Article Cite This: Macromolecules XXXX, XXX, XXX−XXX
Solution Properties of Polysarcosine: From Absolute and Relative Molar Mass Determinations to Complement Activation Benjamin Weber,† Alexander Birke,† Karl Fischer,‡ Manfred Schmidt,*,‡ and Matthias Barz*,† †
Institute of Organic Chemistry, Johannes Gutenberg University Mainz, Duesbergweg 10-14, 55128 Mainz, Germany Institute of Physical Chemistry, Johannes Gutenberg University Mainz, Jakob Welder Weg 11, 55128 Mainz, Germany
‡
S Supporting Information *
ABSTRACT: Polysarcosine (pSar) was one of the first polymers synthesized in a controlled living manner, but it was only recently when it was reconsidered as a promising alternative for poly(ethylene glycol) (PEG) in biomedical applications. Despite receiving more and more attention, very little is known about the solution properties of pSar, such as coil dimensions and thermodynamic interactions. In this article, we report on these properties of pSar with degrees of polymerization 50 < Xn < 400 that were prepared by controlled living ring-opening polymerization. The polymers are characterized by gel permeation chromatography (GPC), MALDI-TOF mass spectrometry, dynamic and static light scattering (SLS), and viscometry. The chain stiffness of pSar in PBS in terms of the Kuhn statistical segment length, lk, was estimated to lk = 1.5 nm by application of the Yamakawa−Fujii wormlike chain theory to the experimentally determined hydrodynamic radii, Rh, thus being higher than lk = 1.1 nm for PEG in PBS. Also, the second virial coefficients, A2, of pSar and PEG in PBS were similar and reflect their good solubility in aqueous solution. While the universal calibration of GPC elution volumes failed for pSar in HFIP utilizing PMMA standards, it worked better in PBS buffer with PEG standards. Alternatively, an Rh−Mw relation is established in the present work, which enables the determination of molar masses of pSar by simple DLS measurements. In addition, it is demonstrated that pSar independent from its chain length (50 < Xn < 400) does not induce any detectable complement activation (C5a) in human serum.
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and architecture.12 Additionally, Ling and co-workers have reported the use of α-amino acid N-thiocarboxyanhydrides (NTA), which show higher storage stabilities but reduced reactivity in amine-initiated ring-opening polymerization.13,14 Comprehensive overviews on polypeptoid synthesis and initial applications may be found in recent reviews.10,12,15−17 pSar possesses comparable properties to PEG, being a nonionic, hydrophilic polymer with exclusive H-bond acceptor properties and thus strictly following Whitesides’ rules for protein resistant materials.18−20 The Kimura lab and our group have demonstrated the use of pSar as a hydrophilic “stealth”like polymer in various applications, e.g. polymeric micelles21−23 and polyplexes24,25 for imaging and therapy. It was shown in several studies that pSar reduces protein-induced aggregation of nanoparticles in solution,24,26−30 as well as in human blood serum,31 and that it suppresses complement activation.26 In addition, protein resistant surfaces may be obtained grafting pSar from and onto surfaces.19,32,33 These properties should ultimately lead to long circulating nanoparticles in in vivo experiments. In contrast to PEG, pSar is based on the endogenous amino acid sarcosine, which occurs
INTRODUCTION Already more than 100 years ago in the beginning of the past century, Herrman Leuchs discovered α-amino acid-N-carboxyanhydrides (NCA) (Leuchs’ anhydrides).1−3 While the NCA polymerization can be performed with primary amines under controlled living conditions until degrees of polymerization of ∼100, transition metal catalysts,3−5 trimethylsilanes,6 ammonium salts,7,8 or using high-vacuum techniques (HVT)2 enable a controlled living polymerization over an even broader range of chain lengths, since the most common side reaction, the activated monomer mechanism (AMM), is suppressed. Thus, whenever the polypeptide occurs exclusively in one solution conformation, e.g., in random coil or α-helix conformation, polypeptides with low dispersities may be obtained and characterized by standard polymer characterization techniques.9 In contrast to α-amino acid NCA, N-substituted glycine NCAs (NNCAs), e.g., sarcosine NCA (N-methylglycine NCA), are intrinsically unable to undergo the AMM, since the acidic N−H proton is simply absent. 