Journal of Drug Delivery Science and Technology 30 (2015) 408e416
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Local delivery of resveratrol using polycaprolactone nanofibers for treatment of periodontal disease c a, Milan Petelin b, Julijana Kristl a, * Spela Zupan ci c a, Sasa Baumgartner a, Zoran Lavri a b
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia Department of Oral Medicine and Periodontology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 April 2015 Received in revised form 14 July 2015 Accepted 14 July 2015 Available online 15 July 2015
Periodontal disease is a biofilm-associated inflammatory disease of the periodontium. Resveratrol (RSV) is a promising natural substance for treatment due to its anti-inflammatory and anti-oxidative effects. RSV lacks efficacy under in vivo conditions due to low solubility and stability. Our special interest was to evaluate the effect of several selected organic solvent mixtures on polycaprolactone (PCL) nanofibers morphology, RSV incorporation and its release using electrospinning method and scanning electron microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and in vitro drug release measurements. The results showed that different organic solvent mixtures and electrospinning parameters strongly influence nanofiber morphology because thick, thin, flat, or circular nanofibers can be produced. RSV incorporated into PCL-nanofibers below 5% is mostly in amorphous form, and at higher loading nanocrystals were seen on the surface of thinner nanofibers, whereas thicker nanofibers can also cover the crystals. PCL nanofibers enabled prolonged release compared to the dissolution of pure RSV in sink condition. A bi-phase release kinetic was the consequence of RSV dissolution and cleavage of hydrogen bonding and hydrophobic interactions between PCL and RSV. The RSV-loaded PCL nanofibers will provide RSV for treatment of periodontal disease in the periodontal pocket even longer due to sustain release, and lowgingival fluid flow. © 2015 Elsevier B.V. All rights reserved.
Keywords: Nanofiber Electrospinning Polycaprolactone Drug loading Controlled release Periodontal diseases
Periodontal disease is a biofilm-associated inflammatory disease of the periodontium [1]. One of key elements for the development of periodontal disease is colonization of teeth and periodontal tissues by pathologic bacteria, which release lipopolysaccharides that activate the immune system. This leads to inflammation that causes damage to the periodontal tissues due to the increased levels of cytokines and matrix metalloproteases [2]. The host immune response is essentially protective, but when a host is susceptible due to various reasons, such as smoking, poorly controlled diabetes, stress, or genetic factors [3], the response is not normal. Hypo- or hyper-responsive inflammatory pathways lead to enhanced tissue destruction and formation of periodontal pockets. The consequence of an inappropriate body response and the presence of chronic inflammation is the development of chronic periodontal disease [4]. Moreover, some recent studies reported
* Corresponding author. E-mail address:
[email protected] (J. Kristl). http://dx.doi.org/10.1016/j.jddst.2015.07.009 1773-2247/© 2015 Elsevier B.V. All rights reserved.
new evidence that periodontal disease could be associated with a systemic oxidative stress state, reduced overall antioxidant capacity, and increased biologic markers for alveolar bone degradation in saliva [4e7]. Periodontal disease is very difficult to treat, because it is a complex disease affected by several factors. Taking these into account, there are three different approaches: (i) removal of dental biofilm with brushing and additional antimicrobial therapy; (ii) modulation of host inflammatory response aiming to suppress inflammation and restore homeostasis; and (iii) regenerative periodontal therapy of a destructed periodontum [8]. Mechanical removal of dental plaque as a gold standard of treatment with additional antimicrobial therapy in the case of persistent periodontitis [1] is not always successful, since disease recurrence is quite common [9]. Because inflammatory response plays a major role in the disease progression and tissue destruction, drugs that can modulate the host inflammatory response represent an important therapeutic approach [10]. The purpose of such therapy is to restore the balance between pro-inflammatory mediators and destructive enzymes on the one hand, and anti-inflammatory
