Synthesis and characterization of zwitterionic peptides derived from natural amino acids and their resistance to protein adsorption

Abstract

Various functional groups can be easily introduced onto the poly(α,β-L-aspartic acid) by using different reagents to open the succinimide ring in polysuccinimide (PSI). In this work, two natural basic amino acids, L-histidine and L-lysine were used as the ring opening reagents to react with PSI. As a result, both positively and negatively charged moieties were introduced onto the same side chain simultaneously, which provides a nano-scale homogenous mixture of balanced charges. The chemical structures of the obtained polymers were confirmed by FT-IR and ¹H NMR spectroscopy. Zeta potential and turbidity measurements were applied to investigate the zwitterionic property of the polymers. Substrates pre-coated with the zwitterionic polymers exhibited good hydrophilicity and anti-protein-adsorption ability. What's more, the in vitro cytotoxicity test suggested that these peptide-based zwitterionic materials had good biocompatibility, indicating their good potential as non-fouling materials in the biomedical applications.

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1 Introduction

Protein adsorption has severely limited the full exploitation of surface-based biomedical devices. The prevention of surface fouling is becoming increasingly important for the development of medical implants,¹ biosensors^2,3 and drug delivery systems.^4,5 It is generally accepted that materials exhibiting high resistance to nonspecific protein adsorption show strong interaction with water. Surface characteristics, including hydrophilicity and charge distribution, are important factors that determine the non-fouling capability of surfaces.^6,7

One of the most widely studied non-fouling materials is poly(ethylene glycol) (PEG) for its high hydrophilicity, flexibility, non-toxicity and non-immunoreaction.^8–13 However, it faces the problem of relatively low stability in the presence of oxygen and transition-metal ions found in most biochemically relevant solutions.^14–16 In addition, studies showed that the PEG surfaces would limit the interactions between carriers and the target tissues and affect the cellular uptake of the loaded cargo.^17,18 Another class of non-fouling materials is the phosphorylcholine materials, but their monomers are not readily available.¹⁹ For these reasons, developing a new type of non-fouling material is meaningful in both academic research and practical applications.

Over the last few years, it has been demonstrated that zwitterionic and mixed-charge surfaces are highly resistant to nonspecific protein adsorption from undiluted blood plasma/serum and to bacterial adhesion/biofilm formation.^20–23 Due to the electrostatically induced hydration a tightly bounded and structured water layer around zwitterionic polymer chains is formed to repel protein adsorption. However, most of the synthesized zwitterionic materials are polyolefins or polyacrylates, which are not biodegradable and biocompatible. These limit their use in biomedical applications. There are only a few works reported to circumvent this problem. Yeo et al.²⁴ reported a zwitterionic chitosan derivative served as a coating material to cationic surfaces to prevent protein adsorption, which could be removed in a pH-responsive manner. In addition, Jiang^25–27 synthesized a series of biodegradable peptides from certain natural amino acids in the form of alternating or randomly mixed charges, which exhibited high resistance to nonspecific protein adsorption. Polypeptides were biodegradable and their final metabolized products were natural amino acids.²⁸ Recently, a mixed-charge polypeptide derivative was developed in our group by amidation of poly(α,β-L-aspartic acid) with L-histidine methyl ester, which exhibited distinct anti-fibrinogen-fouling property.²⁹ However, for this kind of polymer, the positive and negative charges distribute randomly and separately, which cannot ensure a nanometer-scale homogenous mixture of balanced charged groups as the zwitterionic polymers. The latter is crucial for them to achieve a highly resistance to nonspecific protein adsorption.

Polysuccinimide (PSI) can be easily prepared by thermal polycondensation of L-aspartic acid. By using different reagents to open the succinimide ring in PSI, various functional groups can be easily introduced into the side chain of poly(α,β-L-aspartic acid).^30–39 Herein, two natural basic amino acid methyl ester, L-lysine methyl ester and L-histidine methyl ester were used to open the PSI. After ring-opening reaction with PSI and the followed hydrolysis, one basic group (amino group for L-lysine and imidazole group for L-histidine) and one carboxyl group would be introduced onto the side chain of polyaspartamide. Their zwitterionic properties and anti-protein-adsorption abilities were investigated in this paper.

