Magnetite Nanoparticles Can Be Coupled to Microbubbles to Support

Mar 29, 2012 - support not only ultrasound-based imaging methods but also other complementary ... Imaging is a basic need in modern medical diagnostic...
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Magnetite Nanoparticles Can Be Coupled to Microbubbles to Support Multimodal Imaging Torkel B. Brismar,† Dmitry Grishenkov,‡̧ Björn Gustafsson,§ Johan Har̈ mark,⊥ Åsa Barrefelt,† Satya V. V. N. Kothapalli,‡̧ Silvia Margheritelli,∥ Letizia Oddo,∥ Kenneth Caidahl,§ Hans Hebert,⊥ and Gaio Paradossi*,∥ †

Department of Clinical Science, Intervention and Technology at Karolinska Institutet, Division of Medical Imaging and Technology, Stockholm, Sweden and Department of Radiology, Karolinska University Hospital in Huddinge, Sweden ‡̧ Department of Medical Engineering, School of Technology and Health, KTH Royal Institute of Technology, Stockholm, Sweden § Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden ∥ Department of Chemical Sciences and Technologies, Università di Roma Tor Vergata, Rome, Italy ⊥ School of Technology and Health, KTH Royal Institute of Technology and Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden ABSTRACT: Microbubbles (MBs) are commonly used as injectable ultrasound contrast agent (UCA) in modern ultrasonography. Polymer-shelled UCAs present additional potentialities with respect to marketed lipid-shelled UCAs. They are more robust; that is, they have longer shelf and circulation life, and surface modifications are quite easily accomplished to obtain enhanced targeting and local drug delivery. The next generation of UCAs will be required to support not only ultrasound-based imaging methods but also other complementary diagnostic approaches such as magnetic resonance imaging or computer tomography. This work addresses the features of MBs that could function as contrast agents for both ultrasound and magnetic resonance imaging. The results indicate that the introduction of iron oxide nanoparticles (SPIONs) in the poly(vinyl alcohol) shell or on the external surface of the MBs does not greatly decrease the echogenicity of the host MBs compared with the unmodified one. The presence of SPIONs provides enough magnetic susceptibility to the MBs to accomplish good detectability both in vitro and in vivo. The distribution of SPIONs on the shell and their aggregation state seem to be key factors for the optimization of the transverse relaxation rate.

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INTRODUCTION Imaging is a basic need in modern medical diagnostics. The main approaches, such as ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI), and single-photon emission computer tomography (SPECT), are continuously reformulating their benchmarks in terms of imaging resolution, speed, and health impact. Recently, more advanced diagnostic tools have been developed in which two or more imaging methods can be combined. By doing so, the most relevant properties from each modality can be merged together. For instance, the high resolution of CT, and recently of MRI, can be combined with the high sensitivity of SPECT or positron emission tomography (PET) in new imaging methodologies as SPECT-CT,1 PET-CT,2 and PET-MRI.3 To increase further the sensitivity and specificity of modality fusion, an injectable microdevice supporting multimodality imaging approaches would be of great value. In this Article, we will describe a micro/nano-construct that can support multimodal imaging. The two modalities that our device is intended for are US and MRI, but other imaging techniques could be supported by the same concept system. Our approach supporting dual imaging, such as sonography and magnetic resonance, refers to © 2012 American Chemical Society

the implementation of a polymer-shelled ultrasound contrast agent (UCA) with superparamagnetic iron oxide nanoparticles (SPIONs) anchored by means of a chemical coupling of the magnetic nanoparticles to the external surface of the microbubbles (MBs Type A) or by embedding the SPIONs in the polymer matrix constituting the microbubble shell (MBs Type B). Similar approaches, which use poly(D,L-lactide) shelled MBs to support iron oxide nanoparticles, have been investigated by Yang et al.4,5 One of the points addressed in this work is the US performance of this new microdevice. The backscattering efficiency of MBs is generally high because in these structures the difference between the compressibility and density of the scatterer and the surrounding media is maximized. Moreover, the compressibility of a gas is several orders of magnitude higher than that of a particle (such as a red blood cell or solid microparticle). For these reasons, a gas bubble is a unique object whose echogenicity cannot be matched by that of gasless particles of similar size and Received: January 18, 2012 Revised: March 6, 2012 Published: March 29, 2012 1390

