence the
438
duration of this release part, differs for respective samples, according to the different
439
incorporated PTX concentrations. Namely, the higher the incorporated PTX dose, the bigger
440
the overlap of this initial release region between samples of increasing incorporated PTX
441
amounts. Following this initial fast release, the release mechanism of PTX dissolution is
442
presumably for the most part governed by the concentration gradient between the material
443
interior and the release media, and hence its diffusion from the bulk material to the drug
444
dissolution at the material surface, and finally to the bulk solution. This part of PTX is likely
445
to interact with the host materials as well. Presumably this is due to possible hydrogen
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446
bonding (carbonyl and amine groups from PTX, hydroxyl and carboxyl groups from ALG,
447
and hydroxyl and amine groups in TMC). A smaller contribution to the overall dissolution
448
process is due to the lower parts of the samples, where PTX has to either penetrate through all
449
of the above layers or is released sideways from the coatings (see the schematic depiction of
450
this scenario in Figure 7).
451
In addition, two important characteristics can be noted by presenting the release results as the
452
cumulative release drug mass (Figure 6a). Firstly, it is evident that by increasing the PTX
453
amount in the samples, the corresponding amount of released PTX can be increased without a
454
significant effect on the overall release kinetics (see further discussion below). Hence, the
455
proposed preparation process allows us to fine-tune the final released dose of PTX by a very
456
simple method, namely increasing the concentration of PTX in the solution during
457
preparation. The potential therapy using such materials would be similarly efficient in
458
lowering inflammation (targeted PTX action in this case) during the release, whereas the final
459
dose, which is connected with the individual patient (patient’s physical characteristics), can be
460
fine-tuned without significant effect on the overall release kinetics, hence inflammation will
461
be lowered with the same clinical efficiency for the respective patient. Since the main purpose
462
of this study was the preparation of possible clinically applicable multilayered wound
463
dressings for treatment of chronic venous ulcerations, it was important to somehow consider
464
the actual applicability of the proposed preparation approach. To validate our research study,
465
we prepared the same multilayers as on Si-wafer (model nanofilms) also on a clinical used
466
alginate dressing. For this purpose, we incorporated in it the highest PTX concentration used
467
in the model systems (20 mg/mL, Figure 6a) and compared the release kinetics with the
468
model systems for the same concentration. Slightly unexpectedly, the shape of the release
469
profiles of the model films and the clinically more realistic sample are almost identical.
470
Although there is a difference between the overall performance of this sample, since it
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471
overlaps with the release curve for the model system containing a lesser PTX amount (15
472
mg/mL compared to the targeted 20 mg/mL), this result is nevertheless very promising. This
473
difference probably originates from the distribution of PTX after the final rinsing during the
474
spin coating. For the “real” system PTX can be distributed through the whole bulk of the
475
material (both the multilayers and the substrate alginate dressing) in contrast to the model
476
multilayered system, where there is no possible uptake of PTX into the Si-wafer substrate.
477 478
3.2.2 Analysis of the released PTX dependence on the incorporated PTX dose
479
In order to evaluate the possible control over the released mass of PTX in respective samples
480
through the change of the initial concentration of PTX solution to be incorporated, we plotted
481
the released PTX masses at two different times. The chosen time points were after 6h of the
482
release, and after 5 days, and plotted them as a function of the starting PTX concentration
483
(ranging from 1 mg/mL to 20 mg/mL). Figure 6b shows the plotted results. Since we could
484
immediately observe a possible linear trend at both mentioned withdrawal times, we fitted
485
these results using a linear fit. The results are shown as part of Figure 6b. Based on the ~99%
486
and ~97% R2 values, we could confirm that the proposed, relatively simple tailoring of the
487
preparation procedure, is quite effective for controlling the final released amount of PTX,
488
hence providing a great tool for clinicians to adjust the PTX therapeutic dose for individual
489
patients.
490 491
3.2.3 The percentage of released PTX as a function of time
492
Figure 6c shows the results in the form of the percentage of PTX released as a function of
493
time. This percentage was calculated by using the final released amount (after six days when
494
the concentration stabilized). The most important findings from Figure 6c complements the
495
above discussed (section 3.2.1). Namely, not only can the dose be increased (even adjusted)
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by simply increasing the PTX concentration for “loading” into the multilayers, but that
497
although the incorporated PTX amount was increased, all incorporated PTX in the respective
498
samples was released in almost the same time frames. The latter can be seen in the very
499
similar release profiles in Figure 6c, reaching the end point of release after six days. This
500
finding is very important, if we consider potential clinical application, where in accordance
501
with the patients’ physical characteristics (mostly mass and overall health assessment), the
502
dose of a drug has to be adjusted. To put the prepared multi-layered wound dressings into a
503
more comprehensible perspective, we should here again mention that PTX provides an anti-
504
inflammatory activity, which can be provided by our system in a localized manner, namely
505
directly at/in the wound, where the drug can reach high concentrations. The latter could
506
ensure a much-improved efficacy, since the activity of PTX is dose dependant, as well as by
507
lowering the potential degradation of the drug (more likely through other types of
508
administration, where the drug has to cross the liver, the major organ for metabolism, prior to
509
reaching the bloodstream). PTX has a relatively low half-time (45-60 min), which can be
510
overcome by this type of administration. Finally, local delivery would also lead to much less
511
unwanted effects that mostly accompany systemic administration (per os, even intravenous).
