Effect of Linear-Hyperbranched Amphiphilic Phosphate Esters on

Article pubs.acs.org/JAFC

Effect of Linear-Hyperbranched Amphiphilic Phosphate Esters on Collagen Fibers Xuechuan Wang,*,† Xiaoxiao Guo,† Haijun Wang,† and Peiying Guo‡ †

College of Bioresources Chemical and Materials Engineering, Shaanxi University of Science and Technology; Shaanxi Research Institute of Agricultural Products Processing Technology, Xi’an, Shaanxi, People’s Republic of China 710021 ‡ College of Arts and Sciences, Shaanxi University of Science and Technology, Xi’an, Shaanxi, People’s Republic of China 710021 S Supporting Information *

ABSTRACT: The surfactants of the linear-hyperbranched phosphate esters (PAMAMGn-3-Ps) have been constructed through random multibranching esterification of lauroyl chloride and phosphate ester as a branching agent. Subsequently, a series of surfactant products were obtained. Benefiting from the amphiphilic structure with the hydrophilic core and many hydrophobic tails, PAMAMGn-3-Ps were able to self-assemble into nanomicelles in aqueous media. Importantly, the polymers show low critical micelle concentrations (CMCs) and small particle sizes. Here, PAMAMG1-3-P was applied in the collagen fibers of leather to improve the fibers’ distance and mechanical property of collagen fibers. Additionally, the polymers display significant flexibility, which could replace ordinary fatliquor in the future. The result provides a new application of using linear-hyperbranched amphiphilic phosphate esters into traditional leather materials to enhance the performance of collagen fibers. KEYWORDS: PAMAM, phosphorylation, amphiphilic, collagen fibers, cross-linking

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phospholipids can deposit on the surface of collagen fibers and form a lubrication layer. Therefore, the hyperbranched phosphate ester could probably adhere to the collagen fibers and reduce their friction. Furthermore, the PO bond has strong complexation ability to metal, so the hyperbranched phosphate esters could be used to produce “permanent” coordination with Cr3+ in collagen fibers. Thus, we decided to design and synthesize some amphiphilic hyperbranched phosphate esters, which combine the advantages of hyperbranched polymers and phospholipids. In this work, the amphiphilic polymers with a polar phosphate headgroup and hydrophobic aliphatic tail have been synthesized via esterification of dendritic polyamide with lauroyl chloride and subsequent capping reaction of phosphorus pentoxide. The schematic synthetic process of hyperbranched phosphate esters with various generations is shown in Figure 1. We have focused on analyzing the chemical composition and interfacial behavior of these terminal amphiphilic hyperbranched polymers. Here, the linear-hyperbranched amphiphilic phosphate esters have been used as an efficient additive for collagen fibers. The structure of collagen fibers modified with synthetic polymers has been explored, and the interaction between synthetic polymers and collagen fibers has also been investigated.

INTRODUCTION Animal hides containing type I collagen are commercially important as natural frameworks largely utilized in the medical, food, and leather industries.1 The demand for leather continues to grow because of its comfort, softness, extensibility, and so on.2 However, the raw animal hides have so poor flexibility and elasticity that they must be treated through a series of physical and chemical treatments to obtain outstanding performances. Fatliquoring is one of the key operations in the manufacture of leather.3 In the process of fatliquoring, the added natural oils can penetrate into the collagen fibers and reside in the regions between the molecular chain segments. The decreasing intermolecular action makes the collagen fibers more flexible and protects the leather against cracking. However, the problem is that the natural oils are in short supply. On the other hand, natural oils have other limitations. For instance, natural oils are easily oxidized, and the odor and hand-feel of leathers treated with natural oils are not ideal. Therefore, the latest research focuses on developing replacements for natural oils to improve the properties of collagen fibers. Dendritic polymers and hyperbranched polymers, combining the characteristics of functionalized macromolecules and nanoparticles, have attracted significant interest due to their promising properties.4−6 In the past few years, considerable attention was given to grafting modifications with organic molecules, long chains, and so on, which have greatly improved the propertyies of hyperbranched polymers and broadened their application.7−10 However, few studies have been reported on the phosphorylation of hyperbranched polymers. In general, the pentavalent nature of the phosphorus allows introductions of bioactive molecules and extensive modification of the physical and chemical properties of the polymers.11,12 In addition, the hyperbranched phosphate ester is similar to phospholipids in structure. It has been reported that © XXXX American Chemical Society

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EXPERIMENTAL PROCEDURES

Materials. The polyamidoamine dendrimers with different generations (PAMAMG0, PAMAMG1, PAMAMG2) were purchased from Weihai CY Dendrimer Technology Co. N,N-Dimethylformamide Received: Revised: Accepted: Published: A

October 10, 2016 December 13, 2016 December 15, 2016 December 15, 2016 DOI: 10.1021/acs.jafc.6b04482 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Schematic illustration routes of PAMAMGn-3-Ps.

