Effect of Molecular Weight, Crystallinity, and Hydrophobicity on the

Apr 7, 2009 - America, 345 Scarborough Road, Briarcliff Manor, New York 10510. Received October 29, 2008; Revised Manuscript Received February 23, ...
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Biomacromolecules 2009, 10, 1025–1031

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Articles Effect of Molecular Weight, Crystallinity, and Hydrophobicity on the Acoustic Activation of Polymer-Shelled Ultrasound Contrast Agents Ceciel Chlon,† Constant Gue´don,† Bram Verhaagen,‡ William T. Shi,‡ Christopher S. Hall,‡ Johan Lub,† and Marcel R. Bo¨hmer*,† Philips Research Europe, HTC11, 5656 AE, Eindhoven, The Netherlands, and Philips Research North America, 345 Scarborough Road, Briarcliff Manor, New York 10510 Received October 29, 2008; Revised Manuscript Received February 23, 2009

Polymer-shelled microbubbles are applied as ultrasound contrast agents. To investigate the effect of the polymer on microbubble preparation and acoustic properties, polylactides with systematic variations in molecular weight, crystallinity, and end-group hydrophobicity were used. Polymer-shelled cyclodecane filled capsules were prepared by emulsification, and the cyclodecane was removed by lyophilization to obtain hollow capsules. Complete removal of cyclodecane from the microcapsules was only achieved for short chain (about Mw 6000) crystalline polymers. The pressure threshold for acoustic destruction of the microbubbles was found to increase with molecular weight. Noncrystalline polymers showed a higher threshold for destruction than crystalline polymers. Hydrophobically modified short chain crystalline polymers showed the steepest increase in acoustic destruction after the threshold as a function of the applied pressure, which is a favorable characteristic for ultrasound mediated drug delivery. Microcapsules made with such polymers had an inhomogeneous surface including pores through which cyclodecane was lyophilized efficiently.

Introduction Ultrasound microbubble contrast agents are used for the enhancement of ultrasound images, thereby improving the accuracy of diagnoses.1 These microbubbles can be synthesized with a lipid, polymer, or protein as the shell material and have a core consisting of gas. Besides contrast enhancement in ultrasound imaging, these agents have potential therapeutic applications in the field of gene and drug delivery and in sonothrombolysis.2-4 Drugs or genes can be attached to or built into the microbubbles to facilitate image-guided drug delivery. To use such an agent for therapeutic applications, the microbubble needs to be fragmented to release the content. Lipid and albumin-shelled microbubbles are applied in the clinic to support ultrasound imaging. Contrast imaging with these agents is performed at low acoustic pressures, for instance, around 100 kPa at 1 MHz. As gas is compressible and expandable as a function of pressure, this leads to volume changes of the microbubble when ultrasound is applied. The volume changes can lead to diameter changes with for instance a factor of 2.5 The required acoustic threshold pressure to fragment lipid and protein bubbles is also low but occurs over a wide acoustic pressure range, which is due to their broad size distribution.6 Compared to lipid-shelled agents, polymer-shelled agents are stiffer and therefore they show less volume changes if an acoustic pressure is applied. Polymer-shelled microbubbles made * To whom correspondence should be addressed. Fax: +31 40-2744906. Phone: +31 40-2748252. E-mail: [email protected]. † Philips Research Europe. ‡ Philips Research North America.