10 This directly explains why polypeptoids can be prepared using primary amines under controlled living conditions as first reported by Sisido et al. in 1977.11 Luxenhofer and co-workers have performed detailed studies on polymerization of NNCAs, underlining the controlled living character of the polymerization, which provides precise control over polymer length, block sequence, © XXXX American Chemical Society
Received: February 9, 2018 Revised: March 17, 2018
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DOI: 10.1021/acs.macromol.8b00258 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules
washing bottles filled with aqueous NaOH solution. The solvent was evaporated under reduced pressure, yielding a brownish oil as crude reaction product. The oil was dried under reduced pressure (1 × 10−3 mbar for 2 h) to obtain an amorphous solid, free of phosgene and HCl, confirmed by testing against silver nitrate solution. The crude product was redissolved in 40 mL of THF and precipitated with 300 mL of dry hexane. The solution was cooled to −18 °C overnight to complete precipitation. The solid was filtered under dry nitrogen atmosphere and dried in a stream of dry nitrogen for 60−90 min and afterward in high vacuum for 2 h in the sublimation apparatus. The crude product was sublimated at 85 °C and 1 × 10−3 mbar. The product was collected from the sublimation apparatus in a glovebox on the same day. The purified product (110 mmol, 65% yield, colorless crystallites; melting point: 102−104 °C (lit.: 102−105 °C)) was stored in a Schlenk tube at −80 °C and only handled in a glovebox. 1H NMR (300 MHz, CDCl3): δ/ppm = 4.22 (2 H, s, −CH2−CO−), 2.86 (3 H, s, −CH3). Synthesis of P1−P3. Sar-NCA was transferred under nitrogen counter flow into a predried Schlenk tube equipped with a stir bar and again dried in high vacuum for 1 h. Then the NCA was dissolved in dry DMF to yield a solution of 100 mg mL−1 with respect to the NCA. 1/n equivalent of neopentylamine was added to this solution. The solution was stirred at room temperature and kept at a constant pressure of 1.25 bar of dry nitrogen via the Schlenk line to prevent impurities from entering the reaction vessel while allowing CO2 to escape. Completion of the reaction was confirmed by IR spectroscopy (disappearance of the NCA peaks (1853 and 1786 cm−1)). Directly after completion of the reaction, the polymer was precipitated to cold ether and centrifuged (4500 rpm at 4 °C for 15 min). After discarding the liquid fraction, new ether was added, and the polymer was resuspended in a sonic bath. The suspension was centrifuged again, and the procedure was repeated. After DMF removal by the resuspension steps, the polymer was dissolved in water and lyophilized, obtaining a stiff, porous mass of polymer. Synthesis of P4. Sar-NCA was transferred under nitrogen counterflow into a predried Schlenk tube equipped with a stir bar and again dried in high vacuum for 1 h. Then the NCA was dissolved in dry DMF to yield a solution of 100 mg mL−1 with respect to the NCA. 1/100 equiv of neopentylamine was added to this solution. The solution was stirred at room temperature and kept at a constant pressure of 1.25 bar of dry nitrogen via the Schlenk line to prevent impurities from entering the reaction vessel while allowing CO2 to escape. Completion of the reaction was confirmed by IR spectroscopy (disappearance of the NCA peaks (1853 and 1786 cm−1)). Directly after completion of the reaction, the solution was evacuated for 20 min, evaporated solvent was refilled, and another 100 equiv with respect to the initiator was added. After repeating this procedure (4 × 100 in total), the polymer was precipitated to