Figure 2. Detailed scheme for synthesis of PAMAMG1-3-P. (DMF) was dried over calcium hydride and then purified by vacuum distillation. Lauroyl chloride was purchased from Aladdin Chemical Reagent Corp. and distilled. Triethylamine (TEA) was refluxed with phthalic anhydride, potassium hydroxide, and calcium hydride in turn and distilled just before use. Tetrahydrofuran (THF) was dried by refluxing with the fresh sodium benzophenone complex under N2 and

distilled just before use. Methanol and dichloromethane were purchased from Sigma-Aldrich. Phosphorus pentoxide (P2O5) was dried over anhydrous MgSO4. Other reagents were purified by common purification procedures. The goat wet blue was supplied by Liquan Shunji Leather Co., Ltd. B

DOI: 10.1021/acs.jafc.6b04482 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Synthesis of Linear-Polyamidoamine Dendrimers (PAMAMGn-ms). The PAMAMGn-ms were obtained by the esterification of PAMAMGns with lauroyl chloride in DMF. TEA was added as the acceptor of HCl. As the reaction proceeds, TEA hydrochloride precipitates from the solution, and the quantity of deposits corresponds to the consumed lauroyl chloride. To confirm the optimal grafting ratio, we synthesized a series of PAMAMG1-ms by controlling the molar ratio of lauroyl chloride to PAMAMG1. The molar ratios of lauroyl chloride to PAMAMG1 are 8:1 (PAMAMG1-1), 8:2 (PAMAMG1-2), 8:3 (PAMAMG1-3), 8:4 (PAMAMG1-4), 8:5 (PAMAMG1-5), 8:6 (PAMAMG1-6), 8:7 (PAMAMG17), and 8:8 (PAMAMG1-8), respectively. As a typical example of PAMAMG1-3, PAMAMG1 (10.0 mmol) and TEA (30.0 mmol) were dissolved in 40 mL of DMF in a dried three-neck flask fitted with a magnetic stirrer, a dry nitrogen inlet, and a drying tube. The freshly distilled lauroyl chloride (30.0 mmol) was added dropwise into the PAMAMG1 solution, and then the solution was stirred at room temperature for 12 h. After the precipitation of triethylamine hydrochloride had been filtered out, the solvent was distilled off. Subsequently, the residue was then dissolved in 20 mL of dichloromethane and washed with water three times. The oil phase was dried with anhydrous magnesium sulfate salt, and then the pale yellow PAMAMG1-3 in solid state was obtained. Other PAMAMG1-ms were prepared in synthetic steps similar to those for PAMAMG1-3. The synthetic methods of PAMAMG0-ms and PAMAMG2-ms are also similar to that of PAMAMG1-3. The molar ratios of lauroyl chloride to PAMAMGn were controlled from 4:1 to 4:4 for PAMAMG0-ms and from 16:1 to 16:16 for PAMAMG2-ms. The grafting ratio of PAMAMGn-ms was determined by measuring the hydroxyl numbers according to QB/T 12008.3-2009. The grafting ratio was calculated by using the following equation:

ω=

A 0 − A1 A0

To confirm the content of the monoester, disester, and phosphoric acid, the products were added into the 50% methanol aqueous solution, and then a pH glass electrode was inserted. The potentiometric titration curves of pH are shown in Figure S1. The content of monoester, disester, and phosphoric acid was determined by potentiometric titration and calculated by usingeq 2. For PAMAMG1-3-P, the content of phosphomonoester (PME) is 52.68%, that of phosphodisester (PDE) is 39.37%, and that of phosphoric acid (PA) is 7.95%. The yield of PAMAMG0-3-P is 50.38% (monoester), 42.23% (disester), and 7.39% (phosphoric acid), and that of PAMAMG2-3-P is 51.45% (monoester), 41.88% (disester), and 6.67% (phosphoric acid). ω (PME)% =

(V2 − V1) − (V3 − V2) × 100% V1

V1 − (V2 − V1) × 100% V1 V − V2 ω (PA)% = 3 × 100% V1 ω (PDE)% =

(2)

