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Electrospun Blends of Gelatin and Gelatin-dendrimer Conjugates as a Wound Dressing and Drug Delivery Platform Alpana A Dongargaonkar, Gary L. Bowlin, and Hu Yang Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 15 Oct 2013 Downloaded from http://pubs.acs.org on October 20, 2013
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Electrospun Blends of Gelatin and Gelatin-dendrimer Conjugates as a Wound Dressing and Drug Delivery Platform
Alpana A. Dongargaonkar1, Gary L. Bowlin2, Hu Yang1,3,*
1
Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA
2
Department of Biomedical Engineering, University of Memphis, Memphis, TN 38152, USA 3
Massey Cancer Center, Virginia Commonwealth University, Richmond 23298, USA
*Correspondence should be addressed to Hu Yang, Department of Biomedical Engineering, Virginia Commonwealth University, 401 West Main Street, P.O. Box 843067, Richmond, VA 23284. E-mail:
[email protected] 1
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ABSTRACT In this work, we report a new nanofiber construct based on electrospun blends of gelatin and gelatin-dendrimer conjugates. Highly branched star-shaped polyamidoamine (PAMAM) dendrimer G3.5 was covalently conjugated to gelatin via EDC/NHS chemistry. Blends of gelatin and gelatin-dendrimer conjugates mixed with various loading levels of silver acetate (0, 0.83%, 1.65%, and 3.30% w/w) were successfully electrospun into nanofiber constructs (NCs). The NCs were further converted into semi-interpenetrating networks (sIPNs) with photoreactive polyethylene glycol diacrylate (Mn=575 gmol-1) (PEG DA575). They were characterized in terms of fiber morphology, diameter, pore size, permeability, degradation, and mechanical properties. The resulting sIPN NCs retained nanofiber morphology, possessed similar fiber diameters to counterpart NCs, and gained improved structural stability. The sIPN NCs also showed good swelling capacity owing to porous structures and were permeable to aqueous solutions. Silvercontaining sIPN NCs allowed sustained silver release and showed antimicrobial activity against two common types of pathogens—Staphylococcus aureus and Pseudomonas aeruginosa. Incorporation of dendrimers into the gelatin nanofibers through covalent conjugation not only expands drug loading capacity of nanofiber constructs but provides tremendous flexibility for developing multifunctional electrospun dressing materials. Key Words: Electrospinning, Dendrimer, Gelatin-dendrimer hybrid, Nanotechnology, Wound care
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INTRODUCTION The wound healing cascade is a sequence of five typical local events: acute inflammation, chronic inflammation, growth of granulation tissue, foreign body reaction, and remodeling, during which specific “cues”, e.g., cells and bioactive molecules, are involved and orchestrated in a spatial and temporal fashion. Therefore, wound dressing materials are expected to not only provide basic function of covering wounds from infection but possess controlled structures and properties as well as adaptability to accommodate functions essential for wound healing.1 Current FDA-approved wound dressings can be classified into three major categories based on properties and modes of action: passive products that are traditional dressings such as gauze, interactive products that are generally permeable to oxygen and water but impermeable to bacteria and commonly applied to wounds that do not produce significant amounts of exudates, and bioactive products that deliver bioactive substances beneficial to wound healing although some products are composed of substances that inherently promote wound healing. In particular, bioactive products have been developed primarily based on natural polymers including gelatin, collagen, noncollagenous proteins, proteoglycans, alginates, and chitosan.2,
3
Nonetheless, few
existing dressing materials can satisfactorily fulfill multiple purposes or be capable of delivering multiple cues to complement wound healing. Development of effective dressings requires the underlying dressing material to be structurally adaptable and possess controlled characteristics. In this work, we report a novel highly adaptable biocompatible platform composed of electrospun blends of gelatin and gelatin-dendrimer conjugates, which is further intertwined with a cross-linked polyethylene glycol (PEG) network to form semi-interpenetrating networks (sIPNs).4 This new nanofiber construct has adaptable structure and compelling properties. 3
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Gelatin is a biocompatible and degradable substance and has been widely used to develop wound dressings.2 Dendrimers are highly branched macromolecules comprised of a central core, internal branches and a number of reactive surface groups.5-7 It is the presence of numerous surface groups that makes dendrimers suitable carriers for carrying high drug payloads and/or multifunctionality.8-11 Dendrimers can complex with silver to exert higher antimicrobial activity than silver alone.12,
13
PAMAM dendrimers themselves exhibit strong anti-inflammatory
activity.14 Electrospinning enables a high degree of control over a wide range of structural features and properties of the construct such as shape, porosity, modulus, degradation, fiber size and orientation.15 Fabricating polymers into nanofiber constructs with electrospinning to mimic the features of extracellular matrix (ECM) has become an important approach in wound dressing development and drug delivery.16-23 In addition, nanoscale surface features and topography of nanofibers regulate cell behaviors, such as adhesion, proliferation, and infiltration, which are desirable features for wound healing. Incorporation of dendrimers into the gelatin nanofibers through covalent conjugation not only expands drug loading capacity of nanofiber constructs but provides tremendous flexibility for developing multifunctional electrospun dressing materials. Overall, we envision that this unique dendrimer-gelatin nanofiber construct can be tailored to deliver state-of-the-art therapeutics as they are developed to treat different types of wounds including chronic wounds, burns, and skin cancers. As bacterial infection is commonly associated with wound healing, silver as an integral component was incorporated into the electrospun dressing. Silver-containing nanofiber constructs (NCs) on the basis of blends of gelatin (G) and gelatin-dendrimer conjugates (G-D) were fabricated via electrospinning and further processed with PEG diacrylate to form sIPN NCs. They were characterized in terms of fiber morphology, diameter, pore size, permeability, 4
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degradation, and mechanical properties. Furthermore, silver release kinetics and antimicrobial properties of the dressing materials were assessed.
