Poly(3-ethylglycolide): a well-defined polyester matching the hydrophilic hydrophobic balance of PLA

Damiano Bandelli ab, Julien Alex ab, Christian Helbing c, Nico Ueberschaar d, Helmar Görls e, Peter Bellstedt a, Christine Weber ab, Klaus D. Jandt *c and Ulrich S. Schubert *ab
aLaboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstr. 10, 07743 Jena, Germany. E-mail: ulrich.schubert@uni-jena.de
bJena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
cChair of Materials Science (CMS), Department of Materials Science and Technology, Otto Schott Institute of Materials Research, Faculty of Physics and Astronomy, Friedrich Schiller University Jena, Löbdergraben 32, 07743 Jena, Germany. E-mail: k.jandt@uni-jena.de
dMass Spectrometry Platform, Friedrich Schiller University Jena, Humboldtstr. 8, 07743 Jena, Germany
eInstitute of Inorganic and Analytical Chemistry (IAAC), Friedrich Schiller University Jena, Humboldtstr. 8, 07743 Jena, Germany

Received 15th June 2019 , Accepted 5th September 2019

First published on 6th September 2019


The ring opening (co)polymerization of 3-ethyl-1,4-dioxane-2,5-dione (3-ethylglycolide, EtGly) and enantiopure lactide using benzyl alcohol as initiator and the organobase 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene as catalyst yielded polyesters with predictable lactide and EtGly content between 5 and 20 mol%, molar masses around 11[thin space (1/6-em)]000 g mol−1 and dispersities below 1.3. Due to the amorphous nature of the atactic poly(3-ethylglycolide) (PEtGly), dynamic scanning calorimetry revealed increasing glass transition and melting temperatures with an increasing lactide content of the statistical copolymers. Nanoparticles with a diameter of 160 nm and spherical shape were obtained from each polyester by applying the nanoprecipitation method, as confirmed by dynamic light scattering and scanning electron microscopy investigation. The constant hydrophilic to hydrophobic balance (HHB) of PLA and PEtGly was confirmed by fluorescence spectroscopy using pyrene loaded nanoparticles, confirming that a set of materials was obtained suitable to enlighten the effect of crystallinity on nanoparticle degradation.


Introduction

Nanoparticulate drug carriers for delivering therapeutic compounds to target organs in the human body have been the subject of research for several decades.1 In particular, aliphatic polyesters such as polylactide (PLA), poly(lactide-co-glycolide) (PLGA) and polycaprolactone (PCL) are of interest for the encapsulation of hydrophobic drugs due to their biodegradability and high tissue compatibility.2,3 The encapsulation and the release behavior of drugs from polymeric nanoparticles is influenced by a large number of parameters such as temperature,4 molar mass of the polymers,5 additives6 and the shape of the nanomaterials.7 Moreover the particle size as well as the interaction of the encapsulated drug with the nanomaterial matrix, play a crucial role.5,8–13

Since drug release depends on erosion of the particle surface, the diffusion of the drug out of the nanomaterial matrix as well as on the degradation of the nanomaterial,11 it is expected that physico-chemical properties of nanocarrier materials affect the release behavior of drugs.14–17 In combination, these examples show that a multitude of factors contribute to the release kinetics. However, to elucidate the contributions of the individual properties to the performance of the system, systematic studies are required, keeping as many parameters constant as possible. To understand the influence of the thermal properties of the nanoparticle matrix material, other factors such as molar mass, shape, size and the hydrophilic to hydrophobic balance (HHB) should be kept constant. It is, however, not straightforward to decouple particularly the HHB from the thermal properties and the crystallinity for standard polyester materials because an alteration of the latter is frequently accompanied by a change of the former.15,18

The two most common polyesters PLA and PLGA perfectly demonstrate the challenge: Encapsulated guests are released faster from an amorphous PLGA matrix than from a semicrystalline PLA matrix because PLGA is more prone to hydrolysis.19,20 However, PLGA is also less hydrophobic than PLA because glycolide lacks the two methyl substituents of lactide. Is the faster release kinetics from PLGA due to its amorphous nature or due to its increased hydrophilicity? In order to level out the HHB imbalance, we targeted the novel monomer 3-ethyl-1,4-dioxane-2,5-dione (3-ethylglycolide, EtGly) as being an isomer of lactide (Scheme 1). The ring-opening polymerization (ROP) of the cyclic dilactone EtGly, comprising a glycolic unit and a 2-hydroxybutyric unit, should enable access to a polyester featuring the same HHB as PLA. Small amounts of comonomers featuring opposite configuration are known to alter the thermal properties of semicrystalline isotactic PLA formed from enantiopure monomers.21,22 Consequently, the copolymerization of racemic EtGly and enantiopure lactide should enable access to a series of copolymers meeting the requirements as well.


image file: c9py00875f-s1.tif
Scheme 1 Schematic representation of the synthetic pathway yielding polyesters with the same hydrophilicity as PLA.

As organic bases are well-working catalysts, e.g. for the ROP of lactide as well as its copolymerization with glycolide,23–25 the guanidine organobase 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (mTBD)26 was selected as catalyst for the ROP to avoid the toxic standard catalyst tin(II) 2-ethylhexanoate (SnOct2).27 To exclusively vary the thermal properties and the degree of crystallinity (wc), a library of nine polyesters with constant HHB and similar molar mass was targeted. The materials were analyzed by means of differential scanning calorimetry (DSC) and nanoparticles obtained from the corresponding polyesters were characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM) investigations.

