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Synthesis of amphiphilic pullulan-graft-poly(ε-caprolactone) via click chemistry
Journal Pre-proof Synthesis of amphiphilic pullulan-graft-poly(ε-caprolactone) via click chemistry
Layde T. Carvalho, Rodolfo M. Moraes, Gizelda M. Alves, Talita M. Lacerda, Julio C. Santos, Amilton M. Santos, Simone F. Medeiros PII:
S0141-8130(19)33653-0
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.12.207
Reference:
BIOMAC 14235
To appear in:
International Journal of Biological Macromolecules
Received date:
20 May 2019
Revised date:
9 December 2019
Accepted date:
23 December 2019
Please cite this article as: L.T. Carvalho, R.M. Moraes, G.M. Alves, et al., Synthesis of amphiphilic pullulan-graft-poly(ε-caprolactone) via click chemistry, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.12.207
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© 2019 Published by Elsevier.
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Synthesis of amphiphilic pullulan-graft-poly(ε-caprolactone) via click chemistry Layde T. Carvalhoa, Rodolfo M. Moraesa, Gizelda M. Alvesa, Talita M. Lacerdab, Julio C. Santosb, Amilton M. Santosa, Simone F. Medeirosa,* a
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Department of Chemical Engineering, Engineering School of Lorena, University of São Paulo, EEL-USP. Lorena - SP, Brazil. b Department of Biotechnology, Engineering School of Lorena, University of São Paulo, Lorena SP, Brazil.
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*Corresponding author at: Department of Chemical Engineering, Engineering School of Lorena, University of São Paulo, EEL-USP, Estrada Municipal do Campinho s/n, Campinho, Lorena - SP, CEP 1602-810, Brazil. E-mail address: [email protected] (Simone F. Medeiros)
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ABSTRACT
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Chemical modification of natural polymers has been commonly employed for the development of new bio-based materials, aiming at adjusting specific properties such as solubility, biodegradability, thermal stability and mechanical behavior. Among all natural polymers,
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polysaccharides are promising materials, in which biodegradability, processability and bioreactivity make them suitable for biomedical applications. In this context, this work describes the synthesis
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and characterization of a novel amphiphilic pullulan-g-poly(ε-caprolactone) (Pull-g-PCL) graft copolymer. In a first step, pullulan was chemically modified with 2-bromopropionyl bromide to obtain bromo-functionalized pullulan (PullBr). Then, this precursor was modified with sodium azide, leading to azide pullulan (PullN3). In parallel, propargyl-terminated poly(ε-caprolactone) was prepared via ring-opening polymerization (ROP). These preliminary steps involved the synthesis of azide and alkyne compounds, capable of being linked together via alkyne-azide cycloaddition reaction catalyzed by copper (Cu (I)), which leads to Pull-g-PCL. The chemical structures of the polymers were assessed by Proton Nuclear Magnetic Resonance (1H NMR) and Fourier Transform Infrared (FTIR).
1. Introduction Biopolymers can be defined as natural or synthetic substances that are capable of interacting with the biological system for medical treatments. The increasing interest in biopolymers is due to 1
Journal Pre-proof their ability to be well tolerated by the body, without interfering with the normal function of the organism [1,2]. Among the natural biopolymers, polysaccharides are based on monosaccharide units linked together through glycosidic bonds. High biodegradability, processability and bioreactivity make this class of biomaterials very promising for biomedical and food applications [3,4]. More specifically, polysaccharides and their derivatives are often explored for different applications in biomedicine, such as encapsulation of active principles, controlled drug release and tissue engineering [5]. Polysaccharides also exhibit potential for gene delivery, due to its nonionic and hydrophilic properties, which increases the half-life in blood circulation and prevents
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interactions with serum proteins, avoiding the recognition and clearance by the reticuloendothelial cells [6]. The main polysaccharides of interest are cellulose and starch, but other more complex
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carbohydrates, produced by bacteria and fungi, have also been studied, such as xanthan gum,
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pullulan and hyaluronic acid [6,7]. Pullulan is a hydrophilic, biodegradable, nontoxic, odorless and tasteless polysaccharide produced from the fungus Aureobasidium pullulans [8]. The hydroxyl
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groups available on the pullulan’s backbone, which allow different chemical modifications, together with the fact that it is approved by the American Food and Drug Administration (FDA) for a wide
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variety of applications [10], put this versatile material in a prominent position for the development of new systems suitable for the pharmaceutical industry.
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Research in the field of biodegradable polymers has demonstrated the importance of the combination of different biopolymers, aiming to obtain new materials with properties that are not
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possible to obtain in a homopolymer [3,11]. Chemical modification of polymers is a promising method for the development of new biomaterials, aiming at the adjustment of specific properties such as solubility, biodegradability, thermal stability and mechanical behavior [4,12,13]. Currently, chemical modification techniques are important mechanisms for the adaptation of structures and properties of polysaccharides. Through the homogeneous or surface chemical modification of polysaccharides, different functional groups can be introduced, as well as different derivatives capable of forming nanogels, hydrogels and micelles [14]. The modifications frequently applied to polysaccharides are esterification, etherification, oxidation, nucleophilic displacement reactions and grafting reactions [15,16]. Since the 1990s, the chemical modification of pullulan is often reported in literature, aiming at obtaining more versatile grafted polymers. Donabedian and Mc Carthy [17] described the preparation of chemically-modified pullulan via ring-opening polymerization of εcaprolactone and L-lactide using tin octanoate Sn(Oct)2 catalyst. The resulting grafted copolymer exhibited new crystallographic reflections neither seen in pullulan nor poly(ε-caprolactone) and poly(L-lactide). The results indicated that the ring-opening process for all pullulan derivatives 2
Journal Pre-proof appeared to occur via an acyl-oxygen cleavage, producing an ester linkage with the hydroxylterminated backbone. Poly(ɛ-caprolactone) (PCL) is a synthetic polymer that has high potential to be applied in chemical modifications of polysaccharides. Due to the biodegradability and biocompatibility of PCL and its copolymers, they are often used for manufacturing biodegradable sutures, orthopedic fixation systems, artificial skin, tissue engineering supports and for controlled release systems of active compounds [18,19]. The relatively high crystallinity and high hydrophobicity of PCL homopolymer limit its application in active principles delivery systems because of their low in vivo degradation rate. However, in PCL-based blends and copolymers such disadvantages can be
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reduced [20-22].
The present study aims at describing the synthesis of an amphiphilic pullulan-g-poly(ε-
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caprolactone) graft copolymer, which have high potential to be used for the development of core-
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shell-like particles for the controlled release of pharmaceutical ingredients. The copolymer was synthesized via the 1,3-dipolar alkyne-azide cycloaddition reaction catalyzed by copper (Cu (I))
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[23]. Firstly, pullulan was chemically modified with 2-bromopropionyl bromide and then by sodium azide. Propargyl-terminated poly(ε-caprolactone) was prepared in parallel by ring-opening
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polymerization (ROP). Then, propargyl-terminated poly(ε-caprolactone) was grafted into the pullulan’s backbone by alkyne-azide cycloaddition reaction. The amphiphilic graft copolymer
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obtained here seems to be a promising material for the preparation of biocompatible and
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biodegradable particles and for the controlled release of active ingredients.