512
The latter is especially important for drugs like PTX, which, although very effective, possess
513
a wide range of different pharmacodynamic activities. From Figure 6c it also again evident
514
that the multi-layered dressing prepared on the alginate substrate, resembling the more
515
realistic clinically applicable wound dressings, exhibits the same release profile. This once
516
more confirms the above statement that the model and the real system possess a similar
517
release performance.
518 519
3.2.4 Evaluation of the release mechanism
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520
Our objective was also to understand the release kinetics from our nanofilm multi-layered
521
coating systems from the mechanistic perspective. For this purpose, we applied several
522
models to describe the respective release profiles
523
studied different materials and their release performances before57-62. Unfortunately, as simple
524
as our system may seem (schematically depicted in Figure 1 and Figure 6), no single
525
available model was able to describe our profiles completely with the desired statistical
526
weight. Nevertheless, if the obtained release profiles are divided into two sections (dividing
527
the faster initial release from the steadier release at higher release times), fitting with available
528
models get more effective. Such division into two sections can be confirmed also by
529
calculating the first derivatives of the release profiles as shown in Figure 6d. Considering the
530
above discussion and the successfully applied release models (Zero-order and modified
531
Korsmayer-Peppas to account for the initial burst release), the two release regions could be
532
the initial diffusion controlled release (with a burst in the first minutes), and the following
533
second part, where the remainder of the PTX is released in a linear fashion (regardless of the
534
remaining PTX concentration in the samples) (Figure 6e and f).
535
The first derivatives calculated from the release data clearly confirm the above assumptions.
536
Namely, they show that all release profiles show the two release regions, and the distinction
537
point at 360 min of the release (inset graph in Figure 6d). Therefore, addressing the release
538
region before and after this part, seems to be appropriate. Here we have to mention also that
539
for the purpose of fitting, the zero-order kinetics model requires the data to be presented and
540
fitted in the form of the cumulative drug release (in our case this means the released PTX
541
mass until the chosen time). On the other hand, the Korsmayer-Peppas model fits release data
542
in the form of the fraction of the released drug amount in certain time point, which compared
543
with the final released amount (in our case this means the fraction of release PTX at point tx
544
and its comparison with the final release point at t = 8640 min). Due to the latter, the fitted
54-56
. The authors of the present study have
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results are shown in the form of the two respective graphs, exhibiting the mentioned
546
representation types. Table 1 presents the R2 values for respective samples and respective
547
regions, fitted with the mentioned models. As can be immediately deduced from the table,
548
both types of fitting produce excellent results (according to the calculated R2 values for
549
respective fits).
550 551 552
Table 1. R2 values for the fitted release results for all samples for both regions, using the zero-order kinetics model and the modified Korsmayer-Peppas model, respectively. Fast release part Constant release part (zero Sample/R2 value (Korsmayer-Peppas fit) order kinetics fit) 3TMC-3ALG-3PTX_1 0.98617 0.9954 3TMC-3ALG-3PTX_5 0.98726 0.97407 3TMC-3ALG-3PTX_10 0.97587 0.96388 3TMC-3ALG-3PTX_15 0.99158 0.98109 3TMC-3ALG-3PTX_20 0.98387 0.97533 ALG_3TMC-3ALG-3PTX_20 0.98111 0.98021
553 554
The suitability of the used models to describe the release profiles of the prepared samples,
555
makes sense also from the general description of both models. Korsmayer-Peppas derived a
556
simple relationship that describes the release of drugs from polymeric systems, which has
557
been successfully applied to describe release from several modified release dosage forms.
558
Additionally, the generic equation used to describe this model (the Power law) is applicable
559
for times, where release has not yet reached 60% of the overall amount of incorporated drug
560
63, 64
561
coefficient, which is ideal for the samples in this study. Therefore, the mentioned fits well
562
with our system. The second part of the release profile can be well described with a linear fit,
563
which is in pharmacy referred to as the zero-order model 65. In the latter, the release rate of a
564
respective drug is independent of its concentration. Such release is desired especially in
565
certain classes of medicines, like in pain control, heart and blood pressure maintenance etc.,
566
and therefore also in our wound dressing, where maintenance of the anti-inflammatory action
. Finally, a modified version of this model incorporated also the so called “burst release”
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567
for a prolonged time, could lead to an improved healing performance of the chronic venous
568
ulceration. The latter is true also from the clinical perspective, since a prolonged exposure to
569
PTX, and hence its action, improves its overall anti-inflammatory activity 13, 66.