Preparation of Leather. The raw collagen fibers were taken from the spinal part of goat wet blue. The synthesized PAMAMG1-3 (8 g) and PAMAMG1-3-P (8 g) were respectively added to 100 mL of deionized water under stirring with a magnetic bar at 50 °C. The mixtures were applied to the fatliquoring process for the goat wet blue as shown in Table S2. Characterizations. Nuclear magnetic resonance (NMR) analysis was recorded on the ADVANCEIII 400 MHz spectrometers with deuterium oxide (D2O) as solvent. Gel permeation chromatography (GPC) was performed on a Waters 2695 GPC system (300 × 7.8 mm mixed-B and mixed-C column) equipped with a refractive index (RI) detector. DMF containing 0.01 mol/L polystyrene was used as the mobile phase at a flow rate of 0.8 mL/min at 40 °C. Fourier transform infrared spectrometer (FTIR) spectra were recorded on a VECTOR22 instrument by KBr sample holder method. Potentiometric titration was performed using a Metrohm all-purpose 905 Titrino apparatus equipped with a combined 804 Ti Stand and 800 Dosina. Dynamic light scattering (DLS) measurements were performed in aqueous solution using a Zetasizer NANO-ZS90 apparatus equipped with a 4.0 mW laser at λ = 633 nm. All of the samples of 0.2 mg/mL were measured at 20 °C and at a scattering angle of 90°. Transmission electron microscopy (TEM) studies were performed with a FEI Tecnai G2 F20 S-TWIN instrument operated at 200 kV. The concentrations of PAMAMGn-3s and PAMAMGn-3-Ps were 0.5 mg/ mL. The solution was diluted to various desired concentrations of PAMAMGn-3s and PAMAMGn-3-Ps (from 2 mg/mL to 5.0 × 10−5 mg/mL) with a constant pyrene concentration of 6.0 × 10−7 mol/L. The fluorescence spectra of PAMAMGn-3s and PAMAMGn-3-Ps solutions were recorded on an FS5 fluorescence spectrometer (Edinburgh Instruments Co.) to investigate the CMCs, and the pyrene was used as the fluorescence probe. The CMCs were also measured through an automatic surface tension meter (QBZY-1, Shanghai Fangrui Instrument Co., Ltd.) by using the Wilhelmy plate technique with a sandblasted platinum plate as the sensor. The morphologies of collagen fibers were observed by a scanning electron microscope (E-SEM) FEI Q45 (FEI Co.) operated at 5 kV. The structure of collagen fibers was confirmed by attenuated total reflectance fast infrared spectrophotometry (ATR-IR). ATR-IR characterization of leather was carried out using a VERTEX 70, NETZSCH FTIR spectrometer and a ZnSe crystal at a 45° angle of incidence. XPS measurements were performed on an X-ray photoelectron spectrometer (XPS, Kratols Axis Supra) with a monochromatic focused Al Kα X-ray source (1486.6 eV) to determine C, N, O, Cr, and P elements on the slice surface. Peak fitting was performed with the software package Casaxps, and surface elemental stoichiometry was determined from peak-area ratio. The binding energy (BE) of C 1s (284.6 eV) was selected for energy calibration. Xray diffraction data (XRD) were obtained using a 2.2 kW rotating anode X-ray diffractometer (D8 Advance, Bruker, Germany) with a

(1)

The hydroxyl values (A0) of PAMAMG0, PAMAMG1, and PAMAMG2 were measured to be 402.50, 196.00, and 108.00, respectively, and the hydroxyl values (A 1) of PAMAMG0 -3, PAMAMG1-3, and PAMAMG2-3 were measured to be 124.62, 139.59, and 88.99, respectively. According to eq 1, the esterification rates of PAMAMG0-3, PAMAMG1-3, and PAMAMG2-3 are 69.03, 28.78, and 17.60%, respectively. The critical micelle concentrations (CMCs) and particle sizes of PAMAMG1-ns are summarized in Table S1. As seen in Table S1, the particle sizes of PAMAMG1-ns increase with the content of lauroyl chloride. Generally, it is difficult for fatliquor with a size above 400 nm to permeate the collagen fibers. Therefore, the molar ratios of lauroyl chloride to PAMAMGn should be controlled below 8:5. In addition, the lower the CMC is, the more it can lubricate and enlarge the distance between the collagen fibers. PAMAMG1-3 reveals the smallest CMC, indicating that it has the best permeability and emulsification performance. By comparing the particle sizes and CMCs of PAMAMG1-ns, the optimal product was identified as PAMAMG1-3. Next, we chose PAMAMGn-3 for the next phosphorylation. Synthesis of Linear-Polyamidoamine Phosphate Ester Dendrimers (PAMAMGn-3-Ps). Because the PAMAMGn-3s contains hydroxy without grafting. PAMAMGn-3-Ps were prepared by the phosphorylation of PAMAMGn-3s using P2O5 as a reactant. As a typical example of PAMAMG1-3-P, the synthetic route of PAMAMG1-3-P is shown in Figure 2. PAMAMG1-3 (10.0 mmol) was dissolved in 40 mL of THF, and then P2O5 (80.0 mmol) was added slowly into PAMAMG1-3. The mixture was stirred at 60 °C for 6 h. After the esterification reaction, the deionized water (10 mL) was added into the same flask at 65 °C for 3.5 h to improve the content of phosphomonoester. The crude product was dissolved in 20 mL of ethanol and stirred slightly. After standing for an hour, the products were precipitated and filtered. The filtrate was dried under vacuum to obtain some transparent and colorless oil. To prepare PAMAMG0-3-P and PAMAMG2-3-P, 40.0 and 160 mmol of P2O5 were added, respectively. C