cold ether and centrifuged (4500 rpm at 4 °C for 15 min). After discarding the liquid fraction, new ether was added, and the polymer was resuspended in a sonic bath. The suspension was centrifuged again, and the procedure was repeated. After DMF removal by the resuspension steps, the polymer was dissolved in water and lyophilized, obtaining a stiff, porous mass of polymer. Acetylation of Polysarcosine. Polysarcosine was acetylated to circumvent influences of a positively charged chain end as it has been reported in literature.45 Hence, to a solution of pSar in DMF 10 equiv (with respect to the end group) of triethylamine was added. The solution was allowed to stir for 30 min before adding 5 equiv of acetic anhydride. The reaction mixture was stirred at room temperature overnight and precipitated in cold ether as it was described above. After dialysis and lyophilization a fluffy white powder was obtained. 1H NMR (400 MHz, DMSO): δ/ppm = 4.49−3.79 (2n H, br, −CH2− CO−), 3.05−2.64 (3n H, br, −CH3), 0.90−0.77 (9 H, br, −C− (CH3)3). MALDI-TOF Mass Spectroscopy. MALDI-TOF mass spectra46 were recorded using a Bruker (Billerica, MA) Reflex II MALDI-TOF mass spectrometer equipped with a 337 nm N2 laser. Acceleration of the ions was performed with pulsed ion extraction (PIE, Bruker) at a voltage of 20 kV. The analyzer was operated in reflection mode, and
naturally in our body and takes part in the glycine metabolism.34 Although pSar is currently used in several applications,35 very little is known about its solution properties. While Fetsch and Luxenhofer have studied thermal properties of aliphatic polypeptoids, including polysarcosine,36 indicating that polysarcosine is an amorphous polymer, Schlaad and co-workers have reported not only on the solution self-assembly of pSar containing polypeptoids but also on thermal solution properties, e.g., cloud points of polypeptoids.37,38 In addition, Zhang and co-workers were able to tune the cloud points of polypeptoids through copolymerization and a conformational change of the polymer,39 whereas the group of Ling reported on tunable cloud points of pSar-containing copolymers.40 A detailed study on solution properties of pSar itself, however, has not yet been reported in the literature. In addition, besides cytotoxicity very little is known about the biocompatibility of pSar.29 While pSar-based micelles showed enhanced circulation times in mice and the biocompatibility of immunogenic proteins can be improved by conjugation of pSar,41 studies on complement activation induced by pSar in relation to the degree of polymerization have not yet been reported in the literature. Hence in this work, we report on the solution properties of polysarcosine comprising 50 up to 400 repeat units and investigate their acute immunogenicity (complement activation). The selected chain length regime seems to be particularly interesting because pSar3500 (DP = 49) approximately corresponds to PEG2000 (DP = 45) and pSar28000 (DP = 395) to PEG20000 (DP = 454) in terms of number of repeat units.42 These PEGs are commonly used in the design of drug delivery systems, imaging probes, or “theranostics” as protein resistant materials.43,44 In this study pSar polymers are characterized by gel permeation chromatography (GPC), MALDI-TOF mass spectrometry (MALDI-TOF), viscometry, and dynamic (DLS) and static light scattering (SLS) in order to derive an universal relationship between hydrodynamic radius and mass for a fast and accurate determination of molecular weights.
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EXPERIMENTAL SECTION