2. Materials and Methods 2.1. Materials
ε-caprolactone (ε-CL, 97%, Sigma-Aldrich) was purified by vacuum distillation. Pullulan (Pull, food grade) was kindly supplied by Dinaco, Brazil and used as received. The analysis of molar mass and dispersity of commercial pullulan via GPC provided the values of Mn = 120 kDa, Mw = 244 kDa and Đ = 2.03. 2-bromopropionyl bromide (BPB, 97%, Sigma-Aldrich), triethylamine (TEA, ≥99%, Sigma-Aldrich), sodium azide (Synth), tin (II) 2-ethylhexanoate ((Sn(Oct)2, 92.5-100%, Sigma-Aldrich), 1,3,5-trioxane (≥99%, Sigma-Aldrich), copper(I) bromide (CuBr, 98%, Sigma-Aldrich), N,N,N’,N”,N”-pentamethyldiethylenetriamine (PMDETA, 99%, Sigma-Aldrich), propargyl alcohol (99%, Sigma-Aldrich), dimethyl sulfoxide (DMSO-d6, 99%, Sigma-Aldrich), deuterium oxide (D2O, 99.9%, Sigma-Aldrich) and chloroform-d (CDCl3, 99.8%, Sigma-Aldrich) were used as received. Toluene (P.A., Synth) was distilled at atmospheric pressure in the presence of calcium hydride (CaH2). Dimethylsulfoxide (DMSO, P.A., Synth) and N,N3
Journal Pre-proof dimethylformamide (DMF, 99.8%, Synth) were distilled under reduced pressure in the presence of calcium hydride (CaH2). 2.2. Synthesis of propargyl-terminated poly(ε-caprolactone) The propargyl-terminated-PCL was synthesized following the methodology proposed by Amici et al [24] (Scheme 1). 20 g of ε-CL (175 mmol) and 10 ml of toluene were added to a 100 ml round bottom flask. 0.3 g of propargyl alcohol (5.35 mmol) and 0.35 g of Sn(Oct)2 (0.86 mmol) were solubilized in 10 ml of toluene and then added to the reaction flask. The molar proportion between propargyl alcohol and monomer was 1:33 and between propargyl alcohol and the catalyst
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Sn(Oct)2 was 1:0.15, both aiming to obtain PCL with a theoretical molar mass of, approximately, 4000 g.mol-1. The system was magnetically stirred under nitrogen atmosphere for 30 minutes at
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room temperature, heated to 100 °C and then kept under stirring for 8 hours. After this period,
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propargyl-terminated PCL was purified by precipitation in cold diethyl ether, dried under vacuum at 30 °C for 24 hours, and isolated as a white powder. The precipitation procedure was repeated three
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times for the removal of residues from the reaction. The polymerization yield was determined by 1H NMR from the integration of PCL protons (2H, -CH2OC(=O)-, 4.05 ppm) compared to the ε-CL
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protons (2H, -CH2CH2O-. 4,25 ppm). The calculated yield was 96%.
Scheme 1: Synthesis of propargyl-terminated poly(ε-caprolactone) via ring opening polymerization.
2.3. Synthesis of pullulan-Br
1 g of pullulan (6.18 mmol of glucose units) was stirred with 20 ml of DMF in a round bottom flask. After total dissolution, 1.25 g of TEA (12.36 mmol) was added, and the flask was kept in an ice bath, under nitrogen atmosphere and vigorous stirring. 0.67 g of BPB (3.09 mmol) was added dropwise in the solution during a 1-hour period, and the system was sealed. The reaction mixture was stirred for 72 h at room temperature. The product was dialyzed against distilled water for 3 days and then precipitated in a 1:5 diethyl ether/methanol solution. The final product (PullBr, Scheme 2) was dried under vacuum for 24 h.
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triethylamine.
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2.4. Synthesis of pullulan-N3 (PullN3)
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Scheme 2: Synthesis of PullBr via esterification of pullulan with BPB at the presence of
1 g of the previously obtained PullBr (6.18 mmol glucose unit, DS=0.08) was dissolved in
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20 ml of DMF in a round bottom flask. Then, 0.16 g of NaN3 (2.47 mmol) was added to the solution and the mixture was homogenized under nitrogen atmosphere and vigorous stirring. The reaction was stirred for 24 h at 80 °C. The product was dialyzed against distilled water for 3 days and precipitated in a 1:5 diethyl ether/methanol solution. The final product (PullN3, Scheme 3) was dried under vacuum for 24 h.
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Scheme 3: Synthesis of PullN3 from the reaction of PullBr with NaN3.
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2.5. Synthesis of Pull-g-PCL grafted copolymer
The Pull-g-PCL grafted copolymer was synthesized via the alkyne-azide cycloaddition
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reaction (Scheme 4). 0.2522 g of PullN3 was added to a round bottom flask with 0.5006 g of propargyl-terminated PCL and 5 ml of DMF, under nitrogen atmosphere. Meanwhile, 37.18 mg.ml1
of CuBr solution and 44.92 mg.ml-1 of PMDTA solution in DMF were prepared and kept under a
nitrogen atmosphere. The reaction flask was kept in an ice bath, then 3 mL of PMDTA solution and 3 ml of CuBr solution were added into the flask. The reaction was kept for 5 days at 60 °C, under a nitrogen atmosphere. After this time, the copolymer was precipitated in a cold 1:5 diethyl ether/methanol solution.
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Scheme 4: Cycloaddition reaction between azide-funcionalized pullulan (PullN3) and propargyl terminated poly(ε-caprolactone).
2.6. Characterizations
2.6.1. Proton Nuclear Magnetic Resonance (1H NMR) The chemical structures of the polymers as well as the intermediate compounds were assessed by Proton Nuclear Magnetic Resonance (1H NMR) on a Mercury VX 300 spectrometer (Varian, USA) operating at 300 MHz. This technique was also used for analyzing the conversion of ε-CL, as well as for estimating the degree of substitution of PullBr and PullN3. The solvents used were DMSO-d6, CDCl3 or D2O, depending on the specific solubility of each product. 2.6.2. FTIR
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Journal Pre-proof The chemical composition of the polymers was qualitatively assessed by an Spectrum 100 FTIR (PerkinElmer, USA) equipped with an Attenuated Total Reflection accessory (ATR). Diamond crystal was used as the internal reflection element, and resolution of the analysis was 4 cm-1.
2.6.3. Differential Scanning Calorimetry Differential scanning calorimetry (DSC) measurements were performed in a TA Instruments (USA) Q20 equipment. The samples were heated in a rate of 10 °C.min-1, from -80 to 200 °C under a nitrogen atmosphere. Thermograms were obtained by the second heating cycle in order to
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eliminate the influence of water in the samples.
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2.6.4. X-ray photoelectron spectroscopy (XPS)
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XPS was employed to study the surface elemental composition of dried unmodified pullulan, PullBr, PullN3 and Pull-g-PCL. The measurements were obtained with a VSW HA-100 spherical
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analyzer and un-monochrometed Al Ka radiation (hv=1486.6 eV). Survey scans were conducted from 1100 to 0 eV with a scan step of 44 eV. The samples were fixed to a stainless-steel sample
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holder with double-faced conducting tape and analyzed. Curve fitting was performed using
2.6.5. X-ray diffraction
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Gaussian line shapes, and a Shirley background was subtracted from the data.
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X-ray diffraction patterns were obtained by using a PAN-analytical Empyrean ACMS 101 (Malvern, UK) diffractometer at room temperature. The X-ray tube consisted of a target material made of copper (Cu), which emits Kα radiation. The analyzes were performed using a 40 kV accelerating potential and current of 40 mA. The experiments were conducted with a scan range from 10 to 90° (2θ), a step size of 0.02° (2θ) and a counting time of 50 seconds per step.
2.6.6.
Gel Permeation Chromatography (GPC)
Number average molecular weight (Mn) and dispersity (Đ) of the propargyl-terminated PCL was determined by Gel Permeation Chromatography (GPC), using a Breeze (Waters GPC, USA) chromatograph equipped with a 2414 refractive index (RI) detector. THF with 0.3% v/v of triethylamine was used as eluent. The sample was prepared with a concentration of 10 mg.ml -1, filtered on a modified polyvinylidene difluoride (PVDF) membrane filter (0.45 μm) and subsequently injected into the equipment. The solvent flow rate was 1 ml.min-1, and the internal temperature of the columns was 30 °C. Calibration curves were obtained using monodispersed 8
Journal Pre-proof polystyrene standards. For the unmodified commercial pullulan sample, a Liquid Chromatograph (HPLC) with automatic injector (Sil-20A) and a Shimadzu (Japan) refractive index detector, was used. Two Phenomenex, PolySep - SEC GFC P5000 and P3000 columns (3000-400,000 g.mol-1 separation range, 300 x 7.8 mm column dimensions) and one Phenomenex, PolySep - SEC GFC P pre column (35 x 7.8 mm dimension) were used for separation. The pullulan was solubilized in phosphate buffer solution (50 mmol.l-1, pH 7), containing 0.05% (w/v) sodium azide (fungistatic) and 0.05% w/v potassium nitrate, and filtered on modified PVDF polyvinylidene difluoride membrane filter (0.45 μm). The buffer solution was used as a mobile phase, with a fixed flow rate
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of 0.5 ml.min-1 and injection volume of 15 μl.