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Figure 3. 1H NMR (a) and 13C NMR (b) spectra of PAMAMG1 and PAMAMG1-3-P. fixed Cu Kα radiation of 0.154 nm. The sample was scanned in the range of diffraction angle 2θ from 5° to 60° with a scanning rate of 2°/ min. The real lattice space d, which represents the dimension of collagen fibers, can be calculated as d = nλ/(2 sin θ), where λ is the Xray wavelength, θ is half of the diffraction angle, and n is 1. The physical−mechanical property of collagen fibers was examined by using the standard IULTCS methods. The mechanical properties of leather were analyzed using an electronic universal testing machine (UTM2102, Shenzhen Suns Technology Stock Co., Ltd.). The strain rate was 50 mm/min, and each test was carried out on five samples (1 cm × 10 cm) to obtain a mean value. We marked the thickness of the leather as d1 (mm) before fatliquoring and d2 (mm) after fatliquoring. The thickening rate (Tp) can be calculated according to the following equation: Tp =

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d 2 − d1 d1

Table 1. Molecular Weights and Polydispersity Indices of Polymers sample

Mn

PDI

PAMAMG0 PAMAMG0-3 PAMAMG0-3-P PAMAMG1 PAMAMG1-3 PAMAMG1-3-P PAMAMG2 PAMAMG2-3 PAMAMG2-3-P

430 760 860 1050 1490 1990 2280 2880 3440

1.05 1.01 1.02 1.06 1.05 1.37 1.45 1.13 1.42

new absorbance corresponding to the OCO stretching appears at 1738 cm−1, indicating the formation of an ester bond. By comparison with the spectra of PAMAMG1-3 and PAMAMG1-3-P in Figure S2, it can be seen that the absorbance of hydroxyl groups at 3412 cm−1 reduces dramatically after the esterification reaction. Meanwhile, three new absorbance peaks appear at 1268, 1178, and 986 cm−1, which are attributed to the asymmetrical and symmetrical stretchings of PO and P OC, respectively. The 1H NMR and 13C NMR spectra of PAMAMG1 and PAMAMG1-3-P are exhibited in Figure 3. By comparison with

(3)

RESULTS AND DISCUSSION Structure of Linear-Polyamidoamine Phosphate Ester Dendrimers (PAMAMGn-3-Ps). The resulting PAMAMGn-3s and PAMAMGn-3-Ps were characterized by 1H NMR, 13C NMR, GPC, and FTIR. By comparison with the FTIR spectra of PAMAMG1 and PAMAMG1-3 in Figure S2, it was found that the absorbance of hydroxyl groups at 3286 cm−1 reduces dramatically after the esterification reaction. Simultaneously, a D


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Figure 4. Relationship of the intensity ratio (I3/I1) and the concentration of PAMAMGn-3 (a) and PAMAMGn-3-P (b) solutions by steady-state fluorescence measurement.

Table 2. Properties of the PAMAMGn, PAMAMGn-3, and PAMAMGn-3-P Aggregates

a

sample

CMCa (mg × 10−2 /mL)

CMCb (mg × 10−2/mL)

diameter (nm)

PDI

PAMAMG0 PAMAMG1 PAMAMG2 PAMAMG0-3 PAMAMG1-3 PAMAMG2-3 PAMAMG0-3-P PAMAMG1-3-P PAMAMG2-3-P

   3.30 1.52 6.56 3.01 3.50 3.87

   3.40 1.48 6.45 3.20 3.55 3.24

54.9 67.7 172.3 98.5 161.6 289.2 111.7 176.0 348.9

0.285 0.327 0.362 0.395 0.583 0.614 0.425 0.632 0.880

CMC value determined by steady-state fluorescence measurement. bCMC value determined by surface tension measurement.

which corresponds to the tertiary amine (−NH−CH2−CH2− O−) of the phosphorus in terminal protons of PAMAMG1-3-P. The chemical structure of PAMAMG1-3-P was also identified by the 13C NMR spectra. In the 13C NMR spectra of PAMAMG13-P shown in Figure 3b, the characteristic peak of the −C( O)O− group appears at 164.60 ppm, and the signals of methyl and methane are located at 20.40−33.70 ppm, which

the 1H NMR spectra of PAMAMG1 and PAMAMG1-3-P shown in Figure 3a, some new signals appear at 0.64, 0.92, and 1.02− 1.15 ppm in the 1H NMR spectra of PAMAMG1-3-P. These new signals could be assigned to the protons of methyl PAMAM−OOC(CH2)10CH3, the methylene (PAMAM− OOC(CH2)10CH3, and the PAMAM−OOCCH2(CH2)9CH3 respectively. Meanwhile, a new signal is observed at 3.89 ppm, E


