1. Introduction

Nowadays, ω-aminoalkyl dihydrogen phosphates H₂N-(CH₂)_n-OP(O)(OH)₂ (referred to as AAP-n thereafter) and their salts have found limited biomedical applications despite the fact that they possess functional amino and phosphate groups present in many biological molecules, like proteins and nucleic acids. AAP-2, a phospholipid moiety, has been used to stabilise apatite colloid with potential for cellular drug delivery [1]. AAP-3 (or its salt) is an active cosmetic ingredient promoting collagen biosynthesis in the skin, as shown in a US patent [2]. AAP-6 has been condensed with biological molecules like biotin [3] and uridine 5'-monophosphate [4] to form various bioconjugates.

The synthesis of AAP-n involves O-selective phosphorylation of corresponding amino alcohols. Phosphorus oxychloride (POCl₃) is not suitable because it reacts with both hydroxyl and amino groups. Only AAP-3 has been synthesised using this phosphory lation reagent, which reacts with 3-aminopropanol forming a cyclic phosphoramidate chloride initially, followed by a selective hydrolysis of the P(O)-N bond in the six-member ring [2]. A more universal synthesis has been achieved using phosphoric acid [3-6] or pyrophosphoric acid [7] as the phosphorylation reagent. However, a high temperature (140-250 ℃), a long reaction time (18-40 h) and a high vacuum (below 50 mmHg) are usually required for these methods.

The inconvenient synthesis may account for the limited availability of AAP-n. To the best of our knowledge, only AAP-2 is commercially available (e.g. from Sigma-Aldrich). We recently synthesised a series of ammonium salts of AAP-n (n=3, 4, 5, 6) at mild temperatures (0-25 ℃) using POCl₃ as the phosphorylation reagent [8]. The key is to protect the amino group of each amino alcohol with a fluorenylmethyloxycarbonyl (Fmoc) group prior to phosphorylation. We further adopted these ammonium salts as dispersing agents to synthesise hydroxyapatite hydrocolloids [9], showing an increase in the aspect ratio of the colloidal particles with an increase in the carbon number of the dispersant.

In this study, we modified our previous synthesis [8], forming a monosodium salt of each AAP-n (n=3-6, referred to as AAP-n-Na thereafter) which are easy to purify via recrystallisation. This simple synthesis resulted in highly pure AAP-n-Na (purity over 99%), making it possible to finely characterise their chemical structures and compositions. Based on this, the pKa constants of AAP-n series and the cytotoxicity of their sodium salts (AAP-n-Na) were determined and reported for the first time. We believe these basic data are important for further research into and applications of AAP-n and their salts in biomedical engineering, such as the functionalisation of hydroxyapatite, which is the mineral phase of bone.

2. Experimental 2.1. Materials

O-Phosphorylethanolamine (i.e. AAP-2, >98%, TCI, Japan) was used as a control. 3-Amino-1-propanol (>98.5%, J & K Scientific Ltd., Beijing, China), 4-amino-1-butanol (>98%, Chengdu Best Reagent Co., Ltd., Chengdu, Sichuan, China), 5-amino-1-pentanol (>96%, Alfa Aesar, USA) and 6-amino-1-hexanol (>97%, also from J & K Scientific Ltd.) served as amino alcohols (AC-n, n=3-6). Fluorenylmethyloxycarbonyl chloride (Fmoc-Cl, 99%, Asta Tech, Chengdu, Sichuan, China) and POCl₃ (>98%, Kelong Chemical, Chengdu, Sichuan, China) were used as amino-protecting and phosporylating agents, respectively.

2.2. Synthesis of AAP-n-Na

As shown in Fig. 1, the whole synthesis involved three steps, i.e., protecting the amino group, phosphorylating the hydroxyl group and removing the protecting group. The first two steps were described previously [8]. In the third step, each Fmoc-AAP-n (0.025 mol) was dissolved in 30 mL of N, N-dimethylformamide (DMF), into which 150 mL of piperidine/DMF (1:4, v/v) was slowly dripped. After magnetically stirring for 2 h, a resulting white precipitate (which was a mixture of AAP-n and its piperidine salt with a molar ratio of 1:1, see Fig. S1 in Supporting information) was obtained by filtration, and then washed with 30 mL × 3 of ethyl acetate. The washed precipitate was dissolved in 30 mL of water, and the pH was adjusted to about 11.2 using a solution of 0.5 mol/L NaOH. The water phase was extracted with 20 mL × 5 of chloroform. Each extraction proceeded for at least 0.5 h under vigorous stirring to allow piperidine to enter into the organic phase. Afterwards, the pH of the water phase was adjusted to 8.6-8.9 using 0.5 mol/L HCl prior to the addition of 20 mL of ethanol. The mixture was rested at -18 ℃ for 15 h to crystallise AAP-n-Na. Finally, the product was recrystallised the same way, and dried at 60 ℃ for 10 h.

