Article pubs.acs.org/Biomac
Bioadhesive Nanoaggregates Based on Polyaspartamide‑g‑C18/ DOPA for Wound Healing Sooyoun Lim,# Minh Phuong Nguyen,# Youngjin Choi, Jaeyun Kim, and Dukjoon Kim* School of Chemical Engineering, Sungkyunkwan University, Suwon, Kyunggi 16419, Republic of Korea S Supporting Information *
ABSTRACT: Biocompatible adhesive nanoaggregates were synthesized based on polyaspartamide copolymers grafted with octadecylamine (C18) and 3,4-dihydroxyphenylalanine (DOPA), and their adhesive properties were investigated with regard to wound healing. The chemical structure and morphology of the synthesized polyaspartamide-g-C18/DOPA nanoaggregates were analyzed using 1 H-nuclear magnetic resonance spectroscopy (1H NMR), dynamic light scattering (DLS), and transmission electron microscope (TEM). The in vitro adhesive energy was up to 31.04 J m−2 for poly(dimethylacrylamide) gel substrates and 0.1209 MPa for mouse skin, and the in vivo wound breaking strength after 48 h was 1.8291 MPa for C57BL/6 mouse. The MTT assay demonstrated that the synthesized polymeric nanoaggregates were nontoxic. The polyaspartamide-g-C18/DOPA nanoaggregates were in vivo tested to mouse model and demonstrated successful skin adhesion, as the mouse skin was perfectly cured in their dermis within 6 d. As this material has biocompatibility and enough adhesive strength for wound closure, it is expected to be applied as a new type of bioadhesive agent in the human body.
1. INTRODUCTION
Polypeptide is a biopolymer that is biodegradable in the human body. Polysuccinimide (PSI) is a precursor of polyaspartamide synthesized from aspartic acid. As PSI has a penta-ring structure that opens easily in the presence of an amine group (−NH2), the grafting of hydrophobic and hydrophilic pendants is feasible. The resulting ring-opened product, polyaspartamide, is thus a biodegradable, biocompatible, and nontoxic polypeptide polymer. By grafting both hydrophobic and hydrophilic groups on PSI, polyaspartamide may have an amphiphilic nature and thus can be applied as biocompatible polymeric nanoparticles for drug delivery in the human body. Researchers have used dopamine in a hyperbranched polymer system, which was inspired by a mussel adhesive protein containing a catechol group such as DOPA.11,21,22 The hydroxyl group of DOPA plays an important role in adhesion by forming hydrogen bonding with hydrophilic surfaces.23−29 The aim of this study is to create a new type of biocompatible polymeric nanoaggregate. PSI was used as the polymer backbone and was grafted with hydrophobic C18 and hydrophilic DOPA with the ability to bind with skin surface. In a diluted aqueous solution state, C18 and DOPA will be directed inside and outside the micellar (nanoaggregate) structure with adhesive properties, and thus can be used as potential adhesive material in the human body. As several types
Wound healing along with bleeding control is a very important aspect of human health.1−3 In the past, stitches4 were frequently used to suture the injured tissue, because of their simplicity in application for wound closure. As staples5 are quicker and easier to apply than stitches, they have been reported to lessen the wound infection rate more effectively. Even though each method has unique advantages, both methods occasionally lead to tissue trauma and scarring (Figure 1)6−10 and thus the materials should be removed after application.11,12 Polymeric adhesives13 can also be used for wound closure. There are a variety of polymeric adhesives with diverse properties. For example, the fibrin14 generated inside the human body has very good biocompatible properties, but it has relatively weak adhesive characteristics. Although cyanoacrylate15 has strong adhesive strength and can be artificially synthesized, it often shows toxicity in the human body.16 Other types of polymeric adhesives have been used in the human body; however, those usually require activation by heat, light, pH, or chemical reaction.11,17,18 Recently, an aqueous solution of spherical metal oxide nanoparticles has been applied as an adhesive material, with the nanoparticles acting as anchors. As the particle surface has adsorption characteristic of biotissue, this method is very simple and fast for reduction of scarring. However, as inorganic nanoparticles are generally not biodegradable, they remain in the body a long time.19,20 © XXXX American Chemical Society