2 Experimental

2.1 Materials

PSI (M_n = 2.1 × 10⁴, M_w/M_n = 1.3) was synthesized in our laboratory, according to the ref. 29 and 40. The L-histidine methyl ester dihydrochloride (L-His-OMe·2HCl, purity >98%) and L-lysine methyl ester dihydrochloride (L-Lys-OMe·2HCl, purity >98%) were obtained from Tianjin HEOWNS Biochemical Technology Co. Amino propyl triethoxy silane (APTES, purity >99%) was purchased from Alfa Aesar. Triethylamine (TEA, Tianjin Chemical Co.) was purified by distillation before use. Silica wafers and glass slides were obtained from Beijing Top Vendor Science & Technology. They were cleaned and treated as described.⁴¹ Briefly, the silica wafers and glass slides (1.1 cm × 1.1 cm) were submerged in Piranha solution (70/30, v/v, sulfuric acid/hydrogen peroxide) for 20 min, and subsequently rinsed them with water. After this process was repeated, the substrates were sonicated in 50% (v/v) isopropanol/water for 20 min and then washed with water. The substrates were heated at 60 °C in RCA solution (5 : 1 : 1, v/v/v, water/hydrogen peroxide/ammonia solution) for 20 min. Finally, they were washed with water and dried under a stream of nitrogen. Bovine serum albumin (BSA), fibrinogen (FIB) and Fluorescein isothiocyanate (FITC) were purchased from Lianxing Biotechnology Co. Ltd.

2.2 Synthesis of L-histidine grafted α,β-poly(2-hydroxyethyl aspartic acylamino) (His–PHAA) and L-lysine grafted α,β-poly(2-hydroxyethyl aspartic acylamino) (Lys–PHAA)

L-Histidine methyl ester grafted α,β-poly(2-hydroxyethyl aspartamide) (MeO-His–PHAA) was prepared via a ring-opening reaction of PSI. Briefly, PSI (0.97 g, 10 mmol) and L-His-OMe·2HCl (4.84 g, 20 mmol) were dissolved in 10 mL DMSO. Triethylamine (TEA) (7 mL, 50 mmol) was added to the mixture. Grafting reaction was maintained at 80 °C for 24 h and followed by aminolysis by 2-aminoethanol (12 mmol) at room temperature for 12 h. The L-histidine methyl ester grafted α,β-poly(2-hydroxyethyl aspartamide) (MeO-His–PHAA) was obtained by dialysis and lyophilization.

The above obtained MeO-His–PHAA (1.96 g, 10 mmol) was then dissolved in 1 M sodium hydroxide (2 equivalents per ester group) and the same volume of methanol was added. The solution was stirred at room temperature for 8 h.⁴² Hydrochloric acid (1 M) was added to neutralize NaOH and modulate the pH to neutral. His–PHAA was obtained by dialysis and lyophilization. Lys–PHAA was obtained through the same processes as His–PHAA.

2.3 Fourier transform infrared spectra (FT-IR) spectroscopy

FT-IR was measured by Bio-Rad FTS6000 spectrophotometer at room temperature. Polymer samples were prepared by well dispersing the complex in KBr powder and compressing the mixtures to form a plate.

2.4 Nuclear magnetic resonance (NMR) spectroscopy

¹H NMR spectra were recorded on a Varian UNITY-plus 400 spectrometer using D₂O or DMSO-d6 as the solvent. Chemical shifts were reported in ppm.

