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Influence of polydispersity of poly(lactic acid) on particle formation by rapid expansion of supercritical CO2 solutions
J. of Supercritical Fluids 51 (2010) 376–383
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Influence of polydispersity of poly(lactic acid) on particle formation by rapid expansion of supercritical CO2 solutions Muhammad Imran ul-haq, Alberto Acosta-Ramírez, Parisa Mehrkhodavandi, Ruth Signorell ∗ Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1, Canada
a r t i c l e
i n f o
Article history: Received 10 July 2009 Received in revised form 23 October 2009 Accepted 24 October 2009 Keywords: Poly(lactic acid) RESS Supercritical CO2 Nanoscale particles and infrared spectroscopy
a b s t r a c t Poly(lactic acid) (PLA) particles were generated by rapid expansion of supercritical PLA/CO2 solutions (RESS). Two different PLA samples, one with high (PDI = 2.4) and the other one with low (PDI = 1.4) polydispersity but similar number average molecular weight, were compared. After micronization, the polymers were analysed by rapid-scan infrared spectroscopy, scanning electron microscopy, size-exclusion chromatography, differential scanning calorimetry, and NMR spectroscopy. Our investigation reveals that the polydispersity of the polymers strongly affects the size but not the shape of the particles. We found larger particles (∼730 nm) for the PLA with high polydispersity than for the PLA with low polydispersity (∼270 nm). In both cases, spherical particles were formed. Moreover, our results clearly show that PLA with high polydispersity is less suitable for RESS processing because the low-molecular weight chains are depleted over time and process conditions are thus not constant. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Poly(lactic acid) (PLA) and its copolymers are widely used for medical and pharmaceutical applications. Because of their biocompatibility and biodegradability, these polymers are ideal base materials to produce parenteral drug delivery systems, such as microparticles, nanoparticles, slabs, pellets, and in situ formed implants [1–7]. Rapid expansion of supercritical CO2 solutions (RESS) is an attractive method to produce submicron-sized particulate drug delivery systems [8–22]. The process conditions are comparatively mild and the particles are generated free from solvent residues. Several previous investigations were devoted to the micronization of l-poly(lactic acid) and dl-poly(lactic acid) by RESS [8–11] as well as to the formation of particulate drug delivery systems containing PLA [9,12,17–22]. In these drug delivery systems, PLA is either mixed with the drug or coats the drug particles. The goal of using a polymer in combination with the drug is twofold. The presence of the polymer can enable controlled drug release. Furthermore, PLA is used to stabilize the drug particles. Small drug particles are prone to agglomeration and coagulation, which reduces the effects gained by the micronization. As has been demonstrated for various pharmaceuticals (phytosterol, naproxen, and lovastatin) [9,19–21], coating with PLA strongly reduces agglomeration and coagulation. A comparison of the properties of the PLA particles among the vari-
∗ Corresponding author. Tel.: +1 604 822 9064; fax: +1 604 822 2847. E-mail address: [email protected] (R. Signorell). 0896-8446/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2009.10.010
ous RESS studies reveals major differences in the appearance of the particles (size and shape). A likely explanation for these deviations is the use of different PLA samples with varying molecular weights and polydispersities. The studies reported so far used commercially available PLA samples, which usually have broad molecular weight distributions (polydispersity indices between 2 and 4) as a result of the synthetic techniques employed (polycondensation or ring-opening polymerization of lactides with stannous octanoate (Sn(Oct2 )). The aim of the present contribution is to investigate the influence of the polydispersity of PLA on particle formation by RESS. To our knowledge this is the first systematic study of this kind. We compare commercially available PLA of high polydispersity with PLA of low polydispersity produced by living polymerization [23,24] to study the influence of the polydispersity on the particle size distribution, on the particle shape, and on the temporal stability of the particle generation process. 2. Experimental 2.1. Materials In the present contribution we compare two different types of dl-poly(lactic acid) (dl-PLA). Commercially available poly(lactic acid) (PLA1) with a number average molecular weight Mn = 30,000 g/mol and a polydispersity index of PDI = 2.40 was purchased from Sigma–Aldrich. Polymers with low polydispersity are not available commercially and had thus to be synthesized for the present study. PLA2 with a number average molecular
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CO2 gas (Technical grade, 4.0, Praxair) was used without further purification to prepare the supercritical solution. CO2 was chosen as supercritical solvent since it is a non-flammable, inexpensive, and non-toxic solvent. Due to the low critical data (Tcrit = 304 K, pcrit = 7.38 MPa), supercritical CO2 (sc-CO2 ) allows processing at moderate temperatures and pressures.
Nomenclature List of symbols D mean particle diameter (nm) dl racemic mixture of two enantiomers DSC differential scanning calorimetry Mn number average molecular weight (g/mol) PDI polydispersity index PLA1 poly(lactic acid) with broad distribution PLA2 poly(lactic acid) with narrow distribution p0 extraction and pre-expansion pressure (MPa) RESS rapid expansion of supercritical solution SEC size-exclusion chromatography SEM scanning electron microscope T0 extraction and pre-expansion temperature (K) Tg glass transition temperature
2.2. Solubility measurements
weight Mn = 35,000 g/mol and a polydispersity index of PDI = 1.40 was synthesized by living lactide polymerization using a novel indium catalyst [{(NNO)InCl}2 (-OEt)(-Cl)] (1). The details of the synthesis of PLA2 and of the indium catalyst are described in [23,24]. Briefly, the indium catalyst is an alkoxy-bridged dinuclear indium complex. The reaction of a racemic mixture of (±)-H2 (NNO) with two equivalents of KCH2 Ph forms (±)-KH-(NNO) and KOEt [25], which are not separated and upon addition to InCl3 form [{(NNO)InCl}2 (-OEt)(-Cl)] (1) in a one-pot reaction. Alternatively, the reaction between (±)-KH(NNO) and InCl3 gives [(NNO)InCl2 ], which can be converted into (1) by reaction with NaOEt. PLA2 was synthesized in a 20-mL scintillation vial by adding the catalyst (1) (0.0046 mmol) dissolved in CH2 Cl2 (1 mL) to a solution of lactide (rac-LA) (129.3 mg, 0.92 mmol) in CH2 Cl2 (6 mL). The solution was stirred for 16 h at room temperature, and then the volatiles were removed. The residue was dissolved in a minimum amount of CH2 Cl2 and added to cold wet methanol (7 mL, 273 K). The polymer precipitated and was isolated by centrifugation. The supernatant was decanted off, and the polymer was dried under high vacuum for 2 h prior to analysis. The number average molecular weight and polydispersity of both polymers were measured using size-exclusion chromatography (SEC). Triple detection gel permeation chromatography (GPC-LLS) with a Waters liquid chromatograph equipped with a Waters 515 HPLC pump, Waters 717 plus autosampler, Waters Styragel columns (4.6 mm × 300 mm) HR5E (2000–4,000,000), HR4 (5000–500,000), and HR2 (500–20,000), Waters2410 differential refractometer, Wyatt tristar miniDAWN (scattering detector) and a Wyatt Visco Star viscometer were used (flow rate 0.5 mL/min). The samples were dissolved in tetrahydrofuran (ca. 1 mg/mL) and run in conventional mode. Narrow molecular weight polystyrene standards served for calibration. The results are summarized in Table 1 (“before RESS”) together with the solubility in CO2 determined as described below.