Experimental section

Materials

L-Lactide (L-LA; 98%, Aldrich) was recrystallized from ethyl acetate prior to use. (rac)-2-Hydroxybutanoic acid (95%, abcr) and D-lactide (D-LA, 98%, abcr) were utilized without further purification. The reaction solvent toluene (extra dry, Aldrich), the catalyst 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (mTBD, 98%, Aldrich) and the initiator benzyl alcohol (BnOH, anhydrous, 99.8%, Aldrich) were stored under nitrogen. The solvents utilized for column chromatography (ethyl acetate and n-hexane) were distilled before usage. Rf values were determined from pre-coated TLC sheets ALUGRAM® SIL G/UV254 and silica gel 60 was used as packing material for the preparative columns (both delivered from Machery Nagel). Tetrahydrofuran (THF) for nanoparticle preparation was purified in a solvent purification system (SPS; Pure solv EN, InnovativeTechnology). All other chemicals were purchased from standard suppliers and were used without any further purification.

Instruments

Polymerizations were conducted under nitrogen atmosphere in a MBraun UNILab Plus glove box, which was equipped with high efficiency box filters HEPA H13, a UNILab inert gas purification system and a vacuum pump. Proton and carbon nuclear magnetic resonance (1H and 13C NMR) spectra were measured in CDCl3 or CD2Cl2 at room temperature on a 300 MHz Bruker Avance I or 400 MHz Bruker Avance III spectrometer. The residual 1H peak of the deuterated solvent was used for chemical shift referencing. Homonuclear decoupling experiments were performed using a 500 MHz Bruker Avance III HD spectrometer equipped with a BBO Prodigy probe head. Elemental analysis was carried out using a Leco CHN-932. Melting points were determined with a melting point meter MPM-H2 from VWR international GmbH. Infrared (IR) spectra were measured with an IRAffinity-1 CE from Shimadzu equipped with a quest ATR diamond extended range X – single-reflection-ATR cuvette.

The crystallographic data were acquired as follows: The intensity data of EtGly was collected on a Nonius KappaCCD diffractometer, using graphite-monochromated Mo-Kα radiation. Data were corrected for Lorentz and polarization effects; absorption was taken into account on a semi-empirical basis using multiple-scans.28–30 The structure was solved by direct methods (SHELXS31) and refined by full-matrix least squares techniques against Fo2 (SHELXL-9731). All hydrogen atoms were located by difference Fourier synthesis and refined isotropically. All non-hydrogen atoms were refined anisotropically.31 MERCURY32 was used for structure representations.

Gas chromatography (GC) measurements were performed on a Shimadzu system (GC-2010 plus) equipped with an AOC-20s autosampler, a FID detector with a flow rate of 1.86 mL min−1 and a PerkinElmer Elite-5MS column (30 m length, 0.25 mm ID, 0.25 μm film thickness, stationary phase: 5% diphenyl, 95% dimethyl polysiloxane using helium as carrier gas. After split injection (AOC 20i injector, 250 °C) the column oven was heated from 60 to 200 °C with a heating rate of 16 °C min−1. GC-high resolution mass spectrometry (HRMS) measurements were performed on a GC/MS consisting of a Trace 1310 gas chromatograph coupled to a Q-Exactive GC mass spectrometer in EI and CI (methane as reactant gas) ionization mode (Thermo, Bremen, Germany, see ESI for details).

Size-exclusion chromatography (SEC) measurements were performed utilizing a Shimadzu system equipped with a CBM-20A system controller, a LC-10AD pump, a RID-10A refractive index detector and a PSS (Polymer Standards Service GmbH, Mainz, Germany) SDV column with chloroform/triethylamine (NEt3)/iso-propanol (94[thin space (1/6-em)]:[thin space (1/6-em)]4[thin space (1/6-em)]:[thin space (1/6-em)]2) as eluent with a flow rate of 1 mL min−1. The column oven was set to a constant temperature of 40 °C. Polystyrene (PS) samples were used for calibration.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) measurements were carried out using an Ultraflex III ToF/ToF instrument (Bruker Daltonics, Bremen, Germany). The spectrometer was equipped with a Nd-YAG laser. All spectra were recorded in the positive reflector mode. The instrument was calibrated with an external PMMA standard from PSS. trans-2-[3-(tert-Butylphenyl)-2-methyl-2-propenylidene]malononitril (DCTB, Sigma Aldrich) was used as matrix and sodium iodide (Sigma Aldrich) as additional salt. Separate solutions of analyte (10 mg mL−1 in chloroform), DCTB (30 mg mL−1 in chloroform) and sodium iodide (100 mg mL−1 in acetone) were prepared. 5 μL of analyte solution, 15 μL of matrix solution and 5 μL of salt solution were mixed, and 1 μL of the resulting solution was deposited on the target plate according to the dried droplet spotting technique. Electro-spray ionization (ESI) time-of-flight mass spectrometry (MS) measurements of EtGly were performed utilizing a Bruker MicroQTof mass spectrometer.

Thermogravimetric analysis (TGA) was measured under nitrogen atmosphere on a STA Netzsch 449 F3 Jupiter. Data were recorded from 30 to 600 °C at a heating rate of 10 °C min−1. Differential scanning calorimetry (DSC) measurements were performed with a Netzsch 204 F1 Phoenix instrument under a nitrogen atmosphere applying a heating rate of 20 °C min−1 in the first and second run and 10 °C min−1 in the third run. The temperature range was from −20 to 260 °C and the cooling rate between the heating runs was 20 °C min−1.