2.3. Structural characterisation of AAP-n-Na

The Fourier transformed infrared (FTIR) spectra were obtained on a Nicolet 560 IR spectrometer (Nicolet Instruments, USA) using KBr disks, with a resolution of 4 cm^-1 between 400 cm^-1 and 4000 cm^-1. The ¹H, ¹³C and ³¹P nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV Ⅱ 400 MHz NMR spectrometer (Bruker Corp., Switzerland). The high-resolution mass spectra were collected on a Bruker maXis Ⅱ time-of-flight mass spectrometer (Bruker Daltonics, USA).

The C, H and N contents in each AAP-n-Na were analysed on a Euro EA 3000 elemental analyser (Leeman Labs Inc., USA). The P and Na contents were measured on a VG PQ ExCell inductive coupled plasma (ICP) emission spectrometer (TJA Corp., USA), using diluted sample solutions with known accurate concentrations.

2.4. Potentiometric titration of AAP-n-Na

We performed potentiometric titrations on each AAP-n-Na in water to determine its purity and the pKa constants of corresponding AAP-n. Commercial AAP-2 was served as the control to assess the accuracy of the method. Typically, around 0.2 g AAP-n-Na (accurate mass recorded with an analytical balance) in 30 mL of water was titrated with 0.05 mol/L HCl and NaOH standard solutions, respectively. Their accurate concentrations were determined by titration with standard Na₂CO₃ (≥99.95%, Tianjin Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China) or potassium biphthalate (≥99.95%, Kelong Chemical, Chengdu, Sichuan, China). All titrations were performed manually under magnetic stirring at room temperature (25±0.5 ℃). About 5 s after each titrant addition, a stable pH value of the analyte solution was measured with a Sartorius PB-10 pH metre (Sartorius AG, Germany). The detailed condition of each titration is shown in Table S1 in Supporting information.

Since the analyte mass (m_a) and the titrant concentration (C_t) are hard to maintain for all titrations (Table S1), it is inconvenient to compare the titration curves in the form of pH vs. real volume of consumed titrant (V_t). Therefore, V_t was normalised to an equivalent volume (V^*) that was defined as:

(1)

where n_a and M_a stand for moles and molar mass of the analyte, respectively; the plus and minus sign indicate NaOH and HCl as the titrant, respectively. Thus V^* indicates the degree to which the analyte has been neutralised by the titrant. The pH values are plotted against corresponding V^* to obtain the titration curve. In case of 100% purity, the absolute value of V^* (丨V^*丨) equals 1 at the first equivalence point; and the real丨V^*丨at this point in the titration curve stands for the purity of the analyte. According to definition (1), the value of丨V_t/V^*丨is a constant equalling the theoretical titrant volume consumed by the analyte at 100% purity in each dissociation step. These values are listed in Table S1 as well, ranging from 17.35 to 25.58 mL.

Each AAP-n can be regarded as a triprotic acid with three dissociation constants (Ka₁, Ka₂ and Ka₃). The pKa constants equal the corresponding pH values when the concentrations of the acid and its conjugate base are the same (see Fig. 3A in the Results section). This is the case at the half equivalence point of each dissociation step, which can be easily identified in the corresponding titration curve.