3. Results and Discussion
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3.1. Characterization of propargyl-terminated poly(ε-caprolactone)
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Figure 1 illustrates the 1H NMR spectra obtained for ε-CL and for the propargyl-terminated poly(ε-caprolactone), with characteristic resonances (ppm) at 2.30 (2H, -OC(=O)CH2, represented
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by “c”), 1.64 and 1.37 (referring to the aliphatic chain, represented respectively by “d” and “e”), 4.05 (2H, -CH2OC(=O)-, represented by “f”) and 3.64 (2 H, -CH2OH, represented by "g") (Figure
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1-a). In addition, the coupling of propargyl alcohol at the end of poly(ε-caprolactone) was confirmed by the presence of resonances at 2.48 (CH≡C−CH2−, represented by “a”) and 4.68 (2H,
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CH≡C−CH2−, represented by “b”) (Figure 1-b).
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Figure 1. 300 MHz
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H NMR spectra of ε-caprolactone (a) and propargyl-terminated poly(ε-
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caprolactone) (b) in CDCl3.
Figure 2 shows the FTIR spectra of ε-CL and propargyl-terminated poly(ε-caprolactone), while its molar mass and dispersity (Đ) are presented in Table 1.
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Figure 2. FTIR spectra of ε-caprolactone (a) and propargyl-terminated poly(ε-caprolactone) (b).
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FTIR spectra show bands at (cm-1) (i) 1732, related to the stretching of ester carbonyl groups, present in both ε-CL and propargyl-terminated poly(ε-caprolactone), (ii) 3267, related to the
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H-C≡ stretching of propargyl-terminated poly(ε-caprolactone), and (iii) 2947 and 2866, which are
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characteristic of the asymmetric and symmetric stretching of the C-H bond, present in both samples.
caprolactone).
Propargylterminated PCL
X(%)a
Mn(g.mol-1)b
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Sample
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Table 1. Number average molar mass (Mn) and dispersity (Đ) of propargyl-terminated poly(ε-
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Mn(g.mol-1)c
Mn(g.mol-1)a
Đb
Degree of polymerization
3798,8
4319.6
1.45
37.40
Determined by a 1H NMR, b GPC and c Theoretical. The values of number average molecular weight (Table 1) from the polymerization of εcaprolactone were obtained via different methods, such as GPC, 1H NMR and Theoretical, resulting, respectively, in 6438 Da, 4319,6 Da and 3798 Da. The difference between the values occurred due to the method characteristics, for GPC, for example, the standard used is polystyrene which do not have the same chemical structure and, consequently, hydrodynamic volume from PCL. To estimate the degree of polymerization, an integration value of 1.00 was attributed to the 1
H NMR signal at 4.68 ppm (Figure 1-b) from the propargyl unit. Then, the integration of the 11
Journal Pre-proof signals at 4.05 ppm and 3.65 ppm (Figure 1-b) were 36.32 and 1.08, respectively. The sum of these integration values resulted in a 37.40 degree of polymerization. Based on this result, the molar mass of the propargyl-terminated poly(ε-caprolactone) was calculated, multiplying the degree of polymerization by the ε-caprolactone molecular weight and adding the molecular weight of the propargyl unit, present at the end of the polymer chain. The dispersity of the polymer (Đ = 1.45) also indicates that it is suitable to be grafted onto pullulan polymeric chain. Based on both the FTIR and 1H NMR results, it was possible to confirm that propargyl-terminated PCL was successfully synthesized.
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3.2. Preparation of azide-functionalized pullulan
Figure 3 shows the 1H NMR spectra of unmodified pullulan, PullBr and PullN3. 1H NMR
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spectrum of commercial pullulan (Figure 3-a) is in accordance with literature [26-29] exhibiting
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resonances (ppm) at chemical shifts higher than 4.5, arising from anomeric protons in (14) and (16) linked glucose units [29]. Multiplets in chemical shifts from 3.0 to 4.0 can be attributed to
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protons of glucose units. The precise assignments of peaks would require a higher resolution
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equipment and specific experimental conditions for NMR spectra acquiring [29]. The 1H NMR spectra of PullBr (Figure 3-b) exhibited resonances (ppm) 4.35 (-CH(CH3)C(=O)-, represented by "b") and at 2.26 (-CH(CH3)C(=O)-, represented by "c"), which is an indicative of the grafting of
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BPB onto the pullulan polymeric chain. The displacements of the peak “c”, from 2.26 in PullBr (Figure 3-b) to 2.16 in PullN3 (Figure 3-c) is a strong indicative of the desired modification [30]. H NMR spectrum of PullBr (figure 3-b) was used to calculate the molar degree of
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substitution (DS), according to the equation DS = (4*A)/(3*B+A), where A is the integration value of signals from methyl groups in BPB and B is integration value of signals from hydroxyl protons plus hydrogen anomeric protons of pullulan. 4 and 3 refer, respectively, to the number of protons in pullulan (one triplet and one singlet) and to the number of protons in methyl groups in BPB [31]. The calculated DS was 0.08, which means that for each one hundred glucose units in pullulan, eight hydroxyl groups were substituted.
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Figure 3. 300 MHz 1H NMR spectra of unmodified pullulan (a), bromo-functionalized pullulan (PullBr) (b) and azide functionalized pullulan (PullN3) (c) in DMSO-d6. The FTIR spectra of unmodified pullulan, PullBr and PullN3 shown in Figure 4 provide some important complementary information about the chemical modification of pullulan [32]. Firstly, a reduction in the band at 3320 cm-1 characteristic of pullulan hydroxyl groups could be observed after their substitution by BPB molecules and posteriorly by azide groups, confirming the partial replacement of the hydroxyl groups present in the material. The band at 1758 cm -1, 13
Journal Pre-proof characteristic of –C=O binding, appears in Figures 4-b and 4-c, which indicates the partial substitution of hydroxyl groups in pullulan. Finally, the band at 2050 cm-1, related to C-N3 binding, can be clearly evidenced in Figure 4-c, corroborating the results previously reported [33] and
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confirming the functionalization of pullulan with azide groups, as expected.
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functionalized pullulan (c).
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Figure 4. FTIR spectra of unmodified pullulan (a), bromo-functionalized pullulan (b) and azide
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Journal Pre-proof Figure 5. XPS diagrams of unmodified pullulan (a), bromo-functionalized pullulan (PullBr) (b) and azide functionalized pullulan (PullN3) (c). Figure 5 shows the results from XPS analyses for unmodified pullulan, PullBr and PullN3. All samples presented mainly carbon and oxygen, with binding energies at around 280 eV and 530 eV, respectively. For bromo-functionalized pullulan (Figure 5-b) the presence of bromine (Br) can be evidenced by three peaks at 69.0 eV (Br-3d), 187.8 eV (Br-3p) and 255.0 eV (Br-3s). Other small peaks in this region may indicate the presence of impurities, mainly silicon, from the glue used to attach the samples onto the stainless steel holder. These characteristic peaks of Br
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disappeared in PullN3, indicating the substitution of Br by azide groups. The presence of a characteristic peak at binding energy of 400 eV (N1s) (Figure 5-c) confirmed the introduction of N3
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groups in pullulan chains.