Materials and Methods. n-Hexane was distilled from Na/K and ethyl acetate from CaH2. Dimethylformamide (DMF) was purchased from Acros and dried over BaO and molecular sieves (3 Å), fractionally distilled under vacuum at 40 °C, and stored at −80 °C under the exclusion of light. Prior to use, DMF was degassed in vacuum to remove traces of dimethylamine. Hexafluoroisopropanol (HFIP) was purchased from Fluorochem. Diphosgene was purchased from Alfa Aesar, and deuterated solvents were from Deutero GmbH. Other chemicals were purchased from Sigma-Aldrich and used as received unless otherwise stated. 1 H NMR spectra were recorded on a Bruker AC 400 at a frequency of 400 MHz. Two-dimensional NMR spectra as 1H DOSY were recorded on a Bruker Avance III HD 400 at 400 MHz. All spectra were recorded at room temperature (25 °C) and calibrated using the solvent signals. Melting points were measured using a Mettler FP62 melting point apparatus at a heating rate of 2.5 °C min−1. Synthesis of Sarcosine N-Carboxyanhydride. The synthesis of sarcosine NCA was adapted from literature and modified.45 A total of 14.92 g (167.4 mmol) of sarcosine, dried in vacuo for 1 h, was weighed into a predried, three-neck, round-bottom flask. A total of 300 mL of absolute THF was added under a steady flow of nitrogen, and 16.2 mL (134 mmol) of diphosgene was added slowly via syringe, and the nitrogen stream was reduced. The colorless suspension was mildly refluxed for 3 h, yielding a clear solution. Afterward, a steady flow of dry nitrogen was led through the solution for another 3 h into two gas B
DOI: 10.1021/acs.macromol.8b00258 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules Scheme 1. Synthesis of Polysarcosine Starting from the Amino Acid
Figure 1. Standard polymer analysis of (A) HFIP-GPC of P1 (blue), P2 (red), P3 (green), and P4 (yellow). (B) 1H NMR analysis of P1 (blue), P2 (red), P3 (green), and P4 (yellow). dn/dc Measurements. The dn/dc measurements were performed with a self-built Michael interferometer at 20 °C using a laser wavelength λ = 632.8 nm with seven different concentrations in typical concentration range from 0.1 to 3 g/L. The results for the pSar samples in PBS are given in Table 3. The significantly smaller value for sample P4 is theoretically not expected, the most obvious explanation being contamination by a solvent with a low refractive index or other impurities. Repeated measurements gave the same values within 3%. Neither NMR (Figure 2) nor thermogravimetric analyses (see Figure S3) of all samples provided any evidence for traces of solvent or other impurities, which strongly suggests that the smaller dn/dc value of P4 is not an artifact but rather a polymer-related property as will be discussed below. Capillary Viscometry. The experiments were performed at 20 °C using Ubbelohde capillary viscometers for dilution sequences of type Oc with a capillary diameter of 0.46 mm, in combination with AVS310 (Schott, Mainz, Germany). Hagenbach corrections were applied. The viscometric measurements were carried out in DPBS (1X): Dulbecco’s Phosphate Buffered Saline, no calcium, no magnesium; Gibco by Life Technologies. All samples were freed of dust by means of PTFE filters with a pore diameter of 5 μm (Carl Roth GmbH, Germany). HFIP-GPC. Hexafluoroisopropanol (HFIP) gel permeation chromatography (GPC) was performed with HFIP containing 3 g/L potassium trifluoroacetate as eluent at 40 °C and a flow rate of 0.8 mL min−1. The columns were packed with modified silica (PFG columns; particle size: 7 μm, porosity: 100 and 1000 Å). Polymer was detected with a refractive index detector (G 1362A RID, JASCO) and a UV/vis detector (UV-2075 Plus, JASCO). Molecular weights were calculated using a calibration performed with PMMA standards (Polymer Standards Services GmbH) and toluene as internal standard. Elution diagrams were analyzed using WinGPC UniChrome 8.00 (Build 994) software from Polymer Standards Services. Aqueous GPC. Aqueous gel permeation chromatography (GPC) was performed with water containing 100 mM phosphate buffered saline as eluent and a flow rate of 1.0 mL min−1. The columns were packed with HEMA (HEMA Bio columns; particle size: 10 μm, porosity: 40, 100, and 1000 Å). Polymer was detected with a refractive index detector (Agilent Technologies 1260 infinity) and a UV/vis detector (Agilent Technologies 1260 infinity). Molecular weights were calculated using a calibration performed with PEG standards (Polymer Standards Services GmbH). Elution diagrams were analyzed using Agilent Technologies software.