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g/mol with PDI values of 1.02, 1.37, and 1.42 by GPC using linear polystyrene as calibration, respectively. These molecular weights are smaller than the real molecular weights of PAMAMGn-3-Ps because dendritic polymers are normally smaller in size than linear polymers with the same molecular weights and are hardly expanded in solution. Characterization of PAMAMGn-3s and PAMAMGn-3-Ps. In general, an amphiphilic polymer can form a core/shell structure in water, and so do PAMAMGn-3s and PAMAMGn-3Ps.13,14 In the selective solvent of water, PAMAMGn-3-Ps molecules spontaneously self-assemble into nanomicelles driven by the strong hydrophobic/hydrophilic interaction among alkyl tail arms and hyperbranched phosphate ester cores. The inner cores of micelles consist of the hydrophobic linear arms and the shells of micelles composed of the hydrophilic hyperbranched phosphate esters.15,16 Here, we evaluated the CMCs of amphiphilic PAMAMGn-3s and PAMAMGn-3-Ps in water using the pyrene probe fluorescence spectrometry.17,18 I1 (373.0 nm) and I3 (384.0 nm) are the emission intensities of the first and third bands in the fluorescence spectrum of pyrene, respectively. The emission intensity ratio of I3/I1 is very sensitive to the polarity of the medium surrounding pyrene molecules. The smaller the ratio is, the greater the polarity of the medium. The relationships of I3/I1 and the concentration of amphiphilic PAMAMGn-3s and PAMAMGn-3-Ps with different numbers of generations are shown in Figure 4. At low concentrations of PAMAMGn-3s and PAMAMGn-3-Ps, the intensity ratios of I3/I1 remain nearly unchanged, indicating the characteristic of pyrene in a water environment. As the concentrations of PAMAMGn-3s and PAMAMGn-3-Ps increased, the intensity ratio of I3/I1 started to increase dramatically and reached the characteristic level of pyrene in completely hydrophobic environment at a certain concentration of PAMAMGn-3s and PAMAMGn-3-Ps. The phenomenon demonstrates the whole formation of micellization in

Figure 5. Representative TEM microscopic image of PAMAMGn-3s (a) and PAMAMGn-3-Ps (b) micelles.

demonstrates that the lauroyl chloride has been grafted to PAMAMG1. After the terminal hydroxyl groups of PAMAMG1-3 are capped by the phosphate, the signal at 60.00 ppm of PAMAMG1 moved to 64.71 and 69.28 ppm of PAMAMG1-3-P. The electronegativities of oxygen and phosphorus are stronger than that of hydrogen, so the chemical shift of methylene shifts to low field. Furthermore, the signal of PAMAMG1 (−NH− CH2−CH2−O−) at 40.17 ppm shifted to 35.53 ppm in the 13C NMR spectrum of PAMAMG1-3-P. In brief, the above NMR and FTIR results clearly demonstrate that PAMAMG1-3-P was synthesized successfully. The molecular weights and polydispersity indices of PAMAMGns, PAMAMGn-3s, and PAMAMGn-3-Ps are summarized in Table 1, and the GPC curves are shown in Figure S3. As seen in Table 1, a clear shift toward higher molecular weights was observed by comparing the molecular weight of PAMAMGn-3-Ps with those of the intermediate PAMAMGn-3s and the precursor PAMAMGn. The results indicated the corresponding grafting and coupling reactions were successful. The number-averaged molecular weights of PAMAMG0-3-P, PAMAMG1-3-P, and PAMAMG2-3-P were 860, 1990, and 3440

Figure 6. E-SEM microphotographs of collagen fibers treated with different chemicals: (a) without treatment; (b) treated with PAMAMG1-3; (c) treated with PAMAMG1-3-P; (a′, b′, c′) images for panels a, b, and c with a 5000× higher magnification. F


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Figure 7. XPS survey spectra: (a) collagen fibers without treatment; (b) collagen fibers treated with PAMAMG1-3; (c) collagen fibers treated with PAMAMG1-3-P.

generations are shown in Figure S4. The CMCs were also obtained by surface tension measurement and are summarized in Table 2. Obviously, the results obtained by surface tension measurement (CMCb) and steady-state fluorescence measurement (CMCa) reached a good agreement. DLS measurements

solution. From the shape of the curves, the CMCs of the amphiphilic PAMAMGn-3s and PAMAMGn-3-Ps are obtained and summarized in Table 2. In addition, the relationships of the surface tensions and the concentrations of amphiphilic PAMAMGn-3s and PAMAMGn-3-Ps with different numbers of G