Dynamic light scattering (DLS) and ζ-potential measurements were performed on a Zetasizer Nano ZS (Malvern Instruments, Herrenberg, Germany) at 25 °C (λ = 633 nm) at an angle of 173°. For each measurement, 3 × 30 s runs were carried out in triplicates after an equilibration time of 30 s. The mean particle size was approximated as the effective (Z-average) diameter and the width of the distribution as the dispersity index (PDI) of the particles obtained by the cumulants method assuming a spherical shape. Fluorescence spectra were recorded on a Jasco FP-8300 instrument using spectroscopy-grade solvents and quartz cuvettes (1 cm pathway). The device was measuring from 350 to 550 nm with a scan speed of 20 nm min−1 and a data interval of 0.2 nm. Shape and dimensions of the nanoparticles were investigated by scanning electron microscopy (SEM) with an AURIGA 60 CrossBeam® Workstation (Carl Zeiss AG, Oberkochen, Germany). Samples were previously coated with tungsten.

Synthesis of 3-ethyl-1,4-dioxane-2,5-dione (EtGly)

A solution of 2 g (rac)-2-hydroxybutanoic acid (19 mmol) and 2.68 mL triethylamine (NEt3, 19 mmol) in 60 mL diethyl ether was cooled in an ice/sodium chloride bath to −5 °C and stirred for 20 minutes. Subsequently, a solution of 2.18 mL bromoacetyl bromide (25 mmol) in 5 mL diethyl ether was added dropwise within 30 minutes not allowing the reaction mixture to exceed 0 °C. The ice bath was removed and the reaction mixture was allowed to reach room temperature. The reaction mixture was stirred for another five hours at room temperature. Precipitates were removed by filtration and the remaining solution was concentrated under reduced pressure.

The crude yellow oil was dissolved in 1 L acetone and 20 g sodium bicarbonate (0.35 mol) were added in small portions. The reaction mixture was refluxed under vigorous stirring overnight. Subsequent to cooling to room temperature, the reaction mixture was filtered to remove the excess of salts. The solution was concentrated under reduced pressure and diluted with 1 to 2 mL of diethyl ether to precipitate salts and impurities. After filtration, the supernatant was washed with 2 mL distilled water and the aqueous phase was extracted three times with ca. 5 mL of diethyl ether. The combined organic phases were dried over sodium sulfate and evaporated under reduced pressure. The crude oil was purified by column chromatography (SiO2, n-hexane/EtOAc, 5[thin space (1/6-em)]:[thin space (1/6-em)]1, Rf = 0.16) to obtain the product as colorless solid, which contained crystals suitable for crystallographic analysis.

3-Ethyl-1,4-dioxane-2,5-dione (EtGly). Yield = 27%. M.p.: 54.1 °C. 1H NMR [ppm] (CDCl3, 300 MHz): δ = 1.15 (t, 3H, J = 7.38, 7.41 Hz, CH3), 2.11 (m, 2H, CH–[C with combining low line][H with combining low line]2–CH3), 4.88 (dd, 1H, J = 4.94, 2.27, 4.95 Hz, [C with combining low line][H with combining low line]–CH2–CH3), 4.94 (d, 2H, J = 1.80 Hz, CO–[C with combining low line][H with combining low line]2–O) ppm. 13C NMR (CDCl3, 300 MHz): δ = 8.86 (C-1), 24.39 (C-2), 65.32 (C-4), 164.29 (C-5), 165.40 (C-6) ppm. IR [cm−1]: [small nu, Greek, tilde] = 2978 (CH3), 2947 (CH2), 2916 (CH), 1747 (O–C[double bond, length as m-dash]O), 1076 (O–C–C([double bond, length as m-dash]O)); GC-CI-MS: m/z (rel. intensity) = 145.04956 (100; calculation for C6H9O4+ as [M + H]+: 145.04954, error: 0.17 ppm); 116.01054 (5; calculation for C4H4O4+ as [M − CH2 − CH3]+: 116.01041, error: 1.09 ppm); 87.04412 (55; calculation for C4H7O2, [O–(C[double bond, length as m-dash]O)–CH–CH2–CH3 + H]+: 87.04406, error: 0.74 ppm); 59.049215 ([O–(C[double bond, length as m-dash]O)–CH2 + H]+); Elemental anal. Calc. for C6H8O4: C: 50.00; H: 5.60; found: C: 49.93; H: 5.56. Crystal Data for EtGly: C6H8O4, Mr = 144.12 g mol−1, colourless prism, size 0.112 × 0.110 × 0.088 mm3, monoclinic, space group P21, a = 8.0925(9), b = 5.0020(6), c = 8.1097(9) Å, β = 92.838(1)°, V = 327.87(6) Å3, T = −140 °C, Z = 2, ρcalcd = 1.460 g cm−3, μ(Mo-Kα) = 1.24 cm−1, multi-scan, transmin: 0.5867, transmax: 0.7456, F(000) = 152, 3303 reflections in h(−7/10), k(−6/6), l(−10/8), measured in the range 2.51° ≤ Θ ≤ 27.47°, completeness Θmax = 100%, 1451 independent reflections, Rint = 0.0352, 1333 reflections with Fo > 4σ(Fo), 123 parameters, 1 restraints, R1obs = 0.0436, wR2obs = 0.0942, R1all = 0.0504, wR2all = 0.0995, GOOF = 1.120, Flack-parameter 1.7(16), largest difference peak and hole: 0.346/−0.209 e Å−3.

Ring-opening polymerization

All polymerizations were performed at 23 °C in a glovebox under nitrogen atmosphere (<0.1 ppm H2O; <0.1 ppm O2). A stock solution of initiator and catalyst was used to achieve a ratio of [M]/[BnOH]/[mTBD] = 100/1/1 for all polymerizations.

