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A hyperbranched polyester as antinucleating agent for Artemisinin in electrospun nanofibers
European Polymer Journal 60 (2014) 145–152
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Macromolecular Nanotechnology
A hyperbranched polyester as antinucleating agent for Artemisinin in electrospun nanofibers Bonadies Irene a,⇑, Ambrogi Veronica b, Ascione Laura b, Carfagna Cosimo a,b b
Institute for Polymers, Composites and Biomaterials (IPCB), CNR, Via Campi Flegrei 34, 80078 Pozzuoli (Na), Italy Department of Chemical, Materials and Production Engineering (DICMAPI), University of Naples Federico II, P. le Tecchio 80, 80125 Napoli, Italy
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Article history: Received 6 June 2014 Received in revised form 5 September 2014 Accepted 8 September 2014 Available online 18 September 2014 Keywords: Artemisinin Hyperbranched polymer Drug delivery system Electrospinning Core-shell nanofibers Crystallinity inhibition
a b s t r a c t The paper deals with the development of an antinucleating strategy to prevent the crystallization of Artemisinin (ART), a potent natural antimalarial agent, through its encapsulation into core–shell nanofibers, constituted by a core of ART blended with a hyperbranched poly(butylene adipate) (HB), covered with a shell of poly(vinyl pyrrolidone) (PVP). The highly branched polymer acted as an effective antinucleating agent. Scanning and transmission electron microscopy analyses evidenced the regular and homogenous morphology of the core–shell electrospun nanofibers. Attenuated total reflectance spectroscopy, differential scanning calorimetry and X-ray diffraction analysis were utilized to assess the activity and the physical state of ART into the nanofibers. It was demonstrated that ART was successfully encapsulated in electrospun nanofibers and could efficiently retain its amorphous state. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction In China around 2800 BC lived a demigod known as the Divine Ploughman (Sheng Nung). He defined how to cultivate crops of rice and mullet. Amongst the herbs he described, Qinghaosu was indicated as an efficient agent in the treatment of malarial fever. Malaria known as Nue Ji, a foul weather disease, was common in South West China, where it is still endemic and in monsoon season is epidemic. Malaria is a very frequent disease that still causes many deaths in Africa, Asia and South America. Over the time, it has been found that several herbs could fight this disease, including Qinghaosu (Artemisia Annua) [1]. The genus of Artemisia is a member of the family Compositae and includes more than 300 species of annual, biannual and perennial herbs. The name derives from ⇑ Corresponding author. Tel.: +39 0818675063. E-mail addresses: [email protected] (B. Irene), ambrogi@ unina.it (A. Veronica), [email protected] (C. Cosimo). http://dx.doi.org/10.1016/j.eurpolymj.2014.09.005 0014-3057/Ó 2014 Elsevier Ltd. All rights reserved.
Artemis, daughter of Zeus and Leto. Twin sister of Apollo, she is the greek goddess associated with the wildlife of the earth, human and fertility, reproduction and childbirth. The mechanism of action of Artemisinin (ART), the terpene extracted from A. Annua, involves the activation of the molecule by iron with breaking of the peroxide bridge, followed by the formation of a very reactive radical and subsequently by the formation of a covalent bond between the radical and the protein of the parasite [2,3]. In other words, the terpene works powerfully in an iron rich environment such as the red blood cells, through the release of free radicals acting as killer of the malaria parasite. Although Artemisinin has shown excellent permeability across the intestinal mucosa, it has low bioavailability because of its poor aqueous solubility, which can adversely affect its efficacy [4]. ART is extensively metabolized in the liver, thus oral bioavailability is low [5], however thanks to its low molecular weight (MW = 282.3 Da) and a short half-life of 2–3 h [6], ART is a suitable candidate for transdermal drug delivery system (TDDS).
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Transdermal drug delivery is an efficient self-administration method through skin, which allows to bypass the hepatic first-pass effect assuring a sufficient amount of drug for an adequate period of time. Drug delivery through the cutaneous route has several advantages, such as high efficacy and safety by controlling the rate, time, and site of release within the body. This system prevents adverse effects associated with undesired fluctuations in drug concentration and the degradation of non-released drugs within the body [7]. However, when a TDDS is used, one of the most relevant problems is related to drug crystallinity. In fact, the crystals in its formulations may lead to a reduction of drug bioavailability caused by low solubility as well as drug flux through the stratum corneum. Therefore, inhibition of crystallization is imperative to maintain its efficiency and prolong the shelf life of the active ingredient. In this frame, the high crystallinity of ART may represent an obstacle in its application as active agent in TDDS. In a recent paper [8], Shi et al. have described a method to inhibit the recrystallization of ART through its encapsulation in core– shell cellulose acetate/poly(vinyl pyrrolidone) fibers via a coaxially electrospun technique. The composites fibers exhibited a very high efficiency in the delivery of amorphous ART trough skin in TDDS devices. Electrospinning provides the opportunity for direct addition of drugs into the electrospun nanofibers thus improving the encapsulation efficiency, and reduce the burst release via proper selection of drug-polymer–solvent system or electrospinning technique [9]. The high surface area of nanofibers as well as three-dimensional open porous structure help to reduce the constraint towards drug diffusion resulting in a more efficient drug release system [10]. Moreover, electrospun membranes can be cut to any sizes and shapes making them suitable for target clinical application [11]. Electrospinning occurs when a charged polymer solution hold in a syringe, possessing sufficient molecular entanglements, emits from the tip of the needle a charged fluid jet in the presence of an electric field [12]. In fact at a critical voltage, the Coulombic repulsion of the charges overcomes the surface tension of the polymer droplet, and a charged jet is ejected from the tip of the droplet. The jet travels towards a grounded electrode, while the solvent gets evaporated, and the resultant fibers are collected on a grounded target. It is well known that the viscosity of the solution has a deep effect on electrospinning and the resultant fiber morphology. Generally, the viscosity of the solution is related to the extent of polymer molecule chains entanglements within the solution. A jet of low molecular weight fluid breaks up into small droplets, a phenomenon termed electrospraying, while a polymer solution with sufficient chain overlap and entanglements does not break up but undergoes a bending instability that causes a whip-like motion between the capillary tip and the grounded target [13]. In this paper, the key issue was to develop an electrospun fibrous system in which the re-crystallization of this potent antimalarial agent could be inhibited by blending it with a properly selected hyperbranched poly(butylene adipate) (HB). Thanks to its unique topological structure and