The solubility of the unprocessed polymers (Table 1, “before RESS”) in CO2 was determined gravimetrically at 20 MPa and 323 K [26] using a dynamic solubility measurement technique described in detail by Alessi et al. [9,27,28]. In order to determine the quality and reproducibility of the solubility data, the measurements were repeated twice with a reproducibility of ±10%. The solubility of both polymers is given in Table 1 in terms of mass of polymer per mass of supercritical CO2 . As expected, the experimental data show a clear decrease in solubility with decreasing polydispersity. The lower solubility of PLA2 is due the absence of low-molecular weight chains, which are more soluble than high-molecular weight chains. The higher solubility of low-molecular weight chains compared to highmolecular weight chains is generally observed for polymers (see for explanations for example Refs. [30,31] and references therein). A direct comparison of our solubility data with previous literature values is not possible since the polymers used have different molecular weights and different polydispersities (often not specified) [9,17,20]. Furthermore, some studies use dl-PLA and others l-PLA, which have different solubilities [9]. The rather low solubility of our samples, however, is consistent with the high-molecular weights of our samples. 2.3. Particle formation The poly(lactic acid) particles were generated by rapid expansion of supercritical PLA/CO2 solutions [12–15]. A scheme of our RESS setup is shown in Fig. 1 and described in more detail in Ref. [29]. It consists of two units, the mixing unit where the supercritical solution is prepared, and the expansion unit where particle formation happens. The polymer was filled into the extractor together with glass beads and mixed with CO2 up to the desired pressure of p0 = 38 MPa. The total amount of PLA always exceeded its overall solubility. For the present experiments, the extractor and reservoir were connected and had the same pressure p0 and were heated to the same temperature T0 = 333 K. The supercritical solution was then expanded through a small nozzle (diameter of 400 m and length of 250 m) into the expansion chamber (0.08 m3 ). This expansion leads to supersaturation and thus to particle formation. The nozzle was operated in a pulsed mode with opening times between 500 and 600 ms and pulse repetition rates of 1 min−1 . With the same methods as described in Ref. [29], we have ascertained that the spray reached steady state conditions after about 40 ms. In situ particle characterization with infrared spectroscopy as well as particle collection for off-line characterization was performed in the expansion chamber.
Table 1 Number average molecular weights (Mn ) and polydispersity index (PDI) for PLA1 and PLA2 from SEC measurements. Before RESS: data of the original polymers. After RESS: data recorded after the RESS experiments (after run 3, see Section 3). As described in Section 2.2, the solubility in sc-CO2 was determined for the original polymers (see Section 2.2). Glass transition temperature (Tg) for PLA1 and PLA2 from DSC. As described in Section 2.4, residual material from the extractor after depressurization was used for these analyses. Polymers
Before RESS (unprocessed polymers) −1
Mn (g mol PLA1 PLA2
30,000 35,000
)
PDI 2.4 1.4
After RESS (after 3rd run) Solubility g(PLA)/g(CO2 ) −6
7.00 × 10 3.60 × 10−6
Tg (K)
Mn (g mol−1 )
PDI
Tg (K)
324 ± 1 329 ± 1
35,000 35,000
2.0 1.4
331 ± 1 330 ± 1
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Fig. 1. Scheme of the RESS setup: (a) mixing unit and (b) expansion unit. See Ref. [29] for details.
2.4. Characterization methods Mid-infrared spectra of the particles were recorded in situ in the aerosol phase (see Fig. 1) with a Bruker IFS 66v/S Fourier transform infrared spectrometer with a spectral resolution of 2 cm−1 and a time resolution of 30 ms. The spectrometer was equipped with a mid-infrared globar light source, a KBr beam splitter and an MCT detector. In situ measurements allow us to determine the relative amounts of polymer particles in the gas phase directly after expansion. Particle shape and size were determined with scanning electron microscopy (SEM, Hitachi S4700). For this purpose, the aerosol particles were collected onto silica plates located in the expansion chamber (Fig. 1) where they were allowed to settle directly from the aerosol phase. Particles were collected for several hours to ensure that a representative particle ensemble was collected from the gas phase. Additional measurements with a Scanning Mobility Particle Sizer directly sampling the aerosol phase confirmed that the amount of ultrafine particles, which are difficult to collect on a silica plate and thus could significantly bias the SEM size distributions, were negligible. The silica plates with polymers particles were coated with gold (5 nm) in a sputter coater. The average particle sizes were statistically determined from SEM images by image analysis software (ImageJ Version 1.38). Polymer molar mass distributions were measured via sizeexclusion chromatography (SEC) using tetrahydrofuran as eluent. The SEC setup was calibrated against low polydispersity polystyrene standards. The Differential Scanning Calorimetry (DSC) analysis was performed on a TA Instruments DSC Q100 (New Castle, DE, USA) equipped with a liquid nitrogen cooling system. Accurately weighed samples (∼2–5 mg) were hermetically sealed in an alu-
minum pan and heated from 308 to 440 K at a rate of 10 K/min under nitrogen flow. 1 H NMR spectra were recorded with a Bruker Advance-300 spectrometer operating at 300 MHz (1 H). Samples were dissolved in CD2 Cl2 and the spectra were recorded at 298 K. Chemical shifts are reported relative to the residual proton resonance to the deutrated solvent at 5.32 ppm (1 H). The amount of micronized material in the expansion chamber was not very high after the different runs (see Section 3) due to the rather low solubility and the pulsed operation. This was in particular true for PLA2. For this reason, we have performed additional NMR, DSC, and SEC measurements after run 3 using residual material from the extractor walls after depressurization to obtain high quality data (see data in Table 1 and NMR spectra in Fig. 9b). It is important to remark that apart from the higher quality the data from the residual material in the extractor and from the expansion chamber are almost identical. 3. Results The aim of the present contribution is to compare particle formation by RESS of PLA1 and PLA2 with different polydispersity but similar number average molecular weight (see Table 1). We are interested in studying the influence of polydispersity on the particle size, shape, and on the temporal characteristics of the particle generation process by RESS. To investigate the temporal behaviour a series of RESS experiments where the polymer was allowed to dissolve in sc-CO2 for 2 h (1st run), then for another 2 h (total of 4 h, 2nd run), and finally for yet another 12 h (total of 16 h, 3rd run) (see Table 2). All experiments were performed with the same sample and the reservoir was topped up with CO2 after expansion in between the runs. Our expectation was to find major changes with
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Fig. 2. Scheme of the depletion of short polymer chains (thin lines) as a function of time (runs) in sc-CO2 for (a) PLA1 with high polydispersity and (b) PLA2 with lower polydispersity. Long polymer chains are sketched as thick lines.