by emulsification techniques can be obtained with a much narrower size distribution than lipid-shelled ultrasound contrast agents as used in the clinic. This leads to a sharp threshold for activation, which is the destruction of the microbubbles by ultrasound. Apart from the release of a drug payload, this can lead to (temporary) permeation of vessel walls and is therefore interesting for local drug delivery applications. According to Bloch7 and Mehier-Humbert,8 the applied acoustic pressure necessary for the fragmentation of the polymer capsule is higher than for the lipid-shelled agent. This will, however, depend critically on the polymer properties. We recently reported that polymer-shelled microcapsules made of low molecular weight L-polylactide with a fluorinated end group (PLLA-PFO) showed a sharp threshold for activation at low acoustic pressures of about 0.5 MPa at 1 MHz frequency, which is in the same range as the lipid-shelled agent as discussed by Kooiman.9 After the threshold had been reached, the number of microbubbles activated as a function of pressure increased steeply. This property makes this polymer-shelled agent a suitable vehicle for local drug delivery as the content of the agent is released upon destruction by means of ultrasound at an acoustic pressure allowed for diagnostic imaging. In this study, we investigated the influence of the chemical composition of the shell of a polymeric capsule as a tool to control the acoustic properties of the contrast agent. Microcapsules of the linear biodegradable polymer polylactide, both in the L and DL form, PLLA and PDLLA, as well as polylactideco-glycolide) (PLGA) were prepared using an emulsification technique.10 Application of this technique leads to oil-filled capsules from which the core is removed by freeze-drying. The effects of chain length, crystallinity, and the presence of a

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Figure 1. Structural formulas of the polymers with hydrophobic end groups, PLLA-PFO and PDLLA-PFO (A), PLLA-PFH (B), and PLLACx (C), indicating the protons used for verification of the incorporation of the group by NMR.

hydrophobic end group were studied with the aim to investigate the correlation between polymer properties and the suitability of microcapsules made thereof for ultrasound induced drug delivery. Size distributions of the prepared microcapsules were measured before and after freeze-drying. To measure the core content and the shell crystallinity of the capsules (modulated) differential scanning calorimetry (MDSC) was used.11 Electron microscopy analyses were performed to obtain information about the core content and shell morphology. Finally, the acoustic properties are evaluated using an ultrasound setup with a focused 1 MHz transducer, and broadband receiving transducer, which detects the release of gas from the microbubbles, was used to study the destruction of the microcapsules as a function of the applied acoustic pressure.9,12

Experimental Section Materials. Poly-L-lactide (PLLA), poly-DL-lactide (PDLLA), and poly(lactide-co-glycolide) (PLGA) with a lactide to glycolide ratio of 1:1 were gifts from Purac, and an additional PLLA was obtained from Fluka. Low molecular weight polylactides were obtained from Polymer Source. All polymers used in this study are linear. Low molecular weight polymers with hydrophobic end groups were synthesized in house using 1-octanol, 1-octadecanol, pentadecafluoro-1-octanol, or 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanol (all Aldrich) as starting materials. In addition, L- and DL-lactide, stannous 2-ethylhexanoate, and poly(ethylene glycol) (Mw ) 3400) were obtained from Aldrich, chloroform, dichloromethane (DCM), toluene, and diethylether were obtained from Merck, and tetrahydrofuran (THF) was from Acros. Cyclodecane and poly(vinylalcohol), PVA, with a molecular weight of 67 000 were obtained from Fluka. All chemicals were used as received. Synthesis of Low Mw Polylactides with a Hydrophobic End Group. The polymers PLLA-PFH, PLLA-C8, PLLA-C18, and PDLLAPFO (see Figure 1 and Table 1 for the end groups) were synthesized from a molten mixture, according to Lee et al.13 A solution of 0.5 mmol of stannous 2-ethylhexanoate in 0.3 mL of toluene was added to a molten mixture of 0.05 mol of L- or DL-lactide and 4.5 mmol of 1-octanol, 1-octadecanol, pentadecafluoro-1-octanol, or 3,3,4,4,5,5,6,6,6nonafluoro-1-hexanol, respectively, in a nitrogen atmosphere at 135 °C. After stirring for 4 h, the mixture was cooled to room temperature and dissolved in 25 mL of chloroform. All synthesized polymers, dissolved in chloroform, were filtered using a Whatman GD/X syringe filter (0.45 µm), and 100 mL of methanol was added. The precipitate was filtered off and redissolved in 50 mL of dichloromethane. After addition of 250 mL of diethyl ether, the preparation was cooled at 4 °C. A white solid was obtained, which was dried in a desiccator. Polymer Characterization. All polymers were analyzed with GPC. Twenty microliter aliquots of the polylactide samples (concentration approximately 5 µg/µL in THF) were analyzed using an Agilent 1200 HPLC system, consisting of a binary pump, an autosampler, a column