Polymerization kinetics for L-LA, D-LA and EtGly

144 mg monomer (1 mmol) were dissolved in 3.3 mL toluene (c = 0.3 mol L−1). To start the polymerization, 20 μL stock solution containing 1.03 μL BnOH (0.01 mmol), 1.44 μL mTBD (0.01 mmol) and 17.53 μL toluene were added ([M]/[BnOH]/[mTBD] = 100/1/1). Samples were taken after 0, 2, 5, 10, 20, 30, 40, 50, 60, 80, 100 and 120 minutes. Each sample was quenched with a fourfold excess of benzoic acid (0.41 mg, 3.4 mmol) dissolved in chloroform.

The monomer conversion was determined by 1H NMR spectroscopy for L- and D-LA. GC measurements were performed to obtain the conversion of EtGly.

General procedure for the homopolymerization (P1 to P3)

The monomer and toluene were added to a vial and stirred until complete dissolution (c = 0.3 mol L−1). Subsequently, a stock solution consisting of BnOH, mTBD and toluene was used to initiate the polymerization ([M]/[BnOH]/[mTBD] = 100/1/1). After two hours of stirring at room temperature, the reaction mixture was quenched with a four fold excess of benzoic acid dissolved in toluene and a sample was taken to determine the monomer conversion. After precipitation of the polymers in ice-cold methanol, the homopolymers were dried in vacuo and yielded white powders. Further details are provided in the ESI.
PLLA (P1). Conv. = 85%; yield = 66%. 1H NMR (400 MHz, CDCl3): δ/ppm = 1.60 (d, 474H, J = 7.12 Hz, H-1), 5.18 (q, 158H, J = 7.07, 7.13, 7.09 Hz, H-2), 1H NMR (300 MHz, CD2Cl2): δ = 1.56–1.58 (br, 575H, H-1), 5.15–5.19 (br, 158H, H-2), 7.37 (br, 5H, H-3); SEC (CHCl3, PS calibration): Mn = 16 kg mol−1; Đ = 1.06.
PDLA (P2). Conv. = 85%; yield = 71%. 1H NMR (400 MHz, CDCl3): δ/ppm = 1.60 (d, 504H, J = 7.13 Hz, H-1), 5.18 (q, 168H, J = 7.08, 7.12, 7.10 Hz, H-2), 1H NMR (300 MHz, CD2Cl2): δ = 1.56–1.58 (br, 576H, H-1), 5.15–5.21 (br, 168H, H-2), 7.37 (br, 5H, H-3); SEC (CHCl3, PS calibration): Mn = 19 kg mol−1; Đ = 1.06.
PEtGly (P3). Conv. = 97%; yield = 64%. 1H NMR (400 MHz, CDCl3): δ/ppm = 1.05 (t, 210H, J = 7.36, 7.66 Hz, H-1), 1.89–2.11 (br, 140H, H-2), 4.64–4.95 (br, 140H, H-4), 5.09–5.21 (br, 70H, H-3); SEC (CHCl3, PS calibration): Mn = 12 kg mol−1; Đ = 1.19.

General procedure for the statistical copolymerization (P4 to P9)

The statistical copolymers P4 to P9 were obtained as described for the homopolymers P1 to P3. A constant ratio of [M]total[thin space (1/6-em)]:[thin space (1/6-em)][TBD][thin space (1/6-em)]:[thin space (1/6-em)][BnOH] = 100[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1 was kept, while the feed ratio of L- or D-LA and EtGly was varied as indicated in Table 1. All copolymerizations were performed at room temperature for two hours. In an exemplary reaction for P4, 352 mg (2.44 mmol) of L-LA and 19 mg (0.13 mmol) of EtGly were dissolved in 8.56 mL anhydrous toluene. The ROP was initiated by addition of a stock solution containing 2.66 μL BnOH (0.03 mmol), 3.35 μL mTBD (0.03 mmol) and 93.99 μL toluene. The polymerization proceeded at room temperature for two hours. The analyses and purification were performed as described above. Details for P5 to P9 are provided in the ESI.
Table 1 Selected structural characterization data of the synthesized (co)polymers
Sample Polymer mol% LA/EtGly feed Conv.a [%] mol% LA/EtGly NMRb M n [kg mol−1] NMRb M n [kg mol−1] SECc Đ SECc
a Overall monomer conversion determined by integration of suitable signals in the 1H NMR spectra of the reaction solution. The conversion for P3 was determined via GC analysis. b Determined by integration of suitable signals in the 1H NMR spectra of the purified polymers. c Eluent CHCl3, RI detection, PS calibration. Values correspond to the purified polymers.
P1 PLLA 100/0 85 100/0 11 16 1.06
P2 PDLA 100/0 85 100/0 12 19 1.06
P3 PEtGly 0/100 97 0/100 10 12 1.19
 
P4 P(LLA-stat-EtGly) 95/05 75 95/05 11 16 1.11
P5 P(LLA-stat-EtGly) 90/10 74 89/11 11 13 1.20
P6 P(LLA-stat-EtGly) 80/20 69 78/22 10 12 1.23
 
P7 P(DLA-stat-EtGly) 95/05 77 96/04 12 19 1.10
P8 P(DLA-stat-EtGly) 90/10 79 91/09 11 18 1.21
P9 P(DLA-stat-EtGly) 80/20 77 78/22 10 15 1.28


P(LLA-stat-EtGly) (P4). Feed L-LA/EtGly = 95/05; conv = 75%; yield = 74%. 1H NMR (300 MHz, CDCl3): δ/ppm = 1.00–1.07 (br, 12H, H-2), 1.50–1.79 (br, 483H, H-6), 1.92–2.04 (br,11H, H-3), 4.59–4.89 (br, 7H, H-1), 5.14–5.21 (br, 152H, H-4, H-5), 1H NMR (300 MHz, CD2Cl2): δ = 1.00–1.01 (br, 13H, H-2), 1.45–1.63 (br, 536H, H-6), 1.89–2.07 (br, 12H, H-3), 4.63–4.87 (br, 7H, H-1), 5.10–5.19 (br, 151H, H-4, H-5), 7.36 (br, 5H, H-7); SEC (CHCl3, PS calibration): Mn = 16 kg mol−1; Đ = 1.11.