time for PLA1 but not for PLA2, as sketched in Fig. 2. Shorter polymer chains (thin lines in trace a) dissolve better in sc-CO2 than longer polymer chains (thick lines). For this reason, particles formed after the 1st run mainly consist of short chains, while particles formed after the 3rd run consist of longer chains because the short chains are depleted over time. The depletion of short chain polymer in PLA1 is expected to have a strong effect on the particle properties, which will therefore change over time during the RESS process. By contrast, the low polydispersity of PLA2 should guarantee much more stable particle properties during the RESS process, since the depletion of short chain polymers as a function of time is not an issue. PLA2 contains almost no short chain polymers. 3.1. Infrared spectroscopy With infrared spectroscopy, we can characterize the particles directly after formation in the aerosol phase (Fig. 1). This provides information on the total PLA amount in the aerosol phase and on the size of the aerosol particles. Fig. 3 shows the experimental spectra for PLA1 (panel a) and PLA2 (panel b) as a function of time during the RESS process (i.e. for the different runs). As expected both types of PLA show identical infrared band positions. The strongest one is the carbonyl band around 1760 cm−1 , while all other bands are comparatively weak (see insets). Note that the strong saturated bands in the insets are due to gas phase CO2 . For PLA1 we find a pronounced decrease of the absorbance over time (1st run to 3rd run in panel a), which reflects a decrease in the total amount of particulate PLA generated by RESS with increasing Table 2 Mean particle size (d50 ) from SEM for PLA1 and PLA2 as a function of time (number of runs). Pronounced changes with time are found for PLA1 but not for PLA2. Runs of RESS
Total dissolution time in sc-CO2 (h)
d50 (nm) PLA1
d50 (nm) PLA2
1st 2nd 3rd
2 4 16
725 560 325
265 270 260
Fig. 3. Infrared spectrum of PLA particles as a function of time (number of runs) in the region of the carbonyl band and between 1600 and 4400 cm−1 (insets). (a) PLA1 and (b) PLA2. The sharp peaks on the carbonyl absorption are due to water gas phase residues. The strong bands at 2360 and 3620 cm−1 in the insets are from CO2 gas.
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Fig. 4. SEM images of unprocessed PLA (before RESS): (a) PLA1 and (b) PLA2.
time. The reason is the lower solubility of longer chain PLA which increasingly dominate as the short chains are depleted over time as explained in Fig. 2 (see following sections). Consequently less PLA is found in the aerosol phase after the 3rd run than after the 1st run. The infrared spectra shown in the inset also give a first hint that the size of the PLA particles decreases from the 1st to the 3rd run. Elastic scattering causes a pronounced baseline shift towards higher wavelengths in the infrared extinction spectra of aerosols [32]. Since larger particles are better scatterers than smaller ones the larger baseline shift observed in the 1st run compared with the 3rd run indicates a decrease of the average particle size over time. This conclusion is confirmed by the SEM measurements in the following section. Particles generated from PLA2 behave differently (panel b). The infrared spectra show no decrease in the amount of PLA in the aerosol phase as a function of time. Nor do they show any change in scattering of the infrared light over time (inset). In this case, all polymer chains have similar chain lengths from the beginning so that any significant fractionation of different chain lengths over time is impossible (Fig. 2b). PLA2 forms smaller and fewer particles than PLA1 because of its overall lower solubility. More importantly the comparison of the infrared results for the two types of PLA demonstrates that constant particle formation conditions are stable only for PLA2, but not for the polydisperse PLA1. 3.2. Scanning electron microscopy Further information on the amount, size, and shape of polymer particles is gained from SEM images, which also allow us to compare the unprocessed (“before RESS”) PLAs. Fig. 4 reveals that
Fig. 5. SEM images of PLA1 particles as a function of time (number of runs): (a) 1st run and (b) 3rd run.
unprocessed samples of PLA1 (panel a) and of PLA2 (panel b) both consist of coagulated bulk material with essentially no difference between the two different types of PLA. Micronization completely changes the appearance of the polymers as demonstrated in Fig. 5 for PLA1 and in Fig. 6 for PLA2. In both cases RESS processing yields nice spherical polymer particles, but in contrast to the particle’s shape, their size and the amount of particles formed strongly depend on the polydispersity of PLA. The SEM images for PLA1 after the 1st and the 3rd runs in Fig. 5a and b, respectively, confirm the findings from infrared spectroscopy. The higher solubility of shorter chains leads to much larger particles after the 1st run with mean diameters around 730 nm. After the 3rd run when most of the short chains have been depleted and the solubility thus decreased, the mean size drops to about 330 nm. The SEM images for PLA2 in Fig. 6 are also consistent with the infrared results. Compared with PLA1 the particles are smaller and there are fewer of them (same collection time). The mean diameter of PLA2 particles lies around 270 nm independent of time (number of run), which indicates that small oligomers are absent and polymer solubility remains the same even after the 3rd run (Fig. 2b). The particle size distributions extracted from the SEM images of PLA1 (Fig. 5) and PLA2 (Fig. 6) are given in Fig. 7. They summarize the trends observed in the images. The particle size for PLA1 decreases over time because the overall solubility decreases in parallel with the depletion of short polymer chains and a corresponding reduction in polydispersity. By contrast the particle size for PLA2 is stable because the original substance has already a low polydispersity and thus a constant (and low) solubility. Fig. 8 characterizes the particle
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Fig. 6. SEM images of PLA2 particles as a function of time (number of runs): (a) 1st run and (b) 3rd run.
size distributions with three parameters: d50 , d10 , and d90 . d50 is the average diameter (values in Table 2), while d10 (d90 ) is the diameter for which 10% (90%) of the particles have a smaller diameter and hence the remaining 90% (10%) have a larger diameter. d50 as well as the width of the size distribution is constant over time for PLA2. For PLA1 by contrast, d50 decreases by 40% from the 1st to the 3rd run and the distribution becomes narrower. A direct comparison of our results with previous RESS studies on PLA [9,17,19,20] is not possible since the various investigations use different RESS conditions (different nozzle sized, pressures, expansion conditions etc.) and different PLA samples. Sizes, for example, were reported to vary from 300 nm up to ∼10 m. A general trend, however, cannot be derived from these investigations due to varying experimental conditions.