Chlon et al. oven, and a UV detector, equipped with two PLgel mixed-C columns (300 × 7.5 mm, 5 µm particles) in series, run at 45 °C and a flow rate of 1 mL THF per minute. The eluate was monitored using UV absorption at 236 nm. The high molecular weight samples of Fluka and Purac were hardly soluble in THF and were therefore measured in DCM at 30 °C under otherwise the same experimental conditions. The system was calibrated using the L-polylactide standards (PSS-PLAkit), Polymer Standard Services, Mainz, Germany) in THF and in DCM. Standards were run in duplicate, and the average elution volumes (Ve) were calculated and plotted in a calibration curve. Subsequently, the samples were run in duplicate, and the average molecular weights of the synthesized polymers were calculated using their elution volumes and the calibration curve. For the home-synthesized polymers, the presence of the hydrophobic end group was confirmed by 1H NMR. NMR spectra of the polymers were recorded with a Bruker DPX300 spectrometer in deuterated chloroform. The 1H NMR spectra are given in Figure S1A-H in the Supporting Information. For the PLLA-PFO and the PDLLA-PFO samples, the incorporation of the fluorinated end group in the precipitated product can be verified from the ratio of the protons adjacent to the fluorinated carbon atoms and the methane proton of the last lactide bearing the OH end group, marked as “a” and “b” as indicated in Figure 1. If this value equals 2, complete incorporation is achieved. The 1H NMR spectrum of polymer PLLA-PFH revealed that the quartet of the proton of the last lactide Hc at 4.36 ppm partly coincides with the multiplets of protons adjacent to the first CH2, Ha around 4.45 ppm; see the Supporting Information, Figure S1f. Therefore, the incorporation of the end group was analyzed by comparing the integral of these combined signals with that of the signals of the proton adjacent to the last CF2, Hb observed as multiplet at 2.49 ppm; see Figure 1 and Figure S1f. For poly(L-lactic acid) octyl terminated (PLLAC8, x ) 6) and poly(L-lactic acid) octadecyl terminated (PLLA-C18, x ) 16), the terminal methyl protons were used for quantification of the incorporation of the alkyl chain, which should lead to a theoretical value of 3. The obtained values are for all polymers within 10% of the expected values as indicated in the Supporting Information, Figure S1. Capsule Preparation. A 5% solution (w/w) of a polymer in dichloromethane was prepared. To 250 mg of this solution were added 100 mg of cyclodecane and 0.9 g of dichloromethane. The solution was emulsified in 20 g of 0.3% w/w PVA in water, shaken to prepare a premix, and then passed 10 times through an acrodisk 1 µm glass filter. Subsequently, the emulsion was stirred for 1 h to remove the DCM by dissolution in the aqueous phase and subsequent evaporation. After DCM evaporation, the sample was centrifuged at 3000 rpm (Gforce is 968g) for 30 min. The top fraction was retrieved and washed with 5% poly(ethyleneglycol) (PEG, Mw 3400) in water, after which the sample was rapidly frozen at -80 °C in a precooled glass vial. To remove the ice and the cyclodecane fraction, freeze-drying took place in a Christ epsilon 2-6 freeze drier for 20 h at 1.98 mbar followed by 20 h at 0.03 mbar. The shelf temperature of the freeze drier was maintained at -10 °C. After freeze-drying, the system was filled with nitrogen. Samples were stored at 4 °C. For studies of the capsules by MDSC and XPS and studies of the morphology by SEM and TEM, the preparation procedure was carried out without the washing step with 5% of PEG. Particle Size Analysis. A Beckman Coulter Counter (Multisizer 3) was used to measure the particle size distribution as well as the number of particles using a 50 µm aperture tube. An aliquot is mixed with 50 mL of isotonII (Beckman Coulter) from which 100 µL was analyzed in the diameter range of 1.1 to 30 µm. Differential Scanning Calorimetry. Modulated differential scanning calorimetry experiments were performed using a Q1000-TA calorimeter. The freeze-dried samples (about 0.5-1.0 mg) were packed in PerkinElmer aluminum pans. An empty aluminum pan was used as a reference. For the used modulated heating program, the pans were first equilibrated at -50 °C. The heating cycle runs from -50 until 200 °C at an average heating rate of 5 °C/min. The amplitude of the modulation