physical/chemical properties, the selected polymer can engage the antimalarial agent in its branches, preventing ART molecules crystallization. However, due to its structure characterized by a low concentration of entanglements, HB exhibits unsuitable rheological behavior to be electrospun by itself [14]. For this reason, the core–shell configuration has been chosen to obtain HB nanofibers without defects. In a typical coaxial electrospinning process two or more polymer solutions are forced by an electrostatic potential to eject out through different but co-axial capillary channels, resulting in a core–shell structured composite micro or nano-fiber. Thanks to the core–shell structure the drugs can be released in a more controlled way without any initial burst effect tailoring the shell composition and characteristics. The polymer shell also plays an essential role in protecting the drug molecules from direct exposure to the aggressive environments. The most critical step was to obtain electrospun core–shell nanofibers constituted by a core of a blend of ART and HB and a shell constituted by poly(vinyl pyrrolidone) (PVP), an easily electrospinnable polymer. PVP has several potential advantages, as it is a common anti-nucleating agent [15] and it can enhance the inhibition effect of HB on drug crystallization. Moreover, it can increase the surface hydrophilicity of the fibers improving drug water solubility [16,8]. In particular, our attention was devoted to ascertain the prevention of ART crystallization in electrospun nanofibers and to confirm the maintainment of its chemical integrity by evaluating that peroxide bridge, responsible for pharmacological activity, was not destroyed during electrospinning. 1. Experimental 1.1. Materials Artemisinin colorless needles crystalline powder (ART, average Mw = 282,33 Da) was procured from Chemical Point UG and utilized as received. Poly(vinyl pyrrolidone) K = 90 (PVP, average Mw = 360,000 Da) was purchased from Sigma–Aldrich Chemie GmbH (Schnelldorf, Germany). Chloroform (CHCl3) (P99%), methanol (CH3OH) (P99.8%), acetone (P99.9%), dimethylacetamide (DMAc) and tetrahydrofuran (THF) (P99.9%) were used as solvents during the reaction route. All reagents and solvents were purchased from Sigma–Aldrich (Italy) and used without any further purification. The hyperbranched polymer (HB) utilized (Mw (HB) = 37,700 g/mol, Tm = 55 °C) was synthesized by some of the authors according to a procedure described in details in a previous work [17]. 1.2. Preparation of electrospun nanofibers Electrospun core–shell nanofibers (indicated as HA-P fibers) were obtained with an Electrospinning Setup NF103 MECC Co., Ltd. (Fukuoka, Japan) by using a special ultra coaxial nozzle and a plate collector. The drive pipe
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1.3. Physical characterization 1.3.1. Scanning and transmission electron microscopy The morphology of electrospun nanofibers was evaluated by a field-emission scanning electron microscopy (FESEM, QUANTA200, FEI, The Netherlands) after sputtercoating with gold–palladium. Samples were preliminarily dried under vacuum in order to remove residual solvent traces, then directly located on metal stubs to preserve the fiber morphology. The core–shell morphology of coaxial nanofibers was verified by transmission electron microscopy (TEM) with a FEI Tecnai G12 (LAB6 source) equipped with a FEI Eagle 4 K CCD camera (Eindhoven, The Netherlands) operating with an acceleration voltage of 120 kV. Before characterization,
samples of electrospun nanofibers were placed on a copper grid in order to obtain electron micrographs. 1.3.2. Attenuated total reflectance spectroscopy (ATR) A Thermo Fischer Scientific Nicolet 6700 FTIR was utilized to determine the ATR spectra of HB sample and HAP fibers. The ATR was used at a resolution of 4 cm 1 and 16 scans were averaged for each spectrum in a range between 4000–650 cm 1. 1.3.3. Thermogravimetric analysis (TGA) Thermogravimetric analysis was performed in duplicate by TA Q5000 analyzer in order to investigate on the thermal stability of HB and HA-P fibers. Sample weight was about 5–10 mg and test procedure involved a ramp from 40 °C up to 700 °C at a heating rate of 10 °C/min in nitrogen atmosphere. 1.3.4. Differential scanning calorimetry (DSC) The thermal properties of HB, PVP, ART and HA-P fibers were determined under nitrogen atmosphere (flow rate of 50 ml/min) by TA Instrument Q20 differential scanning calorimetry held in sealed aluminium crucibles. Neat materials and HA-P fibers were analyzed according to the same thermal cycle: First heating run at 10 °C/min from 40 °C up to 180 °C; First cooling run at 10 °C/min to 20 °C; Second heating run at 10 °C/min from 20 °C up to 180 °C. Melting (Tm) and crystallization (Tc) temperatures, melting (DHm) and crystallization (DHc) enthalpies were evaluated through three repeated scans for each sample in order to verify the reproducibility of thermal transitions. 1.3.5. X-ray diffraction The physical state of ART contained in the fibers was determined by X-ray diffraction (XRD) analysis. Measurements were carried out on neat ART and HA-P fibers using a Philips XPW diffractometer with Cu Ka radiation (1.542 Å) filtered by nickel. The scanning rate was 0.02°/ s, and the scanning angle was from 5° to 60°. 2. Results and discussion
Scheme 1. Basic design of coaxial electrospinning set-up.
ART was encapsulated in a core–shell fibrous system (HB-PVP) realized by electrospinning in order to inhibit its re-crystallization. To assess the quality of fibers and the real impact of processing parameters on electrospun fiber morphology, the core–shell fiber mat was first analyzed through electron microscopy, SEM and TEM, as reported in Fig. 1. HA-P fibers (Fig. 1a) revealed an uniform structure without beads-on-string morphology, with an average diameter of 500 ± 125 nm. Moreover, it is notable from micrographs that the electrospun nanofibers were not adhered at their junction zone due to the perfect evaporation of solvent during the jet path between the needle and the collector. As a consequence, the fiber shape was maintained throughout its length, even after landing on the collector. This indicates the achievement of the optimal electrospinning conditions, including the distance between
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consists of a central needle (internal diameter of 0.2 mm) surrounded by a concentric annular tube (0.8 mm for internal diameter). 30 wt.% HB solution containing ART (50 wt.% with respect to HB) was used as core material and prepared in THF. The shell material was obtained from a 15 wt.% PVP solution in anhydrous ethanol. Both polymer solutions for core and shell materials were separately fed into the coaxial nozzle from which they were ejected simultaneously (Scheme 1). The flow rates for both polymer solutions were 0.5 mL h 1. The applied voltage value and the distance between the coaxial nozzle and the collector (covered with aluminium foil) were 23 kV and 170 cm, respectively. All the electrospun nanofibers were obtained at room temperature and 35% relative humidity. Image analysis software (Image J 1.47) was utilized to evaluate the electrospun fiber diameter. In order to determine the efficiency of HA-P core–shell fibers in inhibiting ART crystallization, nanofibers were stored at room temperature and 35% relative humidity and characterized after 4 months.