2.5. Cytotoxicity test

AAP-2 was transformed into AAP-2-Na by neutralising it with equimolar NaOH. Each AAP-n-Na (0.1 g, n=2-6) was dissolved in 8 mL of phosphate buffered saline (PBS, pH 7.4, Fisher Scientific, Beijing, China). The pH was adjusted to 7.4 using 1 mol/L HCl and the volume was calibrated to 10 mL. The resulting 1% (w/v) solution was sterilised by filtration through a 0.22-μm filter (Minipore, USA) and then diluted to 0.1%, 0.05% and 0.01% with cell culture medium to obtain AAP-n-Na containing media. Accordingly, the PBS was diluted by 10, 20 or 100 times with the cell culture medium, serving as negative control media. The cell culture medium was Dulbecco’s Modified Eagle medium (DMEM, Fisher Scientific) supplemented with 10% foetal bovine serum (FBS), 0.1% penicillin and 0.1% streptomycin. The equivolume mixture of 30% H₂O₂ and the supplemented DMEM served as a positive control medium.

L929 mouse fibroblasts (West China Centre of Medical Sciences, Sichuan University, China) suspended in 200 μL of AAP-n-Na containing media or control media were seeded at 1000 cells per well into a 96-well plate (Corning, China). To avoid cross contamination, the positive control group was arranged in a separate plate. The medium was refreshed every 2 days. After incubation at 37 ℃ with 95% air and 5% CO₂ for a predetermined period (1, 3, 5 and 7 days, n=6 for each culture condition), cell morphology was observed using an Olympus Ⅸ 71 inverted microscope (Olympus, Japan) followed by assessing cell proliferation by the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. In detail, the medium was removed and 20 μL of 5 mg/mL MTT solution (Sigma, USA) was added. After incubation for additional 4 h in the dark, the supernatant was discarded and 150 μL of dimethyl sulfoxide was added to dissolve the purple formazan crystals generated by the cells. Ten min later, the optical density (OD) of the solution at 570 nm (which is proportional to the viable cell number) was measured on a Multiskan MK3 microplate reader (Thermo Scientific, Shanghai, China).

The relative cell growth rate (RGR) is defined as the percentage of the OD value of each specimen relative to the OD value of the corresponding negative control. According to ISO 10993-5, an RGR of less than 75% indicates some toxicity to cells.

3. Results and discussion 3.1. Chemical structures of AAP-n-Na

As shown in Fig. 2, the FTIR spectra of AAP-n-Na demonstrate absorptions related to phosphate (ν_p=o at 1092 cm^-1, ν_{P-O-C (as)} at 982 cm^-1, ν_{P-O-C (s)} at 815 cm^-1, δ_{O-P-O (as)} at 581 cm^-1 and δ_{O-P-O (s)} at 547 cm^-1) [10, 11] and alkyl ammonium groups (ν_N-H, very broad from 2000 cm^-1 to 3200 cm^-1, δ_{N-H (as)} at 1635 cm^-1 and δ_{N-H (s)} at 1587 cm^-1) [12, 13]. The free amine (ν_N-H at 3422 cm^-1) [12] and hydroxyl (ν_O-H at 2571 cm^-1, from P-OH groups) [10] absorptions are consistent with the non-inner salt form of AAP-n-Na. The relative intensity of -CH₂-absorptions (ν_{C-H (as)} at 2938 cm^-1, ν_{C-H (s)} at 2868 cm^-1 and δ_C-H at 1484 cm^-1) increased with the carbon number in the AAP-n-Na series. The ¹H NMR spectra clearly show every -CH₂-signal from each AAP-n-Na with the same intensity, except that signals e and f from AAP-6-Na overlap and thus show double intensity. Likewise, every carbon in AAP-n-Na displayed a separate signal in the corresponding ¹³C spectrum. All AAP-n-Na demonstrated a single ³¹P signal at about 4 ppm because only one type of phosphate group exists in these molecules. Note that the detailed chemical shifts are summarised in Table S2 in Supporting information.

The high resolution mass spectra of all AAP-n-Na (Fig. S2 in Supporting information) mainly showed one peak from corresponding [AAP-n]^- anions whose absolute m/z value was perfectly consistent with the molar mass (Table 1). The measured elemental compositions also highly agree with the theoretical values.

