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Home > Synthesis and Characterization of PLL-alt-PEG Copolymers
Synthesis and Characterization of PLL-alt-PEG Copolymers
The PEG-crosslinked alternating copolymers MPLL-alt-PEG with a multi-armed PLL polyelectrolyte segment were designed and synthesized. As demonstrated in Figure 1, PEZ was firstly synthesized through ring-opening polymerization of ZLL-NCA using PEI (Mw = 0.8 kDa) with eight peripheral primary amines as a macroinitiator [30]. Subsequently, difunctional SCM-PEG-SCM was introduced to crosslink PEZ and then the crosslinked product PEZ-alt-PEG was obtained under optimized crosslinking conditions [28]. The benzyloxycarbonyl protection groups of PEZ were removed in the presence of anisole and methanesulfonic acid to produce the final copolymer MPLL-alt-PEG. It should be mentioned that the MPLL unit in MPLL-alt-PEG is eight-armed and the degree of polymerization of each arm is five, based on the fact that this multi-armed polypeptide was found to have broad-spectrum antibacterial activity and high selectivity from our previous study [16]. Meanwhile, MPLL-alt-PEG was designed and synthesized at a feeding ratio of SCM-PEG-SCM to MPLL at 1:1, since this feeding ratio is high enough for SCM-PEG-SCM to crosslink most of the MPLL unit which can also leave most of the primary amines free for the efficient electrostatic interactions between cationic MPLL segment and anionic drugs or bacterial membranes.
Copolymers PEZ, PEZ-alt-PEG, and MPLL-alt-PEG were characterized by 1H NMR and the results are shown in Figure 2A-C. As shown in Figure 2B, the integration peaks at 3.4-4.0 ppm, which correspond to methylene groups (-OCH2CH2O-) of the PEG segment, were observed after the crosslinking reaction. Using the integration peaks at 3.4-4.0 ppm of the PEG segment versus the peaks at 1.0-2.0 ppm corresponding to methylene groups of the PEZ (or MPLL) block (-CH2 CH2 CH2CH2NH-), the estimated molar ratios of PEG to PEZ (or MPLL) segments in PEZ-alf-PEG (or MPLL-alt-PEG) were found very close to their feeding ratios (Table 1). Comparing Figure 2C with 2B, the negligible residual benzyl signal at 7.3-7.5 ppm indicated that benzyloxycarbonyl groups were completely removed. These results indicated that the SCM-PEG-SCM crosslinking reaction and benzyloxycarbonyl deprotection of PEZ have proceeded in a controlled manner and MPLL-a-PEG was successfully synthesized.
The molecular weights of PEZ and PEZ-alt-PEG were measured by GPC in DMF using PMMA as the standard, while the molecular weight of MPLL-alf-PEG was determined in a HAc-NaAc buffer (pH of 4.5) using PEG as the standard. As shown in Table 1, the number average molecular weight (Mw) of PEZ is 6.3 kDa, while the Mw of PEZ-alt-PEG increased to 14.3 kDa. After the benzyloxycarbonyl protecting groups were removed, the Mw of the product was decreased to 8.5 kDa. All these results further confirmed the successful synthesis of MPLL-alt-PEG.
Preparation and Characterization of drug-loaded PIC Micelles
To evaluate the feasibility of MPLL-alt-PEG as a drug carrier, the drug-loading capacity, and self-assembly of the copolymers were firstly investigated using methyl orange, an anionic water-soluble dye, as the model compound of small molecule drugs. It was expected that the primary amino groups of the MPLL segment would be protonated at pH 7.4 and carried a positive charge, thus electrostatically entrapped anionic drugs and subsequently self-assembled into PIC micelles with an MPLL/drug polyion complex core and crosslinked PEG outer shell (see illustration in Scheme 1). It revealed that MPLL-alt-PEG exhibited a high MO loading capacity of up to 52.4% ± 0.4%.
