Pyrene-Terminated, Amphiphilic Polypeptide and Its Hydrogen

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Article Cite This: ACS Omega 2018, 3, 4423−4432

Pyrene-Terminated, Amphiphilic Polypeptide and Its HydrogenBonded Interpolymer Complex as Delivery Systems of Doxorubicin Chun-Yi Tsai,† Chin-Hsiang Chung,‡ and Jin-Long Hong*,‡ †

Formosa Chemicals & Fibre Corporation, No. 1, Taisu Industrial Park, Mailiao Township, Yunlin County 63801, Taiwan Department of Materials and Optoelectronic Science, National Sun Yat-sen University, Kaohsiung 80424, Taiwan

‡

S Supporting Information *

ABSTRACT: The intensity ratio between the first (373 nm) and the third (383 nm) vibronic peaks [I1/I3, as the pyrene (Py) scale] of fluorescent Py was used to monitor the critical concentration, drug-loading, and -releasing behaviors of a Pyterminated, amphiphilic polypeptide PPM and its hydrogen-bonded interpolymer complex (HIPC) with poly(acrylic acid) (PAA). Primarily, an amphiphilic PPM with a hydrophobic Py terminal and hydrophilic methoxy-bis(ethylene oxide) pendant groups was synthesized through multiple preparative steps, and the resultant PPM was thoroughly mixed with PAA through a preferable hydrogen bond (H bond) interaction to form HIPC. The emission study suggested that the I1/I3 ratio and the quantum yield (ΦF) are effective in determining the critical concentrations of the aqueous PPM and PPM/PAA solutions. Moreover, the I1/I3 ratio and ΦF were found to be convenient measures for determining the amounts of doxorubicin drugs loaded by and released from the aqueous PPM and PPM/PAA solutions.

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INTRODUCTION Stimuli-responsive polymers have received considerable attention with regard to their potential applications in the biomedical field in the past few decades.1−3 The stimuli include temperature, light, electric fields, pH, and so on, which can be applied in many biomedical applications, such as the delivery systems of smart drugs and genes, tissue engineering scaffolds, and biological separation.4−12 For synthetic materials applied in the field of drug delivery, polypeptides were commonly practiced in view of their structural resemblance to proteins.13,14 In general, the synthetic polypeptides required some chemical modification steps to construct their hydrophilic functional groups, to enhance their water miscibility, and circulation in vivo.15 The polypeptides are also structurally interesting in view of the fact that they are capable of forming hierarchically ordered structures of α-helices and β-sheets stabilized by intra- and intermolecular H-bonds, respectively.16 Moreover, with these inherent amide groups, the polypeptides tend to proceed with H bond interactions with other molecules, generating new complex systems with the desired properties for study. The formation of hydrogen-bonded interpolymer complex (HIPC) between weak polyacids, such as poly(acrylic acid) (PAA) and poly(methacrylic acid), and proton-accepting polymers, such as poly(ethylene oxide), 17−20 poly(acrylamide),21−23 and poly(vinyl ethers),24,25 has been widely studied in the past four decades. Considerable research studies26,27 had been conducted on various aspects of the complex formation process in solutions and interfaces. In general, in an aqueous solution containing mixtures of such © 2018 American Chemical Society

complementary polymers, the interpolymer association by successive H bonds leads to the formation of compact interpolymer complexes with controlled self-assembled structures. With the unique and extendable properties, many HIPC systems have been investigated28−33 for their potential as a platform to deliver drugs in an efficient and controlled manner. The fluorescence spectrum of the pyrene (Py) molecule contains several vibronic peaks; among them, the intensity ratio34,35 between the first (373 nm) and the third (383 nm) vibronic peaks I1/I3 was affected by the polarity of the surrounding solvent media. The I1/I3 ratio (so called Py scale) ranges from low, in nonpolar hydrophobic solvents, to high, in polar hydrophilic solvents. Therefore, this ratio provides a scale for assessing the hydrophobicity of the environment surrounding the Py molecules. The environmental polarity (or hydrophobicity) change upon micelles formation had been accurately predicted through the use of the Py scale. Besides, the large environmental hydrophobicity change during the HIPC formation, which involves conversion from two hydrophilic polymers to a more hydrophobic complex, also facilitated the use of the Py scale in measuring several properties of HIPC.36−44 In this study, a Py-terminated, amphiphilic polypeptide PPM was prepared through multiple synthetic steps (Scheme 1) and was used as a proton-acceptor to H bond with PAA, forming the PPM/PAA HIPC system for investigation. For both PPM Received: January 21, 2018 Accepted: March 29, 2018 Published: April 24, 2018 4423

