Influence of macromolecular structure of novel 2- and 4-armed polylactides on their physicochemical properties and in vitro degradation process
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The objective of this study was to analyse how macromolecular structure of polylactides influences their properties and degradation rate. To achieve this, novel 2- and 4-armed PDLLA and PLLA (noted as 2b and 4b) were synthesized by ring-opening method. 1,4-butanediol and pentaerythritol were used as initiators and stannous octoate was used as catalyst. The obtained polymers were investigated in terms of molecular weight by size exclusion chromatography, thermal properties by differential scanning calorimetry and thermogravimetry, and hydrophilicity by the contact angle measurements. The in vitro degradation test was carried out in PBS solution at 37 °C by means of the mass loss, water uptake, molecular weight and thermal properties changes. The branched polylactides including 2bPDLLA, 4bPDLLA, 2bPLLA and 4bPLLA were successfully synthesized and the average molecular weights were around 40-45 kDa. The numbers of arms in each polymer just slightly influenced the thermal properties and the contact angle. The crystallinity of 4bPLLA was 23 %, whereas for 2bPLLA it was 41 %. The degradation rates of both 2b and 4bPLLA were similar and the degradation process was similar only during first 7 weeks. After this period, the degradation rate of 4bPDLLA increased. Consequently, thermal properties and degradation profiles of the branched polymers would depend on plural factors, such as chain length and crystallinity in branched structure.
KeywordsStar shape polymers PLLA PDLLA Degradation
Biodegradable synthetic polymers have been extensively studied throughout last few decades. Their key advantages are ability to tailor mechanical properties and degradation kinetics. They can be fabricated into various desired shapes and designed with chemical functional groups. Among them are aliphatic polyesters, such as polyglicolide, polylactide and their copolymers, polycaprolactone or polydioxanone, which have attracted considerable attention because of their biocompatibility [1, 2, 3]. Moreover, these polymers degrade by hydrolysis of ester bonds and degradation products are resorbed through the metabolic pathways .
One of the most common aliphatic polyesters used in biomedical application is polylactide (PLA). The basic building block for PLA is lactic acid with an asymmetric carbon atom and exist in two optically active configuration: L(+) and D(−). Optically pure poly(L-lactide) PLLA and poly(D-lactide) PDLA are crystalline with a glass-transition and melt temperature of about 55 °C and 175 °C, respectively, whereas poly(D,L-lactide) PDLLA is amorphous with glass transition temperature slightly lower than for optically pure PLLA and PDLA .
Despite wide use of PLAs, there are some limitations of physicochemical properties, such as lack of controlled degradation based on its high crystallinity. It is known that molecular weight, crystallinity and samples dimensions are the most important parameters which influence the degradation rate, however hydrophilicity, permeability and glass transition temperature can also affect this process [6, 7, 8]. In order to better control degradation of PLAs, many modifications were done, mainly based on linear copolymerization or block copolymerization [9, 10].
Almost twenty years ago, our research group proposed that polymer architecture would also considerably affect the thermal properties of typical aliphatic polyester, poly(ε-caprolactone) (abbreviated as PCL) . Actually, PCL is well-known semi-crystalline polymer and it has melting point around 60 °C. We intended to design new temperature responsive materials using PCL, therefore, that temperature is too high to consider the biomedical application. It was critical to modulate the melting point near body temperature. Then we designed branched PCL to control crystallinity of PCL. As expected, the combination of two kinds of the branched PCL, that were 2-armed and 4-armed PCL, precisely controlled the softening point of their cross-linked materials near body temperature .
So far, there are not many papers describing the influence of polylactides architecture on their properties. Korhonen et al. have synthesized and characterized star-shaped PLAs with 1, 2, 4, 6 and 10 arms . Srisa-ard et al. have analysed influences of arm numbers (1, 4, 6 and 16) and length on the thermal properties . Hao et al. have investigated the 1, 3, 4, 5 and 6-armed polymers in the terms of crystallization kinetics . Tsuji et al. have also analysed the crystallization behaviour of PLAs, but only 1 – and 2-arm linear PLLA . They also investigated the hydrolytic degradation and thermal properties of 1 – and 2-arm PDLLA . Degradation was also investigated by Yuan et al. , but he has studied only linear and hexa-armed PLLA and PDLLA.
In this paper the 2- and 4-armed PLLA and PDLLA was investigated. It is complementary study to the mentioned above. The new materials were synthesized and characterized to show how their new architecture influences their physic-chemical properties and degradation rate.