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Figure 7 shows that the XPS spectra of the collagen fibers contain only signals for the elements of C, N, O, Cr, and P. After the introduction of PAMAMG1-3-P, the bands at 135 and 136 eV can be observed, which are attributed to asymmetric dual peaks of P 2p3/2 and 2p1/2 orbit, respectively. Electrons from the O (KLL) Auger energy level are also evidenced in each spectrum, but the amplitudes of the corresponding peaks are low. The detailed atomic fractions for C, N, O, Cr, and P in various samples are given in Table 3. The results reveal that PAMAMG1 and PAMAMG1-3-P have entered the fibers, and the formations of the clusters are responsible for the cross-linking between fiber bundles. This may be the reason that PAMAMG13 and PAMAMG1-3-P with multiterminal bases can be crosslinked with collagen fiber side chains. Figure 8 shows that the high-resolution XPS spectra of C 1s treated with no polymer, PAMAMG1, and PAMAMG1-3-P have similar XPS C 1s core level spectra. According to the NIST Xray Photoelectron Spectroscopy Database, each curve of the C 1s peaks is fitted with four peaks.21 The peaks are attributed to carbon atom bound to hydrogen (CH), carbon (CC), carbon singly bound to nitrogen and oxygen (CN and C O), and carbon involved in an amide bond (OC), respectively. The binding energies and related bonding type percentage at various chemical states are listed in Table 4. The intensity of the component located at around 288.0 eV originating from the peptide bond does not change the primary structure when PAMAMG1-3 and PAMAMG1-3-P were added to leather. Meanwhile, the positions of the peaks are similar when phosphorus was introduced. The reason is that the phosphorus and hydrogen possess nearly the same electronegativity, so the binding energy does not change significantly. Furthermore, compared to the untreated leather, the peak area of CO/CN increases, but the percentages of CC and CO decline. This illustrated that the cross-linking and chemical process occurred with the breaking of peptide chains and the grafting of extra oxygen-containing groups into the side chains of the collagen molecules. The figure of XRD shows the linear intensity profiles of the collagen fibers versus diffraction angle. The collagen fibers are a kind of natural polymer material with multiple layer structure, which show the periodic change of the structure. Figure 9 shows the XRD intensity profiles of the collagen fiber versus diffraction angle treated with PAMAMG1-3 and PAMAMG1-3-P. From the diffraction peak, the lateral spacing between collagen molecules is obtained by Bragg’s equation (Table 5). Peak 1 in real lattice space represents the characteristic intermolecular lateral distance within collagen fibers.21 Peak 2 stands for diffuse reflection caused by much internal structure of collagen fibers. The position of Bragg reflection approximately at peak 3 relates to the axial rise distance between the amino acid residues along collagen triple helices or helical rise per residue.22 Peaks 4 and 5 represent the N telopeptide and the C telopeptide, respectively. We can see peaks 4 and 5 diffraction peaks partially overlap each other because of close position and similar intensity from Figure 9. When PAMAMG13 and PAMAMG1-3-P were added to leather, diffraction peak 1 became obvious, suggesting that the collagen molecules after the addition of polymers maintained a regular and orderly manner. Meanwhile, by comparison with the blank sample, the lateral spacing of the fibers increased after the addition of polymer. By comparison of Tables 2 and 5, it is obvious that the particle sizes of PAMAMG1-3 and PAMAMG1-3-P are 161.6 and 176.0 nm, but the molecules within microfibrils are purely close

Table 3. Elemental Composition (atm %) Obtained by XPS from Collagen Fibers Treated with PAMAMG1-3 and PAMAMG1-3-P sample

C (1s)

N (1s)

O (1s)

Cr (2p)

P (2p)