2.5 Turbidity and zeta potential measurements

Polymer aqueous solution with the concentration of 1.0 mg mL⁻¹ was prepared for the turbidity measurement. The initial pH of polymer solution was adjusted to pH 2.0 with 1.0 M HCl. The titration was carried out by the stepwise addition of 0.1 M NaOH. The pH values were checked using a Sartorius PB-10 pH meter (Sartorius Instrument, Germany). The turbidity of the solution during the titration was measured by monitoring the absorbance at 380 nm using a Shimadzu UV-2450 spectrophotometer. At the same time, the zeta potential in response to pH values was recorded using a Zetasizer Nano ZS90 malvern instrument.

2.6 His–PHAA and Lys–PHAA modified silicon wafers and glass slides

Silicon wafer and glass slides were treated with a mixture of ethanol (2 mL), APTES (0.4 mL) and (25–28%) ammonia solution (0.1 mL) for 2 h. The substrates were then rinsed with ethanol and water, dried under a stream of nitrogen to afford the amine-functionalized silica wafers and glass slides, named as NH₂–SW and NH₂–GS, respectively.

The silica wafers and glass slides were pre-coated with the polymer His–PHAA (or Lys–PHAA) before the protein adsorption experiments. The NH₂–SW and NH₂–GS were incubated in His–PHAA (or Lys–PHAA) PBS solutions (0.2–4.0 mg mL⁻¹, 10 mM NaCl and pH 7.4) for 5 h at room temperature, followed by water washes (3 × 10 min) and dried under a stream of nitrogen.

2.7 Contact angle measurements

The water contact angles of NH₂–SW and polymer coated silica wafers were measured using a contact angle goniometer (JC2000C Contact Angle Meter, Powereach Co., Shanghai, China) at room temperature. Water droplet (∼4 μL) was delivered to the surface using a microliter syringe. At least five sample readings from different locations on the surface were averaged and the angles reported were reliable to ±3°.

2.8 Protein adsorption measurements

BSA and FIB were labelled with FITC (FITC : protein = 4 : 1) at room temperature for 1 h. The resulting solutions were dialyzed in phosphate buffered saline (PBS) (10 mM, pH 7.4) for 48 h to remove the free FITC molecules. The FITC labelled BSA and FIB (FITC–BSA and FITC–FIB) solutions were stored at 4 °C.

The zwitterionic peptides modified glass slides (polymer concentration was 4 mg mL⁻¹), NH₂–GS, and unmodified glass slides were incubated with FITC–BSA or FITC–FIB solution (0.5 mg mL⁻¹) in a phosphate buffer (10 mM, 150 mM NaCl, pH 7.4) on a shaker at 37 °C for 10 h, followed by extensively washing with pure water to remove loosely adsorbed proteins. The fluorescence microscope images of the FITC–BSA or FITC–FIB adsorption on glass slides were taken by upright fluorescence microscopy (Zeiss Axio Imager Z1, Germany) in dark field.

Besides, the polymer coated silica wafers were incubated with BSA or FIB solution (0.2 mg mL⁻¹) in a phosphate buffer (10 mM, 150 mM NaCl, pH 7.4) on a shaker at 37 °C for 10 h. The amounts of adsorbed protein were determined by measuring the FIB and BSA concentrations before and after the adsorption process via the Bradford method.⁴³ The adsorption capacity of proteins on the polymer-coated surface per area (q, μg cm⁻²) was calculated using the following eqn (1):⁴⁴

q = (C_i − C_f)V/S (1)

where C_i and C_f are the initial protein concentration and the protein concentration in the supernatant after the adsorption, respectively. V and S are the total volume of the solution and the total surface area of the silica wafer, respectively. The reported data were mean values of triplicate samples. NH₂–SW without coating polymer was used as the control.