3.2. pKa constants of AAP-n

The pKa constants of the AAP-n series (n=3-6) were measured by a simple titration method. To the best of our knowledge, only the pKa constants of AAP-2 [14, 15] and AAP-6 [4] have been reported previously. Fig. 3A shows the dissociation equations of AAP-n and the calculation of the dissociation constants. The titration curves, starting from pure acid form (AAP-2, Fig. 3B) or monobasic form (AAP-n-Na, Fig. 3C), all display two typical inflexion points, IP₁ and IP₂, corresponding to AAP-n and [AAP-n]^-, respectively. The third inflexion point (IP₃) corresponding to [AAP-n]^2- is missing. The inflexion point coordinates and the pKa constants obtained from the titration curves are listed in Table 2. The丨V^*丨at the first equivalence point (IP₂ for AAP-2 and IP₁ for AAP-n-Na) represents the purity of the titrand which is above 99% (Table 2). The measured pKa constants of AAP-2 are consistent with reported values [14], showing the reliability of the titration method. However, Mohan and Abbott reported slightly lower values [15]. This might be related to the fact that KNO₃ was added to the titrand solution to adjust the ionic strength up to 0.2 in that study.

The disappearance of the third inflexion point (IP₃, Fig. 3B and C) in the titration curve of each AAP-n is not surprising. Previous researchers have also observed the same phenomenon during base into acid titration of AAP-2 [15] and H₃PO₄ [16]. Hamann theoretically analysed the existing conditions for inflexions near equivalence points in titration curves of polybasic acids [17]. In a typical base into acid titration of a tribasic acid (pKa₁=5, pKa₂=8, pKa₃=11), the third inflexion vanishes when the concentration of the acid or base is less than 0.1 mol/L. In this study, the intervals between neighbouring pKa constants of AAP-n were about 4-5 pH units (Table 2), making each dissociation step occur independently, similar to the typical case proposed by Hamann (where the interval was 3 pH units [17]). Moreover, the initial concentrations of AAP-n-Na ranged from 0.031 mol/L to 0.046 mol/L (Table S1) and the titrant (HCl and NaOH solutions) concentrations were about 0.05 mol/L, i.e. all less than 0.1 mol/L. Therefore, according to the theoretical analyses by Hamann, the third inflexions of all AAP-n should disappear under the conditions used in this study.

3.3. Synthesis art of AAP-n-Na

The FTIR, NMR and high resolution mass spectra, and the elemental analysis results all highly matched the chemical structures of AAP-n-Na (Fig. 2 and Table 1), whose purity was over 99% (Table 2). It can be concluded that the chemical synthesis in this study was very successful. As a matter of fact, selective phosphorylation of the hydroxyl groups in α, ω-amino alcohols is the key to synthesising AAP-n. Previous researchers directly phosphorylated amino alcohols using phosphoric acid or pyrophosphoric acid [3-7]. The selection comes from the higher thermal stability of P(O)-O bond than that of P(O)-N bond at elevated temperatures (140-250 ℃), as suggested in an early patent [5]. We protected the amino group of α, ω-amino alcohols using Fmoc group before phosphorylation, allowing the synthesis of AAP-n and their salts at low temperatures (0-25 ℃) [8]. Although the synthesis involves three steps (Fig. 1), the yields of the first two steps were relatively high (above 80%) and the intermediates were easily to purify. The first intermediates (Fmoc-AC-n) can be recrystallised in ethyl acetate/petroleum ether (7:1, v/v) and the second intermediates (Fmoc-AAP-n) are insoluble in water, allowing purification by water washing [8].

In the third step, the Fmoc group was removed in piperidine/ DMF, which is a routine method [18]. The released amino groups in the resultant AAP-n homologues had similar pK_b constant (2.80-2.97, calculated as pK_b=14 -pKa₃, see Table 2), which approximates the reported pK_b value (2.88) of piperidine [19]. This means that the primary amino group in AAP-n and the secondary amino group in piperidine possess almost the same potential to be protonated with the dihydrogen phosphate group in AAPn. Therefore, free AAP-n (in the form of an inner salt) and its piperidine salt co-precipitated from the DMF solution in a molar ratio of about 1:1 (Fig. S1), once the Fmoc group was removed. The residue of free piperidine was washed away using ethyl acetate.

For purification, each co-precipitate was dissolved in water, and NaOH solution was added to deprotonate the piperidine salt at pH 11.2. The free piperidine was extracted with chloroform, leaving the monosodium and disodium salts of AAP-n (Fig. 1) in the aqueous phase. The molar ratio of both salts was about 1:1 because the pH value 11.2 approximates the pKa3 constant of AAP-n (Fig. 3C and Table 2). The pH value was then adjusted to 8.6-8.9, i.e. close to the pH value at the corresponding IP₂ point (Fig. 3C and Table 2) where AAP-n-Na predominates. Finally, pure AAP-n-Na was obtained by recrystallisation from water/ethanol. Actually, the titration results were used to optimise the purification method.