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Fig. 7. Size distributions of PLA particles extracted from SEM images: (a) PLA1 and (b) PLA2.
transition temperature (Tg). This is the reason why we find the lowest glass transition temperature of 324 K for unprocessed PLA1 (Table 1). Consistent with this, Tg increases to 331 K after the 3rd run. The depletion of short chains and thus the change in the polydispersity over time explains why the particle formation for PLA1 changes with the number of runs (see infrared and SEM results). The corresponding values for Mn , PDI, and Tg for PLA2 do not change during processing (Table 1). It is thus not surprising that particle formation conditions are stable for this polymer (see infrared and SEM results). The lower PDI value of PLA2 compared with PLA1 after the 3rd run also explains why fewer and smaller particles
3.3. SEC, DSC, and 1 H NMR Additional proof that the polydispersity of the polymer causes the difference in particle size and particle number density comes from SEC, DSC, and 1 H NMR measurements. The depletion of lowmolecular weight polymers over time for PLA1 is confirmed by the SEC and DSC measurements of the unprocessed PLA1 (before RESS) and of PLA1 after the 3rd run (after RESS) in Table 1. The polydispersity index decreases from 2.4 to 2.0 and the number average molecular weight increases from 30,000 to 35,000 g/mol, a clear indication of depletion of short chain polymers over time. The same trend is found in the DSC measurements. The thermal stability of polymers depends on their polydispersity. Longer chains lead to higher thermal stability of the polymer and thus higher glass
Fig. 8. Particle size d10 (♦ ), d50 ( 䊉), d90 ( ) of PLA1 and PLA2 after the 1st and the 3rd run extracted from SEM. Empty symbols: PLA1, Filled symbols: PLA2.
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Fig. 9. 1 H NMR of PLA1: (a) unprocessed (before RESS) and (b) after the 3rd run (after RESS). As described in Section 2.4, residual material from the extractor after depressurization was used for this measurement.
(Figs. 5 and 6) are formed from PLA2 after the third run. The DSC analyses also show that both polymers, PLA1 and PLA2, are in an amorphous state. Finally, the depletion of very short oligomers for PLA1 during RESS is also confirmed by the 1 H NMR spectra in Fig. 9. Panel a shows the spectrum of unprocessed PLA1 (before RESS) and panel b the spectrum after the 3rd run (after RESS). The most intense signals are those located at ∼1.55 and ∼5.16 ppm, which corresponded to –CH3 and –CH in the repetitive central groups present in the backbones of the polymer chains [33]. The –CH(e) groups in the backbones of small oligomers (up to the tetramer) appear at different chemical shifts between 5.02 and 5.12 ppm compared with the –CH(d) of longer polymer chains at 5.16 ppm. For PLA1, these signals (between 5.02 and 5.12 ppm) are only present before micronization (panel a) but not after the 3rd run (panel b). This indicates the removal of small oligomeric chains and thus also short chain polymers during the RESS process. The corresponding 1 H NMR spectra of a PLA2 in Fig. 10 are almost identical and do not show any significant contributions from small oligomers not even for the unprocessed material. 4. Summary The present contribution demonstrates that the polydispersity of poly(lactic acid) has a strong influence on particle formation
Fig. 10. 1 H NMR of PLA2: (a) unprocessed (before RESS) and (b) after the 3rd run (after RESS). As described in Section 2.4, residual material from the extractor after depressurization was used for this measurement.
by RESS. Comparing two PLA samples with different polydispersity (PDI = 2.4 and 1.4) but similar number average molecular weight (around 35,000 g/mol) we find that the polymer with high polydispersity forms much larger particles than the polymer with low polydispersity. The reason for this is the higher solubility of the short polymer chains present in the polymer with high polydispersity. The particle shape by contrast is not influenced by the polydispersity. Both polymers form spherical particles. We have also studied the stability of the particle formation as a function of time. Particle formation is stable for the polymer with low polydispersity. For the polymer with the high polydispersity, we find by contrast that the particle properties rapidly change during the RESS process. The particles become smaller with increasing time. The reason lies in the continuous depletion of short polymer chains, which are depleted faster than long chains during the RESS process on account of their higher solubility. This declining overall solubility in sc-CO2 reduces the average particle size in the aerosol phase. While the role played by the molecular weight in the micronization of PLA is well recognized, our investigation clearly demonstrates the additional importance of low polydispersity to ensure stable process conditions in technical applications of RESS. As an example, if PLA-coated drug particles are formed by RESS only a PLA sample with low polydispersity ensures a constant coating thickness during the process. Commercially available PLA unfortu-
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nately has rather high polydispersity. New synthetic approaches such as the living polymerization with a novel catalysts employed in the present study are crucial to improve this situation. Another way at least to reduce the polydispersity of PLA prepared by conventional synthetic methods is to pre-treat the polymer by adding extraction steps in sc-CO2 before micronization by RESS. With this method it is, however, very difficult to reach the same low PDIs as with living polymerization. Acknowledgements The authors are grateful to E. Breininger from the research group of Prof. Dr. Michael Türk who performed the solubility measurements and the SEM analysis with the Imaging analysis software. We also thank A.F. Douglas for help with the initial synthesis of the PLA2 samples. This project was financially supported by the Natural Sciences and Engineering Research Council of Canada, by the Canada Foundation for Innovation, and by the A.P. Sloan Foundation (R.S.). References [1] P.P. DeLuca, R.C. Mehta, A.G. Hausberger, B.C. Thanoo, Biodegradable polyesters for drug and polypeptide delivery, in: M.A. El-Nokaly, D.M. Piatt, B.A. Charpentier (Eds.), Polymeric Drug Delivery Systems, American Chemical Society, Washington, DC, 1993, pp. 53–79. [2] S. Giovagnoli, P. Blasi, M. Ricci, C. Rossi, Biodegradable microspheres as carriers for native superoxide dismutase and catalase delivery, AAPS PharmSciTech. 5 (4) (2004), Article 51. [3] M. Ricci, P. Blasi, S. Giovagnoli, L. Perioli, C. Vescovi, C. Rossi, Leucinostatin-A loaded nanospheres: characterization and in vivo toxicity and efficacy evaluation, Int. J. Pharm. 275 (2004) 61–72. [4] R.A. Jain, The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices, Biomaterials 21 (2000) 2475–2490. [5] C. Wasana, H. Wim, O. Siriporn, Preparation and characterization of Cephalexin loaded PLGA microspheres, Curr. Drug Deliv. 6 (1) (2009) 69–75. [6] H. Onishi, Y. Machida, In vitro and In vivo evaluation of microparticulate drug delivery systems composed of macromolecular prodrugs, Molecules 13 (2008) 2136–2155. [7] T. Musumeci, C.A. Ventura, I. Giannone, B. Ruozi, L. Montenegro, R. Pignatello, G. Puglisi, PLA/PLGA nanoparticles for sustained release of docetaxel, Int. J. Pharm. 325 (2006) 172–179. [8] A. Sane, M.C. Thies, Effect of material properties and processing conditions on RESS of poly(l-lactide), J. Supercrit. Fluids 40 (2007) 134–143. [9] M. Türk, G. Upper, P. Hils, Formation of composite drug–polymer particles by coprecipitation during the rapid expansion of supercritical fluids, J. Supercrit. Fluids 39 (2006) 253–263. [10] K. Matsuyama, K. Mishima, H. Umemoto, S. Yamaguchi, Environmentally Benign formation of polymeric microspheres by rapid expansion of supercritical carbon dioxide solution with a nonsolvent, Environ. Sci. Technol. 35 (2001) 4149–4155.