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Table 1. Overview of Polymers Used in the Study Including the Supplier, End Group, and Molecular Weight Information as Determined by GPCa

a

polymer

supplier

product

end group R

Mw (g/mol)

Mw/Mn

PLGA PLLA PLLA PLLA PLLA PDLLA PDLLA PLLA-PFO PLLA-PFO PLLA-PFO PLLA-PFO PDLLA-PFO PLLA-PFH PLLA-C8 PLLA-C18

Purac Purac Fluka Polymer Source Polymer Source Purac Polymer Source Philips Research Philips Research Philips Research Philips Research Philips Research Philips Research Philips Research Philips Research

Purasorb 125/107 Purasorb PL 81273 P2294.LLA P2301.LLA Purasorb 125/110 P7330.LA2OH

CH3(CH2)11CH3(CH2)11CH3(CH2)9CH3CHCH3OCH3CHCH3OCH3(CH2)11CH3O(CH2)2OCF3(CF2)6CH2CF3(CF2)6CH2CF3(CF2)6CH2CF3(CF2)6CH2CF3(CF2)6CH2CF3(CF2)3CH2CH2CH3(CH2)7CH3(CH2)17-

8000 90900 179700 6100 1700 8100 4400 6500 2400 1900 1300 4000 2800 2400 2500

1.6 1.8 2.6 1.3 1.0 1.8 1.1 1.2 1.1 1.1 1.0 1.2 1.2 1.2 1.1

For the commercially obtained polymers, the end group information was kindly supplied by the manufacturer.

was 1 °C at a period of 60 s. This MDSC program is similar to other programs found in the literature for the analysis of polymeric capsules14-18 and polylactide fibers.19 Scanning Electron Microscopy and Transmission Electron Microscopy. TEM samples were prepared by applying freeze-dried powder onto standard TEM grids (C foil on Cu grid). Subsequently, the abundant powder was removed from the TEM grid, leaving a fraction of the powder on the grid. TEM studies were performed using a Philips CM12, operated at 120 kV in low-dose mode, which involved low beam intensity searching, followed by accurate focusing in a position away from the area of interest before imaging the area of interest using a preset under-focus setting. For SEM, the samples were prepared by redispersing the freezedried capsules in water and applying a drop of the suspension on a silica substrate. After drying, SEM images were recorded on a Philips SEM XL 40 FEG system at a lowest possible acceleration voltage of 1 kV. XRD. Redispersed freeze-dried microcapsules in water were dried on a silica substrate. Samples were measured with a Panalytical X’Pert Pro MPD diffractometer, equipped with a Cu X-ray source and X’Celerator detector. The X-ray scattering was measured between 10 and 60°. Acoustic Activity. The acoustic system used for this study allows for the detection of the broadband signal generated by escaping gas from a microbubble. This is recorded by the scatter of higher harmonic signals from single acoustically activated microcapsules, indicating bubble destruction, and has published before12,20 (see Figure 2 for an overview of the setup). If a microbubble is present in the confocal region of the two transducers and if the gas that escapes from this microbubble generates a signal above the noise level, the signal represents an acoustic event. This sequence is repeated 100 times, and the number of events is the number of times a signal above the noise was counted. This is expressed as the percentile event count. In more detail, this is established using a focused power transducer (Panametrics V392, diameter ) 1.5 in., focal length ) 2.0 in.) with a center frequency of 1.0 MHz and was employed for generating 32 cycles tone-bursts to activate microcapsules. This transducer is used the induce microcapsule destruction and is driven by an arbitrary waveform generator (WaveTek 295) through a 50 dB power amplifier (ENI 2100L). The acoustic pressure amplitude was calibrated using a broadband membrane hydrophone (Onda NTR MHB500B). The behavior of activated microcapsules was examined using a passive acoustic detector. The passive detector is composed of a broadband focused transducer (1.0 in. in diameter and 2.0 in. in focal length) with a center frequency of 5 MHz (Panametrics V307) as well as a high-pass filter of 3.0 MHz (TTE HB5-3M-65B) and a low-noise signal amplifier (40 dB). The filter is employed to remove directly

