Tunable pH-responsive Chitosan-Poly(acrylic acid) electrospun fibers

Subscriber access provided by READING UNIV

Article

Tunable pH-responsive Chitosan-Poly(acrylic acid) electrospun fibers Rui-Yan Zhang, Ekaterina Zaslavski, Gleb Vasilyev, Mor Boas, and Eyal Zussman Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01672 • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Tunable pH-responsive Chitosan-Poly(acrylic acid) electrospun fibers Rui-Yan Zhang, Ekaterina Zaslavski, Gleb Vasilyev, Mor Boas, Eyal Zussman* NanoEngineering Group Faculty of Mechanical Engineering Technion-Israel Institute of Technology Haifa 32000, Israel

Abstract:

The macromolecular organization in system composed of anionic poly(acrylic

acid) (PAA) and cationic chitosan (Cs), with different degrees of deacetylation (DD), under extensive elongational flow, is described. Cs/PAA nanofibers were obtained, and polyelectrolyte complexation only occurred when fibers were immersed in fluid media of a certain pH. Assembled polyelectrolytes complexes formed a pH-triggered system, as demonstrated by reversible change of the swelling degree, by three orders of magnitude, and a change on the elastic modulus, by two orders of magnitude. Both the swelling degree and the elastic modulus proved sensitive to the DD of Cs. Rheological measurements showed that increased DD of Cs resulted in a decrease in viscosity of both pure Cs and precursor Cs/PAA solutions, attributed to repulsive interactions between ionized amino groups in Cs. At the same time, a DD-dependent change in balance between hydrogen bonding and ion-dipole interactions between the components in Cs/PAA, was responsible for the more pronounced viscosity decrease in these solutions.

Keywords: chitosan, degree of deacetylation, polyelectrolyte complex, electrospun nanofiber, pH-responsive, viscosity

1

ACS Paragon Plus Environment

Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 26

INTRODUCTION Polyelectrolytes are a class of polymers with ionizable groups along their backbone, which can be either protonated or deprotonated, depending on the pH of the surrounding medium. When oppositely charged groups of polyelectrolytes interact, they form polyelectrolyte complexes (PECs).1-3 The complexation kinetics and the characteristics of the resulting PECs are influenced by many factors, such as molecular weight, component concentrations, ionic strength of a media, temperature and pH.4 Applications of PECs5,

6

and the effect of

temperature7, 8 and external mechanical9, 10 and electrical11 fields on their behavior have been extensively investigated under different pH conditions. Although the potential of PEC materials on the mesoscale has been illustrated in these studies, the possibility of exploiting their macromolecular arrangement to obtain efficient responsive structures, has not been fully explored yet. Chitosan

(Cs)

is

a

natural

linear

heteropolysaccharide,

composed

of

2-amino-2-deoxy-D-glucopyranose and 2-acetamido-2-deoxy-D- glucopyranose units and can be produced by deacetylation of chitin. It demonstrates several unique properties, such as immunological, antibacterial and wound healing activities,12 and is a favorable cationic polyelectrolyte due to its non-toxic, biodegradable and biorenewable nature.13 The portion of glucosamine monomer residues in Cs, defined as the degree of N-deacetylation (DD), has a marked influence on the solubility, biodegradability, degree of crystallinity and thermal stability of Cs.14-16 Polar groups in Cs macromolecules form a network of intra- and intermolecular hydrogen bonds (H-bonds).17 In addition, as in chitin,18 the oxygen atom in the carbonyl groups can form both single and double H-bonds, which are involved in both intra2


Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

and intermolecular bonding. Mixing cationic polymer, such as Cs, with an anionic polymer, may result in their self-assembly, or spontaneous association, due to strong, but reversible complexation.19-21 An example of complexation was shown for blends of Cs and poly(acrylic acid) (PAA), which formed tunable interactions, switching from hydrogen bonds to ionic interactions.22 However, a cross-linker had to be added to the complex in order to preserve the structure. Using the traditional dip layer-by-layer (LbL) technique, assembly of Cs/alginate polyelectrolytes on titanium substrates via electrodeposition, was demonstrated.23 Using the same technique, complementary electrostatic interactions between Cs and PAA yielded films with remarkable stability, over 28 days, in simulated in vivo conditions (pH 7.4, phosphate buffered saline at 37°C), however, the elastic modulus dropped from 420 kPa to 27 kPa.24 Alternatively, nano-scale polyelectrolyte complex-based fibers can be fabricated in a single-step electrospinning-based process.25 In this method, a polymer solution, typically semidilute,26, 27 is extruded from a spinneret. An external high electric field creates a strong elongational flow in the formed jet, concurrent with rapid evaporation, which results in kinetically controlled macromolecule arrangement in a small-diameter fibrillar structure.28, 29 During electrospinning, the macromolecular chains are stretched and aligned along the fibers axis. Stretching forces acting on polyelectrolytes are stronger when compared to neutral polymers because of the increased charge density along polyelectrolyte backbone during electrospinning.30 Therefore, it is reasonable to assume that more complexes can be formed in the as-spun fibers, when increasing the number of ionized groups, by increasing the DD. In addition, better packaging of the stretched and aligned macromolecules may increase the 3



Page 4 of 26

complex stability, with enhanced switchable properties. The present work studies Cs/PAA macromolecular organization, achieved by applying an electrical field and extensive elongational flow. Spatial chemical interactions in Cs/PAA precursor solutions and electrospun fibers are demonstrated to be Cs DD-dependent. Swelling behavior and mechanical properties of the electrospun fiber mats immersed in aqueous media with different pHs, are analyzed as a function of DD of chitosan. The results presented show the potential of electrospinning in efficiently assembling PECs, as demonstrated by the reversibility of the swelling degree by three orders of magnitude and elastic modulus by two orders of magnitude.