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The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu
Influence of polydispersity of poly(lactic acid) on particle formation by rapid expansion of supercritical CO2 solutions Muhammad Imran ul-haq, Alberto Acosta-Ramírez, Parisa Mehrkhodavandi, Ruth Signorell ∗ Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1, Canada
a r t i c l e
i n f o
Article history: Received 10 July 2009 Received in revised form 23 October 2009 Accepted 24 October 2009 Keywords: Poly(lactic acid) RESS Supercritical CO2 Nanoscale particles and infrared spectroscopy
a b s t r a c t Poly(lactic acid) (PLA) particles were generated by rapid expansion of supercritical PLA/CO2 solutions (RESS). Two different PLA samples, one with high (PDI = 2.4) and the other one with low (PDI = 1.4) polydispersity but similar number average molecular weight, were compared. After micronization, the polymers were analysed by rapid-scan infrared spectroscopy, scanning electron microscopy, size-exclusion chromatography, differential scanning calorimetry, and NMR spectroscopy. Our investigation reveals that the polydispersity of the polymers strongly affects the size but not the shape of the particles. We found larger particles (∼730 nm) for the PLA with high polydispersity than for the PLA with low polydispersity (∼270 nm). In both cases, spherical particles were formed. Moreover, our results clearly show that PLA with high polydispersity is less suitable for RESS processing because the low-molecular weight chains are depleted over time and process conditions are thus not constant. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Poly(lactic acid) (PLA) and its copolymers are widely used for medical and pharmaceutical applications. Because of their biocompatibility and biodegradability, these polymers are ideal base materials to produce parenteral drug delivery systems, such as microparticles, nanoparticles, slabs, pellets, and in situ formed implants [1–7]. Rapid expansion of supercritical CO2 solutions (RESS) is an attractive method to produce submicron-sized particulate drug delivery systems [8–22]. The process conditions are comparatively mild and the particles are generated free from solvent residues. Several previous investigations were devoted to the micronization of l-poly(lactic acid) and dl-poly(lactic acid) by RESS [8–11] as well as to the formation of particulate drug delivery systems containing PLA [9,12,17–22]. In these drug delivery systems, PLA is either mixed with the drug or coats the drug particles. The goal of using a polymer in combination with the drug is twofold. The presence of the polymer can enable controlled drug release. Furthermore, PLA is used to stabilize the drug particles. Small drug particles are prone to agglomeration and coagulation, which reduces the effects gained by the micronization. As has been demonstrated for various pharmaceuticals (phytosterol, naproxen, and lovastatin) [9,19–21], coating with PLA strongly reduces agglomeration and coagulation. A comparison of the properties of the PLA particles among the vari-
∗ Corresponding author. Tel.: +1 604 822 9064; fax: +1 604 822 2847. E-mail address: [email protected] (R. Signorell). 0896-8446/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2009.10.010
ous RESS studies reveals major differences in the appearance of the particles (size and shape). A likely explanation for these deviations is the use of different PLA samples with varying molecular weights and polydispersities. The studies reported so far used commercially available PLA samples, which usually have broad molecular weight distributions (polydispersity indices between 2 and 4) as a result of the synthetic techniques employed (polycondensation or ring-opening polymerization of lactides with stannous octanoate (Sn(Oct2 )). The aim of the present contribution is to investigate the influence of the polydispersity of PLA on particle formation by RESS. To our knowledge this is the first systematic study of this kind. We compare commercially available PLA of high polydispersity with PLA of low polydispersity produced by living polymerization [23,24] to study the influence of the polydispersity on the particle size distribution, on the particle shape, and on the temporal stability of the particle generation process. 2. Experimental 2.1. Materials In the present contribution we compare two different types of dl-poly(lactic acid) (dl-PLA). Commercially available poly(lactic acid) (PLA1) with a number average molecular weight Mn = 30,000 g/mol and a polydispersity index of PDI = 2.40 was purchased from Sigma–Aldrich. Polymers with low polydispersity are not available commercially and had thus to be synthesized for the present study. PLA2 with a number average molecular
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CO2 gas (Technical grade, 4.0, Praxair) was used without further purification to prepare the supercritical solution. CO2 was chosen as supercritical solvent since it is a non-flammable, inexpensive, and non-toxic solvent. Due to the low critical data (Tcrit = 304 K, pcrit = 7.38 MPa), supercritical CO2 (sc-CO2 ) allows processing at moderate temperatures and pressures.
Nomenclature List of symbols D mean particle diameter (nm) dl racemic mixture of two enantiomers DSC differential scanning calorimetry Mn number average molecular weight (g/mol) PDI polydispersity index PLA1 poly(lactic acid) with broad distribution PLA2 poly(lactic acid) with narrow distribution p0 extraction and pre-expansion pressure (MPa) RESS rapid expansion of supercritical solution SEC size-exclusion chromatography SEM scanning electron microscope T0 extraction and pre-expansion temperature (K) Tg glass transition temperature
2.2. Solubility measurements
weight Mn = 35,000 g/mol and a polydispersity index of PDI = 1.40 was synthesized by living lactide polymerization using a novel indium catalyst [{(NNO)InCl}2 (-OEt)(-Cl)] (1). The details of the synthesis of PLA2 and of the indium catalyst are described in [23,24]. Briefly, the indium catalyst is an alkoxy-bridged dinuclear indium complex. The reaction of a racemic mixture of (±)-H2 (NNO) with two equivalents of KCH2 Ph forms (±)-KH-(NNO) and KOEt [25], which are not separated and upon addition to InCl3 form [{(NNO)InCl}2 (-OEt)(-Cl)] (1) in a one-pot reaction. Alternatively, the reaction between (±)-KH(NNO) and InCl3 gives [(NNO)InCl2 ], which can be converted into (1) by reaction with NaOEt. PLA2 was synthesized in a 20-mL scintillation vial by adding the catalyst (1) (0.0046 mmol) dissolved in CH2 Cl2 (1 mL) to a solution of lactide (rac-LA) (129.3 mg, 0.92 mmol) in CH2 Cl2 (6 mL). The solution was stirred for 16 h at room temperature, and then the volatiles were removed. The residue was dissolved in a minimum amount of CH2 Cl2 and added to cold wet methanol (7 mL, 273 K). The polymer precipitated and was isolated by centrifugation. The supernatant was decanted off, and the polymer was dried under high vacuum for 2 h prior to analysis. The number average molecular weight and polydispersity of both polymers were measured using size-exclusion chromatography (SEC). Triple detection gel permeation chromatography (GPC-LLS) with a Waters liquid chromatograph equipped with a Waters 515 HPLC pump, Waters 717 plus autosampler, Waters Styragel columns (4.6 mm × 300 mm) HR5E (2000–4,000,000), HR4 (5000–500,000), and HR2 (500–20,000), Waters2410 differential refractometer, Wyatt tristar miniDAWN (scattering detector) and a Wyatt Visco Star viscometer were used (flow rate 0.5 mL/min). The samples were dissolved in tetrahydrofuran (ca. 1 mg/mL) and run in conventional mode. Narrow molecular weight polystyrene standards served for calibration. The results are summarized in Table 1 (“before RESS”) together with the solubility in CO2 determined as described below.