Materials and methods
L-lactide and D,L-lactide were kindly supplied by the Musashino Chemical Laboratory (Japan) and recrystallized twice from ethyl acetate before use. 1,4-butanediol (>99 %) and pentaerythritol (>98 %) were purchased from Tokyo Chemical Industries (Japan). Tin 2-ethylhexanoate (95 %) and phosphate buffer saline tablets pH = 7.4 were from Sigma Aldrich (Japan and Poland, respectively). All solvents (Wako Pure Chemical Industries, Japan) were analytical grade; only amylene-stabilized chloroform (POCH, Poland) was HPLC grade. All chemicals were used as received.
Synthesis of 2- and 4-armed polylactides
Fourier transform infrared spectroscopy
Chemical structure of synthesized polylactides was analysed by ATR-FTIR (attenuated total reflectance - Fourier Transform Infrared Spectroscopy). For each sample 64 scans in the range of wavenumbers 400–4000 cm−1 with resolution of 4 cm−1 were done and averaged.
Molecular weight measurements
The molecular weights of the polymers were measured by size exclusion chromatography and multi-angle laser light scattering (SEC-MALLS). This method doesn’t require the narrow distributed polymer standards for calibration and is more suitable for star-shaped or branched polymers [19, 20]. The number and weight average molecular weights (Mn and Mw) were determined by modular system Agilent 1200 series HPLC with refractive index detector and 3-angle (48°, 90° and 132°) laser light scattering detector MiniDAWN TREOS (Wyatt Technology). All samples (2 mg/ml) were filtered through PTFE 0.2 μm membrane before analysis to remove the small amount of dust. Measurements were made at 30 °C with PLgel 5 μm MIXED-C column (300 × 7.5 mm). The chloroform was used as the solvent at flow rate of 1 ml/min. The evaluation of the results was made using ASTRA 5.3 software (Wyatt Technology). The specific refractive index PLA in chloroform dn/dc = 0.024 mL/g  was used for calculations.
The thermal properties were analysed using differential scanning calorimeter (DSC) Q200 TA Instruments. Samples of about 6 mg were analysed in alumina pans. The measurements were run according to heat-cool-heat procedure from 0 °C to 200 °C for PLLA and from 0 °C to 80 °C for PDLLA, at heating and cooling rate of 10 °C/min. In order to eliminate the internal stresses the glass transition temperature Tg, melting temperature Tm, crystallization temperature Tc and crystallinity Xc were determined from second heating scan.
Thermal stability of polymers was analyzed using thermogravimetric analyzer Q5000 TA Instruments. The mass of each specimen was 8-10 mg and the reaction environment was flowing nitrogen (25 ml/min). The TG curves were recorded in the temperature range from 50 °C to 450 °C, at heating rate of 10 °C/min. The DSC and TGA results were analyzed in Universal Analysis 2000 software (TA Instruments).
The X-ray diffraction (XRD) analysis for PLLA samples was carried out with using Bruker AXS D8 DISCOVER diffractometer using Cu Kα (0.1542 nm) radiation. The scans were made on PLLA casted films in the 2θ range of 14–26°.
The wettability of the polymers surface was evaluated on the basis of water static contact angle. The measurements were made using a contact angle goniometer OCA20 (Dataphysics). The samples were prepared as thin coatings on the microscope glass slides by dip-coating method. After completely dried, 3 μl of water droplet was dropped on the airside surface of the film at room temperature, and the contact angle was immediately measured. 10 measurements were made for each sample and the results were averaged.
In vitro hydrolytic degradation
In vitro degradation was carried out in phosphate buffered saline (PBS) pH = 7.4 at 37 °C. Degradation medium was changed every week. Polymers were prepared by wet cast technique as follows. Polymers were dissolved in chloroform and poured into Petri dishes and left for drying under chemical hood for 24 h. Then films were dried up in vacuum dryer for one week (50 mbar, 25 °C). Three samples of 10x15mm2 and thickness of 0.35 um for each time point were cut and immersed in 7 ml of PBS. The mass loss and water uptake were calculated gravimetrically, molecular weight and thermal properties changes were analyzed by SEC and DSC according to the procedures described previously. Degraded samples were investigated also by ATR-FTIR and SEM observations. SEM images were performed with using Phenom ProX (PhenomWorld). Samples for microscopic analysis were sputtered with 7 nm gold layer.