3.3. Grafting of PCL onto pullulan chains via alkyne-azide cycloaddition reaction
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Figure 6 shows the 1H NMR spectrum of pullulan-g-PCL. The resonances (ppm) at 5.704.40, from anomeric protons in (14) and (16) linked glucose units of PullN3 (Figure 3-c)
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are also present in pullulan-g-PCL [39]. It is also possible to observe resonances (ppm) at 2.30 (2H, -OC(=O)CH2, represented by “c”), 1.64 and 1.37 (represented respectively by “d” and “e”), and
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4.05 (2H, -CH2OC(=O)-, represented by “f”), which are attributed to protons of propargylterminated PCL (Figure 1-b). In addition, the triplet at 2.48 from the alkyne proton of propargyl
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alcohol (Figure 1-b) displaced after chemical reaction was detected at 4.40 (CH≡C−CH2−, “a”) evidencing the triazole-ring formed from the click chemistry reaction between the azide and the alkyne.
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PCL (b) and pullulan-g-PCL (c) in DMSO-d6.
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Figure 6. 300 MHz 1H NMR spectra of azide functionalized pullulan (a), propargyl-terminated
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In the FTIR spectrum of Pull-g-PCL shown in Figure 7-c, the disappearance of the characteristic band of C-N3 binding at 2050 cm-1 when compared to Figure 7-b evidences the
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grafting of PCL onto pullulan. Also, the appearance of the band at 1758 cm -1, characteristic of –
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C=O stretching vibration of PCL (Figure 7-a) corroborates this statement.
Figure 7. FTIR spectra of azide functionalized pullulan (PullN3) (a), propargyl-terminated PCL (b) and pullulan-g-PCL (c).
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Figure 8. XPS diagrams of azide functionalized pullulan (PullN3) (a) and pullulan-g-PCL (b).
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Figure 8 shows XPS results for propargyl-terminated PCL (Figure 8-a) and pullulan-g-PCL (Figure 8-b). Both samples presented mainly carbon and oxygen atoms, with binding energies at
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around 280 eV and 530 eV, respectively [33]. The N1s peak at 400 eV, evidenced only in PullN 3,
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indicates the success of the grafting reaction. Some nitrogen remained into Pull-g-PCL is related to the regioselective formation of 1,4-disubstituted 1,2,3-triazole products. Moreover, the FTIR
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spectrum, presented in Figure 7-c, did not show any remained azide functions present in the grafted copolymer. According to Nwe and Brechbie the Cu(I)-catalyzed cycloaddition reaction between
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azides and alkynes originates a highly stable compound, having great potential for several in vivo applications [40]. Another important observation in XPS diagrams is that it did not show any
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binding energy signal at 69 eV (Figure 8-b), indicating the absence of residual copper in the Pull-gPCL sample [41]. Lallana et al. mentioned that the main problem related to CuAAC reaction is the presence of remaining Cu (I), which did not occur in this work, once again indicating the potential of this grafted copolymer for future applications in drug delivery systems [42].
Figure 9. X-Ray diffractograms of PullN3 (a), propargyl-terminated PCL (b) and pullulan-g-PCL (c). 18
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Figure 9 shows the X-Ray diffractograms for azide functionalized pullulan (PullN3), propargyl-terminated PCL, and pullulan-g-PCL. Analyzing the diffractograms, the reduce in crystallinity of PCL (Figure 9-b) after it was grafted onto the pullulan’s backbone (Figure 9-c) can be noticed with the decrease of the Gaussian fittings of (110), (111), (200) [43], which are characteristic of PCL-based polymers, when comparing with the propargyl-terminatedfunctionalized PCL. Figure 10 shows the thermograms by DSC of propargyl-terminated PCL and pullulan-gPCL, revealing different thermal behaviors in each case. This analysis was also performed for
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pullulan before and after the modification steps. However, the polysaccharide showed no clear Tg transition (data not shown). This result is corroborated by other data previously reported in the
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literature [27]. The determination of transition temperatures for polysaccharides obtained from
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natural sources is often a challenge, since their properties tend to vary greatly depending on the culture medium and they tend to retain a lot of humidity, especially in the case of a hydrophilic
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polymer such as pullulan. Moreover, pullulan will be decomposed before reaching the melting temperature and the same behavior was previously observed by Wang et al. [44] for cellulose.
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According to the authors, there is no sufficient temperature gap between the temperature required to open the inter-molecular bond and the degradation temperature of the polymer. On the other hand,
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propargyl-terminated PCL shows a well-defined Tm at 56.75 °C (Figure 11-a), in accordance with literature [45, 46]. After grafting, a slight reduction in Tm of PCL segment was observed (Figure
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11-b), which is possibly due to segmental mobility, polarity and rigidity of backbone when compared to alkyne-PCL [47,48]. The Tg measured values were -55.33 °C and -66.50 °C for propargyl-terminated PCL and pullulan-g-PCL, respectively, as shown in Figure 11. It can be observed a reduction of the Tg values after the graft reaction, which occured due to the moisture from the pullulan hydrophilicity, resulting in a plasticizing effect of the copolymer.
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Figure 10. Thermograms of PullN3 (a), propargyl-terminated PCL (b) and pullulan-g-PCL(c).
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After grafting PCL onto pullulan’s backbone, there was a significant reduction in the relative amount of PCL’s enthalpy of fusion which corroborates the statement that its crystallinity
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was affected by the reaction [49]. The reduction of PCL’s relative crystallinity rate, before and after ( )
where
is
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its reaction with pullulan, was determined by the equation
equal to 136 J.g-1, the fusion enthalpy of 100% crystalline PCL [47]. The crystallinity rate of the
4. Conclusions
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alkyne-PCL was 80.2% and of the PCL amount in Pull-g-PCL was 38.4%.
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Herein, an amphiphilic copolymer based on pullulan and poly(ε-caprolactone) was synthesized. The homopolymers were separately synthesized either via chemical modification of pullulan, or via ROP of ε-caprolactone, resulting in an azide functionalized pullulan and propargyl ended PCL, respectively. Afterwards, the blocks were conjugated via CuAAC for the synthesis of amphiphilic Pull-g-PCL. The grafted copolymer was characterized via 1H NMR, FTIR and XPS techniques. Further analyses were conducted to demonstrate the occurrence of variations in the crystallinity index of the materials, such as DSC and XRD techniques. The final copolymers has interesting properties, such as amphiphilic, biodegradability and self-assembly. Besides that, it can be obtained by a methodology that can control the amount and the size of the polymer grafting into the pullulan’s backbone, and guarantee the remaining of hydroxyl groups from pullulan without further modification in the available hydroxyl. The individual components of this new graft copolymer, that is, pullulan and PCL, are biocompatible and biodegradable and have already been used in biomedical systems. Finally, it is important to highlight that the Pull-g-PCL grafted copolymer were 20
Journal Pre-proof carefully prepared considering its future application, in the elaboration of particles for encapsulation of active principles and evaluation as controlled release systems. Some preliminary tests have already been carried out, yielding satisfactory results that will be submitted in a new publication soon. Therefore, Pull-g-PCL has good potential to be applied in biomedical devices, mainly as drug delivery systems.
Acknowledgements
The authors acknowledge Dr. Richards Landers for assistance with XPS analyses, Coordination for
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the Improvement of Higher Education Personnel (CAPES) and São Paulo Research Foundation
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(FAPESP, 2019/04269-0) for the financial support.
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Author Statement Layde T. Carvalho: Methodology, Validation, Investigation, Writing - Review & Editing. Rodolfo M. Moraes: Methodology, Data Curation. Gizelda M. Alves: Data Curation. Talita M. Lacerda: Resources. Julio C. Santos: Resources. Amilton M. Santos: Resources, Project administration. Simone F. Medeiros: Supervision, Funding
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acquisition, Project administration.
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Journal Pre-proof Research Highlights: ► Amphiphilic copolymer based in pullulan was successful synthetized via click chemistry reaction. ► The chemical modification was confirmed by different techniques, such as 1H NMR, FTIR, XPS, XRD and DSC. ► The amphiphilic copolymer obtained is suitable to form core-shell nanoparticles.