In our previous purification method [8], ammonia solution was used to release piperidine from the piperidine salt of each AAP-n. Ammonia (pK_b=4.74) is a weaker alkaline than piperidine (pK_b=2.88). This makes ammonia not a good candidate to deprotonate the piperidine salt. Thus, overdosed ammonia had to be used, which was removed from the resultant ammonium salt of AAP-n by freeze-drying. Since no recrystallisation was involved in that method, the product purity was hard to control. Further to this, it was impossible to obtain pKa constants of AAP-n homologues by titration of their ammonium salts, since the ammonium/ammonia pair can affect the titration curves. Thus, our modification to the synthesis of AAP-n salts, compared with the previous method in the literature [8], is fundamental for applications with AAP-n and their derivatives since we obtained a series of pure AAP-n-Na crystals for the first time, making possible a systematic investigation of their structures and properties.

3.4. An odd-even effect on pKa3 constants of AAP-n

An increase in carbon number generally reduced the acidity of AAP-n because the alkyl group is an electron-donating group. This is consistent with the general increase trend in both pKa₁ and pKa₂ values from AAP-2 to AAP-6 (Table 2). The pKa₁ constant increased by about 0.15 from AAP-2 to AAP-3 and reached a plateau at n≥3; the pKa₂ constant displayed an obvious increase from AAP-2 to AAP-4, and then increased very slowly thereafter. Differently, the pKa₃ constant demonstrated a sharp increase from AAP-2 to AAP-3 and then fluctuated at n≥3, showing an odd-even effect as a function of the chain length. In detail, AAP-n with an even carbon number (e.g. n=4) possesses a lower pKa₃ value than the neighbouring homologues with odd carbon numbers (n=3 and 5). Just taking into consideration the AAP-n series with odd carbon numbers (n=3, 5) or even carbon numbers (n=2, 4, 6), a general increase trend in the pKa₃ constant can be observed as well.

Insight into the steric structures of AAP-n-Na with n=odd (3, 5) and those with n=even (2, 4, 6) can help us to understand the odd-even effect on the pKa₃ value. AAP-3-Na and AAP-4-Na were selected to illustrate the structural difference between the two types of AAP-n-Na. Fig. 4A shows their straight chain structures where all the -CH2-groups are in the trans-conformation to minimise steric hindrance among them. In this case, the terminal ionic groups (ammonium cation and phosphate anion) of AAP-3-Na are in the trans-position relative to the aliphatic chain, while both terminal ionic groups occupy cis-positions due to one more -CH₂-group in AAP-4-Na.

As there is no externally added salt (e.g. NaCl) to stabilise the terminal ions in the water where each AAP-n-Na was dissolved, strong ionic interactions must occur either intramolecularly or intermolecularly. In the former case, the ionic attraction between both terminal groups would bend each molecule to form a ring in a head-to-tail manner, causing server deformations in bond lengths and bond angles within the molecule and exposing their deformed hydrophobic -CH₂-groups to the water (Fig. 4B). This molecular conformation therefore is not stable. A more preferable way to stabilise the terminal ions is to form molecular pairs where every two molecules are packed in a side-by-side and head-to-tail manner (Fig. 4C). As a matter of fact, similar pairwise selfassociation in water has been proposed for other organic inner salts, like _L-carnitine [20]. In this way, every molecule of AAP-n-Na can keep its all-trans conformation (Fig. 4A) and its terminal ions are balanced with counterions from another molecule. The side hydrophobic interaction from the aliphatic chains and the hydrogen bonds (see dashed lines, Fig. 4C) between the ammonium and phosphate terminal groups further stabilise the molecular pairs. The trans-positional terminal groups in AAP-3-Na favours six hydrogen bonds in each molecular pair as predicted by Materials Studio software, while only two hydrogen bonds form within each AAP-4-Na pair due to the cis-positional terminal groups. The extra hydrogen bonds in AAP-3-Na pairs can provide extra stability to their ammonium groups. Just taking intermolecular hydrogen bonding into consideration, the ammonium group in AAP-4-Na is much easier to deprotonate than that in AAP-3-Na, which favours a decrease in the pKa₃ constant from AAP-3-Na to AAP-4-Na. This decreasing effect overwhelms the increasing effect due to one more electron-donating -CH₂-group in AAP-4-Na. Overall, the pKa₃ value of AAP-4 is lower than that of AAP-3. Likewise, AAP-6 displays a lower pKa₃ constant than AAP-5.