Figure 2. Ultrasound setup for single particle detection. See text for description of the components.

transmitted (diffraction-induced) 1.0 MHz acoustic signals from the activation transducer. The transducers were placed confocally in a 3.4 L water tank. A four-channel delay/pulse generator (Stanford Research Systems DG535) was used as the time modulator to synchronize the acoustic detector with the activation ultrasound pulses at a PRF of 2.0 Hz. A digital oscilloscope (LeCroy LT374L) was used to digitize the amplified scattering signals with a sampling frequency of 20 MHz. The ultrasound signal transmission, scattered echo reception, and data transfer were all controlled by a computer via LabView (National Instruments). Each sample vial was reconstituted 5 mL of deionized water to give typically 2 × 108 microbubbles/mL (the exact number is measured on a Coulter Counter Multisizer 3). Each microcapsule suspension was further diluted using a precision pipet to give 2 × 106 microbubbles/ mL. A 30 µL aliquot was diluted in the rectangular test chamber containing 3.4 L degassed and deionized water to yield approximately 17 particles/mL. The amount of the freeze-drying additive is then typically 15 µg in 3.4 L, and previous experiments using reference solutions of solid particles redispersed from a PEG matrix showed no acoustic acitivity.9 The water was kept in circulation with a magnetic stirrer at room temperature. For each insonation amplitude, a percentile event count

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Figure 3. Size distribution before and after freeze-drying (fd) of particles made from PLLA-PFO Mw ) 2400 (A) and PLGA (B).

for all activated microcapsules insonations (n ) 100) was measured by automatically counting amplified echo signals whose rms amplitudes were greater than 2.0 mV. Each event count measurement was repeated independently for at least three times.

Chlon et al.

Figure 4. MDSC data for microcapsules prepared from PDLLA Mw ) 4400, pLLA Mw ) 6100, and Mw ) 179 700 (A) and PLLA-PFO Mw ) 2400, PLLA-C8 Mw ) 2400, and PDLLA-pFO Mw ) 4000 (B). Both graphs have an enlarged view, showing the temperature region of the Tg for PDLLA.

Results and Discussion Particle Processing. Figure 3A shows the particle size distribution before and after freeze-drying for PLLA-PFO Mw ) 2400 capsules. The size distributions before and after freezedrying were very similar with a maximum around 2 µm, and 90% of the capsules were less than 3.0 µm in diameter, indicating that the freeze-drying procedures did not lead to a change in the capsule size and that the capsules are wellredispersible after freeze-drying. Apart from this, redispersed capsules were observed to float. If the same preparation procedure is followed using PLGA instead of PLLA-PFO, the size distribution before freeze-drying is similar to that of PLLAPFO Mw ) 2400 (see Figure 3B), but after freeze-drying, the maximum has shifted to smaller sizes, indicating broken capsules. In addition, it was observed that these particles sedimented. The density of the polymer is higher than that of water, and intact capsules or bubbles have a density lower than that of water. This therefore confirms that the capsules were damaged and do not encapsulate gas. This change in size distribution in combination with sedimentation was also observed for particles made of PLLA-C18. For PLLA-PFO Mw ) 1300, no capsules could be made even before freeze-drying. A minimum molecular weight will exist for the formation of polymer capsules; otherwise, the shell integrity is lost. For all other polymers tested (see Table 1), size distributions before and after freeze-drying were as for PLLA-PFO Mw ) 2400 (results not shown). The observations that size distributions before and after freeze-drying superimpose and that the capsules did not sediment but floated do not necessarily imply that gas-filled microbubbles have been made successfully. If cyclodecane was not removed during freeze-drying, the size distribution was also not expected to change and the particles were still expected to have a density smaller than 1 g/cm3; therefore, additional analysis of residual cyclodecane is necessary. Core Content and Shell Crystallinity. To distinguish between microcapsules filled with cyclodecane and filled with