MATERIALS AND METHODS Materials Polyamidoamine (PAMAM) dendrimer generation 3.5 (G3.5) (core: ethylene diamine (EDA); G = 3.5; [dendri-poly(amidoamine)-(COONa)64]), 10 wt% solution in methanol) was purchased from Dendritech (Midland, MI). 1,1,1,3,3,3 Hexafluoro-2-propanol (HFP) was purchased from TCI America (Portland, OR). Agar, aqueous silver standard for ICP-OES, dimethoxyphenylacetophenone
(DMPA),
1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide
(EDC), ethanol (denatured), ethyl ether (anhydrous), gelatin (porcine skin- type A, ~300 g bloom), ninhydrin, N-hydroxysuccinimide (NHS), peptone, phosphate buffered saline (PBS), poly (ethylene glycol) diacrylate (Mn=575 g/mol, ρ=1.12 g/mL at 25 °C) (PEG DA575), silver acetate, sodium bicarbonate (NaHCO3), sodium chloride, and tryptone were purchased from Sigma-Aldrich (St. Louis, MO).
Synthesis of Gelatin-Dendrimer Conjugates (G-D) Gelatin-dendrimer conjugates were obtained by covalently coupling PAMAM dendrimer G3.5 to gelatin. Briefly, a gelatin solution was prepared by dissolving 20 mg of gelatin in 20 ml of 0.1N NaHCO3 solution and stirring it at 80 °C until a clear solution formed. Upon the removal of methanol from PAMAM dendrimer G3.5 stock solution via rotary evaporation, G3.5 (20.7 5
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mg) was redissolved in 2 ml of deionized water and then reacted with 3 mg of NHS and 5 mg of EDC while stirring for 24 h to yield G3.5-NHS esters. Finally, the G3.5-NHS solution (approximately 2 ml) was added to the gelatin solution (approximately 20 ml), and the mixture was vigorously stirred for 4 h while being kept in an ice water bath. Following centrifugation, the gelatin-dendrimer conjugates in supernatant were collected and precipitated out of cold ether followed by dialysis in a dialysis tubing of 12-14 kDa MWCO and freeze drying. Ninhydrin assay was performed to examine the percentage of primary amines in the gelatin backbone before and after conjugation.24
Fabrication of NCs via Electrospinning All NCs were fabricated under the same electrospinning conditions. In general, 1 g of G/G-D blends alone or mixed with various amounts of silver acetate (Table 1) was suspended in 10 ml of HFP followed by thorough mixing for 24 h on a shaker plate. The electrospinning solution was then loaded into a 10 ml Becton Dickinson syringe and placed in a KD Scientific syringe pump. The syringe pump was set to deliver the solution at a rate of 5 ml/h. A voltage of 25 kV was applied to the 18 gauge blunt-tip needle of the syringe by a high voltage power supply (Spellman CZE1000R) (Spellman High Voltage Electronics Corporation, Hauppauge, NY). A stainless steel mandrel (7.5cm×2.5cm×0.5cm, L×W×T) was placed approximately 125 mm from the needle tip and rotated at ~500 rpm for fiber collection. After electrospinning, the resulting mat was carefully removed from the mandrel, placed in a fume hood for degassing and stored in a desiccator.
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Fabrication of sIPN NCs Rectangular samples (7.5 cm×5 cm) were cut from nanofiber mats. A 2 ml of ethanol solution containing 100 µl of PEG DA575 and 4 mg of DMPA was poured onto the sample and allowed to stay for about 30 min. The samples were then exposed to UV light (UVP Blak-Ray Long Wave Lamp, 100 Watts) for 2 min on each side and then air dried.4
Characterization Scanning electron microscopy images of nanofiber samples were taken on a Zeiss EVO 50 XVP scanning electron microscope (SEM). UTHSCSA Image tool Version 3.0 was utilized to determine fiber diameter on the basis of 30 fibers randomly chosen in the image. Porosity of construct
(n=3)
was
calculated
using
the
following
formula:
Porosity = (1 − ρ scaffold ) / ρ material × 100% where ρ scaffold is calculated construct density and ρ material is known material density (1.41 g/cm3 for collagen). Construct permeability and pore size were determined following a method described by Sell et al.25 Tensile testing was conducted on the MTS Bionix 200®- Mechanical testing system with a 100 N load cell. Dog-bone shaped samples (n=6) were cut out from each construct and then placed in the metal grips of the mechanical testing system to be tested at a rate of 10 mm/min.26 Stress, strain, modulus and energy to break were tested using the MTS Bionix 200 Mechanical Testing System in conjunction with TestWorks 4.0 software.