570
As discussed in the section describing the results from the AFM measurements (Figure 5),
571
the surface morphology, although perhaps different in details for respective samples on the
572
nanoscale, exhibits also very similar features on the surface. These most likely represent holes
573
(pores), allowing for a more effective diffusion of PTX from the interior into the release
574
media. These similar surface features between samples are in agreement with the generally
575
similar release profiles obtained for all samples. Finally, the evaluation of the release profiles
576
and discussing possible mechanisms seems to point also to another important characteristic of
577
the prepared samples. Namely that most likely the drug is generally more or less
578
homogenously distributed throughout all layers (regardless of the polysaccharide of the
579
respective layer, Figure 7), which not only allows a better control over the release, but also
580
ensures a lowered risk of possible local drug overdoses that could appear in case of “pealing”
581
off respective layers, exposing a bigger drug amount to the media in a short time.
582 583
3.2.5 Clinical applicability of such materials based on the in vitro drug release performance
584
Finally, let us consider the suitability of the developed wound dressing system for treatment
585
in a real clinical setting. Patients come to the clinics with different states of their wounds that
586
occur in different shapes and sizes. Both mentioned make it a difficult task for the handling
587
doctor to choose and prepare the optimal therapy, as well as to “technically” perform the best
588
wound care. Our system with its controlled release and tailorability of the drug dose, allow for
589
relative simple on site handling and patient specific wound care individualization.
590
Additionally, controlled PTX release, which is exhibited by our system, allows for a
591
controlled anti-inflammatory action, and the dose tailorability, for a high level of personalized
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592
treatment options. All presented results were also obtained for samples of exact dimensions
593
(namely 1 cm x 1 cm). This means that we know the exact amounts of released PTX also per
594
unit area. This allows for an even more important patient specific optimization, namely the
595
size of the respective wound dressing to be applied for an individual patient, can be easily
596
tailored as well. As an example, based on our system, we calculated the incorporated amount
597
of PTX in two differently sized hypothetical wound dressings (Table 2).
598 599 600 601
Table 2: Total incorporated PTX amounts in the prepared samples in this study and calculated values for a hypothetical wound dressing with the dimensions 10 cm x 10 cm. The values were deduced and hence calculated based on the final release point of respective samples (the completeness of the release was confirmed through IR measurement). Sample/Size of hypothetical dressing 1 cm x 1 cm [mg] 10 cm x 10 cm [mg] 3TMC-3ALG-3PTX_1 0.535 53.5 3TMC-3ALG-3PTX_5 0.967 96.7 3TMC-3ALG-3PTX_10 2.120 212.0 3TMC-3ALG-3PTX_15 4.402 440.2 3TMC-3ALG-3PTX_20 5.275 527.5 ALG_3TMC-3ALG-3PTX_20 4.581 458.1
602 603
3.3
604
In vitro testing based on human tissue derived in vitro cell models (based on skin cells –
605
keratinocytes, fibroblasts, melanocytes, and on immune cells – macrophages or monocytes, as
606
well as a combination thereof) serves as the best indication for developed materials safety and
607
possibly even clinical efficiency for topical, transdermal and open wound applications (either
608
in wound care, drug delivery on and through the skin or in skin tissue engineering). Their use
609
is a direct follow-up to the development of different therapeutic systems, evaluating their
610
increasing complexity with a progressive assessment of desired biochemical response. In
611
comparison with other available studies, which are mostly conducted on either animal-derived
612
or cancer-transformed cells
613
The most important among them is the possibility to obtain results-based conclusions with
614
regard to an actual clinical setting
615
experiments to show the potential of the prepared multilayers to be used in wound care.
Cell based assays
67-70
, using primary human-derived cells has many advantages.
71
. Considering the mentioned, we designed a range of
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616 617
3.3.1 Effects of eluates from PTX loaded and unloaded multilayers on viability of fibroblasts
618
MRC-5 fibroblasts exposed to eluates of PTX loaded and unloaded samples did not present
619
changes in cell density or morphology compared to the untreated cultures in brightfield
620
microscopy (Figure 8A-C).
621 622
Figure 8: Morphology of MRC-5 cells exposed to undiluted eluates of PTX loaded (A) and
623
unloaded samples (B) for 48h compared to untreated cells (C).