Figure 5. XRD diffraction patterns of amorphous and crystalline microcapsules, PDLLA Mw ) 4400, pLLA Mw ) 6100, and Mw ) 179 700 (A) and PLLA-PFO Mw ) 2400, PLLA-C8 Mw ) 2400, and PDLLA-pFO Mw ) 4000 (B).

nitrogen gas, the presence of a melting peak of the core material was assessed using MDSC. In addition, information on glass transitions and melting peaks of the polymeric shell are obtained. Figure 4A represents MDSC data for microcapsules consisting of a PLLA Mw ) 6100 and Mw ) 179 700 and PDLLA Mw ) 4400. The two PLLA samples show a melting peak at about 150-155 °C, which is attributed to the melting of PLLA crystals of the shell.21 For the capsules made of PLLA with high molecular weight, an additional peak at 9 °C exists, which is the melting point of cyclodecane. This melting peak is absent for capsules made of the low molecular weight PLLA, which indicates the successful removal of the cyclodecane. The lower the Mw is, the higher is the percentage of crystallinity of the polymer,21 making the capsule shell less entangled, allowing for easier removal of the cyclodecane core. The capsules made of PDLLA also show the melting peak of the encapsulated cyclodecane at 9 °C. This indicated that the cyclodecane was not completely removed during freezedrying. Apart from the incomplete removal, PDLLA capsules have Tg at 40 °C, as shown in the insert in Figure 4.

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Figure 6. TEM images of microcapsules made from (A) PDLLA Mw ) 4400, (B) PLLA Mw ) 179 700, and (C) pLLA Mw ) 6100.

Figure 7. SEM images of microcapsules made from (A) PDLLA Mw ) 4400, (B) PLLA Mw ) 179 700, and (C) PLLA Mw ) 6100.

Figure 4B presents the reversed heat flow curves of capsules made from three different types of polylactide in the molecular weight range of 2000-4000. Both PLLA samples show the characteristic melting points of the crystalline polymers, indicating maintained crystallinity. The amorphous polymer PDLLAPFO has a Tg at 35 °C. The Tm at 9 °C is only visible for the capsules with amorphous shells, not for the capsules made of crystalline polymers, confirming the observation that cyclodecane can be removed efficiently from capsules made of low molecular weight PLLA. To identify the crystalline phases of the polymer capsules, XRD was performed. Figure 5 presents the results for various polymer capsules. All capsules showed to some extent a broad peak between 10 and 25°, originating from amorphous material. The sharp peaks at 16.5 and 19° originated from the R form of the crystalline polylactide,22 which is only visible for capsules made of PLLA, PLLA-PFO, and PLLA-C8. XRD therefore confirms the crystallinity of the polymer shells, in agreement with the observed melting points of the polymers as detected by MDSC. Electron Microscopy. TEM pictures of microcapsules are given in Figure 6. Transparent microbubbles indicate hollow capsules, and a black core is visible for, at least partially, cyclodecane-filled capsules. Residual cyclodecane is present for microcapsules with amorphous and/or high molecular weight polymer shells, in agreement with the MDSC results. The surface characteristics are shown on SEM pictures (Figure 7), which demonstrate a homogeneous surface for PDLLA Mw ) 4400 and PLLA Mw ) 179 700 capsules, while for PLLA Mw ) 6100, the surface is more irregular and shows some holes. The presence of residual oil and the fragility of the samples made it difficult to obtain meaningful pictures for all samples. However, for all PLLA samples with Mw of 6100 and shorter, irregularities and pores in the shell were observed, whereas capsules consisting of an amorphous polymer or polymer with a high molecular weight (Mw ) 90 900 and 179 700) showed homogeneous shell surfaces. The openings in the shell might be formed in the emulsification process, the freeze-drying process, or during sample preparation for SEM. Indications that inhomogeneities in the shells are already present in early stages of the preparation process were obtained from experiments using cyclooctane instead of cyclodecane (results not shown). With cyclooctane, no capsules could be made using short chain PLLA as cyclooctane was lost in the washing procedure. With PDLLA or high molecular weight PLLA, capsule formation with cyclooctane was possible, but removal of cyclooctane was still incomplete. The weak spots on the surface present for low molecular weight PLLA offer a way out for the cyclooctane in