EXPERIMENTAL SECTION Materials. Chitosan, with a molecular weight of 190 - 280 kDa and deacetylation degree of 82%, and polyacrylic acid, with a molecular weight of 450 kDa, were purchased from Sigma-Aldrich. Formic acid (FA) and glacial acetic acid (AA) were obtained from Frutarom (Israel). All chemicals were of analytical grade and were used without further purification. Ultrapure water, with a resistivity of 18.2 MΩ·cm (Bronstead Purification System), was used for preparation of polymer solutions. To obtain chitosan with various degrees of deacetylation, 1 g chitosan (82% DD) was suspended in 10 mL of 37% aqueous NaOH. The solution was stirred vigorously for 0.5 hour at room temperature, and then subjected to a deacetylation reaction for 0, 1 or 3 hours, at 80 °C. The product underwent a series of washings in distilled deionized water (DI water, from Milli-Q water system) in order to neutralize the basic pH of the solution. Then, the material was dried for 8 hours, at 100 °C. 4


Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

The DD was determined by 1H NMR, as per Hirai et al.31 Briefly, 10 mg Cs were dissolved in 2 mL DCl/D2O (2% (v/v)), at 25 °C. Then, 0.6 mL of the sample was placed in an NMR tube and analyzed. Figure 1a shows the spectra obtained for the neat and treated Cs. The DD was calculated as: DD 1

100%

(1)

where A7 is the integral intensity of the peak at δ = 1.8 ppm, corresponding to protons in the N-acetyl residue (see Figure 1b), and A is a sum of integral intensities of protons in the Cs backbone (three peaks at δ = 2.9 - 4 ppm).

The DD values calculated for neat Cs, and Cs subjected to the deacetylation reaction for 1 and 3 hours, were 82%, 92% and 97%, respectively; the Cs was thus labeled Cs-82, Cs-92, and Cs-97, respectively.

Figure 1. 1H NMR spectra for chitosan incubated with 2% (v/v) DCl/D2O solution for 0, 1 or 3 hours, at 25 °C, resulting in deacetylation degrees (DD) of 82%, 92% and 97% (a). Scheme of chitosan monomer unit, where X is the degree of deacetylation (b).

5



Page 6 of 26

Polyelectrolyte solutions. Stock solutions of Cs, with different DDs, in 60% aqueous FA, and PAA in 90% aqueous AA, were prepared by dissolution under continuous stirring, overnight, at room temperature. Various combinations of the stock solutions (Cs/PAA) were mixed at a 1:2 volume ratio, with continuous stirring, for 1 hour, at ~50 °C. The ternary solvent composition was maintained constant at 4:3:3 FA:AA:water (v/v), as was the total polymer concentration, at 5%wt PAA+Cs, while the Cs/PAA weight ratio varied from 0.02 to 0.4. In this way, the weight fraction of Cs in the solutions ranged from 0.1 to 2.0 %wt. The resulting solutions had pH=0, as measured by CyberScan pH510 (Eutech Instruments, USA) pH-meter. Fibers and films. Cs/PAA nanofibers were obtained by electrospinning. In a typical electrospinning process,32 homogeneous 5 %wt solutions, with a Cs/PAA weight ratio of 1/4 (concentration of Cs is 1 %wt) were utilized. The solutions were labeled as S-82, S-92 and S-97 according to the DD of the Cs. The solutions were spun from a G23 needle under a 2 kV/cm (24 kV and distance between electrodes 12 cm) electrostatic field and a flow rate of 0.5 mL/h, controlled by a Harvard Apparatus PHD 2000 syringe pump. An aluminum disc served as a grounded electrode. A tangential velocity of ~25 m/s was used to collect the nanofibers. The obtained fiber mats were dried in an oven, at 90 °C, for 1 hour. The mats, prepared from solutions S-82, S-92 and S-97, were labelled M-82, M-92 and M-97, respectively. For film preparation, the solutions were cast into a Teflon mold, and the solvent was evaporated over 3 days, at ambient temperature. Finally, the films were dried in an oven, at 90 °C, for 1 hour. The resulting films were labelled as F-82, F-92 and F-97. 6


Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Rheological tests. A Discovery DHR-2 rotational rheometer (TA Instruments, USA) was used to characterize the rheological properties of solutions under steady-state shear flow. Parallel-plate geometry, with a diameter of 40 mm and a gap of 0.5 mm, was applied. Oscillatory shear deformation tests were carried out in the region of linear viscoelastic response of the materials. All rheological measurements were performed at room temperature (25 °C). Due to considerable difficulties in dissolving Cs, solutions of 1 %wt Cs were measured since in this case, the concentration of the Cs is equal to its effective concentration in the Cs/PAA solutions. Scanning electron microscopy (SEM). SEM images were obtained using a Carl Zeiss Ultra Plus high-resolution scanning electron microscope, at an acceleration voltage of 1 kV. The specimens were coated with a thin gold film prior to analysis. FTIR. The chemical structure of the fibers and films was investigated by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR, Thermo, Nicolet 380) and was scanned over a range of 500-4000 cm-1, at a resolution of 2 cm-1. Measurements were performed at room temperature and further analyzed using Origin software. WAXS. Scattering was measurements using a diffractometer Molecular Metrology SAXS system, powered at 45 kV and 0.9 mA, with CuKα radiation (λ = 0.1542 nm). The dried fibers were glued to a two-dimensional sample holder and measured at 25 °C. Wet samples were immersed and sealed in chamber containing water, at various pH levels, and measured at 25 °C. 1D-WAXS curves were constructed by integrating the 2D-patterns obtained at every 15° from the equatorial to meridian direction. The orientation degree of the amorphous phase in the fibers and films was characterized using the Herman’s orientation function, 7



3

&⁄

(

!"# $% &⁄

%

(

Page 8 of 26

1)

(2)

where θ is the angle between the polymer chain and the fiber axis.