DOI: 10.1021/acsomega.8b00124 ACS Omega 2018, 3, 4423−4432

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the hydrophilic methoxy-bis(ethylene oxide) units into the side chains of PPM. The synthesis of Py-NH2 required three steps, starting from the first substitution reaction of Py-OH to yield Py-Br, followed by another substitution reaction to produce PyPh, which was then deprotected by hydrazine to yield the desired Py-NH 2 . MEO2 -N3 was prepared through the substitution reaction between sodium azide (NaN3) and MEO2-OTs, which were prepared from the reaction of MEO2 with tosyl chloride. The involved intermediates were carefully identified by proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIRS, Figures S1−S3, Supporting Information). The characterization of PPM was discussed as follows. The successful click reaction between Py-PPLG and MEO2N3 led to the desired PPM, and the success of the reaction can be verified by the comparison of the FTIR spectra of MEO2-N3, Py-PPLG, and PPM (Figure S4, Supporting Information). The alkyne group of Py-PPLG, with the absorption peak at 2130 cm−1, and the azido group of MEO2-N3, absorbing at 2105 cm−1, are the two functional groups involved in the click reaction. After the click reaction, the spectrum of PPM exhibited no sign of these two absorptions, which indicates that the click reaction must be driven to complete. The resonance peaks of PPM and Py-PPLG were assigned in the 1H NMR (Figure 1). Among the resolved resonances, the

Scheme 1. Syntheses of Initiator Py-NH2, MEO2-N3, and the Ring-Opening Polymerization (ROP) of the Monomer PLGNCA by Py-NH2 and the Following Click Reaction To Obtain PPM

and PPM/PAA employed as the delivery systems of the cancer drug doxorubicin (DOX), the deliberately implanted Py terminal of PPM is the focus of this study. Through analyzing the emission spectrum of the Py terminal, two main research goals can be achieved: first, the critical concentrations of both the systems can be determined. Besides the traditional approach of using the I1/I3 ratio in locating the critical concentration, ΦF of the Py terminal was also effective in characterizing the critical concentration. Second, the DOXdelivery behavior can also be evaluated from the resolved I1/I3 ratio and ΦF. The loading amounts of DOX in both the systems were found to be correlated with the I1/I3 ratio and ΦF, and more interestingly, the DOX-releasing profiles are found to be well-correlated with the I1/I3 curves. The facile association between the Py terminal and the incoming DOX is responsible for this interesting phenomenon, which has never been explored previously and provides a convenient route for accessing the DOX-releasing behavior. Preparation effort as well as different instrumentation analyses were conducted with the purpose to offer new meaning of the traditional I1/I3 ratio with an intention to offer the traditional I1/I3 ratio new application.

Figure 1. 1H NMR spectra of (A) PPM and (B) Py-PPLG (CD2Cl2/ CF3COOD = 1/1 (v/v)).

aromatic protons (Hh of Py-PLG and Hm of PPM) of the Py terminal resonate as weak multiplets in the downfield range from 8.0 to 8.4 ppm. The resonance of the methylene protons (Hc of Py-PLG and Hh for PPM) attached to the α-carbon of the ester side group is a singlet at 2.75 ppm. The integration ratio between the resonances of the aromatic Py protons and the methylene protons can be used to calculate the number average molecular weight (Mn) of PPM and Py-PLG. The resultant Mn’s are 2850 g/mol for Py-PPLG and 5270 g/mol for PPM, which are in good correlation with the values (2900 g/ mol for Py-PPLG and 5100 g/mol for PPM, Figure S5, Supporting Information) determined from matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. These results are nevertheless inconsistent with the gel permeation chromatography (GPC) results (4960 g/ mol for Py-PPLG and 9930 g/mol for PPM, Figure S6). As we

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RESULTS AND DISCUSSION Synthesis of an Amphiphilic PPM. The hydrophobic Py terminal and the hydrophilic methoxy-bis(ethylene oxide) pendant chains are the units responsible for the amphiphilicity of PPM, which was prepared according to the synthetic steps illustrated in Scheme 1. To prepare PPM, the polypeptide PyPPLG precursor needed to be synthesized first. By using amino-functionalized Py-NH245 as the initiator to initiate ROP of the cyclic peptide monomer PLG-NCA,46 the hydrophobic Py terminal was successfully built into the polypeptide PyPPLG precursor. The subsequent click reaction47−49 between the alkyne side groups of Py-PPLG and MEO2-N343 introduced 4424