Odd-even effects have previously been observed, mainly in solid systems where the molecular packing mode depends on the odd or even number of repeating structural units as well. For instance, the melting point of straight-chain aliphatic dicarboxylic acids alternate with the carbon number in the chain [21]. Moreover, many interfacial properties of organic self-assembled monolayers (wettability, surface work function, maximum adhesion, tribological property, and so on) depend on the odd or even carbon number in the molecules composing the monolayer, as reviewed in detail by Tao and Bernasek [22]. The odd-even effect on pKa constant in water observed in this study is a new phenomenon worth investigating further.

3.5. Cytotoxicity and biomedical applications of AAP-n-Na

L929 cells generally grew faster in media with AAP-n-Na than in the negative control media without AAP-n-Na, in particular at day 5 and day 7 (indicated by ^*, Fig. 5). The variation in tested AAP-n-Na concentration had no obvious effect on cell growth. The normallly elongated cell morphology was observed in both negative and AAP-n-Na media (Fig. 6). On the contrary, spherical cell morphology (Fig. 6) and no cell growth (Fig. 5) occurred in the H₂O₂ solution (positive control), which was toxic to cells. It can be concluded that no cytotoxicity is associated with AAP-n-Na at a concentration up to 0.1%. This was further confirmed by another cytotoxicity test which showed that 0.5% of AAP-n-Na in cell culture media is toxic to osteoblast-like MG 63 cells while both 0.01% and 0.1% of AAP-n-Na are safe to the same cells (Figs. S3 and S4 in Supporting information). AAP-3 has been reported to be an active cosmetic ingredient promoting fibroblast proliferation and collagen biosynthesis [2]. Our data further suggest that the AAP-n-Na (n=2-6) family might be suitable for cosmetics. The effective concentration can be as low as 0.01% (Fig. 5).

Previous research has shown that ammonium salts of AAP-n (n=2-6) can be used to functionalise hydroxyapatite (bone mineral) nanoparticles, generating reactive amino groups on them [9]. These functionalised particles might be grafted with polymers, with a final goal to prepare bone-like nanocomposites. AAP-n-Na can be used in the same way. The measured pKa constants can be used to calculate the ionic distribution of AAP-n as a function of pH, which is essential to optimise synthesis art of AAP-n functionalised hydroxyapatite particles where [AAP-n]^- ions should predominate over AAP-n molecules and [AAP-n]^2- ions. Taking AAP-5 for example [AAP-5]^- ions are over 90% in the pH range of 7.4-10.3 (Fig. 3C). Therefore, AAP-5 functionalised hydroxyapatite should be synthesised in this pH range.

This study shows that AAP-n-Na homologues are safe to cells at concentrations lower than 0.1%. When used in vivo, the aminoalkyl chain bonded hydroxyapatites can slowly release free AAP-n molecules (mainly in the form of [AAP-n]^- at biological pH of 7.4, according to the measured pKa constants, see Fig. 3B and C), which can be quickly diluted by the body fluid and the real local [AAP-n]^- concentration can be much lower than 0.1%. This implies the in vivo safety of AAP-n and their salts.

4. Conclusions

We synthesised a series of ω-aminoalkyl sodium phosphates (AAP-n-Na, n=3, 4, 5, 6) with a purity over 99%. Their chemical structures were confirmed by both spectroscopic and elemental analyses. The pKa constants of AAP-n were measured by potentiometric titration, showing a general increase trend with the increase of chain length in AAP-n. An additional odd-even effect on the pKa₃ constant was observed where AAP-n with n=even showed a lower pKa₃ value than the neighbouring AAP-n with n=odd. All AAP-n-Na were non-toxic to both L929 fibroblasts and MG 63 osteoblast-like cells at concentrations up to 0.1% in the cell media. This fundamental study is essential for the applications of AAP-n homologues and their derivatives in biomedical engineering fields.

Acknowledgment

The authors are grateful for financial support from the National Natural Science Foundation of China (No. 50973069).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.03.029.