Swelling tests. Swelling of the films and fibers was quantified by equilibrium swelling ratio. The weight of completely dried samples was measured. Then the samples were immersed, for 1 hour, in water with pH adjusted using aqueous HCl, at room temperature, before being weighed again. The swelling degree (SD) of the samples was calculated as:

SD

+, ++-

100%

(3)

where wd and ww are the weight of dry and swollen samples, respectively.

Solid-state NMR. 1H and 13C NMR measurements were carried out at 500 and 125.8 MHz Larmor frequency using an AVANCE III spectrometer (Bruker, USA) equipped with a 4mm triple-resonance MAS NMR probe, with zirconia rotors spun at 10000± 2 and 5000±2 Hz. Cross polarization (CP) magic angle spinning (MAS) echo experiments were carried out at 5.0 µs π/2 and 10.0 µs π pulse widths, an echo interval τ (200 µs) identical to the rotor period TR; a 1H decoupling level of 100 kHz was employed, Hartmann-Hahn rf levels were matched at 50 kHz, with contact times (ct) of 2 ms for

13

C; relaxation delays of 2 s were employed.

Data were processed with the Dmfit program.33 Mechanical tests. A custom-made horizontal tensile machine, equipped with a Sensotec® 2N load cell (model 31/1435-03), was used to carry out tensile tests. This machine is furnished with a bath, which allows for testing in a fluid medium, with different pH values, at a controlled temperature. Specimens (6±0.5 mm in length and 4±0.5 mm in width) were cut 8


Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

and clamped. The stretching rate was 3.0 mm/min, under displacement control.

RESULTS Rheological characterization. FA and AA are used as co-solvents to efficiently dissolve Cs and PAA, respectively. Figure 2 presents flow curves of pure Cs and mixed polymers dissolved in the ternary FA/AA/water (4:3:3) solvent. All the solutions exhibited pseudoplastic behavior and can therefore be considered as entangled. Increased DD of Cs from 82% to 92% resulted in a ~50% viscosity decrease; a further slight viscosity decrease was registered at 97% DD. It was previously established that the deacetylation process does not lead to a meaningful change in molecular characteristics of chitosan.34 Thus, the increase in DD was presumably the dominant factor underlying the viscosity decrease. The viscosity of pure 5% PAA solution (Figure 2b) (the critical concentration of entanglement formation was lower than 3 %wt), was more than one decimal order lower than the viscosity of the Cs/PAA polyelectrolyte mixture solutions. A DD increase resulted in a decrease in the solutions viscosity with an almost 4-fold viscosity drop when DD of Cs was increased from 82% to 97%.

9


Biomacromolecules

10

0

10

1

(b)

10

Viscosity η, Pas

(a)

Viscosity η, Pas

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 26

1% Chitosan solutions Cs-97 Cs-92 Cs-82

-1

10

10 -1

10

0

10

1

10

. Shear rate γ, 1/s

2

10

3

10

0

5% Solutions PAA S-97 S-92 S-82

-1

-2

10

10

-1

0

10

10

1

10

2

3

10

. Shear rate γ, 1/s

Figure 2. Flow curves of 1% solutions of chitosan with different DDs (a); 5% PAA and polyelectrolyte mixture solutions of Cs/PAA (b) at 25°C.

Figure 3 demonstrates the dependencies of zero-shear rate viscosity, η0, versus Cs/PAA weight ratios in a series of solutions prepared with Cs-82, Cs-92 or Cs-97. Empirically, the dependencies of this kind can be fit with a power function,35

./ .0 + 23 4

(4)

where, ηa is the viscosity of solution of the first component, φ is a function representing a relative concentration of the second component, b and n are fitting parameters.

The viscosity of the 5% PAA solution was used as the parameter ηa, whereas the Cs/PAA weight ratio in the complex solutions was used as a function of the Cs concentration. The values of the parameters b and n rose from 28 to 156 and from 1.6 to 1.9, respectively, with the decline of the DD of Cs, demonstrating a stronger dependence of the viscosity for solutions containing Cs with low DD. When redrawing the plot in log-log scale, it can be seen that the dependencies consist of two parts with different slopes (see inset in Fig. 3). It is thus possible to assume that a transition between concentration regimes takes place at the composition corresponding to the bending point in the curve. Indeed, at low Cs concentrations, 10


Page 11 of 26

it is primarily Cs-PAA interactions that contribute to viscosity, as Cs-Cs interactions are rare. The latter interactions begin to impact the viscosity of the solutions more significantly when a “critical” Cs concentration threshold is overcome. This change of the slope occurs in the vicinity of a Cs/PAA ratio of 0.1 for all of the tested series (marked by a cross-hatch rectangle). For this reason, solutions with a higher Cs/PAA ratio, of 0.2 (i.e., 4 %wt. of PAA and 1 %wt. of Cs), were selected for electrospinning. 30

20

η0, Pa·s

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

10

1

10

0

S-82

10- 1 10- 2

10-1

S-92

10

S-97

0 0.0

0.1

0.2

0.3

0.4

Cs/PAA, wt./wt. Figure 3. Viscosity vs. relative weight fraction of Cs in S-82, S-92 and S-97 solutions. Lines show the fit of Eq. 4 to the data. The inset shows the same data redrawn on a log-log scale.