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ACS Omega know, the GPC analysis is based on the hydrodynamic volume of the polymer chains in the eluting solvent and gives relative molecular weights in correlation to the polystyrene standard used in the analysis. Mn’s from the 1H NMR analysis are therefore more reliable and are used hereafter to calculate the molar concentration of the PPM solution. Critical Concentrations of the Aqueous PPM and PPM/PAA. Aqueous PPM and PPM/PAA (in 1:1 molar ratio) solutions with concentrations ranging from 5 × 10−5 to 5 × 10−3 M were prepared for the evaluation of the critical concentration. Primarily, a visual inspection immediately tells us the difference between the PPM and PPM/PAA solutions. With different concentrations, the PPM solutions are all transparent, which is in wide contrast to the turbid PPM/ PAA solutions. The results from the dynamic light scattering (DLS) analysis (Figure S9, Supporting Information) indicated that the sizes of the particles formed in both the solution systems are different. For the PPM/PAA solutions, the resolved average hydrodynamic diameters (Dh’s) are in the range from 50 to 500 nm, which are comparatively larger than the detected Dh’s (from 40 to 160 nm) of the PPM solutions. Supposedly, the larger aggregated particles of the PPM/PAA solutions are superior in carrying more amount of drug than the small-sized PPM solutions, a point which will be discussed later in this discussion. The reason why critical concentration can be determined by the I1/I3 value is attributed to the large hydrophobicity jump at the moment the assembled structures formed in the aqueous solution. Instead of the traditional way of using the I1/I3 ratio to probe the critical concentration,34,35 we found that ΦF is another convenient parameter effective in determining the critical concentration. The key behind this is the drastic change of the Py terminals of the PPM polymers at the moment the solution concentration reaches the critical value and is discussed below. In an aqueous solution, the hydrophobic Py terminals of the amphiphilic PPM chains undergo a major morphological change when the concentration of PPM reaches the critical micelles concentration (cmc). In dilute aqueous solution before cmc, the Py terminals of the isolated PPM chains are surrounded by a vast majority of water molecules. The emission of the Py terminal should resemble that of the pure Py molecule. As we know, a pure Py molecule is a transitional fluorophore with the aggregation-caused quenching50−52 (ACQ) property, that is, the aggregated Py molecules emit less efficiently than the isolated Py molecules. In the dilute solution, the isolated Py terminal of PPM is virtually exempted from the detrimental ACQ effect, and therefore, it emitted efficiently (Figure 2A). In contrast, at concentrations higher than cmc, the amphiphilic PPM chains formed micelles consisting of an inner hydrophobic core, mainly composed of the aggregated Py terminals, and an outer hydrophilic polypeptide shell. Inside the inner core, the aggregated Py terminals emit less efficiently because of the detrimental ACQ effect. At the critical point that the solution concentration reaches cmc, the aggregation of the Py terminals and the related emission efficiency are subjected to a sudden change. Therefore, ΦF was expected to be effective in the determination of the critical concentration. Adding PAA into the dilute solution of PPM should not change the basic feature of the Py terminals of the PPM chains. In the dilute solution, the Py terminals of the PPM chains are still in the isolated form and are exempted from the detrimental

Figure 2. Emission spectra of the aqueous (A) PPM and (B) PPM/ PAA solutions with different solution concentrations (λex = 340 nm).

ACQ effect. The Py terminals of the PPM chains therefore emitted intensely (Figure 2B). An increase in the solution concentration enhances mutual contacts and the accompanied H bond interactions between the PPM and PAA chains. As the solution concentration reaches the critical aggregation concentration (CAC), interchain H bond interactions between PPM and PAA occur to induce the formation of the interassociated aggregates. The initial hydrophilic functions (i.e., the carboxylic acids of PAA and oxide atoms of PPM) are now participating in the H bond interactions, rendering aggregates with higher hydrophobicity than the PPM and PAA chains in the dilute solution. Within the hydrophobic aggregates, the aromatic Py terminals tend to associate together and therefore emit with an attenuated intensity with the detrimental ACQ effect. In this case, increasing the concentration above CAC should cause a drastic change on ΦF. The corresponding ΦF and I1/I3 values adopted from Figure 2 are illustrated in Figure 3, in which the resolved ΦF and I1/I3 values from the initial dilute solutions are quite constant but decline suddenly after the critical points. Extrapolations of the I1/I3 values resulted in the cmc value of 3.9 × 10−4 M for PPM and a CAC value of 2 × 10−4 M for PPM/PAA, respectively. An analogous extrapolation of ΦF values also gave the same values as those from the I1/I3 ratios, which suggests that ΦF is also effective in locating the critical concentration. To demonstrate the validity of the above values, solution transmittance measurements by the UV−vis spectra were also conducted. Similar to the I1/I3 ratio, the solution transmittances (Figure S7, Supporting Information) of both systems are rather high (∼100%) in dilution solution, but they decrease abruptly when the solution concentration approaches the critical values. Extrapolation of the transmittances resulted in the same cmc 4425

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Table 1. Characteristics of DOX Loading for the PPM and PPM/PAA Systems sample c

PPM/DOX1 PPM/DOX2c PPM/DOX3c PPM/PAA/DOX1d PPM/PAA/DOX2d PPM/PAA/DOX3d PPM/PAA/DOX4d a

DLCa (%)

DLEb (%)