2.9 In vitro cytotoxicity test of zwitterionic polyaspartamides

NIH-3T3 cells were used to investigate the biocompatibility of His–PHAA and Lys–PHAA. NIH-3T3 cells were seeded in a 96-well plate at an initial density of 2 × 10⁴ cells per well in DMEM complete medium. After 24 h of culture, the original medium was replaced with fresh DMEM medium. Then polymer solutions were added to the medium at concentrations ranging from 0 to 500 μg mL⁻¹ (0, 50, 125, 250, and 500 μg mL⁻¹). Each dosage was replicated in 6 wells. Cells in media alone devoid of polymers acted as negative control and wells treated with triton X-100 acted as positive control. At 48 h of further incubation, the DMEM was removed and 100 μL of MTT solution were added to each well and replaced with 200 μL DMSO after 4 h. The absorbance value was measured using a microplate reader (Labsystem, Multiskan, Ascent, Model 354 Finland) at a wavelength of 570 nm. The mean values were used as the final data.

3 Results and discussion

3.1 Synthesis and characterization

His–PHAA and Lys–PHAA were synthesized via an aminolysis reaction of PSI. Scheme 1 illustrates the synthetic route. By using different reagents to open the succinimide ring in PSI, various functional groups can be easily introduced into the side chain of poly(α,β-L-aspartic acid). However, in this system the ring-opening reaction were not completely achieved by L-Lys-OMe·2HCl or L-His-OMe·2HCl due to the steric hindrance. Ethanolamine was used as supplement to make sure the unreacted succinimide units transform to 2-hydroxyethyl aspartic acylamino. After ring-opening reaction with PSI and the followed NaOH hydrolysis, one basic group (amino group for L-lysine and imidazole group for L-histidine) and one carboxyl group were introduced into the side chain of polyaspartamide. Both positively and negatively charged moieties connected onto the same side chain in these polymers to provide a nanometer-scale homogenous mixture of balanced charges. The GPC results showed that before and after the NaOH treatment the molecular weight of polymers didn't dropped much (MeO-His–PHAA: M_n = 2.51 × 10⁴, M_w/M_n = 1.14; His–PHAA: M_n = 2.33 × 10⁴, M_w/M_n = 1.16; MeO-Lys–PHAA: M_n = 2.87 × 10⁴, M_w/M_n = 1.16; Lys–PHAA: M_n = 2.63 × 10⁴, M_w/M_n = 1.17). The chemical structures of polymers were confirmed by FT-IR and ¹H NMR spectroscopy.

Scheme 1 Synthesis of His–PHAA and Lys–PHAA.

Fig. 1 shows the FT-IR spectra of PSI, Lys–PSI, Lys–PHAA, His–PSI and His–PHAA. The spectrum of PSI showed the characteristic absorption bands of an imide ring at 1796 and 1717 cm⁻¹. In spectra B and D, the characteristic absorption bands of the succinimide were still present, suggesting the incomplete ring-opening reaction. After reacting with ethanolamine, the characteristic absorption bands of succinimide disappeared in spectra C and E, and an amide group at around 1660 and 1540 cm⁻¹, as well as a broad band around 3400–2500 cm⁻¹ corresponding to carboxyl group emerged, confirming the complete ring opening reaction of PSI.

Fig. 1 FT-IR spectra of PSI (A), Lys–PSI (B), Lys–PHAA (C), His–PSI (D) and His–PHAA (E).

The ¹H NMR spectrum of Lys–PHAA in D₂O is shown in Fig. 2(a), peaks appearing at 1–2 ppm are attributed to the methylene groups of lysine, indicating the successful conjugation of lysine. It is worthwhile to note that both alpha and epsilon amides in L-Lys-OMe can react with the succinimide ring in PSI. As shown in Fig. 2(a), there is a small peak at 4.2 (i′) ppm, which is ascribed to the hydrogen atom on C_α of lysine after ring-opening reaction of PSI by the α-NH₂. By comparing the integration of methylene protons at 1–2 ppm with methylene protons of the PHAA backbone at 2.5–2.9 ppm, the total lysine grafting ratio was calculated to be as much as 85%. Moreover, the ratio of reacted alpha and epsilon amides was 13 : 72, determined by comparing the integration of i′ with i peaks in Fig. 2(a). Obviously, it is not the α-NH₂ but the ε-NH₂ with better activity which should be owing to the steric hindrance and electronic effect.