blank PAMAMG1-3 PAMAMG1-3-P

71.35 72.81 69.01

6.97 7.77 6.45

21.24 19.16 22.69

0.44 0.25 0.41

0 0 1.44

also indicate that all of PAMAMGn-3s and PAMAMGn-3-Ps form micelles with diameters from 54.9 to 384.9 nm, and the detailed data are summarized in Table 2. Apparently, the average diameter of the micelles increases with the numbers of generation. Additionally, the morphologies of micelles were studied by TEM measurement. The representative images in Figure 5 show that PAMAMGn-3-Ps self-assemble into approximately spherical micelles in water. Effect on Collagen Fibers of PAMAMG1-3-P. The collagen fibers have a macroscopically disordered network structure in nature. Identification of the animal source of leather is generally performed by microscopic observation of characteristic morphology.19 Figure 6 shows the morphologies of the collagen fibers after leather drying with the nofatliquoring, PAMAMG1-3, and PAMAMG1-3-P, respectively, under the same process conditions. Macroscopically, it is obvious that the collagen fiber distance increased after the introduction of PAMAMG1-3 and PAMAMG1-3-P, but the collagen texture does not change. Meanwhile, it seems that the PAMAMG1-3-P leads to greater disorder of collagen fiber aggregation. When the PAMAMG1-3 and PAMAMG1-3-P filled in collagen fibers, two different forces became strengthened. One of the forces is perpendicular to the capillary pressure difference, and the other is parallel to the capillary flow adhesion force. The adhesion force of capillary flow tends to decrease the order of the aggregation of the collagen fibers. The result shows that the flow of capillary flow in the capillary is the precondition of determining the interaction between PAMAMG1-3 or PAMAMG1-3-P and collagen fibers. With the addition of polymers in collagen fibers, the conformations of the peptide chains were likely to change, and the vibration frequencies of the chemical bonds would change as well. Figure S5 of the Supporting Information is the ATR-IR spectra of leather treated with PAMAMG1-3 or PAMAMG1-3-P or untreated natural leather. In Figure S5, for the untreated sample, the sharp absorption peak located at 1638 cm−1 is associated with the CO amide of natural leather in the peptide band (I). The peak at 1551 cm−1 represents the flexural vibration of N−H amide (II) and stretching vibration of C−N amide (II). The peaks located at 1340 cm−1 correspond to CN amide (III).20 Additionally, the absorption band at 3305 cm−1 is due to −OH stretching vibration. The bands at 2933 and 1450 cm−1 provide proof of −CH2− and −CH3 stretching vibrations. As for the samples treated with PAMAM G1 -3 and PAMAMG1-3-P, the functional groups were the same compared with the untreated one, except for the bands at 1731 and 1165 cm−1 and the band at 1033 cm−1. The former suggests the O CO vibration and the latter is associated with the PO alkyl vibration. The similarities between untreated natural leather and the leather treated with polymer in the ATR-IR spectra are caused by the micrometer-scale penetration depth of ATR-IR. H


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Figure 8. XPS C 1s core level spectra of collagen fibers: (a) collagen fibers without treatment; (b) collagen fibers treated with PAMAMG1-3; (c) collagen fibers treated with PAMAMG1-3-P.

introduced. This is because the internal irregular part of the collagen fibers generated more diffraction peaks after the addition of the polymer. Apart from peaks 1 and 2, the distance of peak 3 became smaller after the addition of PAMAMG1-3 and PAMAMG1-3-P, and the reason may be the hydroxyl remnant

to 1.0 nm. The result indicates that PAMAMG1-3 and PAMAMG1-3-P could not enter into the microfibrils and could only distribute within the collagen fibers bundles. Besides, peak 2, corresponding to the amorphous region, becomes stronger after PAMAMG1-3 and PAMAMG1-3-P are I


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Table 4. Binding Energies and Atomic Fractions from the XPS C 1s Core Level Spectra for C Element in the Collagen Fibers Treated with PAMAMG1-3 and PAMAMG1-3-P CH

CC

CO/CN

CO

sample

binding energy (eV)

atomic fractions (%)

binding energy (eV)


binding energy (eV)


binding energy (eV)



282.93 283.18 282.98

19.06 23.74 19.22

284.60 284.60 284.60

48.65 38.97 43.64

286.25 286.01 286.03

26.67 34.33 33.57

288.53 288.39 288.52

5.61 2.97 3.57

Figure 9. X-ray diffraction diagram of collagen fiber intensity: (a) collagen fibers without treatment; (b) collagen fibers treated with PAMAMG1-3; (c) collagen fibers treated with PAMAMG1-3-P.

Table 5. XRD Results for Collagen Fibers Treated with PAMAMG1-3 and PAMAMG1-3-P sample

intermolecular lateral packing (nm)

amorphous region (nm)

helical rise per residue (nm)

N telopeptide (nm)

C telopeptide (nm)