Zupancic et al. / Journal of Drug Delivery Science and Technology 30 (2015) 408e416
mediators and enzyme inhibitors on the other [11]. One of the promising supporting natural substances for the treatment of periodontal disease is resveratrol (RSV). Its antiinflammatory and anti-oxidative effects in periodontal disease were shown by reduced production of NO [12], inhibition of vascular endothelial growth factor, decreased vessel permeability [13], and decreased production of inflammatory cytokines, chemokines, and factors for leucocyte differentiation [14]. RSV acting as an antagonist of the aryl-hydrocarbon receptor could have a positive impact on periodontal tissue regeneration [15], and it was also shown that RSV significantly reduces loss of bone tissue [16]. However, RSV lacks the efficacy under in vivo conditions due to unfavorable biopharmaceutical properties (low solubility, rapid metabolism, and chemical instability). Therefore, the development of a carrier system that would overcome the biopharmaceutical obstacles and fully realize its therapeutic and prophylactic potential is still a technological challenge [17]. Highly efficacious dosage forms for the treatment of periodontal disease would need to specifically fit to the periodontal pockets, stay there for prolonged time periods, be in tight contact with surrounding tissue, and be able to control the release of drug. However, the investigations oriented towards development of such dosage forms are quite rare [8,18,19]. Currently, the most frequently used dosage forms are solutions, gels, films, or chips [18]. Nanofibers present a promising new alternative drug delivery system with the ability to load a drug and tailor drug release profile by a modification of the composition and the morphology of nanofibers [20]. Their large surface-to-volume ratios increase the tendency to adhere to the tissue in the periodontal pocket. In addition, the nanofibers morphology enable diffusion of gingival crevicular fluid through the matrix of the delivery system, which may decrease the tendency of flushing of the delivery system out of the periodontal pockets, as was the case of some of the other low porosity delivery systems, such as films or chips [18,21,22]. The most appropriate materials for the preparation of nanofibers by electrospinning technique and for the delivery of a wide variety of active ingredients are biodegradable and biocompatible polymers, such as chitosan, poly(ethylene oxide), poly(vinylalcohol), poly(lactic-glycolic acid), poly(ε-caprolactone) (PCL). A different solution, process, and environmental parameters influence nanofiber formation and their morphology. Water is used for electrospinning of hydrophilic polymers [22]. Conversely, hydrophobic polymers require a careful selection of organic solvents, single or their mixtures with special additives, such as salts that increase the dispersion conductivity [23]. One of the attractive materials for the preparation of nanofibers is PCL, a biodegradable and biocompatible polymer, that also demonstrated sustained drug release kinetics in previous studies of nanodelivery systems [20]. Although RSV was shown to exhibit positive effects for treatment of periodontal disease, to the best of our knowledge, an appropriate delivery system with this substance for the treatment of this disease has not been yet described in the scientific literature. Therefore, the aim of this research was to prepare PCL nanofibers loaded with RSV that could be administrated into the periodontal pocket and there enable slow release. Our special interest was to evaluate the effect of several selected organic solvent mixtures on nanofibers morphology, RSV incorporation and its release. The physicochemical properties of empty PCL nanofibers and pure RSV were characterized and compared with prepared RSV-loaded PCL nanofibers using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC). The release profiles of RSV from nanofibers were also examined using a USP XXV type II (paddle) method.