The solubility of the unprocessed polymers (Table 1, “before RESS”) in CO2 was determined gravimetrically at 20 MPa and 323 K [26] using a dynamic solubility measurement technique described in detail by Alessi et al. [9,27,28]. In order to determine the quality and reproducibility of the solubility data, the measurements were repeated twice with a reproducibility of ±10%. The solubility of both polymers is given in Table 1 in terms of mass of polymer per mass of supercritical CO2 . As expected, the experimental data show a clear decrease in solubility with decreasing polydispersity. The lower solubility of PLA2 is due the absence of low-molecular weight chains, which are more soluble than high-molecular weight chains. The higher solubility of low-molecular weight chains compared to highmolecular weight chains is generally observed for polymers (see for explanations for example Refs. [30,31] and references therein). A direct comparison of our solubility data with previous literature values is not possible since the polymers used have different molecular weights and different polydispersities (often not specified) [9,17,20]. Furthermore, some studies use dl-PLA and others l-PLA, which have different solubilities [9]. The rather low solubility of our samples, however, is consistent with the high-molecular weights of our samples. 2.3. Particle formation The poly(lactic acid) particles were generated by rapid expansion of supercritical PLA/CO2 solutions [12–15]. A scheme of our RESS setup is shown in Fig. 1 and described in more detail in Ref. [29]. It consists of two units, the mixing unit where the supercritical solution is prepared, and the expansion unit where particle formation happens. The polymer was filled into the extractor together with glass beads and mixed with CO2 up to the desired pressure of p0 = 38 MPa. The total amount of PLA always exceeded its overall solubility. For the present experiments, the extractor and reservoir were connected and had the same pressure p0 and were heated to the same temperature T0 = 333 K. The supercritical solution was then expanded through a small nozzle (diameter of 400 m and length of 250 m) into the expansion chamber (0.08 m3 ). This expansion leads to supersaturation and thus to particle formation. The nozzle was operated in a pulsed mode with opening times between 500 and 600 ms and pulse repetition rates of 1 min−1 . With the same methods as described in Ref. [29], we have ascertained that the spray reached steady state conditions after about 40 ms. In situ particle characterization with infrared spectroscopy as well as particle collection for off-line characterization was performed in the expansion chamber.
Table 1 Number average molecular weights (Mn ) and polydispersity index (PDI) for PLA1 and PLA2 from SEC measurements. Before RESS: data of the original polymers. After RESS: data recorded after the RESS experiments (after run 3, see Section 3). As described in Section 2.2, the solubility in sc-CO2 was determined for the original polymers (see Section 2.2). Glass transition temperature (Tg) for PLA1 and PLA2 from DSC. As described in Section 2.4, residual material from the extractor after depressurization was used for these analyses. Polymers
Before RESS (unprocessed polymers) −1
Mn (g mol PLA1 PLA2
30,000 35,000
)
PDI 2.4 1.4
After RESS (after 3rd run) Solubility g(PLA)/g(CO2 ) −6
7.00 × 10 3.60 × 10−6
Tg (K)
Mn (g mol−1 )
PDI
Tg (K)
324 ± 1 329 ± 1
35,000 35,000
2.0 1.4
331 ± 1 330 ± 1
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Fig. 1. Scheme of the RESS setup: (a) mixing unit and (b) expansion unit. See Ref. [29] for details.
2.4. Characterization methods Mid-infrared spectra of the particles were recorded in situ in the aerosol phase (see Fig. 1) with a Bruker IFS 66v/S Fourier transform infrared spectrometer with a spectral resolution of 2 cm−1 and a time resolution of 30 ms. The spectrometer was equipped with a mid-infrared globar light source, a KBr beam splitter and an MCT detector. In situ measurements allow us to determine the relative amounts of polymer particles in the gas phase directly after expansion. Particle shape and size were determined with scanning electron microscopy (SEM, Hitachi S4700). For this purpose, the aerosol particles were collected onto silica plates located in the expansion chamber (Fig. 1) where they were allowed to settle directly from the aerosol phase. Particles were collected for several hours to ensure that a representative particle ensemble was collected from the gas phase. Additional measurements with a Scanning Mobility Particle Sizer directly sampling the aerosol phase confirmed that the amount of ultrafine particles, which are difficult to collect on a silica plate and thus could significantly bias the SEM size distributions, were negligible. The silica plates with polymers particles were coated with gold (5 nm) in a sputter coater. The average particle sizes were statistically determined from SEM images by image analysis software (ImageJ Version 1.38). Polymer molar mass distributions were measured via sizeexclusion chromatography (SEC) using tetrahydrofuran as eluent. The SEC setup was calibrated against low polydispersity polystyrene standards. The Differential Scanning Calorimetry (DSC) analysis was performed on a TA Instruments DSC Q100 (New Castle, DE, USA) equipped with a liquid nitrogen cooling system. Accurately weighed samples (∼2–5 mg) were hermetically sealed in an alu-
minum pan and heated from 308 to 440 K at a rate of 10 K/min under nitrogen flow. 1 H NMR spectra were recorded with a Bruker Advance-300 spectrometer operating at 300 MHz (1 H). Samples were dissolved in CD2 Cl2 and the spectra were recorded at 298 K. Chemical shifts are reported relative to the residual proton resonance to the deutrated solvent at 5.32 ppm (1 H). The amount of micronized material in the expansion chamber was not very high after the different runs (see Section 3) due to the rather low solubility and the pulsed operation. This was in particular true for PLA2. For this reason, we have performed additional NMR, DSC, and SEC measurements after run 3 using residual material from the extractor walls after depressurization to obtain high quality data (see data in Table 1 and NMR spectra in Fig. 9b). It is important to remark that apart from the higher quality the data from the residual material in the extractor and from the expansion chamber are almost identical. 3. Results The aim of the present contribution is to compare particle formation by RESS of PLA1 and PLA2 with different polydispersity but similar number average molecular weight (see Table 1). We are interested in studying the influence of polydispersity on the particle size, shape, and on the temporal characteristics of the particle generation process by RESS. To investigate the temporal behaviour a series of RESS experiments where the polymer was allowed to dissolve in sc-CO2 for 2 h (1st run), then for another 2 h (total of 4 h, 2nd run), and finally for yet another 12 h (total of 16 h, 3rd run) (see Table 2). All experiments were performed with the same sample and the reservoir was topped up with CO2 after expansion in between the runs. Our expectation was to find major changes with
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Fig. 2. Scheme of the depletion of short polymer chains (thin lines) as a function of time (runs) in sc-CO2 for (a) PLA1 with high polydispersity and (b) PLA2 with lower polydispersity. Long polymer chains are sketched as thick lines.