624 625
However, dehydrogenase activity, as an indication for cell viability, was unchanged in the
626
case of cell exposure to the samples not loaded with PTX (Figure 9). In the PTX loaded
627
samples a biphasic reaction was seen. After 24h, viability was increased at all dilutions
628
compared to untreated cells and after 48h and 72h viability was decreased in cells exposed to
629
undiluted and 1:2 diluted eluates. The difference between untreated cells and the eluates was
630
larger after 72h than after 48h exposure.
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631 632
Figure 9: Dehydrogenase activity as indication for cell viability of MRC-5 cells exposed to
633
undiluted (pure) and diluted eluates of PTX loaded (LEFT) and unloaded samples (RIGTH) at
634
time points 24h, 48h, and 72h. Activity is normalized to untreated cells as 100%
635 636
These results confirm that the PTX released from the multilayers did not significantly reduce
637
the cell numbers but increased the ATP content after 24h and it markedly decreased at later
638
time points. This controversy can be possibly explained by the action of PTX on
639
mitochondria. For example, endogenous ATP generation has been reported by Feyli et al in
640
relation to an increased motility of spermatocytes after exposure to PTX
641
exposures to PTX, reduced ATP levels in hepatocytes were reported before
642
showed a reduction of ATP levels starting at PTX concentrations of 50 µM. Since the
643
material, in which PTX was embedded, did not reduce viability and since PTX is known to be
644
only minimally cytotoxic at concentrations in the range of 2 mM 74, the action on ATP levels
645
is a likely reason for the disparity of microscopic data and ATP content.
72
. At longer
73
. The authors
646 647
3.3.2 Adhesion and proliferation of fibroblasts in direct contact with the multilayers
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648
Upon direct contact with the PTX loaded multilayers, the cell density is decreased. The
649
decrease was moderate after 24h and prominent after 72h. The respective changes can be seen
650
when dehydrogenase activity as an indicator for cell viability (Figure 10A) is used and when
651
cell numbers are determined (Figure 10B). Typical changes in cell morphology are also seen
652
(Figure 11). While fibroblasts cultured on glass showed the typical elongated form (Figure
653
11A), cells on the sample material formed cell clusters, which is not typical for this type of
654
cells. Furthermore, cell density was markedly reduced in these cultures (Figure 11B and C).
655 656
Figure 10: Dehydrogenase activity (A) and cell number (B) of MRC-5 fibroblasts grown on
657
glass surface or on PTX loaded and unloaded multilayers. Activity is normalized to the
658
activity of cells grown on the PTX unloaded multilayers while glass surface serves as
659
reference for optimal growth conditions.
660 661
Figure 11: Morphology of MRC-5 cells grown for 72h on glass surface and PTX loaded and
662
unloaded multilayers. A confluent cell monolayer is seen when cells are grown on glass (A).
663
Rarefication of the cell layer is obvious in the PTX loaded samples (B) while formation of
664
cell clusters is seen MRC-5 cells were grown on unloaded multilayers (C).
665
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666
The poor adhesion of cells to the multilayers may be due to TMC (a chitosan derivative).
667
Chitosan membranes were previously shown to require a surface modification to allow good
668
adhesion and proliferation of fibroblasts
669
chitosan to lower extent than other cell types
670
adhesion and proliferation of fibroblasts as well 77. For example, it was previously found that
671
alginate can dissolve in the growth media through cation exchange (Na+ from the growth
672
media for the Ca2+ from the material), leading to extensive material transformation, even
673
degradation 78. Considering the fact that cells need a stable environment to be attached
674
these results are not that surprising
675
than the cell-growth substrate interaction is the formation of cell clusters. Such clusters were
676
also observed when MRC-5 fibroblasts were seeded on the multilayers (Figure 11C).
75
. It was also found that fibroblasts adhered to 76
. Alginate has to be modified to improve
79-81
,
82
. One indication that cell-cell interactions are stronger
677 678
3.3.3 Biocompatibility on human skin-derived cell culture
679
Considering the above-mentioned results, we chose to perform another safety related testing
680
on human skin derived fibroblasts. Based on the poor adhesion results, there were two main
681
objectives behind our choice of this experimental setup. First, we wanted to assess if the
682
proposed thin film compositions release any toxic degradation products that could negatively
683
influence the cell growth, as well as to evaluate possible local PTX overdoses that could also
684
potentially harm the growing fibroblast cells (especially considering the versatile PTX
685
pharmacodynamic activities). The second objective was to assess the viability of the
686
fibroblasts cell culture from another perspective. For this purpose and in order to obtain as
687
much information as possible with regard to the possible influence of our materials on the
688
fibroblast cells during their targeted application in wound treatment, the actual physiological
689
situation during a potential use of such materials was simulated. This was done through the
690
use of respectively withdrawn samples during the in vitro drug release testing after desired
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691
time intervals (e.g. 1, 5, 10, … minutes), composed of the released PTX amount and possible
692
film degradation products (composed of TMC and ALG), for incubation with fibroblasts for
693
24h. The withdrawn samples during the in vitro drug release testing were pipetted together
694
with the fixed number of cells into plastic containers and incubated until confluence was
695
reached. In order to assess the actual influence of the materials we have developed, control
696
samples (ADMEM + 5 wt.% FBS) were prepared, using only the growth media and cells. The
697
results are shown in Figure 12Error! Reference source not found., where each column
698
represents the viability of fibroblasts after incubation for 24h with consecutively withdrawn
699
samples during the release results (therefore the sample names below the columns in
700
minutes).