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1. Materials and methods 1.1. Materials Trans-RSV was purchased from LGC Standards GmbH, Germany with a declared purity of 99.6%, according to the manufacturer. Potassium dihydrogen phosphate, formic acid, sodium iodide, sodium hydroxide, chloroform (C), dichloromethane (DCM), and acetone (A) (for analysis) were obtained from Merck KGaA, Germany. PCL (MW 70,000e90,000 g/mol) and N,Ndimethylformamide (DMF) were purchased from Sigma Aldrich, Germany. Tetrahydrofuran (THF) was purchased from Carlo Elba, Italy. Acetonitrile of HPLC grade was purchased from J.T. Baker, Netherlands. Ultrapure water obtained from a Milli-Q® UF-Plus apparatus (Millipore Corp., Burlington, MA, USA) was used for the preparation of mobile phases. 1.2. Methods 1.2.1. Preparation of the polymer solutions PCL solutions 10% (w/w) were prepared by dissolving PCL and NaI in different mixtures of solvents (Table 1). The dispersions were stirred with a magnetic stirrer overnight at room temperature to dissolve the polymer. Different quantities of RSV (1, 5, 10, and 20% (w/w) according to dry nanofibers) were added to all solutions and mixed by Ultra Turrax T25 (Janke & Kunkel, IKA Labortechnik, Germany) for 5 min at 15,000 rpm to obtain a homogenous solution. 1.2.2. Conductivity of the polymer solutions The solution conductivity was measured by a MC226 Conductivity Meter and electrode Inlab 741 (Mettler Toledo, Switzerland). 1.2.3. Electrospinning of the polymer solutions The polymer solution was placed in a 20 ml plastic syringe fitted with a metallic needle (inner diameter of 0.8 mm). A syringe pump (Model R-99E, RazelTM) was used to feed at a constant rate. High voltage at the needle was achieved by connection to a voltage generator (model HVG-P60-R-EU, Linari Engineering s.r.l., Italy). The electrospinning parameters of each solution are described in Table 1. 1.2.4. The diameter and morphology of the electrospun nanofibers Nanofibers were examined using a 235 Supra 35VP-24-13 highresolution scanning electron microscope (SEM, Carl Zeiss, Germany) operated at an accelerating voltage of 1 kV with a secondary electron detector; no conductive coating layer was applied before imaging. The obtained images were used to determine the average fiber diameters using ImageJ 1.44p software (NIH, USA) by measuring 50 nanofibers chosen randomly. 1.2.5. FT-IR analysis FT-IR was used to qualitatively characterize the interactions between RSV and PCL. The FT-IR spectra of RSV, and empty and loaded PCL nanofibers with 1, 5, 10, and 20% RSV produced from CA0.03 solvent were characterized with FT-IR spectrometer with an attenuated total reflectance accessory (Nexus, Thermo Nicolet, Madison, USA). Spectra in the range of 600e3600 cm1 with a resolution of 8 cm1 were measured. Each recorded spectrum was an average of 16 scans. 1.2.6. Thermal analysis of nanofibers with RSV Thermal analysis was performed using DSC (Mettler Toledo DSC 1, Switzerland) to determine the physical state of pure RSV and when incorporated in nanofibers. An approximately 5 mg sample
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Table 1 Solvent mixtures used for preparation of PCL nanofibers and electrospinning parameters (U e voltage, d e distance from needle to collector). Sample
Solvents
CA0 CA0.01 CA0.03 DCM/DMF THF/DMF
chloroform:acetone chloroform:acetone chloroform:acetone dichloromethane: dimethylformamide tetrahydrofuran: dimethylformamide
Ratio (w/w)
NaI (%)
U (kV)
3:1 3:1 3:1 3:2 1:1
0 0.01 0.03 0 0.03
15 15 15 12 15
was weighed into an aluminum pan and covered with a lid having a 50 mm pinhole. PCL nanofibers were analyzed using a heating rate of 20 C/min in the temperature range 80 to 300 C. Amorphous RSV was prepared in situ by a modified sequence that comprised of a quick heating segment with 40 C/min in the temperature range 25e300 C, followed by fast cooling with 50 C/min to 20 C, where sample was kept under isothermal conditions for 6 min, followed by final heating from 20 to 300 C with 20 C/min. The measurements were performed in an inert nitrogen atmosphere with a flow rate of 50 mL/min. 1.2.7. Release study RSV release was performed on a fully calibrated dissolution apparatus using the paddle method (USP Apparatus II, VanKel Dissolution Apparatus, model VK 7000, USA). Dissolution studies were performed in 600 ml buffers with a pH of 6.8. To prevent nanofiber floating, 4 4 cm mats were placed to brackets which sank at the bottom of the vessel at the beginning of the experiment. The temperature was maintained at 37 ± 0.5 C and vessel speed was kept at 50 rpm. At each sampling time point 10 ml sample (not replaced) of the dissolution medium was withdrawn, filtered with 0.2 mm filters (Minisart RC 15, Sartorius Stedim Biotech GmbH, Germany) and immediately analyzed. Results are given as percentage of the released RSV, whereas 100% was calculated as a theoretical amount of drug incorporated to the nanofibers. All experiments were performed at least in triplicate. The dissolution medium was protected from the light during experiments unless otherwise written. 