2.0 3.3 3.5 4.3 7.0 10.3 15.7

22 12 7 93 62 47 19

The DLC were calculated using the following formula: DLC (%) =

b

weight of loaded drug × 100% weight of nanoparticles

The DLE were calculated using the following formula: DLE (%) =

weight of loaded drug × 100% weight of drug in feed

c PPM/DOX1 = 2 × 10−3 M/4 × 10−4 M, PPM/DOX2 = 2 × 10−3 M/6 × 10−4 M, PPM/DOX3 = 2 × 10−3 M/7 × 10−4 M. dPPM/PAA/ DOX1 = 2 × 10−3 M/2 × 10−3M/1.7 × 10−3 M, PPM/PAA/DOX2 = 2 × 10−3 M/2 × 10−3 M/2.8 × 10−3 M, PPM/PAA/DOX3 = 2 × 10−3 M/2 × 10−3 M/4.3 × 10−3 M, PPM/PAA/DOX4 = 2 × 10−3 M/2 × 10−3 M/6.9 × 10−3 M.

terminal and DOX. As anticipated, the emission of the Py molecule decreases with the increase of DOX in the solution (Figure 4A). If the reduced emission is due to the formation of

Figure 3. I1/I3 ratio and quantum yield (ΦF) for the aqueous (A) PPM and (B) PPM/PAA solutions with different solution concentrations (data adapted from Figure 2).

and CAC values with those obtained from I1/I3 and ΦF. The critical concentrations determined from I1/I3 and ΦF are therefore accurate. Characterization of DOX Loading. For drug loading, the concentrations of PPM and PAA were fixed at the same value of 2 × 10−3 M. By loading different amounts of DOX, the PPM/ DOX and PPM/PAA/DOX solutions (Table 1) were prepared for the DOX-loading study. Here, the applied concentrations of PPM (2 × 10−3 M) and PPM/PAA (2 × 10−3 M/2 × 10−3 M ) are all higher than the critical values of the respective DOXloading PPM/DOX and PPM/PAA/DOX solutions. Compared to the PPM solution, the PPM/PAA solution is a better drug-loading system because the PPM/PAA solution can carry more DOX drugs without the early precipitation of DOX from the corresponding solution (the ultimate DOX loads of PPM/ PAA and PPM are 6.9 × 10−3 and 7 × 10−4 M, respectively) during the preparative step. This result can be confirmed from the drug-loading content (DLC) and the drug-loading efficiency (DLE) summarized in Table 1. The resolved DLC and DLE values of the PPM/PAA solutions are all higher than those of the PPM solution. For the PPM/PAA/DOX1 solution, a maximum DLE of 93% can be achieved. The large aggregates formed in the PPM/PAA solutions are capable of carrying more DOX compared to the small micelles formed in the PPM solutions. We found in this study that the Py terminals of PPM can preferably associate with the DOX molecules to form an aggregated species with a varied emission behavior. To demonstrate this, a pure Py molecule was used as a model compound to simulate the possible association between the Py

Figure 4. Emission spectra of (A) Py (10−4 M) with different molar ratios of DOX in N,N-dimethyl formamide (DMF, λex = 340 nm) and (B) DOX (10−4 M) with different molar ratios of Py in DMF (λex = 477 nm). 4426

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ACS Omega the Py/DOX complex, we may expect the same influence of Py on DOX. The result illustrated in Figure 4B indicated that the emission of DOX at a longer wavelength region (>500 nm) also decreases with the increase of Py in the solution. Therefore, the corresponding Py/DOX complex formed is inferior in its emission efficiency than the individual Py and DOX. The preferable formation of the Py/DOX complex is possible in considering the molecular geometry of DOX and Py. The fluorescent part of the DOX molecule is a three-ring, planar hydroxyanthraquinone (HA) unit, which is geometrically allowed to approach and pack intimately with the three-ring, planar Py molecule, rendering a well-packed complex with an inferior emission efficiency. Moreover, the electron-deficient carbonyl functions of the HA unit of DOX should facilitate the interaction with the electron-rich Py molecule. The intimate association between Py and DOX results in a Py/DOX complex with the correlated I1/I3 and ΦF as discussed below. As illustrated in Figure S11, the I1/I3 and ΦF values adopted from Figure 4A decline with the same rate upon the increase of the DOX concentration in the solution. As described above, I1/ I3 depends on the hydrophobicity of the environment surrounding the Py molecules, whereas the extent of aggregation affects ΦF of Py. The progressive lowering of the I1/I3 ratio indicates that the hydrophobic Py/DOX complexes preferably form upon the addition of DOX. Within the complex aggregates formed in the solution, the Py molecules are situated in a more hydrophobic environment than the pure Py molecule. Therefore, increasing the DOX content results in a more hydrophobic Py/DOX complex. Similarly, the progressive lowering of the ΦF value also indicates that the extent of aggregation of the Py/DOX complex is enhanced upon the increase of DOX loaded in the solution. It is difficult to draw a theoretical foundation for the correlated I1/I3 and ΦF because complicated factors are involved in the final outcome of the I1/ I3 and ΦF values. Presently, the correlated I1/I3 and ΦF can be treated as empirical results, which can be used for further exploration of the DOX-delivery behavior. As with the Py molecule, the Py terminal of PPM should preferably react with DOX to result in the emission reduction. Therefore, the emission spectra of the aqueous PPM/DOX (Figure 5A) and PPM/PAA/DOX (Figure 5B) solutions illustrate the anticipated trend that the emission intensity of the PPM (or PPM/PAA) solution decreases as the DOX content increases. The decrease of the emission intensity was reflected in the decrease of ΦF values for the PPM/DOX (Figure 6A) and the PPM/PAA/DOX (Figure 6B) solutions. As with the aforementioned Py/DOX case, an association between DOX and the Py terminal of PPM must occur preferably to result in an attenuated emission. The resolved I1/ I3 ratio also exhibits the same descendent trend with the increase of the DOX content in the solution. A lower I1/I3 ratio refers to a more hydrophobic environment surrounding the Py terminals of PPM in solution. A planar DOX molecule must react with the planar Py terminal to result in hydrophobic Py/ DOX complexes with a lower I1/I3 ratio. As discussed above, the PPM/PAA solution is superior in carrying more DOX, forming aggregates with a larger size than the PPM solution. Because the aggregates formed in the PPPM/PAA solution are capable of carrying more hydrophobic Py/DOX complexes, the resolved I1/I3 ratios of the PPM/ PAA/DOX solutions (Figure 6B) are therefore lower than those (Figure 6A) from the PPM/DOX solution. I1/I3 ratio is