The results were evaluated statistically by t-Test - paired comparison of means (KyPlot 2.0 beta 13 software). The comparison of 2b and 4b forms of PLAs, not PDLLA and PLLA was done. Data are expressed as a mean ± standard deviation (SD). Three significance levels were used: *p < 0.05, **p < 0.01, ***p < 0.001.
Result and discussion
Polymers synthesis and characterisation
Molecular weight and thermal properties of 2- and 4-armed PLAs
Obtained results are in agreement with ATR-FTIR spectra analysis. Significantly higher degree of crystallinity was calculated for 2b-poly(L-lactide) than for star-shaped form. Longer polymer chains can easier form crystals, than shorter ones characteristic for 4-armed form of PLLA.
In vitro hydrolytic degradation
The amorphous PDLLA samples characterise more degraded structure. There are visible developed, porous surface of the samples. In the case of semi-crystalline PLLA the changes are smaller. After degradation crystallites become more clear, especially in star-shaped poly(L-lactide). It is an effect of degradation mainly amorphous phase of polymers and superficial character of the degradation process. At SEM images of the samples after degradation smaller crystals for 4bPLLA were observed. That confirmed mentioned previously statement, that bigger crystallites are formed by linear polymer than by 4-armed one. For both polymers, amorphous PDLLA and semi-crystalline PLLA, bigger changes were observed for samples with 4-armed macromolecular architecture, what is in agreement with other degradation results.
The comparison of the degradation rate was done on the basis of analysis the ratio of absorbance of peak characteristic for vibration of C-O-C bonds presented at 1081 cm−1 to absorbance of peak characteristic for vibration of C = O bonds observed at 1743 cm−1(AvCOC/AvC=C). For 4bPDLLA the ratio was equal 1.05 and 0.79, whereas for 2bPDLLA it was 1.02 and 0.82, before and after degradation, respectively. The results have shown, that 4bPDLLA degraded faster than linear PDLLA. Similar dependence was observed also for PLLAs. The AvCOC/AvC=C ratio decrease from 1.17 to 1.15 for 4bPLLA and from 1.20 to 1.19 for 2bPLLA. These small changes is an effect of low degradation rate of semi-crystalline polylactides. These results are very well correlated with the molecular weight changes, which were bigger for amorphous PDLLAs than for PLLAs.
Practically all described and analysed above properties of 2- and 4-armed polylactides are strongly dependent on molecular weight. Therefore, during synthesis the assumption of preparing polymers with similar molar masses, about 40 kDa, which is typical for drug carriers, our future goal. This allowed to eliminate one important variable from analysis. On the other hand, it caused differences in the length of polymer arm, what in our opinion caused differences in properties between linear and star-shaped polylactides. As it was shown by Xie et al. , above some critical arms molecular weight, for polycaprolactones it was 3400 Da, properties of polymers, especially thermal properties and crystallinity, are strongly dependent on arm length, not on the core. It happens, because the significant influence of the central part of macromolecule on arms chains mobility, and properties connected with this effect, is limited only to relatively short arms. Similar relationship is expected also for PLAs. In this manuscript the shortest arm was close to 20,000 Da, therefore the influence of a core structure on the PLAs properties is much lower than the influence of arms chain length.
In the present study novel 2- and 4-armed PDLLA and PLLA were synthesized and characterized. The effect of molecules architecture of the novel polylactides on their properties and degradation were analysed. Due to weaker inter-chain interaction, the 4-armed PLAs have slightly lower Tg, and insignificantly lower thermal stability. The higher crystallinity of 2bPLLA was also observed. There was small difference in the hydrophilicity of 2b and 4b PLAs. The hydrolytic degradation was slightly faster for polylactides of more complex architecture (4bPLAs). This is an effect of few aspects like shorter arm’s length or easier oligomers leaching. It can be also affected by higher content of end-chain hydroxyl groups in 4bPLAs. Moreover, thanks to changing of macromolecular structure also water uptake can be controlled.
All properties mentioned above are very important, especially in the case of biodegradable polymers for biomedical application. The presented results have shown the potential of designing polylactides properties by using different star-shaped architecture.
This work was financially supported by WUT-NIMS Joint Graduate School Program, European Regional Development Fund within the Innovative Operational Program in the frame of project BIO-IMPLANT (Grant No. POIG.01.01.02-00-022/09) and National Centre for Research and Development in the frame of project MentorEye (STRATEGMED1/2333624/4/NCBR/2014).
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