Received: April 24, 2017 Revised: June 19, 2017
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DOI: 10.1021/acs.biomac.7b00584 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
trazolium bromide (MTT) was purchased from Alfa Aesar (Ward Hill, MA, USA). 2.2. Synthesis of Polyaspartamide-g-C18/DOPA. PSI was synthesized via the condensation of L-aspartic acid using o-phosphoric acid as an acid catalyst. L-Aspartic acid was added to the solvent (weight ratio of mesitylene/sulfolane = 7:3) in the presence of ophosphoric acid. The suspension was stirred at 175 °C under N2 atmosphere. The byproduct water was removed using a Dean−Stark trap with a reflux condenser. After 8 h, the reaction product was precipitated with excess methanol and washed several times with water until it was neutralized. The PSI product was dried in a vacuum oven at 70 °C for at least 1 d.31,32 After PSI was synthesized, C18 and DOPA were grafted onto PSI. C18 was added to the PSI/DMF solution at room temperature. The mixture was stirred continuously under N2 atmosphere at 70 °C for 24 h, and DOPA/TEA was then added to the polymer solution. After 24 h, the reaction mixture was precipitated with excess ether and washed several times with isopropanol. The polyaspartamide-g-C18/DOPA product was dried in a vacuum oven.33,34 The scheme of this reaction is shown in Figure 2. 2.3. Characterization. 2.3.1. Molecular Weight. The viscosity average molecular weight was measured using an Ubbelohde viscometer. We prepared polymer solution in DMF at the concentration of 0.5 g dL−1. The viscosity average molecular weight was calculated using eq 1, provided by Neri et al.31
Figure 1. Concept of wound closure system. Suture type: This method produces more scarring. Polymeric nanoparticle type: A droplet of polymer particle solution is spread with a brush. The wound edges come into more complete contact than with the suture method. The particle surface adsorbs onto tissue, and works like an anchor. The possible mechanism is shown in the top of the figure.
n = 3.52 × ηr1.56
(1)
where n and ηr indicate the degree of polymerization and reduced viscosity of the polymer solution. 2.3.2. Chemical Structure. The 1H NMR spectra were recorded on a Fourier transform nuclear magnetic resonance spectroscopy (FTNMR, 500 Hz, Unity Inova, USA). For 1H NMR experiments, the samples were prepared by dissolving polymer in DMSO-d6. The FT-IR spectra were obtained using Fourier transform infrared spectroscopy (FT-IR, Bruker IFS-66/S, Bruker, Germany) with attenuated total reflectance (ATR) accessory. 2.3.3. Particle Size and Shape. The micellar structure of polyaspartamide-g-C18/DOPA was formed in water. The dynamic light scattering (DLS, ELS-Z, OTSUKA, Japan) was employed to measure the size distribution and mean size of polymer nanoaggregates in water. The transmission electron microscopy (TEM, HR-TEM, JEM-2100F, JEOL, Japan) was employed to analyze the morphology, size, and shape of the prepared polymeric aggregates. The polymer solution at 0.1 wt % was dropped on a carbon-coated copper grid. After a few minutes, tungsten solution was added dropwise for staining and then the grid was dried in an oven for measurement. 2.4. Adhesive Strength Measurement. Poly(dimethyl acrylamide) (PDMA) hydrogel substrates were synthesized from DMA in the presence of MBA cross-linker and KPS and TEMED initiators. 1.485 g of DMA, 2.3 g of MBA, and 22.5 μL TEMED were first dissolved in 10.62 g of deionized water. After the solution was stirred at room temperature under nitrogen gas, 41 mg of KPS was subsequently added.35 The resulting solution was poured in the MiniPROTEAN Tetra cell casting module mold to control the substrate thickness of 1.5 mm. To remove unreactive compounds, the PDMA product was washed with DI water and then dried in a vacuum oven.