Figure 5. Emission spectra of the aqueous (A) PPM (2 × 10−3 M) and PPM (2 × 10−3 M)/DOX1 (4 × 10−4 M), DOX2 (6 × 10−4 M), DOX3 (7 × 10−4 M) solutions and (B) PPM (2 × 10−3 M)/PAA (2 × 10−3 M) and PPM (2 × 10−3 M)/PAA (2 × 10−3 M)/DOX1 (1.7 × 10−3 M), DOX2 (2.8 × 10−3 M), DOX3 (4.3 × 10−3 M), DOX4 (6.9 × 10−3 M) solutions (λex = 340 nm).

therefore a convenient measure for determining the amounts of DOX loaded in the carriers. In Vitro DOX Release. In studying the in vitro release of DOX, the PPM/DOX1 and PPM/PAA/DOX1 solutions were selected for the measurements conducted in a phosphatebuffered saline (PBS) solution (pH 7.4). Despite the higher DOX content of PPM/PAA/DOX1 as compared to PPM// DOX1 (1.7 × 10−3 vs 4 × 10−4 M, Table 1), PPM/PAA/DOX1 still has a slower release rate according to the resultant release profiles illustrated in Figure 7. Within 24 h, at 37 °C, more than 90% of DOX had been released from the PPM/DOX1 solution. In contrast, only 70% of DOX was released from the PPM/ PAA/DOX solution after 24 h. As a reverse process of the DOX-loading process, the release of DOX should result in an emission enhancement, as against the emission reduction (Figure 5) observed in the DOXloading study. The emission results in Figure 8 indeed illustrated the expected trend that the emission of PPM/ DOX1 and PPM/PAA/DOX1 gradually increases over the period of 48 h. As more DOXs were released from the carriers over time, higher I1/I3 and ΦF were resolved for both PPM/ DOX1 (Figure 9A) and PPM/PAA/DOX1 (Figure 9B) solutions. Again, as with the small-mass Py/DOX system, the correlation between I1/I3 and ΦF is quite good for both the polymeric systems. In contrast to the ΦF value, the I1/I3 ratio was found to be useful in assessing the release percentage of DOX, obtained from the UV−vis absorption spectra (cf. Experimental Section). 4427

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Figure 6. (A) I1/I3 ratio and ΦF value of the aqueous PPM (2 × 10−3 M) with different concentrations of DOX and (B) PPM (2 × 10−3 M)/PAA (2 × 10−3 M) with different concentrations of DOX.

Figure 8. Emission spectra of the aqueous (A) PPM/DOX1 (PPM/ DOX = 2 × 10−3 M/4 × 10−4 M) and (B) PPM/PAA/DOX1 (PPM/ PAA/DOX = 2 × 10−3 M/2 × 10−3 M/1.7 × 10−3 M) solutions during the DOX release at 37 °C (λex = 340 nm).

Figure 7. Release profile of DOX (solid lines) and the I1/I3 (dashed lines) values adapted from the emission spectra for the aqueous PPM/ DOX1 solution (PPM/DOX = 2 × 10−3 M/4 × 10−4 M) and the aqueous PPM/PAA/DOX1 solution (PPM/PAA/DOX = 2 × 10−3 M/2 × 10−3 M/1.7 × 10−3 M) in PBS (pH 7.4) at 37 °C.