time for PLA1 but not for PLA2, as sketched in Fig. 2. Shorter polymer chains (thin lines in trace a) dissolve better in sc-CO2 than longer polymer chains (thick lines). For this reason, particles formed after the 1st run mainly consist of short chains, while particles formed after the 3rd run consist of longer chains because the short chains are depleted over time. The depletion of short chain polymer in PLA1 is expected to have a strong effect on the particle properties, which will therefore change over time during the RESS process. By contrast, the low polydispersity of PLA2 should guarantee much more stable particle properties during the RESS process, since the depletion of short chain polymers as a function of time is not an issue. PLA2 contains almost no short chain polymers. 3.1. Infrared spectroscopy With infrared spectroscopy, we can characterize the particles directly after formation in the aerosol phase (Fig. 1). This provides information on the total PLA amount in the aerosol phase and on the size of the aerosol particles. Fig. 3 shows the experimental spectra for PLA1 (panel a) and PLA2 (panel b) as a function of time during the RESS process (i.e. for the different runs). As expected both types of PLA show identical infrared band positions. The strongest one is the carbonyl band around 1760 cm−1 , while all other bands are comparatively weak (see insets). Note that the strong saturated bands in the insets are due to gas phase CO2 . For PLA1 we find a pronounced decrease of the absorbance over time (1st run to 3rd run in panel a), which reflects a decrease in the total amount of particulate PLA generated by RESS with increasing Table 2 Mean particle size (d50 ) from SEM for PLA1 and PLA2 as a function of time (number of runs). Pronounced changes with time are found for PLA1 but not for PLA2. Runs of RESS
Total dissolution time in sc-CO2 (h)
d50 (nm) PLA1
d50 (nm) PLA2
1st 2nd 3rd
2 4 16
725 560 325
265 270 260
Fig. 3. Infrared spectrum of PLA particles as a function of time (number of runs) in the region of the carbonyl band and between 1600 and 4400 cm−1 (insets). (a) PLA1 and (b) PLA2. The sharp peaks on the carbonyl absorption are due to water gas phase residues. The strong bands at 2360 and 3620 cm−1 in the insets are from CO2 gas.
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Fig. 4. SEM images of unprocessed PLA (before RESS): (a) PLA1 and (b) PLA2.
time. The reason is the lower solubility of longer chain PLA which increasingly dominate as the short chains are depleted over time as explained in Fig. 2 (see following sections). Consequently less PLA is found in the aerosol phase after the 3rd run than after the 1st run. The infrared spectra shown in the inset also give a first hint that the size of the PLA particles decreases from the 1st to the 3rd run. Elastic scattering causes a pronounced baseline shift towards higher wavelengths in the infrared extinction spectra of aerosols [32]. Since larger particles are better scatterers than smaller ones the larger baseline shift observed in the 1st run compared with the 3rd run indicates a decrease of the average particle size over time. This conclusion is confirmed by the SEM measurements in the following section. Particles generated from PLA2 behave differently (panel b). The infrared spectra show no decrease in the amount of PLA in the aerosol phase as a function of time. Nor do they show any change in scattering of the infrared light over time (inset). In this case, all polymer chains have similar chain lengths from the beginning so that any significant fractionation of different chain lengths over time is impossible (Fig. 2b). PLA2 forms smaller and fewer particles than PLA1 because of its overall lower solubility. More importantly the comparison of the infrared results for the two types of PLA demonstrates that constant particle formation conditions are stable only for PLA2, but not for the polydisperse PLA1. 3.2. Scanning electron microscopy Further information on the amount, size, and shape of polymer particles is gained from SEM images, which also allow us to compare the unprocessed (“before RESS”) PLAs. Fig. 4 reveals that
Fig. 5. SEM images of PLA1 particles as a function of time (number of runs): (a) 1st run and (b) 3rd run.
unprocessed samples of PLA1 (panel a) and of PLA2 (panel b) both consist of coagulated bulk material with essentially no difference between the two different types of PLA. Micronization completely changes the appearance of the polymers as demonstrated in Fig. 5 for PLA1 and in Fig. 6 for PLA2. In both cases RESS processing yields nice spherical polymer particles, but in contrast to the particle’s shape, their size and the amount of particles formed strongly depend on the polydispersity of PLA. The SEM images for PLA1 after the 1st and the 3rd runs in Fig. 5a and b, respectively, confirm the findings from infrared spectroscopy. The higher solubility of shorter chains leads to much larger particles after the 1st run with mean diameters around 730 nm. After the 3rd run when most of the short chains have been depleted and the solubility thus decreased, the mean size drops to about 330 nm. The SEM images for PLA2 in Fig. 6 are also consistent with the infrared results. Compared with PLA1 the particles are smaller and there are fewer of them (same collection time). The mean diameter of PLA2 particles lies around 270 nm independent of time (number of run), which indicates that small oligomers are absent and polymer solubility remains the same even after the 3rd run (Fig. 2b). The particle size distributions extracted from the SEM images of PLA1 (Fig. 5) and PLA2 (Fig. 6) are given in Fig. 7. They summarize the trends observed in the images. The particle size for PLA1 decreases over time because the overall solubility decreases in parallel with the depletion of short polymer chains and a corresponding reduction in polydispersity. By contrast the particle size for PLA2 is stable because the original substance has already a low polydispersity and thus a constant (and low) solubility. Fig. 8 characterizes the particle
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Fig. 6. SEM images of PLA2 particles as a function of time (number of runs): (a) 1st run and (b) 3rd run.
size distributions with three parameters: d50 , d10 , and d90 . d50 is the average diameter (values in Table 2), while d10 (d90 ) is the diameter for which 10% (90%) of the particles have a smaller diameter and hence the remaining 90% (10%) have a larger diameter. d50 as well as the width of the size distribution is constant over time for PLA2. For PLA1 by contrast, d50 decreases by 40% from the 1st to the 3rd run and the distribution becomes narrower. A direct comparison of our results with previous RESS studies on PLA [9,17,19,20] is not possible since the various investigations use different RESS conditions (different nozzle sized, pressures, expansion conditions etc.) and different PLA samples. Sizes, for example, were reported to vary from 300 nm up to ∼10 m. A general trend, however, cannot be derived from these investigations due to varying experimental conditions.