of polymeric nanoaggregates were successfully synthesized from PSI and applied as the controlled drug release carriers in our previous studies,30,32,33 the bioadhesive nanoaggregates developed in this study are expected to have a significant impact on wound healing efficiency contributed not only from controllable biodegradable and bioadhesive strength but also from controllable drug releasing ability. Figure 1 shows the conceptual scheme of the adhesion behavior of polymeric nanoaggregates. PSI, a polypeptide, was used as the backbone of the polymer and C18 and DOPA were grafted onto PSI to control the hydrophobic/hydrophilic balance and to provide the adhesive property.31,32
2. MATERIALS AND METHODS 2.1. Materials. L-Aspartic acid, o-phosphoric acid, mesitylene, sulfolane, N,N-dimethylformamide (DMF), 3,4-dihydroxyphenethylamine hydrochloride (DOPA-HCl), triethylamine (TEA), octadecylamine (C18), N,N-dimethylacrylamide (DMA), N,N′-methylene bis(acrylamide) (MBA), N,N,N′,N′-tetramethylethylenediamine (TEMED), Dulbecco’s modified eagle’s medium (DMEM), and amoxicillin were purchased from Sigma-Aldrich (Missouri, USA). Dimethyl sulfoxide-d6 (DMSO-d6) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Potassium persulfate (KPS) and dimethyl sulfoxide (DMSO) were purchased from Samchun (Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-
Figure 2. Synthetic scheme of polyaspartamide-g-C18/DOPA. B
DOI: 10.1021/acs.biomac.7b00584 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
corresponding molecular weight was 60 000 g mol−1 calculated from eq 1. Figure 4 shows the FI-IR spectra of PSI and polyaspartamideg-C18/DOPA. The presence of an imide ring in PSI was
The lap-shear test was conducted using UTM (QC-508E, COMETECH, Taiwan) to determine the adhesive strength. After the prepared PDMA substrate was cut into 5 cm × 5 mm × 1.5 mm dimension, it was exposed to atmosphere for at least 1 d before the test to have its surface equilibrated with the humidity in the atmosphere. Different amounts of polymer solution up to 50 μL were spread on one side of the PDMA gel and it was covered by the other gel as shown in Figure 3.
Figure 3. Lap-shear test sampling design.
A UTM with a 50 N load cell was used at the pulling rate of 150 mm min−1. The adhesion energy was calculated using eq 2, provided by Johner et al.35,36
Gadh = 3(F /w)2 /(2Eh)
(2) Figure 4. FT-IR spectra of PSI and polyaspartamide-g-C18/DOPA.
where w and h are the width and thickness of the gel. E is young’s modulus of the gel, and F is the applied force on the gel. The skin of a C57BL/6 mouse supplied from Hanlim experimental animal laboratory (Seoul, Korea) was used as the substrate for adhesive strength measurement.25,27 The pieces of skin were cut into dimension of 3 cm × 1 cm × 0.86 mm. Two skin pieces were subsequently stuck together by the polymer solution of 5 wt % (10 μL) with the contact area of 1 cm × 1 cm. The samples were preserved in phosphate-buffered saline (PBS) for 2 h before measuring adhesive strength. The tensile test was measured using a UTM (UTM model 5565, LIoyd, Fareham, UK) under a load of 250 N. 2.5. Cytotoxicity Test. Microtitration (MTT) viability assay was used to test the viability of 293T cells under different polymer concentrations. 96 well microplates containing culture medium were used to grow cells. Each of the microplate wells contained around 1 × 104 cells. After 24 h incubation, the polymer solution at different concentrations was introduced in wells and incubated for 24 h. Subsequently, 20 μL MTT solution (5 mg mL−1) was added to each well. The culture medium was taken away after 4 h incubation. 