1.2.8. RSV analytics The UPLC method was used for analysis of RSV because it was recently reported that it showed better separation between transRSV from cis-RSV and degradation products compared to HPLC or UV/VIS spectroscopy [24]. The trans-RSV was determined by the chromatographic system Acquity UPLC (Waters Corp., USA). A UVVIS photodiode array module (PDA) equipped with a high sensitivity flow cell was used for detection. The column with a precolumn used was Acquity UPLC HSS C18 SB 1.8 mm 2.1 100 mm (Waters Corp., USA). A gradient elution was used to achieve chromatographic separation with a mobile phases A (water, containing 0.1% formic acid and 10% of acetonitrile), and B (acetonitrile, containing 2% of water). The mobile phase in the gradient elution progress was: 0e1 min 0% B, 1.5 min 1.7% B, 2.5 min 9.1% B, 7 min 17.6% B, 8 min 28.3% B, 9e11 min 52.4% B, 11.1e13 min 0% B. The flow rate was set at 0.4 ml/min and the column temperature was maintained at 40 C. The auto-sampler temperature was 4 C, and the injection volume was 5 ml. The analytical run time for each sample was 13 min. A UV scan was obtained from 240 to 370 nm with a resolution 0.6 nm and an area under the curve of trans-RSV peak at 306 nm was chosen for detection and further calculation of the trans-RSV amount. 1.2.9. Statistical analysis The results were statistically analyzed using one-way ANOVA, followed by Turkey's post hoc test (PRISM Software Package,
d (cm)
Flow rate (ml/h)
15 15 15 15 and 20 15 and 20
1.6 1.9e2.2 1.9e2.1 1.9e2.0 1.9e2.0
Version 6, Graphpad Software Inc., San Diego, USA). 2. Results and discussion 2.1. The impact of organic solvent mixture and electrospinning parameters on morphology of empty nanofibers A proper solvent mixture has to be selected to dissolve PCL and RSV and to enable production of nanofibers. Therefore, different solvents were tested and their effect on nanofiber morphology was investigated. It is known that solution parameters influence nanofiber production [22,25], where conductivity is one of the important parameters. The prepared solutions showed various conductivities as presented in Table 2. The mixture of chloroform and acetone (CA0) demonstrated really low conductivity, 1.1 mS/cm, resulting in rough nanofibers with a diameter 1570 ± 1160 nm. The majority of nanofibers were beaded with long, thicker parts and a diameter of approximately 2 mm (Fig. 1a). The beaded nanofiber structure is a consequence of the reduction of the electrical charges carried by the jet, causing the collapse of the entangled polymer chains [26]. The addition of 0.01 or 0.03% NaI increased the conductivity dramatically (Table 2), which resulted in the formation of a thinner and more uniform nanofibers (Fig. 1b, c), as was also recently reported by Potrc et al. [23]. Moreover, addition of salt increases the charge density in the ejected jets and, thus, stronger elongation forces are imposed on them. The self-repulsion of the excess charges under the electrical field results in the electrospun fibers having a substantially straighter shape and smaller diameter [22,27]. Chloroform and acetone have high evaporation rates, which resulted in occasional drying at the needle tip. Therefore, DMF with DCM or THF were also tested for PCL nanofiber production. According to the literature data (Table 3), it is expected that mixtures of these solvents would have a higher boiling point and latent heat as chloroform and acetone, therefore needle clotting is less expected. Besides, conductivity and dielectric constant of solvent mixtures are higher compared to chloroform and acetone (Table 3), which is favorable for nanofiber production. Li et al. produced smooth PCL nanofibers at applied 12 kV from the THF/DMF solvent mixture without the addition of salt [28], whereas at 15 kV the electrospinning resulted in nanofibers with several beads. In our case the addition of 0.03% NaI and consequently increased conductivity resulted in smooth flat nanofibers. The 10% PCL in a DCM/DMF or THF/DMF mixture without addition of NaI had low conductivity, whereas conductivity increased for more than 100-times after addition of NaI (Table 2). The conductivity difference between both solvent mixtures also resulted in significant differences between diameter of produced nanofibers (p < 0.05). On the contrary, the addition of salt decreased the spinnability of the DCM/DMF solution. Here it is important to stress that not only conductivity, but also other solvent parameters influence the success of nanofiber formation, such as dielectric contant, latent heat, and surface tension. In some cases, the distance between the needle and the collector effected the nanofiber morphology. Too small distance resulted in flat nanofibers, whereas an increased distance resulted in a circular
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Table 2 Conductivity of 10% PCL solutions and diameter of nanofibers obtained from different solvent mixtures. Solvents CA0 CA0.01 CA0.03 DCM/DMF THF/DMF
NaI (%)
Conductivity at 25 C (mS/cm)
0 0.01 0.03 0 0.03 0 0.03
1.1 ± 0.2 149.9 ± 3.2 396.7 ± 0.6 6.8 ± 0.3 621.7 ± 0.6 3.2 ± 0.1 534.5 ± 0.7
Diameter of empty nanofibers (nm) ± ± ± ± / / 510 ±
1570 620 480 850
1160 290 140 310
200
Fig. 1. Morphology of nanofibers prepared from 10% PCL in (a) chloroform:aceton 3:1 (CA0), (b) with addition of 0.01% NaI (CA0.01), and (c) 0.03% of NaI (CA0.03).