1.185 1.283 1.257

0.421 0.426 0.433

0.295 0.284 0.283

0.225 0.225 0.221

0.209 0.209 0.206

of polymer cross-link with side-chain carboxyls of aspartic and glutamic acids at a single-helix chain, leading to structural distortion of collagen molecules. The analysis elucidates that the polymer can alter the diffraction signal from the collagen helix but does not destroy collagen fibers. Appropriate mechanical responses are required for any material to be considered for a range of applications, specifically where mechanical stability is an essential prerequisite.23 Tensile strength and tear strength are important physical properties to be considered in the manufacturing applications of leather.24 Collagen fibrils have little strength in either flexion or torsion, but they exhibit high tensile strength and tear strength. Those properties are largely attributable to the presence of the crosslinks between polymers and collagen fibers. The physical and mechanical properties of the collagen fibers are presented in Figure 10, including tensile and tear properties. The physical and mechanical properties were analyzed by the main Chinese standard requirements and ISO standard for garment leather (QB 1872-1993, ISO/FDIS 14931:2003). From the two kinds of different directions of horizontal and vertical, the stresses of the collagen fibers are different. With stretching, the leather become progressively tauter, and the slope of the stress strain curve increases. In Figure 10a,b, when the collagen fibers are too highly aligned like untreated leather,

the strain becomes strong but the stress becomes poor. Meanwhile, the strains of collagen fibers that were treated with PAMAMG1-3-P reduce, and the stress becomes better than that of untreated leather. The results demonstrate that the aggregation phenomenon of collagen fibers decreases and the leather has slightly better performance of tensile strength. This is because the PAMAMG1-3-P can promote more movement between collagen fibers and augment the distance of collagen fibers. Tear strength means the maximum load the sample carries when a gap appears, that is, a deformation of axial tension. The tear strengths of the three samples were higher than the occupation standard of garment leather (≥20 N/mm),25 indicating PAMAMG1-3-P does not destroy the structure of collagen fibers. The physical and mechanical properties of leather fatliquored by PAMAMG1-3-P are improved compared with those of leather treated with PAMAMG1-3 and untreated leather. This is because the permeation of PAMAMG1-3-P into the collagen fibers is anticipated to assist in strong cross-linking with the collagen fibers matrix, paving the way for the formation of a network structure. The mechanical properties of the leather are closely related to the distribution of PAMAMG1-3-P in leather. J


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Figure 10. Physical and mechanical properties of collagen fibers from three samples: (a) stress (transverse) versus strain curves of three samples without treatment (a, blank; b, treated with PAMAMG1-3; c, treated with PAMAMG1-3-P); (b) stress (vertical) versus strain curves of the three samples; (c) tear strength of three samples of the transverse and vertical direction.

PAMAMG1-3 and PAMAMG1-3-P, the thickening rate of leather treated with PAMAMG1-3-P increased obviously. PAMAMG1-3P is used as reinforcement filler in the leather and could penetrate into and fill between collagen fibers. The result shows

In addition to physical and mechanical properties, thickening rate is an index to characterize the fullness of leather. The results of thickening rate of leather are shown in Figure 11. Compared with the thickening rate of leather treated with K


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Figure 11. Thickening rate of leather before and after fatliquoring: (a) collagen fibers without treatment; (b) collagen fibers treated with PAMAMG13; (c) collagen fibers treated with PAMAMG1-3-P.

Figure 12. Schematic showing the effect between collagen fibers and linear-hyperbranched amphiphilic phosphate esters.

ecological leather and have favorable implications in collagen fibers.

that the fatliquoring agent with added PAMAMG1-3-P can improve the filling property of leather. The combination of the above studies clearly indicates that hyperbranched phosphate esters cluster deposit among collagen fibers. The binding of hyperbranched phosphate esters to collagen fibers leads to the decrease in the structural order, but it does not destroy the triple helices of collagen. Meanwhile, the hyperbranched phosphate esters could only distribute within the collagen fiber bundles, but could not reach the intermolecular regions within microfibrils. Furthermore, the hyperbranched phosphate esters could suitably cross-link with collagen fibers to enhance the mechanical properties and flexibility of leather. The effect between collagen fibers and linear-hyperbranched amphiphilic phosphate esters was generally illustrated in consideration of the data in this work, as shown in Figure 12. Conclusion. In summary, we synthesized a series of linearhyperbranched phosphate esters (PAMAMGn-3-Ps), which have low CMCs, strong surface activities, and small particle sizes. PAMAMG1-3-P can cross-link with the collagen fibers without destroying their triple-helix conformation and make the collagen fibers separate from each other, so the leather gets softer and thicker after the introduction of hyperbranched phosphate esters. In addition, PAMAMG1-3-P can greatly improve the tensile and tear strengths of leather. Therefore, the amphiphilic hyperbranched phosphate esters are suitable for

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04482. Tables S1 and S2; Figures S1−S5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(X.W.) E-mail: [email protected]. Phone: 0086 + 15802958501. ORCID

Xiaoxiao Guo: 0000-0001-8039-6647 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

National Natural Science Foundations of China (No. 21276151); Key Scientific Research Group of Shaanxi province (No. 2013KCT-08); Scientific Research Group of Shaanxi University of Science and Technology (No. TD12-04). Notes

The authors declare no competing financial interest. L


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Effect of Linear-Hyperbranched Amphiphilic Phosphate Esters on

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