Figure 8. Acoustic activity of microcapsules prepared from crystalline (A) and amorphous (B) polymers with differences in molecular weight.

the emulsification step and are therefore likely to be present in the capsules formation stage. For the encapsulated cyclodecane, the weak spots help to explain the ease of formation of hollow capsules from these polymers, as cyclodecane can sublime through these openings. Acoustic Activity. The acoustic activity as a function of the applied pressure was measured for all samples. For PLLA and PDLLA samples, the activation curves for different molecular weights are given in Figure 8. In Figure 9, the effect of the presence of a hydrophobic end group on the activation curve for PLLA is shown for two regimes in the chain length. For all polymers, the threshold pressure and slope for acoustic activation are quantified (see Table 2). A linear fit was used, including event counts between 4 and 60% as in ref 9. By fitting through the average value plus the standard deviation and separately through the average value minus the standard deviation, the experimental uncertainties were estimated to be (10% for the threshold and (20% for the slope. For both PLLA and PDLLA, the threshold pressure increases with increasing molecular weight and the slope of the activation curve decreases. For PLLA microbubbles, the threshold value increases with the molecular weight from 0.40 MPa for Mw ) 1700 to 0.88 MPa for Mw ) 179 700. PLLA with Mw ) 90 900 appears to form an exception as no acoustic activation could be detected in the pressure range used. The difference with the highest molecular weight tested could be due to difference in

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Chlon et al.

Figure 9. Acoustic activity of microcapsules made from PLLA with and without a hydrophobic end group for molecular weight below 2000 (A) and for molecular weights around 6000 (B). Table 2. Activation Thresholds in MPa, the Slopes of Activation Curves, and the Square of the Correlation Coefficient for All Polymer Microcapsules Studieda Polymer

Mw

threshold (MPa)

PLGA PLLA PLLA PLLA PLLA PDLLA PDLLA PLLA PFO PLLA PFO PLLA PFO PLLA PFO PDLLA PFO PLLA PFH PLLA-C8 PLLA-C18

8000 90900 179900 6100 1700 8100 4400 6500 2400 1900 1300 4000 2800 2400 2500

no capsules >2 MPa 0.88 0.64 0.40 1.15 0.75 0.64 0.57 0.61 no capsules 0.74 0.55 0.65 no capsules

slope (%/MPa)

R2

7.3 45 77 19 61 93 105 173

0.97 0.99 0.96 0.95 0.99 0.99 0.97 0.96

45 126 100

0.63 0.94 0.98

a Thresholds and slopes were obtained by using a linear fit through event counts between 4 and 60%. Experimental uncertainties are 10% in the threshold and 20% in the slope as discussed in the text.

polydispersity, but because both polymers are hardly acoustically active, we did not investigate this in more detail. In the PLLA molecular weight series, the slope decreases with a factor of 10 compared to the Mw ) 1700 with the Mw ) 179 700 sample (see Table 2). This implies that for high molecular weight PLLA only a small fraction of the microbubbles is acoustically activated in this pressure regime. The results for PDLLA capsules also show the increase in threshold value and decrease in slope with molecular weight of the polymer. Comparing the thresholds for PLLA and PDLLA, it is found that for polymers with Mw of 4400-6100 the threshold values for PDLLA are higher than that of PLLA, 0.75 versus 0.64 MPa. This is, however, not significant. For PDLLA capsules with Mw ) 8100, a threshold value of 1.15 MPa is found, which is significantly higher than that of the acoustically active PLLA capsules. It should be noted that the event counts obtained from PDLLA samples started to decrease after 1 h, while for PLLA, the event counts were stable for at least a day. Two reasons for the trend that the acoustic activity decreases with increasing molecular weight can be identified. First, higher Mw polymers have more chain entanglements, leading to a more