701 702 703
Figure 12: Biocompatibility testing results based on incubation of fibroblasts for 24h with
704
consecutively withdrawn samples during in vitro release testing (on the x-axis the minutes
705
represent the times, at which the samples were withdrawn during the release study).
706
From Figure 12Error! Reference source not found. we can immediately observe that the
707
samples with the variable PTX content in most cases outperform the control sample. In other
708
words, regardless of the PTX concentrations, release after different time intervals, all samples
709
exhibit at least the same viability as the control sample.
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710
This result is important in two regards. Firstly, the as-prepared samples are biocompatible
711
regardless of the used PTX concentration, and do not release any harmful TMC or ALG
712
degradation products that could occur during the same period of time. And secondly, the
713
obtained results seem very promising for the best performing material-drug combinations
714
(especially for the samples 3TMC-3ALG-2PTX_10 and 15) with regard to our desire to
715
prepare novel wound dressings that are not only safe, but show even some activity towards
716
healing promotion. Together with the beneficial activity of PTX in lowering the inflammation
717
locally, these could be a much-desired improvement in local treatment of chronic wounds (in
718
our case the chronic venous ulceration). While the samples with the lower incorporated PTX
719
doses (1 and 5 mg/mL respectively) seem to perform as well, the results for the sample with
720
the highest PTX incorporated mount shows a slightly decreased viability, when compared to
721
the control sample. Although there were still no observable dead cells during testing, this
722
concentration might be over the threshold to induce a positive, healing promoting effect, and
723
should therefore be not exceeded, if practical implementation will follow in the future. Trying
724
to understand the positive influence of PTX on the fibroblast growth, we have to take into
725
account that PTX acts as a competitive nonselective phosphodiesterase inhibitor, which raises
726
intracellular cAMP, activates PKA, inhibits TNF and leukotriene synthesis, and reduces
727
inflammation and innate immunity
728
also shown before that PTX for itself can contribute to an improved wound healing, especially
729
in treating diabetic ulcers 14.
730
Since this experiment showed that the actual released amounts of the drug and/or degradation
731
products, does not cause a harmful effect on the fibroblasts, we are positive that the overall
732
performance of the prepared multilayers is still very much beneficial.
13, 83
. The latter activity is further assessed below. It was
733 734
3.3.4 Effect on LPS-induced inflammation of monocytes
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735
PTX was incorporated into the prepared multilayers mainly due to its versatile
736
pharmacodynamic activities, contributing to an anti-inflammatory effect. To show that the
737
released PTX amounts from the prepared samples can actually lower an inflammatory
738
response, we performed an additional cell based assay using human monocytes and their
739
TNF-α secretion upon stimulation by LPS.
740
We have found that the PTX released from the multilayers was able to suppress secretion of
741
TNF-α induced by endotoxin/lipopolysaccharide (LPS, Figure 13). This effect was
742
significant up to a dilution of 1:10 of the eluates. Eluates produced from the non-loaded
743
samples reduced the LPS-induced cytokine secretion to a much lower extent. Eluates in the
744
absence of LPS stimulation caused no effect on secretion of TNF-α.
745 746
Figure 13: Secretion of TNF-α after exposure to LPS in the presence of PTX loaded samples
747
(LEFT) and PTX only (RIGHT).
748 749
The anti-inflammatory effect of the PTX loaded multilayers could be shown but also eluates
750
from the PTX unloaded samples reduced the LPS-induced secretion of TNF-α. This effect is
751
most probably due to the anti-inflammatory effect of the chitosan derivative TMC and
752
alginate. Nanoparticle formulations containing these two polysaccharides were previously
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753
shown to exhibit prominent anti-inflammatory effects in monocytes and keratinocytes
754
exposed to Propionibacterium acnes 84.
755 756
3.3.5 Evaluation of the samples possible clinical applicability
757
Let us now once more consider the combined results from the cell based assay in regard of a
758
possible clinical applicability of the PTX loaded multilayers. As written above, the described
759
results prove that there are no local overdoses, which is the more important for a drug like
760
PTX with its various activities that could even locally, in higher doses, possibly inflict
761
unwanted effects. These are still possible, but based on the obtained results, there should not
762
be any serious unwanted effect also on other cells, and hence tissues, with which our
763
formulation could come into contact. Although, the adhesion testing was not that promising,
764
we believe that the other rather positive results, including the proof of the inflammation
765
lowering effect in the released doses, outweight this part.