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► Pullulan-g-poly(ε-caprolactone) is a promising candidate for drug delivery systems.
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Layde T. Carvalho, Rodolfo M. Moraes, Gizelda M. Alves, Talita M. Lacerda, Julio C. Santos, Amilton M. Santos, Simone F. Medeiros PII:
S0141-8130(19)33653-0
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.12.207
Reference:
BIOMAC 14235
To appear in:
International Journal of Biological Macromolecules
Received date:
20 May 2019
Revised date:
9 December 2019
Accepted date:
23 December 2019
Please cite this article as: L.T. Carvalho, R.M. Moraes, G.M. Alves, et al., Synthesis of amphiphilic pullulan-graft-poly(ε-caprolactone) via click chemistry, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.12.207
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© 2019 Published by Elsevier.
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Synthesis of amphiphilic pullulan-graft-poly(ε-caprolactone) via click chemistry Layde T. Carvalhoa, Rodolfo M. Moraesa, Gizelda M. Alvesa, Talita M. Lacerdab, Julio C. Santosb, Amilton M. Santosa, Simone F. Medeirosa,* a
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Department of Chemical Engineering, Engineering School of Lorena, University of São Paulo, EEL-USP. Lorena - SP, Brazil. b Department of Biotechnology, Engineering School of Lorena, University of São Paulo, Lorena SP, Brazil.
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*Corresponding author at: Department of Chemical Engineering, Engineering School of Lorena, University of São Paulo, EEL-USP, Estrada Municipal do Campinho s/n, Campinho, Lorena - SP, CEP 1602-810, Brazil. E-mail address: [email protected] (Simone F. Medeiros)
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ABSTRACT
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Chemical modification of natural polymers has been commonly employed for the development of new bio-based materials, aiming at adjusting specific properties such as solubility, biodegradability, thermal stability and mechanical behavior. Among all natural polymers,
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polysaccharides are promising materials, in which biodegradability, processability and bioreactivity make them suitable for biomedical applications. In this context, this work describes the synthesis
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and characterization of a novel amphiphilic pullulan-g-poly(ε-caprolactone) (Pull-g-PCL) graft copolymer. In a first step, pullulan was chemically modified with 2-bromopropionyl bromide to obtain bromo-functionalized pullulan (PullBr). Then, this precursor was modified with sodium azide, leading to azide pullulan (PullN3). In parallel, propargyl-terminated poly(ε-caprolactone) was prepared via ring-opening polymerization (ROP). These preliminary steps involved the synthesis of azide and alkyne compounds, capable of being linked together via alkyne-azide cycloaddition reaction catalyzed by copper (Cu (I)), which leads to Pull-g-PCL. The chemical structures of the polymers were assessed by Proton Nuclear Magnetic Resonance (1H NMR) and Fourier Transform Infrared (FTIR).
1. Introduction Biopolymers can be defined as natural or synthetic substances that are capable of interacting with the biological system for medical treatments. The increasing interest in biopolymers is due to 1
Journal Pre-proof their ability to be well tolerated by the body, without interfering with the normal function of the organism [1,2]. Among the natural biopolymers, polysaccharides are based on monosaccharide units linked together through glycosidic bonds. High biodegradability, processability and bioreactivity make this class of biomaterials very promising for biomedical and food applications [3,4]. More specifically, polysaccharides and their derivatives are often explored for different applications in biomedicine, such as encapsulation of active principles, controlled drug release and tissue engineering [5]. Polysaccharides also exhibit potential for gene delivery, due to its nonionic and hydrophilic properties, which increases the half-life in blood circulation and prevents
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interactions with serum proteins, avoiding the recognition and clearance by the reticuloendothelial cells [6]. The main polysaccharides of interest are cellulose and starch, but other more complex
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carbohydrates, produced by bacteria and fungi, have also been studied, such as xanthan gum,
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pullulan and hyaluronic acid [6,7]. Pullulan is a hydrophilic, biodegradable, nontoxic, odorless and tasteless polysaccharide produced from the fungus Aureobasidium pullulans [8]. The hydroxyl
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groups available on the pullulan’s backbone, which allow different chemical modifications, together with the fact that it is approved by the American Food and Drug Administration (FDA) for a wide
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variety of applications [10], put this versatile material in a prominent position for the development of new systems suitable for the pharmaceutical industry.
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Research in the field of biodegradable polymers has demonstrated the importance of the combination of different biopolymers, aiming to obtain new materials with properties that are not
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possible to obtain in a homopolymer [3,11]. Chemical modification of polymers is a promising method for the development of new biomaterials, aiming at the adjustment of specific properties such as solubility, biodegradability, thermal stability and mechanical behavior [4,12,13]. Currently, chemical modification techniques are important mechanisms for the adaptation of structures and properties of polysaccharides. Through the homogeneous or surface chemical modification of polysaccharides, different functional groups can be introduced, as well as different derivatives capable of forming nanogels, hydrogels and micelles [14]. The modifications frequently applied to polysaccharides are esterification, etherification, oxidation, nucleophilic displacement reactions and grafting reactions [15,16]. Since the 1990s, the chemical modification of pullulan is often reported in literature, aiming at obtaining more versatile grafted polymers. Donabedian and Mc Carthy [17] described the preparation of chemically-modified pullulan via ring-opening polymerization of εcaprolactone and L-lactide using tin octanoate Sn(Oct)2 catalyst. The resulting grafted copolymer exhibited new crystallographic reflections neither seen in pullulan nor poly(ε-caprolactone) and poly(L-lactide). The results indicated that the ring-opening process for all pullulan derivatives 2
Journal Pre-proof appeared to occur via an acyl-oxygen cleavage, producing an ester linkage with the hydroxylterminated backbone. Poly(ɛ-caprolactone) (PCL) is a synthetic polymer that has high potential to be applied in chemical modifications of polysaccharides. Due to the biodegradability and biocompatibility of PCL and its copolymers, they are often used for manufacturing biodegradable sutures, orthopedic fixation systems, artificial skin, tissue engineering supports and for controlled release systems of active compounds [18,19]. The relatively high crystallinity and high hydrophobicity of PCL homopolymer limit its application in active principles delivery systems because of their low in vivo degradation rate. However, in PCL-based blends and copolymers such disadvantages can be
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reduced [20-22].
The present study aims at describing the synthesis of an amphiphilic pullulan-g-poly(ε-
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caprolactone) graft copolymer, which have high potential to be used for the development of core-
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shell-like particles for the controlled release of pharmaceutical ingredients. The copolymer was synthesized via the 1,3-dipolar alkyne-azide cycloaddition reaction catalyzed by copper (Cu (I))
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[23]. Firstly, pullulan was chemically modified with 2-bromopropionyl bromide and then by sodium azide. Propargyl-terminated poly(ε-caprolactone) was prepared in parallel by ring-opening
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polymerization (ROP). Then, propargyl-terminated poly(ε-caprolactone) was grafted into the pullulan’s backbone by alkyne-azide cycloaddition reaction. The amphiphilic graft copolymer
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obtained here seems to be a promising material for the preparation of biocompatible and
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biodegradable particles and for the controlled release of active ingredients.