Fiber formation and macromolecular analysis. Figure 4 shows the SEM images of M-82, M-92 and M-97 fibers which were of a mean diameter (± standard deviation) of 0.35±0.1 µm, 0.25±0.07 µm and 0.18±0.05 µm, respectively.

11



Page 12 of 26

Figure 4. SEM images of the Cs/PAA fibers: M-82 (a), M-92 (b) and M-97 (c).

IR spectra of pure PAA, pure Cs-82 and electrospun fibers mat M-82 are presented in Figure 5. PAA showed a prominent peak at 1697 cm-1, ascribed to C=O stretching deformation in carboxylic groups of the acid, whereas, in Cs-82, the C=O peak was situated at 1700 cm-1, which is ascribed to the acetylated amine group. In addition, the Cs spectrum featured a peak at 1651 cm-1, corresponding to an amine, and a broad peak at 1552 cm-1, corresponding to amide I and amide II. Residual peak of acidic solvents appears at 1718 cm-1. Peaks observed for PAA and Cs-82 were also registered in M-82, with minor shifts. For M-82, the C=O peak was at 1700 cm-1 and the other main peaks were shifted to 1631 cm-1 and 1541 cm-1. These shifts should be ascribed to the formation of H-bonds. At the same time, the peaks at 1552 and 1404 cm-1 attributed to asymmetrical and symmetrical stretching of charged COO– groups, as well as the peak at 1630 cm-1 due to formation of NH3+ groups, were not resolved because of overlapping. Nevertheless, ionic interactions resulting in PEC formation were not expected at this pH level since PAA is completely deionized.

12


Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 5. FTIR spectra of Cs-82 and PAA powders, and electrospun fibers, M-82. Cs and PAA powders were dissolved in the FA/AA/water mixture and then dried prior to measurements.

WAXS. It is known that Cs has a semi crystalline structure, in which each chitosan chain takes an extended two-fold helix conformation, forming a stacked sheet structure.17 After mixing with PAA and electrospinning chitosan crystallinity was lost. The wide-angle scattering pattern and profile of cast films and electrospun fibers demonstrated an amorphous halo with a major peak at 2θ=18° and an additional shoulder at 2θ=37°, similar to PAA.36 For the electrospun fibers, the diffraction intensity at the equator is slightly higher than the intensity measured at the meridian (see Figure 6). The asymmetry of an amorphous halo of this kind is usually associated with orientation in amorphous phase.37 In contrast, the patterns of the cast films showed symmetrical rings, which indicate the absence of orientation in amorphous phase. 13



(a)

Page 14 of 26

(b)

Figure 6. Typical 2D-WAXS patterns of fiber mats (M-97) (a) and cast films (F-97) (b).

As the degree of deacetylation increased, the orientation factor in fibers exhibited a mild and monotonous increase (Figure 7). The orientation factor for fibers M-82 was determined as 0.16, which is comparable with amorphous orientation in electrospun Nylon 6,6 fibers.38 For cast films, the orientation factor was close to 0 and independent of DD. The obvious increase in macromolecular orientation indicated that macromolecules in M-97 underwent more intense stretching due to higher charge density along the Cs backbone at high DDs. In addition, the viscosity of solution S-97 was the lowest at high shear rates. Such high shear rates, created during electrospinning,27 should result in higher degree of polymer chains alignment.

14


Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Figure 7. The Herman’s orientation factor, f , as a function of DD of Cs in electrospun fiber mats and cast films. Solid state NMR. NMR spectroscopy has proven one of the most powerful tools for obtaining details of the hydrogen bonds in polymer systems. The 13C NMR chemical shift of the carboxyl carbon is thought to be the most sensitive to the spatial arrangement of the nuclei comprising the hydrogen bond, since the electronic structure of the carboxyl carbon is greatly affected by the nature of the hydrogen bond. In fact, it was reported that the formation of hydrogen bonds causes a downfield shift of the carboxyl carbon of peptides in solution.39 Carboxyl protons involved in hydrogen bonding have been shown to affect this chemical shift. Figure 1s (Supporting Information) shows the

13

C CP/MAS NMR spectra of PAA powder,

PAA fibers, the Cs/PAA fiber mat (M-97) and film (F-97). For PAA powder, two peaks were identified, the first appearing at around 40 ppm and corresponding to the PAA backbone carbons, and the second at 180 ppm, corresponding to the carboxyl carbons.40 Qualitatively, the spectrum of PAA fibers was identical to that of PAA powder. Cast film F-97, composed of Cs/PAA showed three additional peaks at 60 ppm, 75 ppm and 100 ppm, corresponding to carbons located on the Cs backbone.41 The peak at approximately 22 ppm corresponded to the 15