cross-section in case of DCM/DMF (Fig. 2). However, nanofibers from THF/DMF remained flat in cross-section. The cross-sectional shape of a fiber depends mainly on the rate of solvent evaporation and on the transport of solvent molecules through the drying surface of the jet. If the solvent evaporation is fast and the diffusion of the solvent through the hardened PCL is enabled when the jet is approaching the collector, the jet often shrinks homogeneously and dry fibers with a circular cross-section are formed. At the other hand, in the case when a strong skin is formed on the jet surface, the remaining solvent diffuses slowly. As the evaporation progresses, the skin remaines as a hollow tube, collapsed into a flat structure at the end [26]. It can be concluded that the tested solvents are suitable for PCL nanofiber formation. The resulting nanofiber morphology depended on the selected solvent mixtures and electrospinning conditions, especially on solution conductivity and the distance between the needle and collector. Nanofibers produced from polymeric dispersions with low conductivity (CA0 and DCM/DMF) were significantly thicker (p < 0.05) compared to nanofibers produced by higher conductive solutions (CA0.03 and THF/DMF). Mixing CA enabled formation of a circular-shaped nanofibers and the adding of salt to increase condutivity resulted in thinner nanofibers. Interestingly, the mixture of DCM/DMF enabled production of circular-shaped nanofibers without the addition of NaI, whereas in the THF/DMF mixture NaI was necessary at the given conditions. The formed product had a flat morphology even if the distance was
Fig. 2. Nanofibers produced at different distances between needle and collector formed from 10% PCL in solvent mixtures (a) DCM/DMF and (b) THF/DMF.
increased. Thus, the addition of salt in some organic solvent can be essential for successful production of thin and smooth nanofibers. After successful PCL nanofiber preparation, our further intention was to incorporate RSV.
2.2. The impact of solvent mixture on RSV loading in nanofibers To prepare nanofibers with RSV for the treatment of periodontal disease 1e20% of RSV was incorporated. RSV was fully dissolved up to 5% in all prepared solvents, whereas in the case of 10 and 20% of RSV its solubility in some PCL dispersions was exceeded (Table 4). The average diameters with standard deviations of empty and RSVloaded nanofibers are presented in Tables 2 and 4. It has to be pointed out that dissolved RSV in PCL solutions did not impact the solution conductivity; therefore, the empty nanofibers diameter did not varied much in comparison to RSV-loaded nanofibers. Only 20% of RSV loading leads to an increased nanofiber diameter. RSV was successfully incorporated into nanofibers with 1% and 5% loading without visible crystals on the nanofiber surface and the
Table 3 Physical properties of used solvents. Solvent
Mw
bp ( C)a
ε at 20 Ca
Dipolea
Conductivity (mS/cm)
Latent heat (kJ/mol)a
Surface tension at 20 C (mN/m)a
Abs viscosity at 25 C (mPa s)a
Acetone Chloroform DCM DMF THF
58 119 85 73 72
56 61 40 153 66
20.6 4.8 9.1 36.7 7.6
2.9 1.15 1.8 3.8 1.75
0.25