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Fig. 1. Electron microscopy images of the core–shell HA-P fibers: SEM before (a) and after (b) aging; (c) TEM before aging.
the tip and the collector [18]. In order to analyse the efficiency of core–shell morphology in inhibiting ART crystallization, SEM analysis was also performed on aged samples and reported in Fig. 1b. As it is possible to observe the difference between samples before and after aging involves only the junction zone, as a slight melting effect due to storage condition. TEM analysis was carried out to study the core–shell structure of the nanofibers. Image contrast is based on the differences in scattering mechanisms of distinct phases within a specimen (i.e. differences in density), depending on the state of order (amorphous or crystalline) of the material [19]. Taking these considerations into account, the interface between core and shell materials and the presence of crystalline elements were expected to be evidenced through TEM observations. A representative micrograph of HA-P fiber is reported in Fig. 1c: the HB core containing ART molecules was uniformly encapsulated by the PVP shell, and the average core diameter was 300 ± 50 nm. As described in the introduction, the pharmacological activity of ART is related to its peroxide bridge that is responsible for the formation of a very reactive radical. The maintainment of ART chemical integrity molecules in nanofibers after the electrospinning process was evaluated by spectroscopy analysis. In particular, ATR detection of neat ART and HA-P fibers in the range 950–750 cm 1, before and after aging was employed to identify the vibrational band at 881 cm 1 associated to the endo-peroxide bridge of the molecule [20]. The recorded spectra of ART and HA-P fibers (Fig. 2), revealed the presence of the characteristic band centered at 881 cm 1 both before and after aging, without any appreciable shifting of the peak due to the thermal treatment. This result is of paramount importance, since it indicates that the core–shell nanofibers containing ART, even after aging, were still potentially capable of accomplishing their activity. A further investigation through ATR spectroscopy was finalized to determine the possible interactions between ART and polymer phases thanks to which the drug re-crystallization could be inhibited. Infrared spectroscopy is an effective tool to monitor the interactions between the species contained in a complex structure, such as the core–shell HA-P fibers. In particular, FTIR is very sensitive to hydrogen bondings, which are evidenced by the so-called red shift to lower frequencies of the vibration of groups involved in the formation of the
hydrogen bonds. ATR spectra of HB, ART, PVP and HA-P fibers were analyzed and reported in Fig. 3. The band of the stretching vibrations of AC@O due to the lactone in ART, was observed at 1733 cm 1, which shifted toward lower wave numbers in HA-P fibers (1728 cm 1). This change could be due to hydrogen bonds between HB matrix and ART. HB polyester is characterized by the presence of cyclic branches, ether bonds as well as short hydroxyl terminated branches in its chains [17] that can favourably interact with AC@O groups of ART. This results is encouraging, since it is reported in literature [21,22] that hydrogen bonds among drugs and polymer matrixes could enhance solubilization of drugs into polymer and therefore prevent crystallization into electrospun nanofibers. Moreover, from Fig. 3 it is evident that both the virgin PVP AOH stretching (3428 cm 1) and ART AC@O vibrations (1733 cm 1) moved to lower wave numbers in HA-P fibers (3387 cm 1 and 1728 cm 1, respectively). Since stronger intermolecular bondings may give rise to broad and intense bands of AOAH stretching vibrations, which are often overlaid with peaks due to Fermi resonance interactions [23], the formation of hydrogen bonds between PVP (AOH groups) and ART (AC@O groups) may also be hypothesized. Thermal stability of neat HB, ART, PVP and HA-P fibers were investigated through TGA measurements and reported in Fig. 4. In Table 1 the temperature at 5% weight loss (T5% wt.loss) and the temperature at maximum degradation rate (Tmax.deg.rate), defined as the maximum of the weight loss derivative curves, are also reported for HB, ART, PVP and HA-P fibers. The thermal degradation process of the nanofibers consisted of three main steps. In the temperature range between 180 and 300 °C, the degradation peak visible in the derivative TG traces of HA-P fibers was related to the ART degradation; in the second range between 300 and 390 °C, it was relative to HB matrix and finally in the range between 390 and 460 °C it was associated to the PVP matrix. The shift to higher value of T5% wt.loss for HA-P fibers with respect to neat ART was a proof of the higher thermal stability of ART thanks to the presence of polymer matrixes. It is well known that the for the realization of an effective TDDS system is important to avoid the drug crystallization because of crystals lead to the reduction of drug solubility and thus bioavailability. The main goal of this
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Fig. 3. ATR spectra of neat HB, ART, PVP, and HA-P fibers in the range 2000–600 cm
paper is just to avoid ART crystallization by encapsulating it in core–shell electrospun fibers so it is basic to evaluate the thermal properties of nanofibers. In order to determine the material transition, DSC analysis was carried out on neat HB, ART, PVP and HA-P fibers, respectively. In Table 2, the melting and crystallization temperatures (TmI and TmII, derived from the first and the second heating scan and Tc, respectively), the enthalpies of fusion and crystallization (DHmI, DHmII, derived from the first and the second heating scan, respectively, and DHc) are reported for HB, ART and HA-P fibers before and after aging and HA-P fibers isothermally treated at 140 °C for 1 h (named HA-P fibers iso 140 °C) before and after aging. PVP is amorphous and for this reason the thermogram and the thermal values were not reported. The neat ART used for the fibers exhibited a sharp melting endothermic peak at 154 °C (TmI) and a crystallization exothermic peak at 93 °C (Tc) as shown in Fig. 5.
1
. Inset: ATR spectra in the range 4000–600 cm
1
.
HB sample displayed a single melting peak at TmI = 55 °C that in the second heating scan became more complex, splitting into two peaks. This behavior is linked to the complex architecture structure of HB that includes linear and branched moieties, corresponding to different melting process [17]. In order to evaluate the effect of electrospun nanofibers on thermal properties of ART, DSC analysis was carried out on HA-P fibers prior and after aging; results are reported in Fig. 6. The PVP matrix in the fiber did not exhibit any melting peak or phase transition, being an amorphous polymer. Only a broad endotherm peak between 60 °C and 130 °C, appeared as a result of dehydration both in the nanofibers prior and after aging because the sample was stored at room condition [24]. As a validation, it was possible to observe that in the second heating scan the dehydration peak disappeared. The absence of ART melting peak in the nanofibers, before and after aging, denotes the good
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Fig. 2. ATR spectra of neat ART, HA-P fibers before aging and HA-P fibers after aging.
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Fig. 4. TG (a) and derivative TG (b) traces of HB, ART, PVP and HA-P fibers.
Table 1 T5% wt.loss and Tmax.deg.rate values for HB, ART, PVP and HA-P fibers.
T5% wt.loss (°C) Tmax.deg.rate (°C)
HB
ART
PVP
HA-P fibers
314 355–375
197 204–252
398 435
210 (207–252)-373–437
efficiency of core–shell nanofibers in the inhibition of ART crystallization. This is likely to be related to the presence of HB branched structure that hinders its structural reorganization.
The analysis in isothermal conditions at T = 140 °C for 1 h was also carried out on HA-P fibers before and after aging to be sure that at a temperature close to Tm, ART did not reorganize and restructure into its crystalline form (not shown here). The absence of any melting peaks of ART in core–shell nanofibers indicated that ART molecules were dispersed in an amorphous state in HB matrix. An additional proof that confirm the efficacy of core–shell fibers to inhibit ART re-crystallization is the X-ray powder diffraction pattern.XRD diffractograms are reported in Fig. 7. The neat ART exhibited various distinct peaks at 2h of 7.33° 11.83°, 14.67°, 15.64°, 16.66°, 18.20°, 20.09° and
Table 2 TmI, TmII, Tc, DHmI, DHmII, DHc, of neat HB, ART, HA-P fibers before and after aging and HA-P fibers isothermally analyzed at 140 °C before and after aging.