766
Therefore, we can say that we have prepared formulations with variable PTX content, which
767
exhibit a dual positive pharmacotherapeutic effect. Namely PTX acts by lowering the
768
inflammation locally while the complete samples add to the overall wound healing
769
performance.
770 771 772
4
773
We have prepared a model and “real” multi-layered wound dressing system, composed from
774
two biocompatible polysaccharides and a potent anti-inflammatory drug. The combination of
775
physic-chemical and functional methods, as well as several cell assays, has shown the
776
potential of the mentioned to be used in future treatment of chronic wounds. Since the used
777
drug pentoxifylline was already proven efficient for systemic treatment of the chronic venous
CONCLUSIONS
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778
ulceration, we have reason to believe that the prepared wound dressings could significantly
779
improve the treatment of the latter by local controlled delivery of the drug and an overall
780
healing promotion. This was confirmed using viability and immune response related studies
781
in vitro on human skin fibroblasts and monocytes, respectively.
782
The developed preparation procedure allows for a tailorable treatment by the adjustment of
783
the initial incorporated pentoxifylline dose, as well as “cutting” of the multi-layered dressing
784
to the desired wound size. An even easier on-site handling would be possible with the
785
production of the developed wound dressings in the form of a pentoxifylline impregnated
786
role, which the handling clinician just cuts to the appropriate size on site to suit the specific
787
patient needs.
788 789
5
790
This work has been partially financed by the Slovenian National Agency (grant numbers: P3-
791
0036, I0-0029 and P2-0118). The authors are grateful to Dr. D. Reishofer, from the
792
Technical University of Graz/Austria for his help in regard of profilometry measurements.
ACKNOWLEDGEMENT
793 794
6
795
The
CONFLICT OF INTEREST authors
state
no
conflict
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of
interest.
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7
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53. Powers, J. G.; Higham, C.; Broussard, K.; Phillips, T. J., Wound healing and treating wounds: Chronic wound care and management. Journal of the American Academy of Dermatology 2016, 74, (4), 607-25. 54. Delfour, M. C., Drug Release Kinetics from Biodegradable Polymers via Partial Differential Equations Models. Acta Appl Math 2012, 118, (1), 161-183. 55. Yin, C.; Li, X. C., Anomalous diffusion of drug release from a slab matrix: Fractional diffusion models. Int J Pharm 2011, 418, (1), 78-87. 56. Aguzzi, C.; Cerezo, P.; Salcedo, I.; Sanchez, R.; Viseras, C., Mathematical models describing drug release from biopolymeric delivery systems. Mater Technol 2010, 25, (3-4), 205-211. 57. Maver, U.; Bele, M.; Jamnik, J.; Gaberšček, M.; Planinšek, O., A fast and simple method for preparation of calcium carbonate-drug composites for fast drug release. Mater Res Bull 2013, 48, (1), 137-145. 58. Nadrah, P.; Maver, U.; Jemec, A.; Tišler, T.; Bele, M.; Dražić, G.; Benčina, M.; Pintar, A.; Planinšek, O.; Gaberšček, M., Hindered Disulfide Bonds to Regulate Release Rate of Model Drug from Mesoporous Silica. ACS Appl. Mater. Interfaces 2013, 5, (9), 3908-3915. 59. Žužek Rožman, K.; Pečko, D.; Šturm, S.; Maver, U.; Nadrah, P.; Bele, M.; Kobe, S., Electrochemical synthesis and characterization of Fe 70Pd 30 nanotubes for drug-delivery applications. Mater Chem Phys 2012, 133, (1), 218-224. 60. Ukmar, T.; Maver, U.; Planinšek, O.; Pintar, A.; Kaučič, V.; Godec, A.; Gaberšček, M., Guest-host van der Waals interactions decisively affect the molecular transport in mesoporous media. J Mater Chem 2012, 22, (3), 1112-1120. 61. Ukmar, T.; Maver, U.; Planinšek, O.; Kaučič, V.; Gaberšček, M.; Godec, A., Understanding controlled drug release from mesoporous silicates: Theory and experiment. J Control Release 2011, 155, (3), 409-417. 62. Maver, T.; Maver, U.; Mostegel, F.; Griesser, T.; Spirk, S.; Smrke, D.; StanaKleinschek, K., Cellulose based thin films as a platform for drug release studies to mimick wound dressing materials. Cellulose 2015, 22, (1), 749-761. 63. Korsmeyer, R. W.; Gurny, R.; Doelker, E.; Buri, P.; Peppas, N. A., Mechanisms of potassium chloride release from compressed, hydrophilic, polymeric matrices: effect of entrapped air. J Pharm Sci 1983, 72, (10), 1189-91. 64. Peppas, N. A., A model of dissolution-controlled solute release from porous drug delivery polymeric systems. J Biomed Mater Res 1983, 17, (6), 1079-87. 