Figure 9. I1/I3 ratio and ΦF values during the release of DOX from the aqueous PPM/DOX1 (PPM/DOX = 2 × 10−3 M/4 × 10−4 M) and PPM/PAA/DOX1 (PPM/PAA/DOX = 2 × 10−3 M/2 × 10−3 M/1.7 × 10−3 M) solutions at 37 °C.

Interestingly, the correlation between I1/I3 and the cumulative release percentage is rather high as we inspected the I1/I3 data (dashed line) included in Figure 8. The use of the I1/I3 ratio in determining the DOX-releasing process has never been attempted previously and is rather convenient as it only required an in situ emission measurement. Here, the consistence between the I1/I3 ratio and the cumulative release percentage is due to the sensitive hydrophobicity change involved in the dissociation of the Py/DOX complex during the release course. As discussed above, within the aggregated Py/ DOX domains, the incorporated DOX drugs contribute to the hydrophobic environment surrounding the Py terminals, and

the detachment of DOX from the Py/DOX aggregates should result in the lowering of the hydrophobicity and the corresponding increase in the I1/I3 ratio. The I1/I3 ratio of the Py/DOX complex must be rather sensitive to the environmental hydrophobicity change to have a good correlation with the DOX-release profile from the UV−vis absorbance measurements.

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CONCLUSIONS An amphiphilic polypeptide PPM was successfully prepared by ROP of the peptide monomer PLG-NCA in the presence of the 4428

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Calcd: C, 83.09; H, 4.18; N, 3.88. Found: C, 83.15; H, 4.23; N, 3.83. Preparation of Py-NH2. A solution of Py-Ph (0.3 g, 0.83 mmol) and hydrazine hydrate (0.268 mL, 5.5 mmol) in ethanol (10 mL) was stirred at 65 °C for 24 h under argon. The resultant precipitate was filtered off and washed several times with ethanol. The solid was then dried under vacuum to obtain the final product (0.22 g, 73%). 1H NMR (500 MHz, DMSOd6): δ 8.4−7.8 (m, 9H), 4.49 (s, 2H, Ha) (Figure S2); Anal. Calcd: C, 88.28; H, 5.67; N, 6.06. Found: C, 88.19; H, 5.74; N, 6.02. Synthesis of 1-(2-Methoxyethoxy)-2-azidoethane (MEO2-N3). Into an ice-cooled solution of MEO2 (18.3 mL, 150 mmol) and tosyl chloride (21.2 mL, 180 mmol) in THF (200 mL), triethylamine (25 mL, 180 mmol) was added dropwise over a period of 20 min. The solution mixture was then gradually warmed to room temperature and stirred overnight. The resultant solution was diluted with water (200 mL), and the pH of the resultant solution was adjusted to be neutral by adding NaHCO3. Sodium azide (11.8 g, 180 mmol) was then added, and the reaction mixtures were refluxed overnight. After cooling to room temperature, the mixtures were extracted by Et2O. The combined organic phase was dried over MgSO4, filtered, and evaporated to obtain a yellow oil. The final product was obtained by eluting from CH2Cl2/ methanol (v/v = 15/1). 1H NMR (500 MHz, CDCl3): δ 2.89− 2.80 (m, 6H, Hb,c,d), 2.72 (s, 3H, Ha), 2.32 (t, 2H, He) (Figure S3). ROP of PLG-NCA to Prepare Py-PPLG. The solution of PLG-NCA (5.612 g, 26.57 mmol) in dry DMF (25 mL) was stirred and bubbled with argon for 30 min before the solution of Py-NH2 (0.03 g, 0.13 mmol) in DMF (1 mL) was injected through a syringe. The reaction mixture was stirred for 2 days at room temperature, and the resultant polymer was precipitated from Et2O and dried in a vacuum oven. 1H NMR (500 MHz, CD2Cl2 + CF3COOD (1/1, v/v)): δ 8.8 (broad, Hf), 8.0−8.4 (m, 9H, Hh), 4.8−4.9 (s, 3H, Hb,e), 4.5 (broad, 2H, Hg), 2.7− 2.8 (broad, 2H, Hc), 2.6 (s, 1H, Ha), 2.1−2.5 (broad, 2H, Hd) (Figure 1). MALDI-TOF: Mn 2900 g/mol, Mw 3270 g/mol, polydispersity 1.12 (Figure S6); GPC (DMF): Mn 4960 g/mol, Mw 6150 g/mol, polydispersity 1.23 (Figure S7). Preparation of PPM by Click Reaction. The solution of Py-PPLG (1 g, 0.37 mmol) and CuBr (0.1 g, 0.68 mmol) in dry DMF (30 mL) was subjected to three freeze/pump/thaw cycles before the addition of MEO2-N3 (3 g, 20 mmol). Three more freeze/pump/thaw cycles of the resultant mixtures were further conducted before PMDETA (0.3 mL, 1.73 mmol) was added through a syringe. The reaction mixtures were then carefully degassed through three freeze/pump/thaw cycles and were heated at 60 °C for 24 h. After cooled to room temperature, the resultant solution mixtures in DMF were passed through a neutral alumina column to remove the copper catalysts. The eluent was concentrated and precipitated from Et2O. The resultant solid was filtered off and dried under vacuum at room temperature to obtain the final product. 1H NMR (500 MHz, CD2Cl2 + CF3COOD (1/1, v/v)): δ 8.8 (broad, Hk), 8.6 (s, 1H, Hf), 8.0−8.4 (m, 9H, Hm), 5.5−5.6 (broad, 2H, Hg), 5.0 (s, 2H, He), 4.8 (s, 1H, Hj), 4.5 (s, 2H, Hl), 4.15 (s, 2H, Hd), 3.9 (s, 4H, Hb,c), 3.65 (s, 3H, Ha), 2.75 (broad, 2H, Hh), 2.1−2.5 (broad, 2H, Hi) (Figure 1). MALDI-TOF: Mn 5199 g/mol, Mw 5780 g/mol, polydispersity 1.13 (Figure S6); GPC (DMF): Mn 9930 g/mol, Mw 12 900 g/mol, polydispersity 1.30 (Figure S7).