As o versatile cytokine family, type I IFNs including IFN-a and IFN-|3 are nowadays acting as promising therapeutic drugs with antibacterial, antiviral, antitumor, and immunomodulatory activities [33,34], Approved by FDA for clinical treatment, type I IFNs exert definite therapeutic effects on malignant tumor, chronic hepatitis B, hepatitis C, chronic myelogenous leukemia, and other diseases [35,36]. There fore, recombinant human interferon-a-2b was chosen in our study as a model therapeutic protein for its potential in treating multifactorial diseases. In the present study, the loading capacity and encapsulation efficiency of IFN in MPLL-alt-PEG micelles were determined to be 16.2% ± 0.3% and 81.0% ± 0.2%, respectively. TEM me a sure events showed that the drug-loaded micelles adopted spherical morphologies (Figure 3A), and dynamic light scattering (DLS) measurements (Figure 3b and Table 2) indicated that the hydrodynamic diameters of M PLL-alt-PE G increased from 6.2 ± 1.8 to 40-80 nm upon encapsulation of MO or IFN with narrow size distributions (polydispersity index < f.3), indicating that MO or IFN was successfully incorporated into the polymeric micelles. Free MPLL-alt-PEG demonstrated a positive zeta potential in a 0.01 M phosphate buffer (pH 7.4) at 25 °C, while the zeta potentials of both MO-loaded micelles and IFN-loaded micelles approached zero (Table 2), further indicating that anionic molecules were encapsulated into the polymeric micelles via It should be mentioned that since the isoelectric point of most proteins was in the acidic range 4-6 [37], PLL-based polymers are effective for pH-responsive protein release [24]. At physiological pH, IFN molecules (isoelectric point 5.9) carrying net multiple negative charges interact electrostatically with the cationic amino termini of the MPLL segment in MPLL-alt-PEG. Once IFN-loaded micelles are transferred to an acidic microenvironment, such as infectious site [38], the charging state of IFN will be changed from net negative to net positive, while the amine side-chains of MPLL remain positively charged. Therefore, the charge switching of loaded proteins may weaken the electrostatic interactions with the MPLL-alt-PEG host, inducing accelerated drug release. As shown in Figure 4, the release rate of IFN from the copolymer at an acidic environment was much higher than that at pH 7.4, showing a pH-sensitive release behavior. The release rate of IFN was faster at pH 4.5 than that at pH 5.5, which further indicated that the disassembly process of IFN/MPLL-alt-PEG micelles was dependent on the acidity of the microenvironment. The result suggested that, in the area with more bacteria and their metabolites, antimicrobial MPLL-alt-PEG and The antimicrobial activities of MPLL-alt-PEG against Gram-positive bacteria MRSA and Gram-negative bacteria P. aeruginosa were evaluated using MPLL as a control. MIC assays were conducted to determine the minimum drug concentration that inhibited bacterial growth. Figure 5A, B demonstrated the optical density of MRSA and P. Perugino in MHB after the cells were treated with different concentrations of MPLL and MPLL-alt-PEG copolymers for 18 h. It was found that the growth inhibition of both bacterial strains was dose-dependent and the antimicrobial activity increased with increasing polymer concentration. As shown in Figure 5A, B, MPLG-tilt-PEG showed MIC values of approximately 3.8 and 7.5 卩M against MRSA and P. aeruginosa, respectively. In contrast, the MICs of MPLL were 7.5 and 15.0 卩M, respectively. These results indicated that crosslinking the low-molecular-weight MPLL with PEG realized a higher antimicrobial activity. In this study, P aeruginous.osa is harder to inhibit than MRSA, which may probably ascribe to the presence of an outer membrane, fewer anionic phospholipids on the membrane, and additional defense mechanisms that might be absent in Gram-positive bacteria [15,16]. It should be mentioned that, though the encapsulation of TFN may shield the amino groups of MPLL-alt-PEG from bidding with anionic bacterial membranes, the pH-sensitive release behavior of IFN/MPLL-alt-PEG enable the polymer to be quickly liberated for binding bacteria at an acidic infectious microenvironment and thus exhibiting antimicrobial activity.
In addition to the high anti-bacterial activity, enormous selectivity for pathogens over mammal cells is also necessary to construct a potential antimicrobial agent. Siscr hemolysis is the most identified side effect of antimicrobial peptides and polymers [39], the hemolytic activity of the MPLL-based copolymers before and after PEGylation was investigated by incubating them with 5% freshly mouse red blood cells suspension at different concentrations. As shown in Figure 5C, both MPLL and MPLL-alt-PEG copolymers exhibited negligible hemolysis activity (less than 5%) with HC50 values, the concentration at which 50% of red blood cells are lysed, greater than 1000 卩g，mL-1. The HC50 values of both copolymers were obviously greater than those of other natural and synthetic polypeptides [40,41], indicating a high degree of selectivity for microbes over mammal red blood cells.
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