Fig. 2 The ¹H NMR spectra of Lys–PHAA (a) in D₂O, His–PHAA (b) and an expansion of the methine region (c) in DMSO-d6.

In Fig. 2(b), the peaks appearing at 7.2 and 8.6 ppm are attributed to the proton of the imidazole group in histidine. The histidine grafting ratio was 32% determined by comparison of the integrations of imidazole protons with methylene protons of the PHAA backbone.

Unlike most other polypeptides prepared by complicated ring-opening polymerization of amino acid N-carboxyanhydrides (NCAs), the product obtained by hydrolysis of PSI is a mixture of two isomers, α- and β-, due to the similar energetics of attacking either carbonyl. Wolk⁴⁵ has analysed the two isomers by one- and two-dimensional NMR techniques. The results indicated that polyaspartates contain the proportion of β to α linkage is 3 to 1 under a variety of hydrolysis conditions. Fig. 2(c) shows an expansion of the methine region, showing resolution of α and β methane protons in His–PHAA. The ratio of β- to α-linkage is 3 : 1, which is consistent with Wolk's conclusion.

3.2 Zwitterionic property measurement

The coexistence of both weak acidic and basic units along the polymer chain results in forming zwitterionic fragments. Isoelectric point (pI) is an important characteristic of the aqueous solution of polyampholytes. To detect the electrophoretic mobility and locate the pI, the zeta potential and optical transmittance were monitored on His–PHAA and Lys–PHAA solutions at various pH values.

As shown in Fig. 3(a), the zeta potential of Lys–PHAA decreases from +8 mV (pH 2.0) to −30 mV (pH 10.5) as the pH increases. The similar changes were seen for His–PHAA in Fig. 3(b). During the charge changing from positive to negative, the point, at which the net charge is zero, is defied as the isoelectric point (pI). The pIs determined by the electrophoretic mobility of Lys–PHAA and His–PHAA in aqueous solutions are 5.17 and 3.46, close to the theoretical values as 5.57 and 3.91, respectively. When the acid/base molar ratio is 1, pI can be calculated by the formula:⁴⁶

pI = 1/2(pK_a + pK_b) (2)

where pK_a and pK_b are dissociation constants for the negative and positive charges, respectively. In this case, pK_a = 2.18 and pK_b = 8.95 for Lys–PHAA, and pK_a = 1.82 and pK_b = 6.00 for His–PHAA. It deserves to mention that in polymer Lys–PHAA, the slight differences in pK_a of the ε-NH₂ and α-NH₂ can be neglected though both of them react with PSI.

Fig. 3 Zeta potential and optical transmittance (λ = 380 nm) of Lys–PHAA (a) and His–PHAA (b) aqueous solutions at different pHs.

The pH-induced solubility transitions of His–PHAA and Lys–PHAA were characterized by turbidity measurement. Fig. 3 shows the pH dependence of the transmittance of His–PHAA aqueous solution at λ = 380 nm. It is evident that His–PHAA possesses the typical characteristics of a polyampholyte. The transmittance of polymer solution is around 80% at either high or low pH.

The modified polyaspartamide derivatives are composed of both negatively and positively charged groups on one repeat unit, bearing opposite charges at pH values far high or below the pI. At low pH, His–PHAA molecule possesses uncharged carboxylic groups and positively protonated imidazole groups, which make the polymer positively charged and molecularly dissolved in aqueous solution due to the repulsion between imidazole groups. At high pH, imidazole groups are deprotonated and the carboxylic groups are ionized, so the polymer shows negative charge. Although the imidazole groups are deprotonated and rendered hydrophobic, the ionization of carboxylic groups can also make His–PHAA dissolve and keep the aqueous solution transparent. Near the pI, obvious precipitation occurs due to the coexistence of positive and negative charges, and the intra- and inter-molecular electrostatic interactions result in a rapid decrease in light transmittance. The strong electrostatic interaction is supposed to be the dominant effect for the aggregation of His–PHAA through turbidity measurements. The turbidity results are consistent with those from the zeta potential measurements. During the whole process from acidic to basic conditions, the zeta potential is close to zero at pH between 3 and 4. The precipitation occurring in this range of pH is induced by the electrostatic interaction between positive amino groups and negative carboxylic moieties.