Nanoparticle preparation

P1 to P9 were dissolved in THF (see Table 3 for details). 0.5 mL of the polymer solution were dropped into 5 mL of milliQ water while stirring at a rate of 1000 rpm. THF was removed by stirring the nanoparticle suspension in an open glass vial overnight and DLS and zeta potential measurements were performed. The pyrene loaded nanoparticles were prepared using 1900 μL of polymeric THF solutions and 100 μL of a pyrene stock solution in THF (c(pyrene) = 1 mg mL−1 for P1 and P2 and c(pyrene) = 0.3 mg mL−1 for P3 to afford a constant pyrene content of 1 mass% in the suspensions). Nanoparticle formulations from the THF mixtures were performed as described above. After DLS measurements, the nanoparticle suspensions of P1 to P3 were diluted 100 times and fluorescence spectra were measured (λex = 339 nm).

Results and discussion

Monomer synthesis

The synthesis of the heterocyclic monomer EtGly was accomplished in a two-step procedure adopted from well-known procedures published for similar monomers (Scheme 1).33–36 (rac)-2-Hydroxybutanoic acid was acylated with bromoacetyl bromide to yield the linear precursor in the presence of triethylamine (NEt3) as scavenger for the formed HBr. The cyclization was afforded by refluxing the crude linear precursor with sodium bicarbonate overnight under highly diluted conditions to promote an intramolecular nucleophilic substitution. As typically observed for such syntheses,36 the purified EtGly was obtained in low yields (27%). Detailed mass spectrometry analysis pointed towards a possible presence of macrocyclic impurities that could not be quantified and might have been produced during GC-MS analysis at temperatures above 250 °C (see ESI). However, NMR spectroscopy supported the purity of the monomer, not indicating any diastereomers or meso forms. X-ray crystallography finally confirmed the identity of EtGly, which crystallized in a slightly twisted boat conformation (Fig. 1). The substitution of the ethyl moiety in bowsprit position allowed the endocyclic atoms to adopt bond angles according to their hybridization (Table S1). EtGly was assigned to the space group P21 and the intermolecular interactions are primarily dominated by van der Waals forces.
image file: c9py00875f-f1.tif
Fig. 1 Section of the packing of the crystal structures of the monomer EtGly obtained by X-ray scattering experiments.

Ring-opening polymerization

Employing mTBD as catalyst, kinetic studies were performed during the ROP of L-LA, D-LA and EtGly in toluene (c = 0.3 mol L−1) at room temperature. Benzyl alcohol was selected as initiator, keeping the initial ratio of [M]/[BnOH]/[mTBD] constant at 100/1/1 for all the polymerizations, as the targeted polyesters should feature a similar molar mass. The conversions of L-LA and D-LA were determined via1H NMR analyses by integration of the methine protons peaks assigned to the monomers and the polymers. Due to overlapping signals in the 1H NMR spectra, the conversion of EtGly was determined via GC investigation using the solvent as internal standard, although a complete baseline separation has not been realized.

The non-linear first-order kinetic plot depicted in Fig. 2 revealed that the apparent polymerization rate decreased throughout the course of the ROP in a similar fashion as reported for other monofunctional hydrogen bond acceptor catalysts such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).37–39 As expected, L-LA and D-LA featured the same kinetic behavior during the polymerization process but the polymerization rate of EtGly was significantly increased. Considering the fact that EtGly represents a monosubstituted glycolide, the latter is consistent with the increased reactivity of glycolide compared to, e.g., lactide.23 As reported for the Sn(Oct)2 catalyzed ROP of methylglycolide,40 a nucleophilic attack at the monomer would preferably take place at the unsubstituted acyl moiety of the dioxanedione ring.


image file: c9py00875f-f2.tif
Fig. 2 Kinetic studies of the homopolymerization of L-LA, D-LA and EtGly conducted in toluene ([M] = 0.3 mol L−1) at room temperature using BnOH as initiator and mTBD as catalyst ([M][thin space (1/6-em)]:[thin space (1/6-em)][mTBD][thin space (1/6-em)]:[thin space (1/6-em)][BnOH] = 100[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1). Left: First-order kinetic plot. Center: Evolution of the molar mass with monomer conversion. Right: Overlay of the SEC elugrams (CHCl3, RI detection, PS calibration).

SEC traces from kinetic samples revealed unimodal molar mass distributions with dispersity values (Đ) below 1.08 for the PLAs and below 1.21 for PEtGly. The molar masses of all three polyesters increased in a linear fashion with monomer conversion during the course of the ROP. The molar masses of the homopolymers were hence well controlled using the same polymerization conditions.

The end group fidelity for the polymerization of EtGly was evaluated by means of MALDI-ToF-MS analysis on the sample taken after two minutes, corresponding to a conversion of 26% (Mn,theo = 3.8 kg mol−1). The most abundant peak at m/z = 1715.6 was assigned to sodiated PEtGly chains initiated by benzyl alcohol and terminated by a proton as depicted in Fig. 3. The distance between two peaks of the main series of Δm/z = 144 corresponds to one EtGly repeating unit. Less abundant signals were assigned to chains with a carboxylic acid α- and a hydroxy ω-end group, possibly caused by water initiation or by deactivation of zwitterionic intermediates by water. Signals related to cyclic polymer chains caused by intramolecular transesterification could not be assigned.


image file: c9py00875f-f3.tif
Fig. 3 MALDI-ToF MS analysis of the sample taken after two minutes during the polymerization of EtGly. Left: Full mass spectrum. Center: Zoom into the m/z region from 1700 to 1875. Right: Overlay of the calculated and measured isotopic patterns and assignment of the most abundant peak.