2. Materials and Methods 2.1. Materials
ε-caprolactone (ε-CL, 97%, Sigma-Aldrich) was purified by vacuum distillation. Pullulan (Pull, food grade) was kindly supplied by Dinaco, Brazil and used as received. The analysis of molar mass and dispersity of commercial pullulan via GPC provided the values of Mn = 120 kDa, Mw = 244 kDa and Đ = 2.03. 2-bromopropionyl bromide (BPB, 97%, Sigma-Aldrich), triethylamine (TEA, ≥99%, Sigma-Aldrich), sodium azide (Synth), tin (II) 2-ethylhexanoate ((Sn(Oct)2, 92.5-100%, Sigma-Aldrich), 1,3,5-trioxane (≥99%, Sigma-Aldrich), copper(I) bromide (CuBr, 98%, Sigma-Aldrich), N,N,N’,N”,N”-pentamethyldiethylenetriamine (PMDETA, 99%, Sigma-Aldrich), propargyl alcohol (99%, Sigma-Aldrich), dimethyl sulfoxide (DMSO-d6, 99%, Sigma-Aldrich), deuterium oxide (D2O, 99.9%, Sigma-Aldrich) and chloroform-d (CDCl3, 99.8%, Sigma-Aldrich) were used as received. Toluene (P.A., Synth) was distilled at atmospheric pressure in the presence of calcium hydride (CaH2). Dimethylsulfoxide (DMSO, P.A., Synth) and N,N3
Journal Pre-proof dimethylformamide (DMF, 99.8%, Synth) were distilled under reduced pressure in the presence of calcium hydride (CaH2). 2.2. Synthesis of propargyl-terminated poly(ε-caprolactone) The propargyl-terminated-PCL was synthesized following the methodology proposed by Amici et al [24] (Scheme 1). 20 g of ε-CL (175 mmol) and 10 ml of toluene were added to a 100 ml round bottom flask. 0.3 g of propargyl alcohol (5.35 mmol) and 0.35 g of Sn(Oct)2 (0.86 mmol) were solubilized in 10 ml of toluene and then added to the reaction flask. The molar proportion between propargyl alcohol and monomer was 1:33 and between propargyl alcohol and the catalyst
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Sn(Oct)2 was 1:0.15, both aiming to obtain PCL with a theoretical molar mass of, approximately, 4000 g.mol-1. The system was magnetically stirred under nitrogen atmosphere for 30 minutes at
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room temperature, heated to 100 °C and then kept under stirring for 8 hours. After this period,
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propargyl-terminated PCL was purified by precipitation in cold diethyl ether, dried under vacuum at 30 °C for 24 hours, and isolated as a white powder. The precipitation procedure was repeated three
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times for the removal of residues from the reaction. The polymerization yield was determined by 1H NMR from the integration of PCL protons (2H, -CH2OC(=O)-, 4.05 ppm) compared to the ε-CL
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protons (2H, -CH2CH2O-. 4,25 ppm). The calculated yield was 96%.
Scheme 1: Synthesis of propargyl-terminated poly(ε-caprolactone) via ring opening polymerization.
2.3. Synthesis of pullulan-Br
1 g of pullulan (6.18 mmol of glucose units) was stirred with 20 ml of DMF in a round bottom flask. After total dissolution, 1.25 g of TEA (12.36 mmol) was added, and the flask was kept in an ice bath, under nitrogen atmosphere and vigorous stirring. 0.67 g of BPB (3.09 mmol) was added dropwise in the solution during a 1-hour period, and the system was sealed. The reaction mixture was stirred for 72 h at room temperature. The product was dialyzed against distilled water for 3 days and then precipitated in a 1:5 diethyl ether/methanol solution. The final product (PullBr, Scheme 2) was dried under vacuum for 24 h.
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triethylamine.
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2.4. Synthesis of pullulan-N3 (PullN3)
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Scheme 2: Synthesis of PullBr via esterification of pullulan with BPB at the presence of
1 g of the previously obtained PullBr (6.18 mmol glucose unit, DS=0.08) was dissolved in
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20 ml of DMF in a round bottom flask. Then, 0.16 g of NaN3 (2.47 mmol) was added to the solution and the mixture was homogenized under nitrogen atmosphere and vigorous stirring. The reaction was stirred for 24 h at 80 °C. The product was dialyzed against distilled water for 3 days and precipitated in a 1:5 diethyl ether/methanol solution. The final product (PullN3, Scheme 3) was dried under vacuum for 24 h.
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Scheme 3: Synthesis of PullN3 from the reaction of PullBr with NaN3.
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2.5. Synthesis of Pull-g-PCL grafted copolymer
The Pull-g-PCL grafted copolymer was synthesized via the alkyne-azide cycloaddition
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reaction (Scheme 4). 0.2522 g of PullN3 was added to a round bottom flask with 0.5006 g of propargyl-terminated PCL and 5 ml of DMF, under nitrogen atmosphere. Meanwhile, 37.18 mg.ml1
of CuBr solution and 44.92 mg.ml-1 of PMDTA solution in DMF were prepared and kept under a
nitrogen atmosphere. The reaction flask was kept in an ice bath, then 3 mL of PMDTA solution and 3 ml of CuBr solution were added into the flask. The reaction was kept for 5 days at 60 °C, under a nitrogen atmosphere. After this time, the copolymer was precipitated in a cold 1:5 diethyl ether/methanol solution.
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Scheme 4: Cycloaddition reaction between azide-funcionalized pullulan (PullN3) and propargyl terminated poly(ε-caprolactone).
2.6. Characterizations
2.6.1. Proton Nuclear Magnetic Resonance (1H NMR) The chemical structures of the polymers as well as the intermediate compounds were assessed by Proton Nuclear Magnetic Resonance (1H NMR) on a Mercury VX 300 spectrometer (Varian, USA) operating at 300 MHz. This technique was also used for analyzing the conversion of ε-CL, as well as for estimating the degree of substitution of PullBr and PullN3. The solvents used were DMSO-d6, CDCl3 or D2O, depending on the specific solubility of each product. 2.6.2. FTIR
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Journal Pre-proof The chemical composition of the polymers was qualitatively assessed by an Spectrum 100 FTIR (PerkinElmer, USA) equipped with an Attenuated Total Reflection accessory (ATR). Diamond crystal was used as the internal reflection element, and resolution of the analysis was 4 cm-1.
2.6.3. Differential Scanning Calorimetry Differential scanning calorimetry (DSC) measurements were performed in a TA Instruments (USA) Q20 equipment. The samples were heated in a rate of 10 °C.min-1, from -80 to 200 °C under a nitrogen atmosphere. Thermograms were obtained by the second heating cycle in order to
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eliminate the influence of water in the samples.
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2.6.4. X-ray photoelectron spectroscopy (XPS)
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XPS was employed to study the surface elemental composition of dried unmodified pullulan, PullBr, PullN3 and Pull-g-PCL. The measurements were obtained with a VSW HA-100 spherical
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analyzer and un-monochrometed Al Ka radiation (hv=1486.6 eV). Survey scans were conducted from 1100 to 0 eV with a scan step of 44 eV. The samples were fixed to a stainless-steel sample
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holder with double-faced conducting tape and analyzed. Curve fitting was performed using
2.6.5. X-ray diffraction
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Gaussian line shapes, and a Shirley background was subtracted from the data.
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X-ray diffraction patterns were obtained by using a PAN-analytical Empyrean ACMS 101 (Malvern, UK) diffractometer at room temperature. The X-ray tube consisted of a target material made of copper (Cu), which emits Kα radiation. The analyzes were performed using a 40 kV accelerating potential and current of 40 mA. The experiments were conducted with a scan range from 10 to 90° (2θ), a step size of 0.02° (2θ) and a counting time of 50 seconds per step.
2.6.6.
Gel Permeation Chromatography (GPC)
Number average molecular weight (Mn) and dispersity (Đ) of the propargyl-terminated PCL was determined by Gel Permeation Chromatography (GPC), using a Breeze (Waters GPC, USA) chromatograph equipped with a 2414 refractive index (RI) detector. THF with 0.3% v/v of triethylamine was used as eluent. The sample was prepared with a concentration of 10 mg.ml -1, filtered on a modified polyvinylidene difluoride (PVDF) membrane filter (0.45 μm) and subsequently injected into the equipment. The solvent flow rate was 1 ml.min-1, and the internal temperature of the columns was 30 °C. Calibration curves were obtained using monodispersed 8
Journal Pre-proof polystyrene standards. For the unmodified commercial pullulan sample, a Liquid Chromatograph (HPLC) with automatic injector (Sil-20A) and a Shimadzu (Japan) refractive index detector, was used. Two Phenomenex, PolySep - SEC GFC P5000 and P3000 columns (3000-400,000 g.mol-1 separation range, 300 x 7.8 mm column dimensions) and one Phenomenex, PolySep - SEC GFC P pre column (35 x 7.8 mm dimension) were used for separation. The pullulan was solubilized in phosphate buffer solution (50 mmol.l-1, pH 7), containing 0.05% (w/v) sodium azide (fungistatic) and 0.05% w/v potassium nitrate, and filtered on modified PVDF polyvinylidene difluoride membrane filter (0.45 μm). The buffer solution was used as a mobile phase, with a fixed flow rate
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of 0.5 ml.min-1 and injection volume of 15 μl.