HB ART
TmI (°C)
TmII (°C)
Tc (°C)
DHmI (J/g)
DHmII (J/g)
DHc (J/g)
55 154
45–52 153
28 93
72.4 73
49 54
51 56
4 2
24 37
19 28
13 17
7 9
24 24
20 18
15 15
HA-P fibers HA-P fibers after aging
47 50
HA-P fibers iso 140 °C HA-P fibers iso 140 °C after aging
48 49
42 43 36–44 39–46
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Fig. 7. XRD patterns of neat ART, HB and HA-P fibers. In the inset, XRD measurements of HB and HA-P fibers.
Fig. 6. DSC traces of HA-P fibers before and after 4 months aging.
22.09° as evidenced in the inset of Fig. 7 [25,26,8,27], which clearly disappeared in the electrospun fibers, confirming that ART lost its crystallinity, becoming amorphous. The only intense peaks in the spectrum of HA-P fibers correspond to the diffraction angle at 21.35° and 24.55 associated to HB sample.
Fig. 8. XRD patterns of HA-P fibers before and after 4 months aging.
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Fig. 5. DSC traces of HB and ART samples.
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As a confirmation of their tendency to amorphization, HA-P fibers were aged for 4 months and analyzed through XRD. The pattern, reported in Fig. 8, shows the good efficiency to inhibit the crystallization of ART by means of HB and the core–shell structure. Indeed, X-ray diffraction results were consistent with the results obtained from calorimetric measurements.
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4. Conclusion One of the major drawback of ART is its crystallinity, which causes reduction of solubility through the skin. In order to prevent the crystallization during the fabrication and storage process of TDDS, a synthesized hyperbranched polyester was utilized as anti-nucleating agent to obtain core–shell electrospun nanofibers including ART. The well-structured core–shell nanofibers have been revealed by SEM and TEM analysis. Thanks to the hyperbranched structure, ART molecules could be solubilized within the core of nanofibers, thus preventing crystallization, as revealed by DSC and X-ray diffraction analysis. Moreover, it was demonstrated that the amorphous state of ART was maintained also after forced aging conditions. ATR spectroscopy confirmed the presence of peroxide bridge, which was responsible for chemical activity of this antimalarial drug. In addition, ART thermal stability was improved thanks to the core–shell structure. As result of this preliminary study, it can be concluded that the use of a hyperbranched polyester as antinucleating agent for Artemisinin in electrospun nanofibers is an effective strategy to preserve the pharmacological activity of the drug and not to compromise ART bioavailability. This is an important step forward for the future realization of TDDS based on novel materials. Acknowledgement Dr. Donatella Duraccio is gratefully acknowledged for the support on the X-ray diffraction analysis of samples. References [1] Klayman DL. Science 1985;228:1049–55. DOI: 10.1126/science. 3887571.
[2] Wu Y. Acc Chem Res 2002;35:255–9. http://dx.doi.org/10.1021/ ar000080b. [3] Moles P, Oliva M, Safont VS. J Phys Chem A 2006;110:7144–58. http://dx.doi.org/10.1021/jp0574089. [4] Titulaer HAC, Lugt J, Zuidema CB. Int J Pharm 1991;69:83–92. http:// dx.doi.org/10.1016/0378-5173(91)90213-8. [5] Sahoo NG, Kakran M, Li L, Judeh Z, Müller RH. Mater Sci Eng, C 2011;31:391–9. DOI: 10.1016/j.msec.2010.10.018. [6] Chan KL, Yuen KH, Jinadasa S, Peh KK, Toh WT. Planta Med 1997;63(1):66–9. [7] Zamani M, Prabhakaran MP, Ramakrishna S. Int J Nanomed 2013;8:2997–3017. http://dx.doi.org/10.2147/IJN.S43575. [8] Shi Y, Zhang J, Xu S, Dong A. J Biomat Sci: Polym Ed 2012;24:551–64. [9] Zeng J, Yang L, Liang Q, et al. J Control Release 2005;105(1–2):43–51. http://dx.doi.org/10.1016/j.jconrel.2005.02.024. [10] Luo X, Xie C, Wang H, Liu C, Yan S, Li X. Int J Pharm 2012;425(1– 2):19–28. http://dx.doi.org/10.1016/j.ijpharm.2012.01.012. [11] Xie C, Li X, Luo X, et al. Int J Pharm 2010;391(1–2):55–64. http:// dx.doi.org/10.1016/j.ijpharm.2010.02.016. [12] Ramakrishna S, Fujihara K, Teo W-E, Lim e T-C, Ma Z. An Introduction to Electrospinning and nanofibers. World Scientific Publishing Co. Pte. Ltd.; 2005. [13] McKee MG, Wilkes GL, Colby RH, Long TE. Macromolecules 2004;37:1760–7. http://dx.doi.org/10.1021/ma035689h. [14] Tonhauser C, Wilms D, Korth Y, Frey H, Friedrich C. Macromol Rapid Commun 2010;31:2127–32. http://dx.doi.org/10.1002/marc. 201000473. [15] Shahzad Y, Shah SNH, Ansari MT, et al. Chin Sci Bull 2012;57:1685–92. http://dx.doi.org/10.1007/s11434-012-5094-2. [16] Yu DG, Zhu LM, White K, Branford-White C. Health 2009;1(2):67–75. http://dx.doi.org/10.4236/health.2009.12012. [17] Samperi F, Battiato S, Puglisi C, Scamporrino A, Ambrogi V, Ascione L, et al. J Polym Sci, Part A: Polym Chem 2011;49:3615–30. http:// dx.doi.org/10.1002/pola.24800. [18] Buchko CJ, Chen LC, Shen Y, Martin DC. Polymer 1999;40(26):7397–407. http://dx.doi.org/10.1016/S0032-3861(98) 00866-0. [19] Vesely D. Polym Eng Sci 1996;36:1586–93. http://dx.doi.org/ 10.1002/pen.10555. [20] Moroni L, Gellini C, Muniz Miranda M, salvi PR, Foresti ML, Innocenti M, Loglio F, Salvetti E. J Raman Spectrosc 2008;39:276–83. http:// dx.doi.org/10.1002/jrs.1880. [21] Ansari MT, Haneef M, Murtaza G. Adv Clin Exp Med 2010;19:745–54. [22] Yu DG, Gao LD, White K, Branford-White C, Lu WY, Zhu LM. Pharm Res 2010;27:2466–77. http://dx.doi.org/10.1007/s11095-010-0239y. [23] Giri N, Natarajan RK, Gunasekaran S, Shreemathi S. Arch App Sci Res 2011;3:624–30. [24] Turner DT, Schwartz A. Polymer 1985;26:757–62. http://dx.doi.org/ 10.1016/0032-3861(85)90114-4. [25] Lipp R. J Pharm Pharmacol 1998;50:1343–9. http://dx.doi.org/ 10.1111/j.2042-7158.1998.tb03357.x. [26] Shahzad Y, Shah SNH, Ansari MT, Riaz R, Safdar A, Hussain T, Malik M. Chin Sci Bull 2012;57:1685–92. http://dx.doi.org/10.1007/ s11434-012-5094-2. [27] Sahoo NG, Kakran M, Abbas A, Judeh Z, Li L. Adv Power Tech 2011;22:458–63. http://dx.doi.org/10.1016/j.apt.2011.03.010.