Swelling Studies 7
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To mimic clinical conditions, simulated wound fluid (SWF) was used for swelling studies. SWF consisted of 50% calf serum and 50% maximum recovery diluent (0.1% w/v peptone and 0.9% w/v sodium chloride).27 Samples (2.5×2.5 cm, n=3) were weighed (Wd) and then immersed in 5 ml of SWF at room temperature. At pre-determined time points up to 48h, the samples were taken out of the fluid, blot dried and weighed. Equilibrium swelling ratio (%) is calculated as
Ws × 100 , where Wd is the mass of dry sample and Ws is the mass of swollen Wd
sample when no further weight increase was observed.
In Vitro Degradation Studies Construct degration at 37 °C in three types of media including SWF, Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and conditioned cell medium was investigated. Conditioned cell medium was DMEM supplemented with 10% FBS conditioned by confluent BJ-hTERT fibroblasts for 24 h. Samples (1×1 cm, n=3) were immersed in 1.5 ml of medium for various lengths of time (6, 12, or 24h), taken out, centrifuged, freeze dried, and weighed. Mass loss (%) is defined as
Wo − Wd ×100 , where Wo is Wo
the initial mass of the dry construct and Wd is the mass of the residue after incubation and freezedrying.
Silver Release Studies
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Silver-containing nanofiber constructs (2.5×2.5 cm) were incubated in 20 ml of PBS at 37°C. At pre-determined time points, 10 ml of PBS was collected for silver content analysis. Meanwhile, 10 ml of fresh PBS was added to maintain the volume of the medium for continuous observation. Silver content was quantified by inductively coupled plasma-optical emission spectroscopy (ICP-OES).28,
29
The Peppas-Sahlin equation was applied for data fitting to
understand silver release mechanism.30
Antimicrobial Activity Assessment The antimicrobial activity of silver-containing nanofiber scaffolds against two common wound pathogens—gram positive Staphylococcus aureus (strain N315) and gram negative
Pseudomonas aeruginosa (strain PA01)—was assessed. Sample scaffold (2.5×2.5 cm) was incubated in a test tube containing 10 ml of 105 diluted bacterial solution inoculated in SWF at 37 °C. Aliquots (100 µl) were withdrawn at 4 h, 24 h, and 48 h and then plated on luria agar plates. The plates were incubated overnight at 37 °C. The bacterial colonies present on the plate were counted and expressed as cfu/ml (colony-forming units per milliliter).
Statistical Analysis Statistical analysis was based on analysis of variance (ANOVA) using SigmaPlot 12. Holm-Sidak method was applied for subgroup pairwise comparison. A p-value of less than 0.05 was considered statistically significant.
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RESULTS Preparation of Electrospun Blends of Gelatin and Gelatin-Dendrimer Conjugates Nearly 25% surface amine groups of PAMAM dendrimer G3.5 was activated with EDC/NHS chemistry to form G3.5-NHS ester. Lysine and arginine constitute 13~16% of total amino acids in gelatin. They readily react with NHS esters and diminish in quantity as a result of coupling reaction. According to the ninhydrin assay, the percentage of free amines in G-D conjugates was only 44% of those found in unmodified gelatin, confirming the success of coupling of dendrimer to gelatin. There are many factors that affect structure of nanofiber constructs and properties, such as voltage applied, mandrel rotating speed, air gap distance, diameter of the needle and the rate at which the polymer solution is delivered.31 To examine effects of silver content in the polymer blends on nanofiber structure and properties, the electrospinning conditions were kept stable. According to SEM images (Figure 1, top panel), blends of gelatin and gelatin-dendrimer conjugates alone or containing various amounts of silver acetate were successfully electrospun to form nanofiber constructs under the conditions specified in this work. The gelatin-based NCs were further treated with PEG DA575 in the presence of photoinitiator DMPA and formed an sIPN NC upon UV light exposure.4 The resultant sIPN NCs retain nanofiber morphology (Figure 1, bottom panel). The fiber diameter of the NCs ranges from 3.20 to 5.89 µm, and that of the sIPN NCs varies from 4.08 to 6.98 µm (Figure 2). According to statistical analysis, the mean fiber diameter of each type of silver-containing NCs (G/G-D/Ag(1), G/G-D/Ag(2), and G/GD/Ag(3)) did not change significantly after having been cross-linked with PEG DA575. In contrast, the effect of cross-linking on non-silver-containing NC G/G-D/Ag(0) was more 10
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pronounced. The mean fiber diameter of NC G/G-D/Ag(0) is 3.20 µm. After forming an sIPN with PEG DA575, the mean fiber diameter significantly increases by 52% to 4.85 µm (p