3. Results and Discussion
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3.1. Characterization of propargyl-terminated poly(ε-caprolactone)
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Figure 1 illustrates the 1H NMR spectra obtained for ε-CL and for the propargyl-terminated poly(ε-caprolactone), with characteristic resonances (ppm) at 2.30 (2H, -OC(=O)CH2, represented
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by “c”), 1.64 and 1.37 (referring to the aliphatic chain, represented respectively by “d” and “e”), 4.05 (2H, -CH2OC(=O)-, represented by “f”) and 3.64 (2 H, -CH2OH, represented by "g") (Figure
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1-a). In addition, the coupling of propargyl alcohol at the end of poly(ε-caprolactone) was confirmed by the presence of resonances at 2.48 (CH≡C−CH2−, represented by “a”) and 4.68 (2H,
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CH≡C−CH2−, represented by “b”) (Figure 1-b).
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Figure 1. 300 MHz
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H NMR spectra of ε-caprolactone (a) and propargyl-terminated poly(ε-
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caprolactone) (b) in CDCl3.
Figure 2 shows the FTIR spectra of ε-CL and propargyl-terminated poly(ε-caprolactone), while its molar mass and dispersity (Đ) are presented in Table 1.
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Figure 2. FTIR spectra of ε-caprolactone (a) and propargyl-terminated poly(ε-caprolactone) (b).
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FTIR spectra show bands at (cm-1) (i) 1732, related to the stretching of ester carbonyl groups, present in both ε-CL and propargyl-terminated poly(ε-caprolactone), (ii) 3267, related to the
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H-C≡ stretching of propargyl-terminated poly(ε-caprolactone), and (iii) 2947 and 2866, which are
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characteristic of the asymmetric and symmetric stretching of the C-H bond, present in both samples.
caprolactone).
Propargylterminated PCL
X(%)a
Mn(g.mol-1)b
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Sample
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Table 1. Number average molar mass (Mn) and dispersity (Đ) of propargyl-terminated poly(ε-
99.67
6438
Mn(g.mol-1)c
Mn(g.mol-1)a
Đb
Degree of polymerization
3798,8
4319.6
1.45
37.40
Determined by a 1H NMR, b GPC and c Theoretical. The values of number average molecular weight (Table 1) from the polymerization of εcaprolactone were obtained via different methods, such as GPC, 1H NMR and Theoretical, resulting, respectively, in 6438 Da, 4319,6 Da and 3798 Da. The difference between the values occurred due to the method characteristics, for GPC, for example, the standard used is polystyrene which do not have the same chemical structure and, consequently, hydrodynamic volume from PCL. To estimate the degree of polymerization, an integration value of 1.00 was attributed to the 1
H NMR signal at 4.68 ppm (Figure 1-b) from the propargyl unit. Then, the integration of the 11
Journal Pre-proof signals at 4.05 ppm and 3.65 ppm (Figure 1-b) were 36.32 and 1.08, respectively. The sum of these integration values resulted in a 37.40 degree of polymerization. Based on this result, the molar mass of the propargyl-terminated poly(ε-caprolactone) was calculated, multiplying the degree of polymerization by the ε-caprolactone molecular weight and adding the molecular weight of the propargyl unit, present at the end of the polymer chain. The dispersity of the polymer (Đ = 1.45) also indicates that it is suitable to be grafted onto pullulan polymeric chain. Based on both the FTIR and 1H NMR results, it was possible to confirm that propargyl-terminated PCL was successfully synthesized.
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3.2. Preparation of azide-functionalized pullulan
Figure 3 shows the 1H NMR spectra of unmodified pullulan, PullBr and PullN3. 1H NMR
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spectrum of commercial pullulan (Figure 3-a) is in accordance with literature [26-29] exhibiting
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resonances (ppm) at chemical shifts higher than 4.5, arising from anomeric protons in (14) and (16) linked glucose units [29]. Multiplets in chemical shifts from 3.0 to 4.0 can be attributed to
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protons of glucose units. The precise assignments of peaks would require a higher resolution
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equipment and specific experimental conditions for NMR spectra acquiring [29]. The 1H NMR spectra of PullBr (Figure 3-b) exhibited resonances (ppm) 4.35 (-CH(CH3)C(=O)-, represented by "b") and at 2.26 (-CH(CH3)C(=O)-, represented by "c"), which is an indicative of the grafting of
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BPB onto the pullulan polymeric chain. The displacements of the peak “c”, from 2.26 in PullBr (Figure 3-b) to 2.16 in PullN3 (Figure 3-c) is a strong indicative of the desired modification [30]. H NMR spectrum of PullBr (figure 3-b) was used to calculate the molar degree of
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substitution (DS), according to the equation DS = (4*A)/(3*B+A), where A is the integration value of signals from methyl groups in BPB and B is integration value of signals from hydroxyl protons plus hydrogen anomeric protons of pullulan. 4 and 3 refer, respectively, to the number of protons in pullulan (one triplet and one singlet) and to the number of protons in methyl groups in BPB [31]. The calculated DS was 0.08, which means that for each one hundred glucose units in pullulan, eight hydroxyl groups were substituted.
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Figure 3. 300 MHz 1H NMR spectra of unmodified pullulan (a), bromo-functionalized pullulan (PullBr) (b) and azide functionalized pullulan (PullN3) (c) in DMSO-d6. The FTIR spectra of unmodified pullulan, PullBr and PullN3 shown in Figure 4 provide some important complementary information about the chemical modification of pullulan [32]. Firstly, a reduction in the band at 3320 cm-1 characteristic of pullulan hydroxyl groups could be observed after their substitution by BPB molecules and posteriorly by azide groups, confirming the partial replacement of the hydroxyl groups present in the material. The band at 1758 cm -1, 13
Journal Pre-proof characteristic of –C=O binding, appears in Figures 4-b and 4-c, which indicates the partial substitution of hydroxyl groups in pullulan. Finally, the band at 2050 cm-1, related to C-N3 binding, can be clearly evidenced in Figure 4-c, corroborating the results previously reported [33] and
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confirming the functionalization of pullulan with azide groups, as expected.
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functionalized pullulan (c).
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Figure 4. FTIR spectra of unmodified pullulan (a), bromo-functionalized pullulan (b) and azide
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Journal Pre-proof Figure 5. XPS diagrams of unmodified pullulan (a), bromo-functionalized pullulan (PullBr) (b) and azide functionalized pullulan (PullN3) (c). Figure 5 shows the results from XPS analyses for unmodified pullulan, PullBr and PullN3. All samples presented mainly carbon and oxygen, with binding energies at around 280 eV and 530 eV, respectively. For bromo-functionalized pullulan (Figure 5-b) the presence of bromine (Br) can be evidenced by three peaks at 69.0 eV (Br-3d), 187.8 eV (Br-3p) and 255.0 eV (Br-3s). Other small peaks in this region may indicate the presence of impurities, mainly silicon, from the glue used to attach the samples onto the stainless steel holder. These characteristic peaks of Br
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disappeared in PullN3, indicating the substitution of Br by azide groups. The presence of a characteristic peak at binding energy of 400 eV (N1s) (Figure 5-c) confirmed the introduction of N3
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groups in pullulan chains.