Unlike His–PHAA, the transmittance of Lys–PHAA, maintained at around 90% without significant changes during the titration. This is due to the strong hydrophilicity of Lys–PHAA compared with His–PHAA. For Lys–PHAA, although there are equal amounts of the positively and negatively charged groups in the polymer, the electrostatic interaction near the pI is not enough to induce a micro phase separation.

3.3 Contact angle measurement

The fundamental principle of the non-fouling behavior of zwitterionic materials lies in their strong capacity to form a hydration layer at the surface via electrostatic interaction. Surface hydrophilicity is one of the most important factors in determining antifouling property of surface. The hydrophilicity of the polymer coated surfaces was evaluated by contact angle measurement, which was commonly used to assess the changes in the hydrophilicity and interfacial energy of substrate surface. Fig. 4 presents the contact angle of NH₂–SW and Lys–PHAA or His–PHAA coated silica wafers. The contact angle of NH₂–SW is 54°, while the contact angle of Lys–PHAA and His–PHAA coated silica wafers are around 19° and 35°, respectively. It was found that the contact angle decreased significantly after the modification of peptide derivatives. The decrease of contact angle indicated that a highly hydrophilic surface was created by the zwitterionic polymers. What's more, Lys–PHAA could modify the hydrophilicity of silica wafers more significantly, which was in accordance with the result of transmittance test.

Fig. 4 Contact angles for the amine-functionalized and Lys–PHAA or His–PHAA coated silica wafers.

3.4 Protein adsorption

Fibrinogen is the first adsorbed protein during the blood coagulation process.⁴⁷ Effectively reducing the adsorption of fibrinogen can greatly inhibit the occurrence of the blood coagulation.⁴⁸ BSA has also been used as a model protein to investigate cell-adhesion and blood compatibility.^49,50 It is well known that protein adsorption is strongly influenced by the surface properties of biomaterials. Recently, surface modification via adsorption of polyampholytes has evoked great interest and resulted in completely different behaviors compared with the unmodified substrates.

In this study, the ability of zwitterionic peptides, His–PHAA and Lys–PHAA, to control bio-fouling was assessed by measuring BSA and FIB adsorption on zwitterionic peptides modified glass slides. The pI of BSA and FIB are 4.7 and 5.5, respectively. For analysis, the NH₂–GS, unmodified and His–PHAA or Lys–PHAA modified glass slides were incubated in FITC–BSA or FITC–FIB solution and then observed under a fluorescence microscope. As a green fluorescent protein was used, fluorescence intensity was directly correlated with the amount of protein adsorbed. For both FIB and BSA adsorptions, the His–PHAA (Fig. 5(a3) and (b3)) and Lys–PHAA (Fig. 5(a4) and (b4)) modified GS showed much lower fluorescence intensity as compared with that on the NH₂–GS (Fig. 5(a1) and (b1)) and unmodified GS (Fig. 5(a2) and (b2)). It revealed that the surfaces modification of zwitterionic polymers improved their ability to resist both FIB and BSA adsorption significantly.

Fig. 5 Protein resistant evaluation (FIB for a1–a4 and BSA for b1–b4) of the polymers modified surfaces and the control surfaces using fluorescence microscopy: (a1 and b1) NH₂–GS; (a2 and b2) unmodified GS; (a3 and b3) His–PHAA modified GS; (a4 and b4) Lys–PHAA modified GS.