Page 16 of 26

CH3 of the acetyl group, which is negligible due to high DD. Akbey et al.40 distinguished between different arrangements of non-hydrogen bonded acidic protons (free carboxylic acid protons) at 178 ppm and protons hydrogen-bonded between two carboxylic acid groups (intra- or inter-chain hydrogen bonding) at 184 ppm; the merged peak at 180 ppm is of our interest. Deconvolution of the peak (Table 1) shows that the free COOH relative intensity for F-97 is 7% higher than that of PAA fibers, meaning, that introduction of Cs partially disrupted intra- or inter-chain H-bonds in PAA, in favor of free COOH that can interact with Cs. Comparison of the spectra of electrospun fiber mat M-97 and of the film F-97 revealed a further decrease in the relative intensity of the peak at 183.1 ppm. Thus, the contribution of this peak is 17%, which is half of that measured in PAA powder (34%). Of note, the free COOH peak of the fiber mat was 0.7 ppm narrower than that of the cast film, likely due to higher polymer matrix homogeneity in fibers obtained by the electrospinning process. Table 1. The best-fitting chemical shifts of the 13C peaks of the carboxyl carbons in the PAA and PAA-Cs complex.

Peak corresponding to intra- or inter-chain H-bonds (dimer)

Peak corresponding to free carboxyl group

Sample Position (ppm)

Width (ppm)

Intensity (%)

Position (ppm)

Width (ppm)

Intensity (%)

PAA powder

183.1

3.1

34

179.7

6.6

66

PAA fibers

183.1

3.1

30

179.7

6.0

70

F-97

183.1

3.6

23

179.4

6.5

77

M-97

183.1

3.0

17

179.3

5.8

83

16


Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Swelling behavior. Results of swelling tests of the Cs/PAA films and fiber mats, at different pH conditions, are shown in Figure 8. For fibers, only moderate swelling of ~ 200% was observed at pH > 2.4. A sharp increase in SD was observed at pH=2.1-2.4 ranged from 1300% to 2200%, depending on the DD. It should be mentioned that fiber diameters remained almost constant at pH>2.4, whereas, below this critical pH value, the fibers became highly swollen and diameters increased significantly. Films SD was practically independent of pH value and was in the range of 200% to 250%.

Figure 8. Swelling degree, SD of Cs/PAA fiber mats and films in water, at different pH values.

Reversibility of swelling behavior was demonstrated by alternately immersing fibers in water at pH 1.0 versus 6.0 (Figure 9). Fibers swelled significantly at pH 1.0, and shrank back at pH 6.0. The swelling behavior of all type of fibers was reversible in this pH range.

17



Page 18 of 26

Figure 9. Swelling degree of Cs/PAA fiber mats, sequentially immersed in water at pH 1.0 and then pH 6.0. Mechanical properties. Tensile tests in aqueous media with different pH values, were conducted to evaluate the pH-responsive behavior of the mechanical properties of the fiber mats. Figure 10a shows stress-strain curves of the mats at pH 1.0, conditions under which all the mats were quite weak, with low strength and stiffness. When raising the pH to 6.0, mechanical strength increased (Figure 10b), as a function of the DD (see results in Table 2). For sample M-97, the strength and Young’s modulus increased by ~ 400-fold and 500-fold, respectively, and only increased by ~ 30 and 6.6 times, respectively, for sample M-82. (a)

(b)

Figure 10. Typical stress-strain curves of the fiber mats at (a) pH=1.0 and (b) pH=6.0. 18


Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

Table 2. Mechanical properties of electrospun fiber mats at different pH. pH

1.0

6.0

Sample

Young’s modulus, kPa

Tensile Elongation Strength, kPa at break, %

M-82

8.0±0.4

9.1±0.6

130±15

M-92

3.6±0.5

6.1±0.5

190±21

M-97

1.2±0.4

3.4±0.6

150±18

M-82

53.1±9.8

290.9±48

341±24

M-92

71.0±11

342.5±75

217±34

M-97

576±25

1310±210

144±27

DISCUSSION Cs solutions demonstrate a ~70% decrease in viscosity with a DD increase from 82% to 97%. At lower DDs, more hydrogen bonds are formed, enabling formation of a denser network, which leads to a viscosity increase. In contrast, at pH=0, the repulsive interactions between the completely ionized42 amino groups hinder formation of a dense, entangled network, and thus, viscosity decreases. Although the acetylated amino group is bulky and may hinder hydrogen bonding,43 the joint effect of these interactions, acting simultaneously, can explain the decrease in viscosity as the DD of Cs increases. The Kuhn segment length of Cs increases from 50 Å to 180Å as DD rises,43 meaning, increasing DDs are accompanied by increased chains rigidity.44 On the other hand, PAA at low pH is fully protonated, and its persistence length is low, approximately 4Å.45, 46 Although complexation of PAA and Cs has already been reported,22 electrospinning provides formation of microstructure within the fibers, without the need for cross-linkers. The Cs and PAA form interpenetrating networks of rigid Cs with flexible PAA harnessed with H-bond/ion-dipole interactions, and under the strong elongation flow, the network is stretched and elongated.28, 47 19