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Macromolecular Nanotechnology
A hyperbranched polyester as antinucleating agent for Artemisinin in electrospun nanofibers Bonadies Irene a,⇑, Ambrogi Veronica b, Ascione Laura b, Carfagna Cosimo a,b b
Institute for Polymers, Composites and Biomaterials (IPCB), CNR, Via Campi Flegrei 34, 80078 Pozzuoli (Na), Italy Department of Chemical, Materials and Production Engineering (DICMAPI), University of Naples Federico II, P. le Tecchio 80, 80125 Napoli, Italy
a r t i c l e
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Article history: Received 6 June 2014 Received in revised form 5 September 2014 Accepted 8 September 2014 Available online 18 September 2014 Keywords: Artemisinin Hyperbranched polymer Drug delivery system Electrospinning Core-shell nanofibers Crystallinity inhibition
a b s t r a c t The paper deals with the development of an antinucleating strategy to prevent the crystallization of Artemisinin (ART), a potent natural antimalarial agent, through its encapsulation into core–shell nanofibers, constituted by a core of ART blended with a hyperbranched poly(butylene adipate) (HB), covered with a shell of poly(vinyl pyrrolidone) (PVP). The highly branched polymer acted as an effective antinucleating agent. Scanning and transmission electron microscopy analyses evidenced the regular and homogenous morphology of the core–shell electrospun nanofibers. Attenuated total reflectance spectroscopy, differential scanning calorimetry and X-ray diffraction analysis were utilized to assess the activity and the physical state of ART into the nanofibers. It was demonstrated that ART was successfully encapsulated in electrospun nanofibers and could efficiently retain its amorphous state. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction In China around 2800 BC lived a demigod known as the Divine Ploughman (Sheng Nung). He defined how to cultivate crops of rice and mullet. Amongst the herbs he described, Qinghaosu was indicated as an efficient agent in the treatment of malarial fever. Malaria known as Nue Ji, a foul weather disease, was common in South West China, where it is still endemic and in monsoon season is epidemic. Malaria is a very frequent disease that still causes many deaths in Africa, Asia and South America. Over the time, it has been found that several herbs could fight this disease, including Qinghaosu (Artemisia Annua) [1]. The genus of Artemisia is a member of the family Compositae and includes more than 300 species of annual, biannual and perennial herbs. The name derives from ⇑ Corresponding author. Tel.: +39 0818675063. E-mail addresses: [email protected] (B. Irene), ambrogi@ unina.it (A. Veronica), [email protected] (C. Cosimo). http://dx.doi.org/10.1016/j.eurpolymj.2014.09.005 0014-3057/Ó 2014 Elsevier Ltd. All rights reserved.
Artemis, daughter of Zeus and Leto. Twin sister of Apollo, she is the greek goddess associated with the wildlife of the earth, human and fertility, reproduction and childbirth. The mechanism of action of Artemisinin (ART), the terpene extracted from A. Annua, involves the activation of the molecule by iron with breaking of the peroxide bridge, followed by the formation of a very reactive radical and subsequently by the formation of a covalent bond between the radical and the protein of the parasite [2,3]. In other words, the terpene works powerfully in an iron rich environment such as the red blood cells, through the release of free radicals acting as killer of the malaria parasite. Although Artemisinin has shown excellent permeability across the intestinal mucosa, it has low bioavailability because of its poor aqueous solubility, which can adversely affect its efficacy [4]. ART is extensively metabolized in the liver, thus oral bioavailability is low [5], however thanks to its low molecular weight (MW = 282.3 Da) and a short half-life of 2–3 h [6], ART is a suitable candidate for transdermal drug delivery system (TDDS).
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Transdermal drug delivery is an efficient self-administration method through skin, which allows to bypass the hepatic first-pass effect assuring a sufficient amount of drug for an adequate period of time. Drug delivery through the cutaneous route has several advantages, such as high efficacy and safety by controlling the rate, time, and site of release within the body. This system prevents adverse effects associated with undesired fluctuations in drug concentration and the degradation of non-released drugs within the body [7]. However, when a TDDS is used, one of the most relevant problems is related to drug crystallinity. In fact, the crystals in its formulations may lead to a reduction of drug bioavailability caused by low solubility as well as drug flux through the stratum corneum. Therefore, inhibition of crystallization is imperative to maintain its efficiency and prolong the shelf life of the active ingredient. In this frame, the high crystallinity of ART may represent an obstacle in its application as active agent in TDDS. In a recent paper [8], Shi et al. have described a method to inhibit the recrystallization of ART through its encapsulation in core– shell cellulose acetate/poly(vinyl pyrrolidone) fibers via a coaxially electrospun technique. The composites fibers exhibited a very high efficiency in the delivery of amorphous ART trough skin in TDDS devices. Electrospinning provides the opportunity for direct addition of drugs into the electrospun nanofibers thus improving the encapsulation efficiency, and reduce the burst release via proper selection of drug-polymer–solvent system or electrospinning technique [9]. The high surface area of nanofibers as well as three-dimensional open porous structure help to reduce the constraint towards drug diffusion resulting in a more efficient drug release system [10]. Moreover, electrospun membranes can be cut to any sizes and shapes making them suitable for target clinical application [11]. Electrospinning occurs when a charged polymer solution hold in a syringe, possessing sufficient molecular entanglements, emits from the tip of the needle a charged fluid jet in the presence of an electric field [12]. In fact at a critical voltage, the Coulombic repulsion of the charges overcomes the surface tension of the polymer droplet, and a charged jet is ejected from the tip of the droplet. The jet travels towards a grounded electrode, while the solvent gets evaporated, and the resultant fibers are collected on a grounded target. It is well known that the viscosity of the solution has a deep effect on electrospinning and the resultant fiber morphology. Generally, the viscosity of the solution is related to the extent of polymer molecule chains entanglements within the solution. A jet of low molecular weight fluid breaks up into small droplets, a phenomenon termed electrospraying, while a polymer solution with sufficient chain overlap and entanglements does not break up but undergoes a bending instability that causes a whip-like motion between the capillary tip and the grounded target [13]. In this paper, the key issue was to develop an electrospun fibrous system in which the re-crystallization of this potent antimalarial agent could be inhibited by blending it with a properly selected hyperbranched poly(butylene adipate) (HB). Thanks to its unique topological structure and