200 μL of DMSO was applied to each well to dissolve Formazan crystals for further incubation for 20 min at 37 °C. After that, the UV−vis absorption measurements were implemented for all well microplates to detect UV absorption intensity at 570 nm wavelength.33,37,38 2.6. In Vivo Wound Healing Test. The Hematoxylin and Eosin (H&E) staining method was used to test the wound healing effect of polymer nanoaggregates. Both C57BL/6 mouse and Sprague−Dawley (SD) rats (Hanlim experimental animal laboratory, Seoul, Korea) were used in this experiment. First, open wounds were created on the skin of the 6 mice and 6 rats. The length of the wound was 1 cm and the laceration was through the skin until the subcutaneous tissue. After that, mice and rats were divided into two groups (3 mice or rats/ group) each. While the wounds of the first mouse or rat group were sutured, those of the second one were treated with the polymer dispersed solution. After the polymer solution was sterile, it was spread on the wounds of mice and rats using a brush. After 6 d, the wounds were cut out and observed by the H&E staining method. In the other experiment, in order to measure the wound breaking strength,25 we created open wounds on the C57BL/6 mouse and the polymer solution was applied on the wounds. After 48 h, the skin was cut in dimensions of 5 cm × 5 mm × 0.86 mm and the sections of skin were soaked in phosphate-buffered saline (PBS) for preservation. The wound breaking strength was measured using a UTM (UTM model 5565, LIoyd, Fareham, UK) under a load of 250 N.
apparent at 1727, 1393, 1217, and 1163 cm−1. The IR bands appearing at 1649, 1535, and 1460 cm−1 are attributed to the amide group, benzene ring in DOPA, and the −CH2− chain in the C18 group, respectively. Appearance of those IR bands confirmed that DOPA and C18 were well grafted onto the PSI backbone. Figure 5 shows the 1 H NMR spectra of PSI and polyaspartamide-g-C18/DOPA. The methane and methylene protons in PSI are displayed at 5.4−5.1, 3.3−3.1, and 2.8−2.6 ppm. The characteristic proton peaks from C18 and DOPA grafted groups are apparent in 1H NMR spectra, confirming that the grafting reaction was successful. The intensity of the proton peaks was used to calculate the degree of substitution of C18 and DOPA, and the results are shown in Table 1. 3.2. Particle Size and Shape. Polymeric nanoaggregates were formed in the aqueous phase. The average size of polymeric aggregates was measured via DLS and TEM. As shown in Figure 6 (TEM images), the solution contains the spherical polymer aggregate grains. Based on DLS results in Table S1, the average size of the C20D50 sample was larger than that of C20D100 sample at the same concentration. In addition, the amount of C20D100 aggregates was more than that of C20D50. The aggregate size was the largest at 5 wt % and the smallest at 0.1 wt %. This indicates that the size of polymer aggregates is significantly affected by the concentration of polymer solution. 3.3. Adhesion Behavior. Adhesion energy was measured using UTM. The adhesive properties of the particles originated from a specific functional group in spherical particles, DOPA, which interacts with PDMA molecules. In order to determine the optimal amount of polymer solution for the highest adhesive strength with PDMA gel in 5 mm × 10 mm dimension (contact area), different amounts of polymer solution were applied dropwise to the hydrogel surface. As shown in Figure 7A, the adhesion energy increased from 13.25 ± 4.42 to 31.04 ± 6.8 J m−2 with increasing polymer solution content in the region below 10 μL, because the adhesive strength increases with the number of particles bound to the PDMA gel surface. In the adhesive content ranging from 10 to 20 μL, the adhesive strength, however, decreased with increasing polymer solution content. In this large range of
3. RESULTS AND DISCUSSION 3.1. Chemical Structure. The condensation polymerization method was used to synthesize PSI from L-aspartic acid. The reduced viscosity of the synthesized PSI was 27, and the C
DOI: 10.1021/acs.biomac.7b00584 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Figure 5. 1H NMR spectra of (A) PSI and (B) Polyaspartamide-g-C18/DOPA.