The polymerization conditions were hence applied for the synthesis of the respective homopolymers PLLA (P1), PDLA (P2) and PEtGly (P3) as well as two series of statistical copolymers comprising 5, 10 and 20 mol% EtGly that are based on PLLA (P4 to P6) and PDLA (P7 to P9), respectively. For all polymerizations, the ratio of [M][thin space (1/6-em)]:[thin space (1/6-em)][mTBD][thin space (1/6-em)]:[thin space (1/6-em)][BnOH] was kept constant at 100[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1 to ensure a similar molar mass of the (co)polyesters (see Table 1). After two hours, the expected monomer conversions were reached for the homopolymers, whereas the overall monomer conversions for the copolymer series ranged from 70 to 80%. Theoretical molar masses between 10 kg mol−1 and 12 kg mol−1 were hence expected. End group analyses by integration of end group and backbone signals in the 1H NMR spectra confirmed these molar masses (Fig. S9–S17). It should be stated that the theoretical molar mass of PEtGly (P3) might be biased by the GC analyses (see above), explaining the deviation of the expected (Mn = 14 kg mol−1) and achieved (Mn = 10 kg mol−1) molar mass values.

The successful incorporation of EtGly into PLLA as well as PDLA was also evident from the 1H NMR spectra, as is exemplarily demonstrated by the overlay shown in Fig. 4. The methine and methylene proton region of the spectra proved to be suitable for the estimation of the copolymer compositions as the methylene proton signals assigned to the EtGly repeating units (peak 1 in Fig. 4) were separated from the overlapping methine proton signals (peaks 4 and 6 in Fig. 4). The resulting values calculated from these peak integrals are in good agreement with the feed ratio of the monomers (compare Table 1), demonstrating that the copolymer composition can be easily tailored.


image file: c9py00875f-f4.tif
Fig. 4 1H NMR spectra of PLLA (P1, 400 MHz, CDCl3), PEtGly (P3, 400 MHz, CDCl3), the copolymer P6 (300 MHz, CDCl3) and schematic representation of the assignment of the signals to the structure of the polyesters.

Unimodal and narrow molar mass distributions (Đ < 1.22) were evident from SEC analysis of the reaction solutions (Fig. S7 and S8). The molar mass distributions were retained subsequent to precipitation for the homopolymers P1 to P3 (1.06 < Đ < 1.19). Although precipitation into ice-cold methanol is a common purification procedure for the ROP of lactides,26 an additional high molar mass distribution appeared in the SEC traces of the purified copolymers. In general, the peak was more prominent in polymers with higher EtGly content, in particular for the PLLA based copolymers. As the peak area corresponded to less than 8% and the overall dispersity never exceeded Đ = 1.3, a minor influence on the properties of the copolymers is assumed.

Stereochemistry of the homopolymers P1 to P3

Proceeding slowly at room temperature, organic bases such as DBU can induce epimerization of lactide.41 Although no or negligible amounts of epimerization were observed after two days at room temperature, i.e. at conditions that are typically applied for a DBU catalyzed ROP,26 other organic base catalysts result in partial loss of the stereoinformation of the monomer upon ROP.42 Less commonly used than DBU (pKa = 16.8 in THF), the basicity of mTBD is increased (pKa = 17.9 in THF).43 To the best of our knowledge, it has not yet been investigated whether the configuration of lactide is retained during a mTBD catalyzed ROP.

Single frequency homonuclear decoupled 1H NMR spectroscopy experiments44 were hence conducted (Fig. 5). The selective decoupling of the methyl group protons of PLLA P1 and PDLA P2 resulted in a collapse of the quartet splitting of the methine proton signal. The resulting singlet corresponds to iii tetrads,44 verifying the presence of isotactic chains. The decoupling experiment on the PEtGly P3, which was obtained from the racemic EtGly monomer, showed a different result. After irradiation at the methylene proton frequency of the ethyl moiety, the overlapping triplets assigned to the protons at the stereocenters collapsed to at least four singlets. Both enantiomers should have therefore been statistically incorporated during the polymerization process, suggesting the presence of an atactic polyester.


image file: c9py00875f-f5.tif
Fig. 5 Selective homonuclear decoupled 1H NMR experiment (500 MHz, CDCl3) and schematic representation of the structures of the polyesters P1 to P3. Left: Collapsed signals of the methine protons of P1. Center: Collapsed signals of the methine protons of P2. Right: Collapsed signal of methine proton of P3.

Thermal properties in bulk

As the stereochemistry of all monomers was retained during the ROP process, the copolymerization of enantipure L-LA or D-LA with defined fractions of the racemic EtGly should result in variations of the crystallinity of the resulting copolymers, while keeping the chain length as well as the HHB constant. Subsequent to TGA analysis to ensure the thermal stability of the materials (Fig. S18), the bulk polyesters were thus investigated by means of DSC in the temperature range from −20 to 260 °C. After a first heating run at 20 °C min−1, two additional heating runs were performed at 20 °C min−1 and 10 °C min−1, respectively, to detect transitions of second and first order at optimum measurement conditions. As the glass transitions remained well detectable at the lower heating rate, the first and third heating runs are in the focus of the following discussion.