physical/chemical properties, the selected polymer can engage the antimalarial agent in its branches, preventing ART molecules crystallization. However, due to its structure characterized by a low concentration of entanglements, HB exhibits unsuitable rheological behavior to be electrospun by itself [14]. For this reason, the core–shell configuration has been chosen to obtain HB nanofibers without defects. In a typical coaxial electrospinning process two or more polymer solutions are forced by an electrostatic potential to eject out through different but co-axial capillary channels, resulting in a core–shell structured composite micro or nano-fiber. Thanks to the core–shell structure the drugs can be released in a more controlled way without any initial burst effect tailoring the shell composition and characteristics. The polymer shell also plays an essential role in protecting the drug molecules from direct exposure to the aggressive environments. The most critical step was to obtain electrospun core–shell nanofibers constituted by a core of a blend of ART and HB and a shell constituted by poly(vinyl pyrrolidone) (PVP), an easily electrospinnable polymer. PVP has several potential advantages, as it is a common anti-nucleating agent [15] and it can enhance the inhibition effect of HB on drug crystallization. Moreover, it can increase the surface hydrophilicity of the fibers improving drug water solubility [16,8]. In particular, our attention was devoted to ascertain the prevention of ART crystallization in electrospun nanofibers and to confirm the maintainment of its chemical integrity by evaluating that peroxide bridge, responsible for pharmacological activity, was not destroyed during electrospinning. 1. Experimental 1.1. Materials Artemisinin colorless needles crystalline powder (ART, average Mw = 282,33 Da) was procured from Chemical Point UG and utilized as received. Poly(vinyl pyrrolidone) K = 90 (PVP, average Mw = 360,000 Da) was purchased from Sigma–Aldrich Chemie GmbH (Schnelldorf, Germany). Chloroform (CHCl3) (P99%), methanol (CH3OH) (P99.8%), acetone (P99.9%), dimethylacetamide (DMAc) and tetrahydrofuran (THF) (P99.9%) were used as solvents during the reaction route. All reagents and solvents were purchased from Sigma–Aldrich (Italy) and used without any further purification. The hyperbranched polymer (HB) utilized (Mw (HB) = 37,700 g/mol, Tm = 55 °C) was synthesized by some of the authors according to a procedure described in details in a previous work [17]. 1.2. Preparation of electrospun nanofibers Electrospun core–shell nanofibers (indicated as HA-P fibers) were obtained with an Electrospinning Setup NF103 MECC Co., Ltd. (Fukuoka, Japan) by using a special ultra coaxial nozzle and a plate collector. The drive pipe
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1.3. Physical characterization 1.3.1. Scanning and transmission electron microscopy The morphology of electrospun nanofibers was evaluated by a field-emission scanning electron microscopy (FESEM, QUANTA200, FEI, The Netherlands) after sputtercoating with gold–palladium. Samples were preliminarily dried under vacuum in order to remove residual solvent traces, then directly located on metal stubs to preserve the fiber morphology. The core–shell morphology of coaxial nanofibers was verified by transmission electron microscopy (TEM) with a FEI Tecnai G12 (LAB6 source) equipped with a FEI Eagle 4 K CCD camera (Eindhoven, The Netherlands) operating with an acceleration voltage of 120 kV. Before characterization,
samples of electrospun nanofibers were placed on a copper grid in order to obtain electron micrographs. 1.3.2. Attenuated total reflectance spectroscopy (ATR) A Thermo Fischer Scientific Nicolet 6700 FTIR was utilized to determine the ATR spectra of HB sample and HAP fibers. The ATR was used at a resolution of 4 cm 1 and 16 scans were averaged for each spectrum in a range between 4000–650 cm 1. 1.3.3. Thermogravimetric analysis (TGA) Thermogravimetric analysis was performed in duplicate by TA Q5000 analyzer in order to investigate on the thermal stability of HB and HA-P fibers. Sample weight was about 5–10 mg and test procedure involved a ramp from 40 °C up to 700 °C at a heating rate of 10 °C/min in nitrogen atmosphere. 1.3.4. Differential scanning calorimetry (DSC) The thermal properties of HB, PVP, ART and HA-P fibers were determined under nitrogen atmosphere (flow rate of 50 ml/min) by TA Instrument Q20 differential scanning calorimetry held in sealed aluminium crucibles. Neat materials and HA-P fibers were analyzed according to the same thermal cycle: First heating run at 10 °C/min from 40 °C up to 180 °C; First cooling run at 10 °C/min to 20 °C; Second heating run at 10 °C/min from 20 °C up to 180 °C. Melting (Tm) and crystallization (Tc) temperatures, melting (DHm) and crystallization (DHc) enthalpies were evaluated through three repeated scans for each sample in order to verify the reproducibility of thermal transitions. 1.3.5. X-ray diffraction The physical state of ART contained in the fibers was determined by X-ray diffraction (XRD) analysis. Measurements were carried out on neat ART and HA-P fibers using a Philips XPW diffractometer with Cu Ka radiation (1.542 Å) filtered by nickel. The scanning rate was 0.02°/ s, and the scanning angle was from 5° to 60°. 2. Results and discussion
Scheme 1. Basic design of coaxial electrospinning set-up.
ART was encapsulated in a core–shell fibrous system (HB-PVP) realized by electrospinning in order to inhibit its re-crystallization. To assess the quality of fibers and the real impact of processing parameters on electrospun fiber morphology, the core–shell fiber mat was first analyzed through electron microscopy, SEM and TEM, as reported in Fig. 1. HA-P fibers (Fig. 1a) revealed an uniform structure without beads-on-string morphology, with an average diameter of 500 ± 125 nm. Moreover, it is notable from micrographs that the electrospun nanofibers were not adhered at their junction zone due to the perfect evaporation of solvent during the jet path between the needle and the collector. As a consequence, the fiber shape was maintained throughout its length, even after landing on the collector. This indicates the achievement of the optimal electrospinning conditions, including the distance between
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consists of a central needle (internal diameter of 0.2 mm) surrounded by a concentric annular tube (0.8 mm for internal diameter). 30 wt.% HB solution containing ART (50 wt.% with respect to HB) was used as core material and prepared in THF. The shell material was obtained from a 15 wt.% PVP solution in anhydrous ethanol. Both polymer solutions for core and shell materials were separately fed into the coaxial nozzle from which they were ejected simultaneously (Scheme 1). The flow rates for both polymer solutions were 0.5 mL h 1. The applied voltage value and the distance between the coaxial nozzle and the collector (covered with aluminium foil) were 23 kV and 170 cm, respectively. All the electrospun nanofibers were obtained at room temperature and 35% relative humidity. Image analysis software (Image J 1.47) was utilized to evaluate the electrospun fiber diameter. In order to determine the efficiency of HA-P core–shell fibers in inhibiting ART crystallization, nanofibers were stored at room temperature and 35% relative humidity and characterized after 4 months.