the ions were detected using a microchannel plate detector. Mass spectra were processed by the X-TOF 5.1.0 software (Bruker, Billerica, MA). A solvent-free sample preparation was performed using trans-2[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) as the matrix and sodium trifluoroacetate as the cationizing salt. Calibration was carried out using a C60/C70 fullerene mixture. Infrared (IR) spectroscopy was performed on a Jasco FT/IR-4100 with an ATR sampling accessory (MIRacle, Pike Technologies), and Spectra Manager 2.0 (Jasco (Gross-Umstadt, Germany)) was used for integration. Dynamic Light Scattering. Polymer solutions were dissolved overnight to yield a solution of 10 mg/mL in PBS (137 mM NaCl, 2.7 mM KCl, 12 mM HPO42− and H2PO4−). After transfer to a dust-free flow box, all samples were filtered (Millipore GHP 0.2 μm) into dustfree cylindrical scattering cells (Suprasil, 20 mm diameter, Hellma, Mühlheim, Germany). Dynamic light scattering measurements (DLS) were performed using a Uniphase He/Ne laser (l = 632.8 nm, 22 mW), a ALV-SP125 goniometer, a ALV/High QE APD-Avalanche photodiode with fiber optical detection, a ALV 5000/E/PCIcorrelator, and a Lauda RC-6 thermostat unit at 20 °C. Angular dependent measurements of typically 15° steps were carried out in the range 30° ≤ q ≤ 150°. For data evaluation experimental intensity correlation functions were transformed into amplitude correlation functions applying the Siegert relation extended to include negative values after baseline subtraction by calculation g1(t) = SIGN(G2(t))· SQRT(ABS((G2(t) − A)/A). All field correlation functions usually showed monomodal decay and were fitted by a sum of two exponentials g1(t) = a exp(−t/b) + c exp(−t/d) to take polydispersity into account, Average apparent diffusion coefficients Dapp were calculated by applying q2Dapp = (ab−1 + cd−1)/(a + c), resulting in an angular-dependent diffusion coefficient Dapp or reciprocal hydrodynamic radius ⟨1/Rh⟩app, according to formal application of Stokes− Einstein law. By extrapolation of ⟨1/Rh⟩app to q = 0, z-average hydrodynamic radii were obtained (uncorrected for c-dependency). Static Light Scattering. Static light scattering (SLS) measurements were performed with an ALV-SP86 goniometer, a Uniphase HeNe laser (25 mW output power at λ = 632.8 nm wavelength), and an ALV/High QE APD avalanche diode fiber-optic detection system. The dilute polymer solutions in PBS with five concentrations (pSar: 2 ≤ c ≤ 10 g L−1; PEG: 10 ≤ c ≤ 50 g L−1) were measured from 30° to 150° in steps of 5°. Prior to measurement the solutions were filtered through 0.2 μm pore size GHP filters (Millipore). C
DOI: 10.1021/acs.macromol.8b00258 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules
Figure 2. MALDI-TOF mass spectrometry for P1 (blue) and P2 (red). C5A Complement Activation. 2 μL of a polymer solution (1 mg mL−1) was incubated with 18 μL of serum for 1 h at 37 °C. Polymerinduced activation of the complement system was assessed by applying the MicroVue C5a EIA kit by quidel (San Diego, CA) according to the manufacturer’s protocol.
and the secondary amine end group. These findings are in line with the reports in the literature.12,47,48 Number-average molar masses Mn of 4300 and 6400 were detected for P1 and P2, respectively. The dispersities obtained by MALDI-TOF (Mw/ Mn = 1.01) are extremely small and should be taken with caution, since it is well-known that higher molar mass species within a sample are underrepresented in the mass spectra due to mass discrimination leading to artificial narrow molar mass distributions.49 Alternatively, the number-average degrees of polymerization of all pSar samples were determined by 1H NMR (see Figure 1 and Table 1), although the result for P4 should be considered with care, since the initiator to background ratio in 1H NMR is very low. P1 was found to have a Mn of 4350 g mol−1 (DP = 60), P2 of 6850 g mol−1 (DP = 95), P3 of 13 200 g mol−1 (DP = 185), and P4 of 37 300 g mol−1 (DP = 500). The results for samples P1 and P2 agree with those by MALDI TOF within experimental error of ±5%. The experimentally determined molar masses correspond reasonably well to those calculated from the M/I ratio within the experimental uncertainty of ±10%. In the next step, the weight-average molar masses Mw of all four pSar samples were determined by static light scattering (SLS). The resulting Zimm plots are shown in Figure 3 and yield the weight-average molar mass Mw and the second virial coefficient A2 according to
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RESULTS AND DISCUSSION For a detailed characterization of solution properties four pSar (DP = 50/100/200/400) polymers have been synthesized. Polymerizations were conducted employing neopentylamine as initiator for the ring-opening polymerization (ROP) of sarcosine NCA (see Scheme 1). The degree of polymerization was adjusted by the monomer-to-initiator ratio (M/I). Polymerizations were carried out in purified DMF (