65. Vanbommel, E. M. G.; Dezentje, R. F. R.; Crommelin, D. J. A.; Fokkens, J. G., Drug Release Kinetics from the Gradient Matrix System - Mathematical-Modeling. Int J Pharmaceut 1991, 72, (1), 19-27. 66. Ubbink, D. T.; Santema, T. B.; Stoekenbroek, R. M., Systemic wound care: a metareview of cochrane systematic reviews. Surgical technology international 2014, 24, 99-111. 67. Uklejewski, R.; Rogala, P.; Winiecki, M.; Kedzia, A.; Ruszkowski, P., Preliminary Results of Implantation in Animal Model and Osteoblast Culture Evaluation of Prototypes of Biomimetic Multispiked Connecting Scaffold for Noncemented Stemless Resurfacing Hip Arthroplasty Endoprostheses. Biomed Res Int, doi: 10.1155/2013/689089 2013. 68. Santillan, G.; Morelli, S.; Katz, S.; Mondelo, N.; Boland, R.; Puche, R.; Roldan, E., Action of the novel bisphosphonate lidadronate in animal models and osteoblast-like cells. Bone 2004, 34, S69-S70. 69. Ma, R.; Tang, S.; Tan, H.; Lin, W.; Wang, Y.; Wei, J.; Zhao, L.; Tang, T., Preparation, characterization, and in vitro osteoblast functions of a nanohydroxyapatite/polyetheretherketone biocomposite as orthopedic implant material. Int J Nanomedicine 2014, 9, 3949-61.
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70. Gyorgyey, A.; Ungvari, K.; Kecskemeti, G.; Kopniczky, J.; Hopp, B.; Oszko, A.; Pelsoczi, I.; Rakonczay, Z.; Nagy, K.; Turzo, K., Attachment and proliferation of human osteoblast-like cells (MG-63) on laser-ablated titanium implant material. Materials science & engineering. C, Materials for biological applications 2013, 33, (7), 4251-9. 71. Czekanska, E. M.; Stoddart, M. J.; Ralphs, J. R.; Richards, R. G.; Hayes, J. S., A phenotypic comparison of osteoblast cell lines versus human primary osteoblasts for biomaterials testing. J Biomed Mater Res A 2014, 102, (8), 2636-43. 72. Feyli, S. A.; Ghanbari, A.; Keshtmand, Z., Therapeutic effect of pentoxifylline on reproductive parameters in diabetic male mice. Andrologia 2017, 49, (1). 73. Massart, J.; Robin, M. A.; Noury, F.; Fautrel, A.; Letteron, P.; Bado, A.; Eliat, P. A.; Fromenty, B., Pentoxifylline aggravates fatty liver in obese and diabetic ob/ob mice by increasing intestinal glucose absorption and activating hepatic lipogenesis. British journal of pharmacology 2012, 165, (5), 1361-74. 74. Teicher, B.; Holden, S.; Herman, T.; Epelbaum, R.; Pardee, A.; Dezube, B., Efficacy of pentoxifylline as a modulator of alkylating agent activity in vitro and in vivo. Anticancer research 1991, 11, (4), 1555-1560. 75. Luna, S. M.; Silva, S. S.; Gomes, M. E.; Mano, J. F.; Reis, R. L., Cell adhesion and proliferation onto chitosan-based membranes treated by plasma surface modification. J Biomater Appl 2011, 26, (1), 101-16. 76. Carvalho, C. R.; Lopez-Cebral, R.; Silva-Correia, J.; Silva, J. M.; Mano, J. F.; Silva, T. H.; Freier, T.; Reis, R. L.; Oliveira, J. M., Investigation of cell adhesion in chitosan membranes for peripheral nerve regeneration. Materials science & engineering. C, Materials for biological applications 2017, 71, 1122-1134. 77. Sarker, B.; Singh, R.; Silva, R.; Roether, J. A.; Kaschta, J.; Detsch, R.; Schubert, D. W.; Cicha, I.; Boccaccini, A. R., Evaluation of fibroblasts adhesion and proliferation on alginate-gelatin crosslinked hydrogel. PloS one 2014, 9, (9), e107952. 78. Maver, T.; Gradišnik, L.; Kurečič, M.; Hribernik, S.; Smrke, D. M.; Maver, U.; Kleinschek, K. S., Layering of different materials to achieve optimal conditions for treatment of painful wounds. Int J Pharmaceut 2017, 529, (1–2), 576-588. 79. Hersel, U.; Dahmen, C.; Kessler, H., RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials 2003, 24, (24), 4385-4415. 80. Puppi, D.; Chiellini, F.; Piras, A.; Chiellini, E., Polymeric materials for bone and cartilage repair. Progress in Polymer Science 2010, 35, (4), 403-440. 81. Shin, H.; Jo, S.; Mikos, A. G., Biomimetic materials for tissue engineering. Biomaterials 2003, 24, (24), 4353-4364. 82. Lee, K. Y.; Mooney, D. J., Alginate: properties and biomedical applications. Prog Polym Sci 2012, 37, (1), 106-126. 83. Babaei, S.; Bayat, M.; Nouruzian, M.; Bayat, M., Pentoxifylline improves cutaneous wound healing in streptozotocin-induced diabetic rats. European Journal of Pharmacology 2013, 700, (1–3), 165-172. 84. Friedman, A. J.; Phan, J.; Schairer, D. O.; Champer, J.; Qin, M.; Pirouz, A.; BlecherPaz, K.; Oren, A.; Liu, P. T.; Modlin, R. L.; Kim, J., Antimicrobial and anti-inflammatory activity of chitosan-alginate nanoparticles: a targeted therapy for cutaneous pathogens. J Invest Dermatol 2013, 133, (5), 1231-9.