3.3. Grafting of PCL onto pullulan chains via alkyne-azide cycloaddition reaction
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Figure 6 shows the 1H NMR spectrum of pullulan-g-PCL. The resonances (ppm) at 5.704.40, from anomeric protons in (14) and (16) linked glucose units of PullN3 (Figure 3-c)
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are also present in pullulan-g-PCL [39]. It is also possible to observe resonances (ppm) at 2.30 (2H, -OC(=O)CH2, represented by “c”), 1.64 and 1.37 (represented respectively by “d” and “e”), and
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4.05 (2H, -CH2OC(=O)-, represented by “f”), which are attributed to protons of propargylterminated PCL (Figure 1-b). In addition, the triplet at 2.48 from the alkyne proton of propargyl
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alcohol (Figure 1-b) displaced after chemical reaction was detected at 4.40 (CH≡C−CH2−, “a”) evidencing the triazole-ring formed from the click chemistry reaction between the azide and the alkyne.
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PCL (b) and pullulan-g-PCL (c) in DMSO-d6.
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Figure 6. 300 MHz 1H NMR spectra of azide functionalized pullulan (a), propargyl-terminated
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In the FTIR spectrum of Pull-g-PCL shown in Figure 7-c, the disappearance of the characteristic band of C-N3 binding at 2050 cm-1 when compared to Figure 7-b evidences the
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grafting of PCL onto pullulan. Also, the appearance of the band at 1758 cm -1, characteristic of –
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C=O stretching vibration of PCL (Figure 7-a) corroborates this statement.
Figure 7. FTIR spectra of azide functionalized pullulan (PullN3) (a), propargyl-terminated PCL (b) and pullulan-g-PCL (c).
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Figure 8. XPS diagrams of azide functionalized pullulan (PullN3) (a) and pullulan-g-PCL (b).
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Figure 8 shows XPS results for propargyl-terminated PCL (Figure 8-a) and pullulan-g-PCL (Figure 8-b). Both samples presented mainly carbon and oxygen atoms, with binding energies at
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around 280 eV and 530 eV, respectively [33]. The N1s peak at 400 eV, evidenced only in PullN 3,
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indicates the success of the grafting reaction. Some nitrogen remained into Pull-g-PCL is related to the regioselective formation of 1,4-disubstituted 1,2,3-triazole products. Moreover, the FTIR
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spectrum, presented in Figure 7-c, did not show any remained azide functions present in the grafted copolymer. According to Nwe and Brechbie the Cu(I)-catalyzed cycloaddition reaction between
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azides and alkynes originates a highly stable compound, having great potential for several in vivo applications [40]. Another important observation in XPS diagrams is that it did not show any
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binding energy signal at 69 eV (Figure 8-b), indicating the absence of residual copper in the Pull-gPCL sample [41]. Lallana et al. mentioned that the main problem related to CuAAC reaction is the presence of remaining Cu (I), which did not occur in this work, once again indicating the potential of this grafted copolymer for future applications in drug delivery systems [42].
Figure 9. X-Ray diffractograms of PullN3 (a), propargyl-terminated PCL (b) and pullulan-g-PCL (c). 18
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Figure 9 shows the X-Ray diffractograms for azide functionalized pullulan (PullN3), propargyl-terminated PCL, and pullulan-g-PCL. Analyzing the diffractograms, the reduce in crystallinity of PCL (Figure 9-b) after it was grafted onto the pullulan’s backbone (Figure 9-c) can be noticed with the decrease of the Gaussian fittings of (110), (111), (200) [43], which are characteristic of PCL-based polymers, when comparing with the propargyl-terminatedfunctionalized PCL. Figure 10 shows the thermograms by DSC of propargyl-terminated PCL and pullulan-gPCL, revealing different thermal behaviors in each case. This analysis was also performed for
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pullulan before and after the modification steps. However, the polysaccharide showed no clear Tg transition (data not shown). This result is corroborated by other data previously reported in the
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literature [27]. The determination of transition temperatures for polysaccharides obtained from
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natural sources is often a challenge, since their properties tend to vary greatly depending on the culture medium and they tend to retain a lot of humidity, especially in the case of a hydrophilic
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polymer such as pullulan. Moreover, pullulan will be decomposed before reaching the melting temperature and the same behavior was previously observed by Wang et al. [44] for cellulose.
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According to the authors, there is no sufficient temperature gap between the temperature required to open the inter-molecular bond and the degradation temperature of the polymer. On the other hand,
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propargyl-terminated PCL shows a well-defined Tm at 56.75 °C (Figure 11-a), in accordance with literature [45, 46]. After grafting, a slight reduction in Tm of PCL segment was observed (Figure
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11-b), which is possibly due to segmental mobility, polarity and rigidity of backbone when compared to alkyne-PCL [47,48]. The Tg measured values were -55.33 °C and -66.50 °C for propargyl-terminated PCL and pullulan-g-PCL, respectively, as shown in Figure 11. It can be observed a reduction of the Tg values after the graft reaction, which occured due to the moisture from the pullulan hydrophilicity, resulting in a plasticizing effect of the copolymer.
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Figure 10. Thermograms of PullN3 (a), propargyl-terminated PCL (b) and pullulan-g-PCL(c).
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After grafting PCL onto pullulan’s backbone, there was a significant reduction in the relative amount of PCL’s enthalpy of fusion which corroborates the statement that its crystallinity
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was affected by the reaction [49]. The reduction of PCL’s relative crystallinity rate, before and after ( )
where
is
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its reaction with pullulan, was determined by the equation
equal to 136 J.g-1, the fusion enthalpy of 100% crystalline PCL [47]. The crystallinity rate of the
4. Conclusions
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alkyne-PCL was 80.2% and of the PCL amount in Pull-g-PCL was 38.4%.
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Herein, an amphiphilic copolymer based on pullulan and poly(ε-caprolactone) was synthesized. The homopolymers were separately synthesized either via chemical modification of pullulan, or via ROP of ε-caprolactone, resulting in an azide functionalized pullulan and propargyl ended PCL, respectively. Afterwards, the blocks were conjugated via CuAAC for the synthesis of amphiphilic Pull-g-PCL. The grafted copolymer was characterized via 1H NMR, FTIR and XPS techniques. Further analyses were conducted to demonstrate the occurrence of variations in the crystallinity index of the materials, such as DSC and XRD techniques. The final copolymers has interesting properties, such as amphiphilic, biodegradability and self-assembly. Besides that, it can be obtained by a methodology that can control the amount and the size of the polymer grafting into the pullulan’s backbone, and guarantee the remaining of hydroxyl groups from pullulan without further modification in the available hydroxyl. The individual components of this new graft copolymer, that is, pullulan and PCL, are biocompatible and biodegradable and have already been used in biomedical systems. Finally, it is important to highlight that the Pull-g-PCL grafted copolymer were 20
Journal Pre-proof carefully prepared considering its future application, in the elaboration of particles for encapsulation of active principles and evaluation as controlled release systems. Some preliminary tests have already been carried out, yielding satisfactory results that will be submitted in a new publication soon. Therefore, Pull-g-PCL has good potential to be applied in biomedical devices, mainly as drug delivery systems.
Acknowledgements
The authors acknowledge Dr. Richards Landers for assistance with XPS analyses, Coordination for
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the Improvement of Higher Education Personnel (CAPES) and São Paulo Research Foundation
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(FAPESP, 2019/04269-0) for the financial support.
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Journal Pre-proof
Author Statement Layde T. Carvalho: Methodology, Validation, Investigation, Writing - Review & Editing. Rodolfo M. Moraes: Methodology, Data Curation. Gizelda M. Alves: Data Curation. Talita M. Lacerda: Resources. Julio C. Santos: Resources. Amilton M. Santos: Resources, Project administration. Simone F. Medeiros: Supervision, Funding
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Journal Pre-proof Research Highlights: ► Amphiphilic copolymer based in pullulan was successful synthetized via click chemistry reaction. ► The chemical modification was confirmed by different techniques, such as 1H NMR, FTIR, XPS, XRD and DSC. ► The amphiphilic copolymer obtained is suitable to form core-shell nanoparticles.
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► Pullulan-g-poly(ε-caprolactone) is a promising candidate for drug delivery systems.
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