Py-NH2 initiator. PPM was then mixed with PAA to form the PPM/PAA HIPC system. The Py terminal of PPM in this study provides a convenient measure for determining the critical concentration of the aqueous PPM and PPM/PAA solutions. The consistent results from the I1/I3 ratio and ΦF of the Py terminals gave the same critical concentration. The H bond interaction between PPM and PAA enhances the formation of aggregates with particle size larger than that of PPM. The larger particles formed in the PPM/PAA solution also help loading more DOX drug compared to the smaller micelles formed in the PPM solution. Because of the preferable formation of the hydrophobic DOX/Py aggregates, the I1/I3 ratio and ΦF of the Py terminal can be used to determine the amounts of DOX loaded in the aqueous PPM and PPM/PAA solutions. The DOX loading decreases the I1/I3 and ΦF values; therefore, the reverse DOXreleasing process results in the increase of I1/I3 and ΦF. The I1/ I3 and ΦF curves were found to be correlated with the release profiles of DOX in both PPM/DOX1 and PPM/PAA/DOX1 solutions.

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EXPERIMENTAL SECTION Chemicals and Materials. 1-Pyrenemethanol, phthalimide potassium salt, hydrazine hydrate, N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99%), propargyl alcohol (99%), and L-glutamic acid (99%) from Acros; PAA (Mw ≈ 5000) and phosphate buffer solution (1 M, pH 7.4) from Sigma; and sodium azide (NaN3, 99%) from Alfa were used directly. A solution of DOX hydrochloride (10 mg, 98%) (from Sigma) and triethyl amine (15 mg) in 20 mL of tetrahydrofuran (THF) was gently stirred at room temperature for 24 h to remove HCl. The suspended salt was filtered off before rotary evaporation to remove the residual THF solvent. THF was refluxed over sodium and benzophenone for 2 days before distillation for use. DMF was refluxed and distilled over CaH2 under nitrogen atmosphere to a flask containing alumina before use. Copper bromide (CuBr, 98%) from Aldrich was purified by washing with glacial acetic acid overnight, followed by washing with absolute ethanol and diethyl ether (Et2O), and then dried under vacuum. The monomer PLG-NCA was prepared according to the previous procedures,53,54 and the detailed preparation steps are given in the Supporting Information. The syntheses of Py-NH2, MEO2N3, and the polymer PPM are illustrated in Scheme 1 and detailed as follows: Preparation of Py-Br. A solution of Py-OH (0.5 g, 2.15 mmol) and PBr3 (0.22 mL, 2.36 mmol) in dry THF (5 mL) was stirred at room temperature for 30 min under argon. After filtration, the residue was washed several times with Et2O. The resultant Py-Br was filtered off and dried under vacuum at room temperature (0.4 g, 80%). 1H NMR (500 MHz, dimethyl sulfoxide (DMSO)-d6): δ 8.7−8.0 (m, 9H), 5.04 (s, 2H, Ha) (Figure S2). Preparation of Py-Ph. A solution of Py-Br (0.4 g, 1.35 mmol) and phthalimide potassium salt (0.37 g, 2.03 mmol) in dry DMF (10 mL) was stirred at 110 °C for 24 h under argon. The resultant solution mixtures were concentrated by rotary evaporation and were redissolved in dichloromethane. The whole solution was then washed with aq 10% NaHCO3 solution (2 × 50 mL) and water (2 × 50 mL). The collected organic layer was then distilled off to yield the final solid product (0.35 g, 87.5%). 1H NMR (500 MHz, DMSO-d6): δ 8.6−7.7 (m, 13H, Hpy,b,c), 5.53 (s, 2H, Ha) (Figure S2); Anal. 4429