The optimal concentration of the polymer solution is another important factor with regard to adhesion strength. Different concentrations of 10 μL polymer solution were dropped onto the hydrogel surface and the adhesive energy was measured. The results are shown in Figure 7C,D for C20D50 and C20D100 systems, respectively. As the polymer solution concentration decreased, the number of particles adsorbed on the PDMA gel surface was reduced (Table S1) and thus the adhesion energy was reduced. The effect of polymer concentration on adhesive energy was the most clearly observed for the C20D100 sample in the concentration ranging between 2.5 and 5 wt %. When its concentration decreased from 5 to 2.5 wt %, the size of the particle decreased (Table 2), but the number of particles remained almost the same (Table S1).35 This suggested that larger particle size is more favorable for adhesion because of the mechanical strength effect rather than the surface area effect in this concentration range. In the relatively lower concentration range between 0.5 and 2.5 wt %, however, the number of nanoaggregates seems to have a more significant effect on adhesion energy. Even though polyaspartamide-g-EA (PHEA) possesses many hydroxy (−OH) groups on its backbone that may interact with PDMA via hydrogen bonding, its adhesive strength was not as high as that of other DOPA-grafted samples including C10D10, C20D50, and C20D100, and even Ludox TM-50. The mouse skins were used as the substrate for lap-shear testing. As shown in Figure 8, the adhesive strengths of C20D100, C20D50, and C20D10 samples are 0.1209 ± 0.0006, 0.073 ± 0.008, and 0.013 ± 0.003 MPa, respectively. The
Table 1. Molar Feed (MF) Ratio and Corresponding Substitution Degree (SD) of C18 and DOPA in Polyaspartamide-g-C18/DOPA sample namea
MF ratio of C18
SD of C18
MF ratio of DOPA
SD of DOPA
C20D50 C20D100
0.2/1.0 0.2/1.0
16 16
0.5/1.0 1.0/1.0
25 37
a CxDy; x means feed % ratio of C18 and y means feed % ratio of DOPA.
amount polymer solution, the free particles not involved in chemical bonding with the PDMA substrate disturb the adsorption. The polymer solution was even overflowing outside the substrate surface beyond its amount of 25 μL. Thus, in this experimental range, 10 μL was the optimal amount of polymer solution for the best adhesion. The adhesive strengths of C20D100, C20D50, and C10D10 samples were compared with those of a couple of reference adhesive samples such as Ludox TM-50,35 a typical inorganic nanoparticle based adhesive, and polyaspartamide-g-ethanol amine (PHEA), a typical polymer based adhesive. Ludox TM50 dispersed aqueous solution at 50 wt % (15 nm radius of silica nanoparticles) and PHEA with 80% ethanol amine were prepared and applied in this experiment, as the adhesive strength of the sample was quite effective at this preparation condition.35,39 As shown in Figure 7B, Ludox TM-50 and PHEA systems show lower adhesion strength than the polymer nanoaggregate systems synthesized in this study.
Figure 6. TEM images of polyaspartamide-g-C18/DOPA aggregates formed at 0.1 wt % polymer (C20D50) concentration. D
DOI: 10.1021/acs.biomac.7b00584 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Figure 7. Lap shear adhesion test, Gadh: (A) different amounts of 5 wt % C20D100 polymer; (B) different materials; (C) different concentrations of C20D50 polymer; (D) different concentrations of C20D100 polymer.
Table 2. Average Sizes of Nanoparticle of Polymeric Micelles sample name
5.0 wt %
2.5 wt %
1.0 wt %
0.5 wt %
0.1 wt %
C20D50 C20D100
319.6 nm 261.7 nm
255.9 nm 236.8 nm
256.5 nm 219.1 nm
257.0 nm 213.3 nm
248.1 nm 220.0 nm
Figure 8. Lap-shear adhesion test result for mouse skins treated with different samples (p value