robust capsule shell, which is harder to break upon an ultrasound pressure. Second, for shells made from crystalline higher Mw polymers, the surface is more homogeneous than for low Mw polymers, resulting in capsules that are not completely gas-filled. The less gas there is available, the less acoustic response is found. As shown in Table 2, the synthesized polymers with a hydropbobic end group all have a threshold for activation of 0.6 ( 0.06 MPa, except for PDLLA-PFO, which has a higher threshold (0.74 MPa, very similar as PDLA Mw ) 4400), supporting the indications that PDLLAs have higher thresholds for activation than PLLAs. Except for PDLLA-PFO, for which the linear fit had a poor correlation coefficient, all samples have a steep slope of the activation curve. The highest slope (172%/ MPa) is obtained for the lowest molecular weight (1900) that could be used to obtain capsules (see also Figure 9). For low molecular weight PLLA microcapsules, the threshold pressure for activation is the same or slightly above that of lipid-shelled microbubbles for which it is in the range of 0.2-0.4 MPa.23 Especially PLLAs with a hydrophobic end group show a steep slope of the activation curve after the threshold is reached. Stability of Microcapsules with Porous Shells. Capsules, for which it was confirmed that cyclodecane was completely removed, were observed to encapsulate gas for at least 3 months after redispersion as the microbubbles keep floating to the top of the container. If pores are present, they do not necessarily allow for the inflow of water, as this depends on the interfacial tension of the shell material according to Laplace’s law:

∆P ) 2γ/R where ∆P is the Laplace pressure, γ is the surface tension, R is the radius of curvature, which is in a cylindrical pore given by rp/cos θ, with contact angle θ, and rp the pore radius. Thompson et al.24 have shown that contact angles of polylactides are close to 90° and, if a fraction of fluorinated polylactide was mixed in, increasing to 110°. Modifying polylactides with hydrophobic end groups therefore renders their surfaces more hydrophobic, preventing the entrance of water into the pores, leading to more stable microbubbles. The weak spots will have an effect on the behavior of the microbubbles upon application of ultrasound. Current models25 start from shell properties, treating the shell as a homogeneous, viscoelastic material. For lipid-shelled microbubbles, the shell homogeneity has been studied for instance by Borden,26 and depending on the precise preparation technique, homogeneous and inhomogeneous shells can be prepared. Single bubble observations with extremely fast cameras show that equally sized microbubbles of the same overall shell composition can respond to ultrasound in different ways, indicating different surface structures. For polymer-shelled microbubbles, regular expansion and contraction as a response to ultrasound is usually not observed. When gas escapes through the shell, the weakest spots are essential.7,27 Other polymer-shelled microbubbles have inhomogeneous shells; see for instance Straub et al.28 Optical single bubble observations of a polymer-shelled agent27 also indicate the importance of weak spots in the shell, and we think that the shell inhomogeneity plays a more prominent role in the acoustic activation of polymer-shelled microbubbles as assumed until now.

Conclusion The acoustic activation of polylactide-shelled microbubbles decreases with chain length, and crystalline PLLA shows lower

Polymer-Shelled Ultrasound Contrast Agents

activation thresholds than amorphous PDLLA. Short chain, hydrophobized PLLA microcapsules form ultrasound contrast agents with a well-defined pressure threshold for activation and a steep increase in the activation with increasing acoustic pressure. The presence of weak spots in the shell is essential for the complete removal of cyclodecane by sublimation and does in the case of hydrophobic polymers not impair the stability of the hollow microcapsules after redispersion. Acknowledgment. This work was supported by innovation subsidies collaborative projects by the Dutch ministry of economic affairs under nr IS042035. We would like to thank Marcel Verheijen (TEM), Rene´ Bakker (XRD), Hetty de Barse and Monique Vervest (SEM), Eef Dirksen (GPC), and Monique Mulder (synthesis of PLLA-PFH). Supporting Information Available. NMR spectra of the polymers synthesized in house, indicating the protons used for determining the ratio for the incorporation of the end groups as given in the Experimental Section. This material is available free of charge via the Internet at http://pubs.acs.org.

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