DOI: 10.1021/acsomega.8b00124 ACS Omega 2018, 3, 4423−4432

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ACS Omega

sulfate standard solution (10 −4 M in 1 N H 2 SO 4 ). Experimentally, ΦF was determined by comparing with a quinine sulfate standard solution (10−4 M in 1 N H2SO4) according to the following equation

Preparation and Characterization of the Aqueous PPM, PPM/PAA, PPM/DOX, and PPM/PAA/DOX Nanoparticles. The aqueous PPM, PPM/PAA, PPM/DOX, and PPM/PAA/DOX solutions were prepared by stepwise additions, with the corresponding solutions thoroughly mixed by stirring for 2 h, of the calculated amounts of PPM, PAA, and DOX in deionized water. The DOX-loaded nanoparticle solutions were then dialyzed against deionized water (100 mL) for 12 h, by using a dialysis bag (MWCO = 1000 g/mol, Orange Scientific), to remove the unloaded DOX. The amounts of DOX loaded into the nanoparticles were determined from an absorbance calibration curve of DOX in deionized water (Figure S10A). The DLC and DLE were calculated using the following formula DLC (%) =

weight of loaded drug × 100% weight of nanoparticles

weight of loaded drug DLE (%) = × 100% weight of drug in feed

⎛ PL ⎞⎛ A ⎞ ΦF = Φreference⎜ S ⎟⎜ R ⎟(nS2 /nR 2) ⎝ PL R ⎠⎝ AS ⎠

where Φ is the quantum yield, PL is the area under the emission peak, A is the absorbance at the excitation wavelength, and n is the refractive index. The subscript R denotes the respective values of the reference quinine sulfate and the subscript S denotes the respective values of the sample. The particle sizes in deionized water were measured by DLS on a Brookhaven 90 plus spectrometer at room temperature. A He−Ne laser operating at 633 nm was used as the light source.

■

(1)

Er (%) =

+ V0Cn

mDOX

× 100%

ASSOCIATED CONTENT

S Supporting Information *

(2)

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00124. FTIR and 1H NMR spectra of small-mass intermediates, Py-PPLG, and PPM; mass spectra, GPC curves, transmittance and I 1/I 3 ratio, and hydrodynamic diameters of PPM and PPM/PAA; absorbance calibration curve of DOX; I1/I3 ratio and ΦF of Py/DOX; and TEM images of PPM, PPM/DOX, PPM/PAA, and PPM/PAA/DOX (PDF)

In Vitro Drug Release. The release behavior of DOX from the carrier solution was performed in 100 mL PBS (pH 7.4, Sigma) at 37 °C. A 5 mL volume of the DOX-loaded solution was sealed in a dialysis bag (MWCO = 1000 g/mol, Orange Scientific). At designated time intervals, 1 mL of the release media was removed for emission spectral analysis, to determine the I1/I3 ratio and ΦE values, and for UV−vis spectra, to determine the amounts of DOX released, by the absorbance calibration curve, in PBS solution at pH 7.4 (Figure S10B). At the same time, an equal volume of fresh media was added to the release system. The cumulative release percentage of DOX was calculated according to the following equation55,56 n−1 Vt∑1 C i

(4)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +886-7-52520004065 (J.-L.H.). ORCID

(3)

Jin-Long Hong: 0000-0003-1066-5818

where mDOX is the amount of DOX encapsulated in the solution particles, V0 is the volume of the release media (V0 = 100 mL), Vt is the volume of the replaced media (Vt = 1 mL), and Cn is the concentration of DOX in the sample. Characterization. The FTIR spectra were recorded with a Bruker Tensor 27 FTIR spectrophotometer; 32 scans were collected at a spectral resolution of 1 cm−1. The solid powders were homogeneously blended with KBr before being pressed to make pellets for measurement. The 1H NMR spectra were recorded by a Varian Unity VXR-500 MHz instrument. The mass spectra were obtained from a Bruker Autoflex III MALDITOF, and DCTB (trans-2-[3-(4-tert-butylphenyl)-2-methyl-2propenylidene]malononitrile) was used as the matrix. The relative molecular weights of the polymers were determined through GPC using the Waters 510 high-performance liquid chromatography system, with DMF as the eluent and at a flow rate of 0.8 mL min−1. The molecular weight calibration curve was obtained from the polystyrene standards. The UV−vis absorption and transmittance spectra were recorded with a JASCO V-770 spectrophotometer. The emission spectra were obtained from a LabGuide X350 fluorescence spectrophotometer using a 450 W Xe lamp as the continuous light source. A small quartz cell with the dimensions of 0.2 × 1.0 × 4.5 cm3 was used to accommodate the solution sample for absorption, transmittance, and emission spectra. ΦF is defined as the ratio of the number of photons emitted to the number of photons absorbed, which was determined by comparing with a quinine

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We appreciate the financial support from the Ministry of Science and Technology, Taiwan, under the contract no. 05B40651-1 MOST105-2221-E-110-091-MY2.

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REFERENCES

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