In general, no significant differences were found upon comparison of the results obtained from the two copolymer series based on PLLA and PDLA, respectively (Table 2). Fig. 6 exemplarily depicts the results of the PLLA copolymer series obtained in the third heating run, and further overlays are provided in the ESI (Fig. S19–S21). Like PLLA and PDLA, the copolymers comprising EtGly represent semicrystalline materials. The typical double fusion events of PLA due to cold crystallization45 were evident during DSC analysis of the copolymers as well. On the other hand, the PEtGly homopolymer P3 was amorphous, which is in line with its atactic structure.


image file: c9py00875f-f6.tif
Fig. 6 DSC thermograms of the polyesters based on PLLA. The thermograms are shifted vertically for clarity. The measurements were performed from −20 to 260 °C (third heating run, heating rate 10 °C min−1, cooling rate 20 °C min−1).
Table 2 Thermal properties of the polyesters estimated from DSC analysis
Polymer mol% EtGly T g[thin space (1/6-em)]a [°C] T c [°C] T m [°C] ΔHf [J g−1] ΔHc [J g−1] w c[thin space (1/6-em)]c [%] T c [°C] T m [°C] ΔHf [J g−1] ΔHc [J g−1] w c[thin space (1/6-em)]c [%]
Third heating runb First heating rund
a Inflection value. b Heating rate 10 °C min−1. c Degree of crystallinity calculated according to eqn (1) using the ΔHf of fully crystalline PLA from literature (image file: c9py00875f-t4.tif (PLA) = 93 J g−1).46 d Heating rate 20 °C min−1.
P1 PLLA 0 56 95 151/163 51 −33 19 158/165 64 69
P2 PDLA 0 55 97 149/163 47 −37 11 157/166 68 73
P3 PEtGly 100 29
P4 P(LLA-stat-EtGly) 5 53 108 142/150 35 −33 2 79 143/151 38 −20 20
P5 P(LLA-stat-EtGly) 11 52 135/140 4 5 92 119/139 42 −2 43
P6 P(LLA-stat-EtGly) 22 48 109/121 32 −4 31
P7 P(DLA-stat-EtGly) 4 53 113 143/150 30 −30 0.4 97 155 50 −15 38
P8 P(DLA-stat-EtGly) 9 51 119 137/142 6 −4 2 95 121/143 42 −12 31
P9 P(DLA-stat-EtGly) 22 48 109/121 30 −4 28


Irrespective of the stereochemistry of the PLA, the glass transition temperatures Tg of the copolymers decreased in a linear fashion with increasing EtGly content, showing that the amorphous domains of the semicrystalline PLA-based materials are strongly affected by incorporation of the racemic comonomer (Fig. 7A). Similar observations hold true regarding the melting temperatures Tm of the two types of crystalline domains (Fig. 7B). As the Tm of polymers is directly correlated to their crystalline dimensions,47,48 this indicated that an increase of the amorphous EtGly content disturbed the PLA-crystal formation, resulting in smaller PLA crystals. Although the melting temperature Tm,1 of the crystallites formed during cold crystallization differed in the first and third heating runs, the higher Tm,2 remained constant (Fig. S22). It should, however, be stated that the initially semicrystalline copolymers comprising 20 mol% of EtGly were not able to recrystallize during the repeated heating and cooling cycles performed during the DSC measurements. In addition, the copolymers featuring 10 mol% EtGly did not recrystallize during the second heating run performed at a heating rate of 20 °C min−1, demonstrating that the crystallization of the materials with higher EtGly fraction is slow.


image file: c9py00875f-f7.tif
Fig. 7 Influence of the molar fraction of EtGly on the thermal properties of the copolyester series based on PLLA (P1, P3 to P6) and on PDLA (P2, P3, P7 to P9) as determined via DSC analysis (cooling rates: 20 °C min−1, heating rate in the first run: 20 °C min−1, heating rate in the second run: 20 °C min−1, heating rate in the third run: 10 °C min−1). A: Glass transition temperature Tg. B: Melting temperatures Tm of both crystallites determined in the third heating run. C: Enthalpy of fusion ΔHf normalized by the ΔHf of fully crystalline PLA (image file: c9py00875f-t3.tif (PLA) = 93 J g−1).46 D: Degree of crystallinity wc calculated according to eqn (1).

The enthalpies of crystallization ΔHc and the enthalpies of fusion ΔHf were determined from the respective peak areas of the DSC thermograms to deduce structure–property relationships regarding the degree of crystallinity wc of the two copolymer series. Higher EtGly content resulted in significantly decreased ΔHf (Fig. 7C). The fact that ΔHf is lower in the third heating run than for the polymers without any additional thermal pre-treatment again points towards low crystallization rate of the polyesters. As the PEtGly homopolymer P3 represented an amorphous sample, we assumed that the EtGly mers alone do not contribute to the crystallinity of the copolymers. The degree of crystallinity above the cold crystallization temperature has hence been roughly estimated by normalization with the enthalpy of fusion of PLA featuring infinite crystal thickness image file: c9py00875f-t1.tif. However, such a value would be of little significance for the polymer performance at room or body temperature. To assess the actual degree of crystallinity wcviaeqn (1), ΔHc was hence taken into account.49

 
image file: c9py00875f-t2.tif(1)

The endo- and exothermal peaks in the DSC thermograms were sometimes rather broad, and the resulting wc values should hence be considered with caution. However, Fig. 7D clearly shows that the crystallinity of PLA was significantly reduced already by incorporation of small fractions of EtGly, although being strongly affected by the thermal prehistory of the samples.