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Fig. 1. Electron microscopy images of the core–shell HA-P fibers: SEM before (a) and after (b) aging; (c) TEM before aging.
the tip and the collector [18]. In order to analyse the efficiency of core–shell morphology in inhibiting ART crystallization, SEM analysis was also performed on aged samples and reported in Fig. 1b. As it is possible to observe the difference between samples before and after aging involves only the junction zone, as a slight melting effect due to storage condition. TEM analysis was carried out to study the core–shell structure of the nanofibers. Image contrast is based on the differences in scattering mechanisms of distinct phases within a specimen (i.e. differences in density), depending on the state of order (amorphous or crystalline) of the material [19]. Taking these considerations into account, the interface between core and shell materials and the presence of crystalline elements were expected to be evidenced through TEM observations. A representative micrograph of HA-P fiber is reported in Fig. 1c: the HB core containing ART molecules was uniformly encapsulated by the PVP shell, and the average core diameter was 300 ± 50 nm. As described in the introduction, the pharmacological activity of ART is related to its peroxide bridge that is responsible for the formation of a very reactive radical. The maintainment of ART chemical integrity molecules in nanofibers after the electrospinning process was evaluated by spectroscopy analysis. In particular, ATR detection of neat ART and HA-P fibers in the range 950–750 cm 1, before and after aging was employed to identify the vibrational band at 881 cm 1 associated to the endo-peroxide bridge of the molecule [20]. The recorded spectra of ART and HA-P fibers (Fig. 2), revealed the presence of the characteristic band centered at 881 cm 1 both before and after aging, without any appreciable shifting of the peak due to the thermal treatment. This result is of paramount importance, since it indicates that the core–shell nanofibers containing ART, even after aging, were still potentially capable of accomplishing their activity. A further investigation through ATR spectroscopy was finalized to determine the possible interactions between ART and polymer phases thanks to which the drug re-crystallization could be inhibited. Infrared spectroscopy is an effective tool to monitor the interactions between the species contained in a complex structure, such as the core–shell HA-P fibers. In particular, FTIR is very sensitive to hydrogen bondings, which are evidenced by the so-called red shift to lower frequencies of the vibration of groups involved in the formation of the
hydrogen bonds. ATR spectra of HB, ART, PVP and HA-P fibers were analyzed and reported in Fig. 3. The band of the stretching vibrations of AC@O due to the lactone in ART, was observed at 1733 cm 1, which shifted toward lower wave numbers in HA-P fibers (1728 cm 1). This change could be due to hydrogen bonds between HB matrix and ART. HB polyester is characterized by the presence of cyclic branches, ether bonds as well as short hydroxyl terminated branches in its chains [17] that can favourably interact with AC@O groups of ART. This results is encouraging, since it is reported in literature [21,22] that hydrogen bonds among drugs and polymer matrixes could enhance solubilization of drugs into polymer and therefore prevent crystallization into electrospun nanofibers. Moreover, from Fig. 3 it is evident that both the virgin PVP AOH stretching (3428 cm 1) and ART AC@O vibrations (1733 cm 1) moved to lower wave numbers in HA-P fibers (3387 cm 1 and 1728 cm 1, respectively). Since stronger intermolecular bondings may give rise to broad and intense bands of AOAH stretching vibrations, which are often overlaid with peaks due to Fermi resonance interactions [23], the formation of hydrogen bonds between PVP (AOH groups) and ART (AC@O groups) may also be hypothesized. Thermal stability of neat HB, ART, PVP and HA-P fibers were investigated through TGA measurements and reported in Fig. 4. In Table 1 the temperature at 5% weight loss (T5% wt.loss) and the temperature at maximum degradation rate (Tmax.deg.rate), defined as the maximum of the weight loss derivative curves, are also reported for HB, ART, PVP and HA-P fibers. The thermal degradation process of the nanofibers consisted of three main steps. In the temperature range between 180 and 300 °C, the degradation peak visible in the derivative TG traces of HA-P fibers was related to the ART degradation; in the second range between 300 and 390 °C, it was relative to HB matrix and finally in the range between 390 and 460 °C it was associated to the PVP matrix. The shift to higher value of T5% wt.loss for HA-P fibers with respect to neat ART was a proof of the higher thermal stability of ART thanks to the presence of polymer matrixes. It is well known that the for the realization of an effective TDDS system is important to avoid the drug crystallization because of crystals lead to the reduction of drug solubility and thus bioavailability. The main goal of this
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Fig. 3. ATR spectra of neat HB, ART, PVP, and HA-P fibers in the range 2000–600 cm
paper is just to avoid ART crystallization by encapsulating it in core–shell electrospun fibers so it is basic to evaluate the thermal properties of nanofibers. In order to determine the material transition, DSC analysis was carried out on neat HB, ART, PVP and HA-P fibers, respectively. In Table 2, the melting and crystallization temperatures (TmI and TmII, derived from the first and the second heating scan and Tc, respectively), the enthalpies of fusion and crystallization (DHmI, DHmII, derived from the first and the second heating scan, respectively, and DHc) are reported for HB, ART and HA-P fibers before and after aging and HA-P fibers isothermally treated at 140 °C for 1 h (named HA-P fibers iso 140 °C) before and after aging. PVP is amorphous and for this reason the thermogram and the thermal values were not reported. The neat ART used for the fibers exhibited a sharp melting endothermic peak at 154 °C (TmI) and a crystallization exothermic peak at 93 °C (Tc) as shown in Fig. 5.
1
. Inset: ATR spectra in the range 4000–600 cm
1
.
HB sample displayed a single melting peak at TmI = 55 °C that in the second heating scan became more complex, splitting into two peaks. This behavior is linked to the complex architecture structure of HB that includes linear and branched moieties, corresponding to different melting process [17]. In order to evaluate the effect of electrospun nanofibers on thermal properties of ART, DSC analysis was carried out on HA-P fibers prior and after aging; results are reported in Fig. 6. The PVP matrix in the fiber did not exhibit any melting peak or phase transition, being an amorphous polymer. Only a broad endotherm peak between 60 °C and 130 °C, appeared as a result of dehydration both in the nanofibers prior and after aging because the sample was stored at room condition [24]. As a validation, it was possible to observe that in the second heating scan the dehydration peak disappeared. The absence of ART melting peak in the nanofibers, before and after aging, denotes the good
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Fig. 2. ATR spectra of neat ART, HA-P fibers before aging and HA-P fibers after aging.
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Fig. 4. TG (a) and derivative TG (b) traces of HB, ART, PVP and HA-P fibers.
Table 1 T5% wt.loss and Tmax.deg.rate values for HB, ART, PVP and HA-P fibers.
T5% wt.loss (°C) Tmax.deg.rate (°C)
HB
ART
PVP
HA-P fibers
314 355–375
197 204–252
398 435
210 (207–252)-373–437
efficiency of core–shell nanofibers in the inhibition of ART crystallization. This is likely to be related to the presence of HB branched structure that hinders its structural reorganization.
The analysis in isothermal conditions at T = 140 °C for 1 h was also carried out on HA-P fibers before and after aging to be sure that at a temperature close to Tm, ART did not reorganize and restructure into its crystalline form (not shown here). The absence of any melting peaks of ART in core–shell nanofibers indicated that ART molecules were dispersed in an amorphous state in HB matrix. An additional proof that confirm the efficacy of core–shell fibers to inhibit ART re-crystallization is the X-ray powder diffraction pattern.XRD diffractograms are reported in Fig. 7. The neat ART exhibited various distinct peaks at 2h of 7.33° 11.83°, 14.67°, 15.64°, 16.66°, 18.20°, 20.09° and
Table 2 TmI, TmII, Tc, DHmI, DHmII, DHc, of neat HB, ART, HA-P fibers before and after aging and HA-P fibers isothermally analyzed at 140 °C before and after aging.