Page 20 of 26

Since the flexibility of PAA is rather high, massive amounts of its intramolecular hydrogen bonds may remain intact and withstand the high shear stress that develops along the electrospun jet. After stretching, as is evident from the WAXS results, and formation of a confined structure in the fibers,38 inter-molecular bonds between PAA and Cs may be reformed. Decreased viscosity resulting from increased DD, as implied by rheological measurements, and higher charge density, results in extensive stretching and thinner electrospun fiber diameters.30 The NMR spectroscopic analysis supported the above observations, as the relative intensity of the peaks indicated higher content of free COOH groups in Cs/PAA fibers in comparison to the powder, suggesting that electrospinning destroys some of the PAA intra/inter-chain hydrogen bonding and thus increases free carboxyl groups that can eventually interact with other species in their environment. In addition, the peak width of free COOH was smaller for Cs/PAA fibers as compared with film. Improved homogeneity implied by narrowing of the free carboxyl peak, may arise from enhanced chain ordering during electrospinning. These results demonstrate the effect of Cs on PAA intra/inter-chain hydrogen bonding, by increasing the amount of free COOH groups that can interact with protonated amine groups of Cs at higher pH values, and further manipulated using the electrospinning technique. Previous reports have demonstrated that swelling behaviors of polyelectrolyte complexes are highly dependent on the pH.48 It is believed that macromolecular packaging is beneficial to the pH-dependent swelling behavior, since swelling behavior is determined by the strength of electrostatic interaction between chitosan and PAA.25 At pH > 2.0, both negatively charged 20


Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

carboxyl COO- and positively charged NH3+ ions coexist,42 thus, a strong electrostatic interaction will be favored rather than H-bonding with water, which will result in deswelling due to water diffusion out. On the other hand, at lower pH regimes (< 2.0), there are no electrostatic interactions between the two polyelectrolytes, due to PAA deionization.49 The presence of hydrogen bonds then enables water to diffuse into the loose intermolecular structures, leading to significant swelling. Among the electrospun fibers, the swelling degree of M-97 can reach about 2000%, illustrating that SD can be greatly promoted with the increase in DD of Cs in fibers. Mechanical properties of electrospun fibers can be determined by the degree of swelling at pH=1.0. The polymers begin to swell after immersion in acidic fluid media and take on a jelly-like consistency. Weak interactions between the macromolecules and the high amount of water serving as a plasticizer, results in low Young’s modulus. As the DD increases, more water diffuses and fewer strong Cs-Cs hydrogen bonds remain, leading to a lower Young’s modulus; therefore M-82 fibers demonstrated enhanced mechanical properties. An opposite trend was observed at pH=6.0, where both tensile strength and Young’s modulus of the fibers mat rose with the increase in DD (see Table 2). In this case, Cs-PAA ionic interactions should be taken into account and indeed, M-97 fibers demonstrated enhanced mechanical properties.

CONCLUSIONS Fibers comprised of Cs and PAA, with different degrees of deacetylation of Cs, were processed under extensive elongational flow. Fibers remained stable for several weeks after several cycles of exposure to alternating pH, despite lack of crosslinkeres. Investigation of 21



Page 22 of 26

spatial hydrogen bonds of the fibers revealed a lower degree of PAA intra- and inter-molecular

interactions.

It

is

assumed

that

the

relatively

rigid

Cs

forms

H-bonds/ion-dipole interactions with the relatively flexible PAA and thereby hinders the formation of intra- and inter-molecular interactions in PAA. Fiber swelling and mechanical properties were found to be pH-dependent and also affected by the deacetylation degree of Cs. This profile could have a potential application in the drug delivery and controlled-drug release arenas, as well in responsive coatings for nanodevices.

AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected]

ORCID Eyal Zussman: 0000-0002-4310-6548 ACKNOWLEDGEMENTS The authors thank to Dr Shifi Kababya for the help with SSNMR results interpretation and to Dr. Ira Benshir for the SSNMR measurements. The authors also thank the Russell Berrie Nanotechnology Institute (RBNI) at the Technion. E.Z. acknowledges the support of the Winograd Chair of Fluid Mechanics and Heat Transfer at Technion.

REFERENCES (1) Chollakup, R.; Smitthipong, W.; Eisenbach, C. D.; Tirrell, M. Phase Behavior and Coacervation of Aqueous Poly(acrylic acid)−Poly(allylamine) Solu3ons. Macromolecules (Washington, DC, U. S.) 2010, 43 (5), 2518-2528. (2) Dobrynin, A. V.; Colby, R. H.; Rubinstein, M. Scaling Theory of Polyelectrolyte Solutions. Macromolecules (Washington, DC, U. S.) 1995, 28 (6), 1859-1871. (3) Dobrynin, A. V.; Rubinstein, M. Theory of polyelectrolytes in solutions and at surfaces. Prog. Polym. Sci. 2005, 30 (11), 1049-1118. (4) Philipp, B.; Dautzenberg, H.; Linow, K. J.; Kotz, J.; Dawydoff, W. Poly-electrolyte complexes - recent developments and open problems. Prog. Polym. Sci. 1989, 14 (1), 91-172. (5) Hammond, P. T. Engineering Materials Layer-by-Layer: Challenges and Opportunities in Multilayer Assembly. AIChE J. 2011, 57 (11), 2928-2940. 22


Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(6) De Geest, B. G.; Sukhorukov, G. B.; Mohwald, H. The pros and cons of polyelectrolyte capsules in drug delivery. Expert Opin. Drug Delivery 2009, 6 (6), 613-624. (7) Clarke, K. C.; Dunham, S. N.; Lyon, L. A. Core/Shell Microgels Decouple the pH and Temperature Responsivities of Microgel Films. Chem. Mater. 2015, 27 (4), 1391-1396. (8) Dautzenberg, H.; Gao, Y. B.; Hahn, M. Formation, structure, and temperature behavior of polyelectrolyte complexes between ionically modified thermosensitive polymers. Langmuir 2000, 16 (23), 9070-9081. (9) Ariga, K.; Mori, T.; Hill, J. P. Mechanical Control of Nanomaterials and Nanosystems. Advanced Materials (Weinheim, Germany) 2012, 24 (2), 158-176. (10) Cranford, S. W.; Ortiz, C.; Buehler, M. J. Mechanomutable properties of a PAA/PAH polyelectrolyte complex: rate dependence and ionization effects on tunable adhesion strength. Soft Matter 2010, 6 (17), 4175-4188. (11) Kim, S. J.; Yoon, S. G.; Lee, K. B.; Park, Y. D.; Kim, S. I. Electrical sensitive behavior of a polyelectrolyte complex composed of chitosan/hyaluronic acid. Solid State Ionics 2003, 164 (3), 199-204. (12) Anitha, A.; Sowmya, S.; Kumar, P. T. S.; Deepthi, S.; Chennazhi, K. P.; Ehrlich, H.; Tsurkan, M.; Jayakumar, R. Chitin and chitosan in selected biomedical applications. Prog. Polym. Sci. 2014, 39 (9), 1644-1667. (13) Dash, M.; Chiellini, F.; Ottenbrite, R. M.; Chiellini, E. Chitosan-A versatile semi-synthetic polymer in biomedical applications. Prog. Polym. Sci. 2011, 36 (8), 981-1014. (14) Bodnar, M.; Hartmann, J. F.; Borbely, J. Preparation and characterization of chitosan-based nanoparticles. Biomacromolecules 2005, 6 (5), 2521-2527. (15) Lamarque, G.; Viton, C.; Domard, A. Comparative study of the second and third heterogeneous deacetylations of α-and β-chitins in a multistep process. Biomacromolecules 2004, 5 (5), 1899-1907. (16) Nam, Y. S.; Park, W. H.; Ihm, D.; Hudson, S. M. Effect of the degree of deacetylation on the thermal decomposition of chitin and chitosan nanofibers. Carbohydr. Polym. 2010, 80 (1), 291-295. (17) Ogawa, K.; Yui, T.; Okuyama, K. Three D structures of chitosan. Int. J. Biol. Macromol. 2004, 34 (1-2), 1-8. (18) Kameda, T.; Miyazawa, M.; Ono, H.; Yoshida, M. Hydrogen bonding structure and stability of alpha-chitin studied by C-13 solid-state NMR. Macromol. Biosci. 2004, 5 (2), 103-106. (19) Jeong, S. I.; Krebs, M. D.; Bonino, C. A.; Samorezov, J. E.; Khan, S. A.; Alsberg, E. Electrospun Chitosan-Alginate Nanofibers with In Situ Polyelectrolyte Complexation for Use as Tissue Engineering Scaffolds. Tissue Eng., Part A 2011, 17 (1-2), 59-70. (20) Berger, J.; Reist, M.; Mayer, J. M.; Felt, O.; Peppas, N. A.; Gurny, R. Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. Eur. J. Pharm. Biopharm. 2004, 57 (1), 19-34. (21) Hamman, J. H. Chitosan Based Polyelectrolyte Complexes as Potential Carrier Materials in Drug Delivery Systems. Mar. Drugs 2010, 8 (4), 1305-1322. (22) Wang, H. F.; Li, W. J.; Lu, Y. H.; Wang, Z. L. Studies on chitosan and poly(acrylic acid) interpolymer complex. I. Preparation, structure, pH-sensitivity, and salt sensitivity of complex-forming poly(acrylic acid): Chitosan semi-interpenetrating polymer network. J. Appl. Polym. Sci. 1998, 69 (8), 1679-1679. (23) Wang, Z. L.; Zhang, X. Q.; Gu, J. M.; Yang, H. T.; Nie, J.; Ma, G. P. Electrodeposition of alginate/chitosan layer-by-layer composite coatings on titanium substrates. Carbohydr. Polym. 2014, 103, 38-45. (24) Gates, S. J.; Shukla, A. Layer-by-Layer Assembly of Readily Detachable Chitosan and Poly(acrylic acid) Polyelectrolyte Multilayer Films. J. Polym. Sci., Part B: Polym. Phys. 2017, 55 (2), 127-131. (25) Boas, M.; Gradys, A.; Vasilyev, G.; Burman, M.; Zussman, E. Electrospinning polyelectrolyte complexes: pH-responsive fibers. Soft Matter 2015, 11 (9), 1739-1747. (26) Reneker, D. H.; Yarin, A. L.; Zussman, E.; Xu, H. Electrospinning of nanofibers from polymer solutions and melts. Adv. Appl. Mech. 2007, 41, 43-195. 23