381
Fig. 7. Size distributions of PLA particles extracted from SEM images: (a) PLA1 and (b) PLA2.
transition temperature (Tg). This is the reason why we find the lowest glass transition temperature of 324 K for unprocessed PLA1 (Table 1). Consistent with this, Tg increases to 331 K after the 3rd run. The depletion of short chains and thus the change in the polydispersity over time explains why the particle formation for PLA1 changes with the number of runs (see infrared and SEM results). The corresponding values for Mn , PDI, and Tg for PLA2 do not change during processing (Table 1). It is thus not surprising that particle formation conditions are stable for this polymer (see infrared and SEM results). The lower PDI value of PLA2 compared with PLA1 after the 3rd run also explains why fewer and smaller particles
3.3. SEC, DSC, and 1 H NMR Additional proof that the polydispersity of the polymer causes the difference in particle size and particle number density comes from SEC, DSC, and 1 H NMR measurements. The depletion of lowmolecular weight polymers over time for PLA1 is confirmed by the SEC and DSC measurements of the unprocessed PLA1 (before RESS) and of PLA1 after the 3rd run (after RESS) in Table 1. The polydispersity index decreases from 2.4 to 2.0 and the number average molecular weight increases from 30,000 to 35,000 g/mol, a clear indication of depletion of short chain polymers over time. The same trend is found in the DSC measurements. The thermal stability of polymers depends on their polydispersity. Longer chains lead to higher thermal stability of the polymer and thus higher glass
Fig. 8. Particle size d10 (♦ ), d50 ( 䊉), d90 ( ) of PLA1 and PLA2 after the 1st and the 3rd run extracted from SEM. Empty symbols: PLA1, Filled symbols: PLA2.
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Fig. 9. 1 H NMR of PLA1: (a) unprocessed (before RESS) and (b) after the 3rd run (after RESS). As described in Section 2.4, residual material from the extractor after depressurization was used for this measurement.
(Figs. 5 and 6) are formed from PLA2 after the third run. The DSC analyses also show that both polymers, PLA1 and PLA2, are in an amorphous state. Finally, the depletion of very short oligomers for PLA1 during RESS is also confirmed by the 1 H NMR spectra in Fig. 9. Panel a shows the spectrum of unprocessed PLA1 (before RESS) and panel b the spectrum after the 3rd run (after RESS). The most intense signals are those located at ∼1.55 and ∼5.16 ppm, which corresponded to –CH3 and –CH in the repetitive central groups present in the backbones of the polymer chains [33]. The –CH(e) groups in the backbones of small oligomers (up to the tetramer) appear at different chemical shifts between 5.02 and 5.12 ppm compared with the –CH(d) of longer polymer chains at 5.16 ppm. For PLA1, these signals (between 5.02 and 5.12 ppm) are only present before micronization (panel a) but not after the 3rd run (panel b). This indicates the removal of small oligomeric chains and thus also short chain polymers during the RESS process. The corresponding 1 H NMR spectra of a PLA2 in Fig. 10 are almost identical and do not show any significant contributions from small oligomers not even for the unprocessed material. 4. Summary The present contribution demonstrates that the polydispersity of poly(lactic acid) has a strong influence on particle formation
Fig. 10. 1 H NMR of PLA2: (a) unprocessed (before RESS) and (b) after the 3rd run (after RESS). As described in Section 2.4, residual material from the extractor after depressurization was used for this measurement.
by RESS. Comparing two PLA samples with different polydispersity (PDI = 2.4 and 1.4) but similar number average molecular weight (around 35,000 g/mol) we find that the polymer with high polydispersity forms much larger particles than the polymer with low polydispersity. The reason for this is the higher solubility of the short polymer chains present in the polymer with high polydispersity. The particle shape by contrast is not influenced by the polydispersity. Both polymers form spherical particles. We have also studied the stability of the particle formation as a function of time. Particle formation is stable for the polymer with low polydispersity. For the polymer with the high polydispersity, we find by contrast that the particle properties rapidly change during the RESS process. The particles become smaller with increasing time. The reason lies in the continuous depletion of short polymer chains, which are depleted faster than long chains during the RESS process on account of their higher solubility. This declining overall solubility in sc-CO2 reduces the average particle size in the aerosol phase. While the role played by the molecular weight in the micronization of PLA is well recognized, our investigation clearly demonstrates the additional importance of low polydispersity to ensure stable process conditions in technical applications of RESS. As an example, if PLA-coated drug particles are formed by RESS only a PLA sample with low polydispersity ensures a constant coating thickness during the process. Commercially available PLA unfortu-
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nately has rather high polydispersity. New synthetic approaches such as the living polymerization with a novel catalysts employed in the present study are crucial to improve this situation. Another way at least to reduce the polydispersity of PLA prepared by conventional synthetic methods is to pre-treat the polymer by adding extraction steps in sc-CO2 before micronization by RESS. With this method it is, however, very difficult to reach the same low PDIs as with living polymerization. Acknowledgements The authors are grateful to E. Breininger from the research group of Prof. Dr. Michael Türk who performed the solubility measurements and the SEM analysis with the Imaging analysis software. We also thank A.F. Douglas for help with the initial synthesis of the PLA2 samples. This project was financially supported by the Natural Sciences and Engineering Research Council of Canada, by the Canada Foundation for Innovation, and by the A.P. Sloan Foundation (R.S.). References [1] P.P. DeLuca, R.C. Mehta, A.G. Hausberger, B.C. Thanoo, Biodegradable polyesters for drug and polypeptide delivery, in: M.A. El-Nokaly, D.M. Piatt, B.A. Charpentier (Eds.), Polymeric Drug Delivery Systems, American Chemical Society, Washington, DC, 1993, pp. 53–79. [2] S. Giovagnoli, P. Blasi, M. Ricci, C. Rossi, Biodegradable microspheres as carriers for native superoxide dismutase and catalase delivery, AAPS PharmSciTech. 5 (4) (2004), Article 51. [3] M. Ricci, P. Blasi, S. Giovagnoli, L. Perioli, C. Vescovi, C. Rossi, Leucinostatin-A loaded nanospheres: characterization and in vivo toxicity and efficacy evaluation, Int. J. Pharm. 275 (2004) 61–72. [4] R.A. Jain, The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices, Biomaterials 21 (2000) 2475–2490. [5] C. Wasana, H. Wim, O. Siriporn, Preparation and characterization of Cephalexin loaded PLGA microspheres, Curr. Drug Deliv. 6 (1) (2009) 69–75. [6] H. Onishi, Y. Machida, In vitro and In vivo evaluation of microparticulate drug delivery systems composed of macromolecular prodrugs, Molecules 13 (2008) 2136–2155. [7] T. Musumeci, C.A. Ventura, I. Giannone, B. Ruozi, L. Montenegro, R. Pignatello, G. Puglisi, PLA/PLGA nanoparticles for sustained release of docetaxel, Int. J. Pharm. 325 (2006) 172–179. [8] A. Sane, M.C. Thies, Effect of material properties and processing conditions on RESS of poly(l-lactide), J. Supercrit. Fluids 40 (2007) 134–143. [9] M. Türk, G. Upper, P. Hils, Formation of composite drug–polymer particles by coprecipitation during the rapid expansion of supercritical fluids, J. Supercrit. Fluids 39 (2006) 253–263. [10] K. Matsuyama, K. Mishima, H. Umemoto, S. Yamaguchi, Environmentally Benign formation of polymeric microspheres by rapid expansion of supercritical carbon dioxide solution with a nonsolvent, Environ. Sci. Technol. 35 (2001) 4149–4155.
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