Nanoparticle formulation

To formulate particles small enough for biological applications and of uniform size, the nanoprecipitation method was applied.50,51 For this purpose, the polyesters were dissolved in THF and dropped rapidly into an excess of pure water under vigorous stirring. The organic solvent was allowed to evaporate overnight, producing aqueous nanoparticle suspensions without the need for additional stabilizers. At constant polymer concentration of 0.5 mg mL−1 in the suspension, the polyesters based on PLA yielded nanoparticles featuring hydrodynamic diameters Dh between 150 and 170 nm, as indicated by DLS measurements (Fig. 8 and Fig. S23). Although PEtGly P3 produced larger nanoparticles under these conditions, the nanoparticle size has been tailored by decreasing the polymer concentration. It was thus possible to formulate nanoparticles from all polyesters with a uniform mean hydrodynamic diameter around 160 nm. The zeta potentials are always strongly negative (ζ ≈ −30 mV), indicating the stability of the particle suspensions (Table 3).
image file: c9py00875f-f8.tif
Fig. 8 DLS size distributions of the PDLA based nanoparticles prepared from P2 and P7 to P9 with hydrodynamic diameters Dh ≈ 160 nm. The full lines represent the intensity-weighted data, the dotted lines represent the number-weighted data.
Table 3 Nanoparticle size distributions and ζ-potentials determined by DLS measurementsa
Polymer c(P) in THF [mg mL−1] c(P) in H2O [mg mL−1] ζ [mV] D h [nm] PDI
a Prepared by dropping 0.5 mL THF solution into 5 mL water.
P1 5 0.5 −32 147 0.12
P2 5 0.5 −31 173 0.16
P3 1.5 0.15 −29 156 0.13
P4 5 0.5 −30 147 0.10
P5 5 0.5 −29 167 0.10
P6 5 0.5 −33 157 0.10
P7 5 0.5 −26 160 0.12
P8 5 0.5 −26 147 0.11
P9 5 0.5 −29 150 0.11


Analysis of the nanoparticles by means of SEM indicated a spherical shape of the nanoparticles formulated from each polyester (Fig. 9 and Fig. S24). The majority of the particles featured diameters below 100 nm, which is most likely due to the drying process prior to the SEM measurements.


image file: c9py00875f-f9.tif
Fig. 9 SEM images of dried nanoparticles prepared from P2 and P7 by nanoprecipitation. Scale bars represent 1 μm.

The tailor-made polyesters hence feature similar molar masses, different thermal properties and are suited to obtain nanoparticles of similar size. Although the copolymer design should ensure a constant HHB throughout the polymer library, the fluorescent probe pyrene52–55 was applied to provide in addition an experimental evidence for the fact. For this purpose, homopolymer nanoparticles formed from P1 to P3 were loaded with pyrene by nanoprecipitation using a THF solution of polymer and pyrene (Table S2). The final pyrene concentration (c ≈ 10−7 M) was set according to protocols that are well-established for the determination of critical micelle concentrations.56,57 Because the polymer concentration had to be varied in order to obtain nanoparticles of similar sizes, the ratio of pyrene to polymer was kept constant at 1 wt% to account for any quenching effects that might occur inside the nanoparticles. Fluorescence spectroscopy was utilized to judge the hydrophilicity of the pyrene environment via the vibrational fine structure of the emission spectrum. As depicted in Fig. 10, the I1/I3 ratio is very similar for all nanoparticle suspensions (I1/I3 ≈ 1.3), confirming that the polymers indeed feature a constant HHB.


image file: c9py00875f-f10.tif
Fig. 10 Normalized fluorescence spectra of pyrene loaded nanoparticles formed from P1 to P3 (λex = 339 nm).

Conclusion

To understand the influence of the thermal properties and the crystallinity of a polyester matrix material on the release of encapsulated drugs, the (co)polymerization of the novel lactide isomer 3-ethyl-1,4-dioxane-2,5-dione (EtGly) represented the basis for the development of a series of polyesters matching the HHB of PLA. The stereochemistry of the enantiopure lactide monomers was retained during the mTBD catalyzed ROP performed at room temperature, resulting in a straightforward method to decrease the degree of crystallinity of well-defined PLLA and PDLA already upon incorporation of 5 to 20 mol% of the racemic EtGly in statistical copolymers. The copolymers featuring similar molar masses were suited to obtain stable nanoparticles with diameters around 160 nm and negative zeta potentials via nanoprecipitation. Fluorescence spectroscopy of the homopolymer nanoparticles using pyrene as probe confirmed the constant HHB of the polyester library.

Having established materials with constant HHB, molar mass and nanoparticle size, our future research will be focused on studying the release kinetics of encapsulated guest molecules from our tailor-made nanomaterials. The further expansion towards other polymers with constant HHB but altered crystallinity would ultimately answer the question if and how the thermal properties of a polymer matrix play a crucial role during drug release.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors thank Nicole Fritz for ESI and MALDI-ToF mass spectrometry analysis. The work was supported by the DFG-funded Collaborative Research Centre PolyTarget (SFB 1278, projects A06 and Z01). We gratefully acknowledge the additional financial support of the Thüringer Ministerium für Wirtschaft, Wissenschaft und Digitale Gesellschaft (Thuringian Ministry for Economic Affairs, Science and Digital Society, ProExzellenz II, NanoPolar) and the Deutsche Forschungsgemeinschaft (DFG), grant references INST 275/389-1 FUGG and INST 275/331-1 FUGG.

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Footnotes

Electronic supplementary information (ESI) available: 1H and 13C NMR spectra, FT-ATR-IR transmittance spectrum, GC chromatogram and CI mass spectrum of EtGly; SEC traces of reaction mixture and purified polymers P1 to P9; detailed experimental descriptions, 1H NMR spectroscopy and SEC of P1 to P9; additional TGA and DSC thermograms, DLS size distributions and SEM micrographs of the nanoparticles and detailed summary of the nanoparticle characterization. See DOI: 10.1039/c9py00875f
Both authors contributed equally.

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