HB ART
TmI (°C)
TmII (°C)
Tc (°C)
DHmI (J/g)
DHmII (J/g)
DHc (J/g)
55 154
45–52 153
28 93
72.4 73
49 54
51 56
4 2
24 37
19 28
13 17
7 9
24 24
20 18
15 15
HA-P fibers HA-P fibers after aging
47 50
HA-P fibers iso 140 °C HA-P fibers iso 140 °C after aging
48 49
42 43 36–44 39–46
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Fig. 7. XRD patterns of neat ART, HB and HA-P fibers. In the inset, XRD measurements of HB and HA-P fibers.
Fig. 6. DSC traces of HA-P fibers before and after 4 months aging.
22.09° as evidenced in the inset of Fig. 7 [25,26,8,27], which clearly disappeared in the electrospun fibers, confirming that ART lost its crystallinity, becoming amorphous. The only intense peaks in the spectrum of HA-P fibers correspond to the diffraction angle at 21.35° and 24.55 associated to HB sample.
Fig. 8. XRD patterns of HA-P fibers before and after 4 months aging.
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Fig. 5. DSC traces of HB and ART samples.
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As a confirmation of their tendency to amorphization, HA-P fibers were aged for 4 months and analyzed through XRD. The pattern, reported in Fig. 8, shows the good efficiency to inhibit the crystallization of ART by means of HB and the core–shell structure. Indeed, X-ray diffraction results were consistent with the results obtained from calorimetric measurements.
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4. Conclusion One of the major drawback of ART is its crystallinity, which causes reduction of solubility through the skin. In order to prevent the crystallization during the fabrication and storage process of TDDS, a synthesized hyperbranched polyester was utilized as anti-nucleating agent to obtain core–shell electrospun nanofibers including ART. The well-structured core–shell nanofibers have been revealed by SEM and TEM analysis. Thanks to the hyperbranched structure, ART molecules could be solubilized within the core of nanofibers, thus preventing crystallization, as revealed by DSC and X-ray diffraction analysis. Moreover, it was demonstrated that the amorphous state of ART was maintained also after forced aging conditions. ATR spectroscopy confirmed the presence of peroxide bridge, which was responsible for chemical activity of this antimalarial drug. In addition, ART thermal stability was improved thanks to the core–shell structure. As result of this preliminary study, it can be concluded that the use of a hyperbranched polyester as antinucleating agent for Artemisinin in electrospun nanofibers is an effective strategy to preserve the pharmacological activity of the drug and not to compromise ART bioavailability. This is an important step forward for the future realization of TDDS based on novel materials. Acknowledgement Dr. Donatella Duraccio is gratefully acknowledged for the support on the X-ray diffraction analysis of samples. References [1] Klayman DL. Science 1985;228:1049–55. DOI: 10.1126/science. 3887571.
[2] Wu Y. Acc Chem Res 2002;35:255–9. http://dx.doi.org/10.1021/ ar000080b. [3] Moles P, Oliva M, Safont VS. J Phys Chem A 2006;110:7144–58. http://dx.doi.org/10.1021/jp0574089. [4] Titulaer HAC, Lugt J, Zuidema CB. Int J Pharm 1991;69:83–92. http:// dx.doi.org/10.1016/0378-5173(91)90213-8. [5] Sahoo NG, Kakran M, Li L, Judeh Z, Müller RH. Mater Sci Eng, C 2011;31:391–9. DOI: 10.1016/j.msec.2010.10.018. [6] Chan KL, Yuen KH, Jinadasa S, Peh KK, Toh WT. Planta Med 1997;63(1):66–9. [7] Zamani M, Prabhakaran MP, Ramakrishna S. Int J Nanomed 2013;8:2997–3017. http://dx.doi.org/10.2147/IJN.S43575. [8] Shi Y, Zhang J, Xu S, Dong A. J Biomat Sci: Polym Ed 2012;24:551–64. [9] Zeng J, Yang L, Liang Q, et al. J Control Release 2005;105(1–2):43–51. http://dx.doi.org/10.1016/j.jconrel.2005.02.024. [10] Luo X, Xie C, Wang H, Liu C, Yan S, Li X. Int J Pharm 2012;425(1– 2):19–28. http://dx.doi.org/10.1016/j.ijpharm.2012.01.012. [11] Xie C, Li X, Luo X, et al. Int J Pharm 2010;391(1–2):55–64. http:// dx.doi.org/10.1016/j.ijpharm.2010.02.016. [12] Ramakrishna S, Fujihara K, Teo W-E, Lim e T-C, Ma Z. An Introduction to Electrospinning and nanofibers. World Scientific Publishing Co. Pte. Ltd.; 2005. [13] McKee MG, Wilkes GL, Colby RH, Long TE. Macromolecules 2004;37:1760–7. http://dx.doi.org/10.1021/ma035689h. [14] Tonhauser C, Wilms D, Korth Y, Frey H, Friedrich C. Macromol Rapid Commun 2010;31:2127–32. http://dx.doi.org/10.1002/marc. 201000473. [15] Shahzad Y, Shah SNH, Ansari MT, et al. Chin Sci Bull 2012;57:1685–92. http://dx.doi.org/10.1007/s11434-012-5094-2. [16] Yu DG, Zhu LM, White K, Branford-White C. Health 2009;1(2):67–75. http://dx.doi.org/10.4236/health.2009.12012. [17] Samperi F, Battiato S, Puglisi C, Scamporrino A, Ambrogi V, Ascione L, et al. J Polym Sci, Part A: Polym Chem 2011;49:3615–30. http:// dx.doi.org/10.1002/pola.24800. [18] Buchko CJ, Chen LC, Shen Y, Martin DC. Polymer 1999;40(26):7397–407. http://dx.doi.org/10.1016/S0032-3861(98) 00866-0. [19] Vesely D. Polym Eng Sci 1996;36:1586–93. http://dx.doi.org/ 10.1002/pen.10555. [20] Moroni L, Gellini C, Muniz Miranda M, salvi PR, Foresti ML, Innocenti M, Loglio F, Salvetti E. J Raman Spectrosc 2008;39:276–83. http:// dx.doi.org/10.1002/jrs.1880. [21] Ansari MT, Haneef M, Murtaza G. Adv Clin Exp Med 2010;19:745–54. [22] Yu DG, Gao LD, White K, Branford-White C, Lu WY, Zhu LM. Pharm Res 2010;27:2466–77. http://dx.doi.org/10.1007/s11095-010-0239y. [23] Giri N, Natarajan RK, Gunasekaran S, Shreemathi S. Arch App Sci Res 2011;3:624–30. [24] Turner DT, Schwartz A. Polymer 1985;26:757–62. http://dx.doi.org/ 10.1016/0032-3861(85)90114-4. [25] Lipp R. J Pharm Pharmacol 1998;50:1343–9. http://dx.doi.org/ 10.1111/j.2042-7158.1998.tb03357.x. [26] Shahzad Y, Shah SNH, Ansari MT, Riaz R, Safdar A, Hussain T, Malik M. Chin Sci Bull 2012;57:1685–92. http://dx.doi.org/10.1007/ s11434-012-5094-2. [27] Sahoo NG, Kakran M, Abbas A, Judeh Z, Li L. Adv Power Tech 2011;22:458–63. http://dx.doi.org/10.1016/j.apt.2011.03.010.