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Table of contetns graphic
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Figure 1: Schematic depiction of the prepared multi-layered films. 186x152mm (300 x 300 DPI)
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Figure 2: Layer thickness and roughness (a), and QCM-D dry mass (b) of multilayer polysaccharide nanofilms assembled from the oppositely charged TMC and ALG. 379x266mm (300 x 300 DPI)
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Figure 3: Water contact angle of multilayer polysaccharide nanofilms assembled from the oppositely charged TMC and ALG. 290x474mm (300 x 300 DPI)
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Figure 4: ATR-IR spectra of multilayer polysaccharide nanofilms prepared (a) without and (b) with PTX incorporation at different concentrations. The spectra of multilayers built on alginate dressing and pure PTX drug is shown in (b). 293x499mm (300 x 300 DPI)
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Figure 5: AFM height images of multilayer polysaccharide nanofilms, incorporated with PTX at different concentrations. Top: 1 x 1 cm2 and bottom: 5 x 5 cm2. 55x17mm (300 x 300 DPI)
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Figure 6: Results from the in vitro release study: a) PTX mass as a function of time, b) linearity testing of the dependence between the drug loading and released PTX mass, c) the % of release PTX as a function of time, d) first derivatives of the release data for all samples, e) fitting of the release profiles up to 360 min using the Zero order model, and f) fitting of the release data from 360 min to 8640 min using the Korsmayer-Peppas model. 208x236mm (300 x 300 DPI)
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Figure 7: Schematic depiction of the influence of the final multilayer preparation step on PTX distribution in the sample. Black arrows show the presumable burst like release, whereas the red arrows present the slower release after 360 min. 318x113mm (150 x 150 DPI)
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Figure 8: Morphology of MRC-5 cells exposed to undiluted eluates of PTX loaded (A) and unloaded samples (B) for 48h compared to untreated cells (C). 35x6mm (300 x 300 DPI)
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Figure 9: Dehydrogenase activity as indication for cell viability of MRC-5 cells exposed to undiluted (pure) and diluted eluates of PTX loaded (LEFT) and unloaded samples (RIGTH) at time points 24h, 48h, and 72h. Activity is normalized to untreated cells as 100% 168x95mm (150 x 150 DPI)
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Figure 10: Dehydrogenase activity (A) and cell number (B) of MRC-5 fibroblasts grown on glass surface or on PTX loaded and unloaded multilayers. Activity is normalized to the activity of cells grown on the PTX unloaded multilayers while glass surface serves as reference for optimal growth conditions. 58x17mm (300 x 300 DPI)
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Figure 11: Morphology of MRC-5 cells grown for 72h on glass surface and PTX loaded and unloaded multilayers. A confluent cell monolayer is seen when cells are grown on glass (A). Rarefication of the cell layer is obvious in the PTX loaded samples (B) while formation of cell clusters is seen MRC-5 cells were grown on unloaded multilayers (C). 36x6mm (300 x 300 DPI)
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Figure 12: Biocompatibility testing results based on incubation of fibroblasts for 24h with consecutively withdrawn samples during in vitro release testing (on the x-axis the minutes represent the times, at which the samples were withdrawn during the release study). 297x119mm (150 x 150 DPI)
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Figure 13: Secretion of TNF-α after exposure to LPS in the presence of PTX loaded samples (LEFT) and PTX only (RIGHT). 327x129mm (150 x 150 DPI)
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