In order to quantitatively evaluate the protein resistant behavior, the zwitterionic polymers were pre-coated on the silica surfaces. Amino modified silica wafers served as water-insoluble platforms carrying positive charges at pH 7.4. While at pH 7.4, His–PHAA and Lys–PHAA bear negative charges as shown in Fig. 3. The polymer His–PHAA or Lys–PHAA could be coated on the wafers due to the electrostatic interactions. The protein adsorption measurements were carried out in 10 mM PBS buffer to maintain a constant pH value during the adsorption process. The amount of protein adsorbed was quantified and compared with those on the unmodified silica wafers and amine-functionalized silica wafers without polymer coatings.

As shown in Fig. 6(a), the FIB adsorption on unmodified silica wafers and the amine-functionalized silica wafers were 68 and 73 μg cm⁻², respectively. While it was 4–32 μg cm⁻² for His–PHAA coated wafers and 2–34 μg cm⁻² for Lys–PHAA coated wafers depended on the pre-coated polymer concentrations. The amine-functionalized silica wafers adsorbed more protein compared with unmodified SW, which was probably due to abundantly positive charges of the NH₂–SW substrate. However, the charge is not the main factor caused the protein adsorption. Zwitterionic polymer modified surface can form a hydration layer via electrostatic interactions,⁵¹ which prevents the adsorption of protein. The reason why less protein adsorbed to Lys–PHAA-modified surfaces compared with His–PHAA-modified surfaces is the grafting density of the Lys–PHAA is higher than that of His–PHAA and this effect is combined with the ability of protein to undergo van der Waals interactions with the less hydrophilic His–PHAA coated surface than with Lys–PHAA. Similar BSA adsorption data are shown in Fig. 6(b). Amino-modified silicon used herein was only a surface model. The specific method for modifying the surface can be made according to the specific situation of the surface material, including the physical adsorption and chemical bonding.

Fig. 6 The adsorption amount of FIB (a) and BSA (b) on the unmodified silica wafers, amine-functionalized silica wafers and Lys–PHAA or His–PHAA coated silica wafers.

3.5 Biocompatibility evaluation

An in vitro cytotoxicity test (MTT assay) was performed using the NIH-3T3 cells to evaluate the biocompatibilities of Lys–PHAA and His–PHAA. The corresponding results are shown in Fig. 7. The cell viability was compared with the negative control cells that had been incubated in the DMEM media. The cytotoxicity of the polymers Lys–PHAA and His–PHAA towards NIH-3T3 cells was evaluated at concentrations ranging from 0 to 500 μg mL⁻¹. Polymers did not show significant cytotoxicity against NIH-3T3 cells. Even at a high concentration (500 μg mL⁻¹), the viabilities were still higher than 90% for both Lys–PHAA and His–PHAA after 48 h incubation. These results suggest that the polymers are of low cytotoxicity, and have potential use in biomedical applications.

Fig. 7 Cell viability of NIH-3T3 cells after exposure to the polymers for 48 h at different dose of Lys–PHAA or His–PHAA.

Conclusions

Two polyaspartamide derivatives, His–PHAA and Lys–PHAA, were successfully synthesized by ring-opening reaction of PSI with basic natural amino acid derivatives. In both His–PHAA and Lys–PHAA, one positively and one negatively charged moieties were connected onto the same repeat unit to provide a homogenous mixture of charges. Zeta potential and turbidity measurements revealed the zwitterionic property of the polymers. Silica wafer pre-coated by the zwitterionic polymers exhibited good hydrophilicity and anti-protein adsorption ability. What's more, the in vitro cytotoxicity test suggested that these peptide-based zwitterionic polymers had good biocompatibility, indicating their good potential as non-fouling materials in biomedical applications.

Acknowledgements

This work was funded by NSFC (51203079), the Natural Science Foundation of Tianjin (14JCYBJC18100), PCSIRT (IRT1257) and NFFTBS (J1103306).

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