Page 24 of 26

(27) Yarin, A. L.; Koombhongse, S.; Reneker, D. H. Bending instability in electrospinning of nanofibers. J. Appl. Phys. (Melville, NY, U. S.) 2001, 89 (5), 3018-3026. (28) Greenfeld, I.; Arinstein, A.; Fezzaa, K.; Rafailovich, M. H.; Zussman, E. Polymer dynamics in semidilute solution during electrospinning: A simple model and experimental observations. Phys. Rev. E 2011, 84 (4), 041806 (29) Greenfeld, I.; Fezzaa, K.; Rafailovich, M. H.; Zussman, E. Fast X-ray Phase-Contrast Imaging of Electrospinning Polymer Jets: Measurements of Radius, Velocity, and Concentration. Macromolecules (Washington, DC, U. S.) 2012, 45 (8), 3616-3626. (30) McKee, M. G.; Hunley, M. T.; Layman, J. M.; Long, T. E. Solution rheological behavior and electrospinning of cationic polyelectrolytes. Macromolecules (Washington, DC, U. S.) 2006, 39 (2), 575-583. (31) Hirai, A.; Odani, H.; Nakajima, A. Determination of Degree of Deacytelation of chitosan by H-1 NMR Spectroscopy. Polym. Bull. 1991, 26 (1), 87-94. (32) Theron, A.; Zussman, E.; Yarin, A. L. Electrostatic field-assisted alignment of electrospun nanofibres. Nanotechnology 2001, 12 (3), 384-390. (33) Massiot, D.; Fayon, F.; Capron, M.; King, I.; Le Calve, S.; Alonso, B.; Durand, J. O.; Bujoli, B.; Gan, Z. H.; Hoatson, G. Modelling one- and two-dimensional solid-state NMR spectra. Magn. Reson. Chem. 2002, 40 (1), 70-76. (34) Chou, S. F.; Lai, J. Y.; Cho, C. H.; Lee, C. H. Relationships between surface roughness/stiffness of chitosan coatings and fabrication of corneal keratocyte spheroids: Effect of degree of deacetylation. Colloids Surf., B 2016, 142, 105-113. (35) Dreval’, V. E.; Vasil’ev, G. B.; Litmanovich, E. A.; Kulichikhin, V. G. Rheological properties of concentrated aqueous solutions of anionic and cationic polyelectrolyte mixtures. Polym. Sci. Ser. A 2008, 50 (7), 751-756. (36) Yamaguchi, H.; Nakanishi, S.; Iba, H.; Itoh, T. Amorphous polymeric anode materials from poly(acrylic acid) and tin(II) oxide for lithium ion batteries. J. of Power Sources 2015, 275, 1-5. (37) Si, Y.; Yu, J. Y.; Tang, X. M.; Ge, J. L.; Ding, B. Ultralight nanofibre-assembled cellular aerogels with superelasticity and multifunctionality. Nat. Commun. 2014, 5, 5802. (38) Arinstein, A.; Burman, M.; Gendelman, O.; Zussman, E. Effect of supramolecular structure on polymer nanofibre elasticity. Nat. Nanotechnol. 2007, 2 (1), 59-62. (39) Asakawa, N.; Kuroki, S.; Kurosu, H.; Ando, I.; Shoji, A.; Ozaki, T. Hydrogen-Bonding Effect on C-13 NMR Chemical-Shifts of L-Alanine Residue Carbonyl Carbons of Peptides in the solid-State. J. Am. Chem. Soc. 1992, 114 (9), 3261-3265. (40) Akbey, U.; Graf, R.; Peng, Y. G.; Chu, P. P.; Spiess, H. W. Solid-State NMR Investigations of Anhydrous Proton-Conducting Acid-Base Poly(acrylic acid)-Poly(4-vinyl pyridine) Polymer Blend System: A Study of Hydrogen Bonding and Proton Conduction. J. Polym. Sci., Part B: Polym. Phys. 2009, 47 (2), 138-155. (41) Heux, L.; Brugnerotto, J.; Desbrieres, J.; Versali, M.-F.; Rinaudo, M. Solid state NMR for determination of degree of acetylation of chitin and chitosan. Biomacromolecules 2000, 1 (4), 746-751. (42) Silva, C. L.; Pereira, J. C.; Ramalho, A.; Pais, A.; Sousa, J. J. S. Films based on chitosan polyelectrolyte complexes for skin drug delivery: Development and characterization. J Membrane Sci 2008, 320 (1-2), 268-279. (43) Anthonsen, M. W.; Varum, K. M.; Smidsrod, O. Solution Properties of Chitosans - Conformation and Chain Stifness of Chitosans with Different Degrees of N-Acetylation. Carbohydr. Polym. 1993, 22 (3), 193-201. (44) Terbojevich, M.; Cosani, A.; Conio, G.; Marsano, E.; Bianchi, E. Chitosan - chain Rigidity and Mesophase Formation. Carbohydr. Res. 1991, 209, 251-260. (45) Cranford, S. W.; Buehler, M. J. Variation of Weak Polyelectrolyte Persistence Length through an Electrostatic Contour Length. Macromolecules (Washington, DC, U. S.) 2012, 45 (19), 8067-8082. 24


Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(46) Takahashi, T.; Takayama, K.; Machida, Y.; Nagai, T. Characteristics of Polyion complexes of chitosan with Sodium Alginate and Sodium Polyacrylate. Int. J. Pharm. (Amsterdam, Neth.) 1990, 61 (1-2), 35-41. (47) Vasilyev, G.; Burman, M.; Arinstein, A.; Zussman, E. Estimating the Degree of Polymer Stretching during Electrospinning: An Experimental Imitation Method. Macromol. Mater. Eng. 2017, 302 (8), 1600554. (48) Hiller, J.; Mendelsohn, J. D.; Rubner, M. F. Reversibly erasable nanoporous anti-reflection coatings from polyelectrolyte multilayers. Nat. Mater. 2002, 1 (1), 59-63. (49) Choi, J.; Rubner, M. F. Influence of the degree of ionization on weak polyelectrolyte multilayer assembly. Macromolecules (Washington, DC, U. S.) 2005, 38 (1), 116-124.

25



Page 26 of 26

For Table of Contents Only

Tunable pH-responsive Chitosan-Poly(acrylic acid) electrospun fibers

Rui-Yan Zhang, Ekaterina Zaslavski, Gleb Vasilyev, Mor Boas, Eyal Zussman

26


Tunable pH-responsive Chitosan-Poly(acrylic acid) electrospun fibers

Recommend Documents