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B isothiocianate (RBITC), (3-aminopropyl)trimethoxysilane (APTMS), sodium cyanoborohydride (NaBH3CN), and sodium metaperiodate (NaIO4) were Sigma Aldrich products. Low-molecular-weight chitosan (CHIT), with Brookfield viscosity 20 000 cP, number-average molecular weight of 50 000 ± 5000 g/mol, and PVA with number-average molecular weight determined by membrane osmometry of 30 000 ± 5000 g/mol and weight-average molecular weight of 70 000 ± 10 000 g/mol determined by static light scattering, were purchased from Sigma-Aldrich, Milan, Italy. An acetylation degree of chitosan of 15% (mol/mol repeating units) was determined by 1H NMR at 300 MHz (Bruker Advance, Germany). Inorganic acids and bases were reagent grade products from Carlo Erba, Milan, Italy. Water was Milli-Q purity grade (18.2 MΩ·cm), produced with a deionization apparatus (PureLab, Steroglass, PG, Italy). Methods. Synthesis of SPIONs. SPIONs (Fe3O4) with an average particle size of 8−10 nm were prepared using controlled coprecipitation.43,44 In brief, 5 mL of an aqueous solution of 1 M FeCl3·6H2O, 0.5 M FeCl2·4H2O, and 0.4 M HCl served as a source of iron. The magnetite particles were coprecipitated under vigorous mechanical stirring (2000 rpm) by adding the iron-containing solution to 50 mL of 0.5 M NaOH. After heating the alkaline solution to 80 °C, the reaction was carried out for 30 min under a N2 atmosphere to prevent oxidation. The particles were collected by sedimentation with the help of a large magnetic stirring bar, washed with degassed water and ethanol, and vacuum-dried. Fabrication of MB. Synthesis of PVA-based MB has been reported elsewhere.21 In brief, an aqueous PVA solution (2% w/v) was added with sodium metaperiodate to split selectively the head-to-head sequence contained in the PVA chains. Shorter chains were obtained with aldehydes as terminal groups.21 The acetalization reaction between these groups and hydroxyl groups present in the polymer chains was carried out at pH 5.5 at room temperature under high shear stirring, using an ultraTurrax homogenizer equipped with a Teflon tip, at 8000 rpm for 2 h. Aldehyde groups remaining on the MB surface after this cross-linking reaction were used for further modifications. SPIONs Silanization. To link SPIONs covalently onto the surface of the MBs, we modified the nanoparticles via silanization to introduce amino groups that were able to react with aldehyde groups of MBs via reductive amination. For silanization, 100 mg of SPIONs was washed once with methanol (20 mL), thereafter with a mixture of methanol and toluene (20 mL; 1:1 v/v), and finally with toluene alone (20 mL). SPIONs were then dispersed into toluene (20 mL), and 0.5 mL of APTMS [3 mM in a methanol/toluene (1:1 v/v) mixture] was added to the SPION suspension. The suspension was further refluxed at 110 °C for 24 h under a N2 flow and vigorous stirring. The modified particles were magnetically collected, washed with methanol three times, and vacuum-dried. Fabrication of MBs/SPIONs/CHITox (Type A). Typically, the weight ratio between MBs and silanized SPIONs was 1:2 (w/w). The SPIONs, sonicated for 90 min in an US bath (“Ultrasound cleaner”, CP104, CEIA, Italy) at a concentration of 20 mg/mL, were added to 10 mg of MBs. Reductive amination at pH 5.0 with NaBH3CN was used for coupling the reactive aldehydes on the MBs shell with amino groups of the APTMS moiety. The suspension was gently shaken for 5 days and exchanged with Milli-Q water. Chitosan oxidation was carried out by dissolving the polymer in water at a concentration of 1% (w/v) at pH 5.5, oxidizing the C2 and C3 carbons of the chitosan-repeating unit for 1 day with NaIO4 (feed molar ratio GlcN/NaIO4 1:0.5, where GlcN indicates the glucosamine-repeating unit in the chitosan chain). Following conjugation with silanized SPIONs, the oxidized-repeating units of chitosan are coupled to hydroxyl groups of the PVA shells by mixing a dispersion of 5 mg of MBs with 13 mL of the chitosan solution. Fabrication of MBs:SPIONs (Type B). Unmodified SPIONs were physically embedded in the shell by exploiting the favorable interaction between iron oxide nanoparticles and PVA45,46 following a patent developed in the consortium of the European project, 3MICRON.47 In brief, SPIONs were resuspended in water at a concentration of 5 mg/mL and sonicated 90 min with a US bath (see above). SPIONs (20 mg/mL) were added during the PVA shell formation. After 2 h of

concentration. In fact, it was the observation of minibubbles when injecting saline at cardiac US imaging that led to the development of gas bubbles as a contrast agent for US.6 Most likely, gas bubbles will continue to be the focus for further developments of UCAs but with outer stabilizing shells counteracting rapid dissolution and enabling functionalization. Lipid and polymer MBs were investigated as UCAs. The lipid shells of marketed MB are stabilized by hydrophobic gases such as perfluorocarbon7 or sulfur hexafluoride8 contained in the core of the MBs. Their size is characterized by a rather broad distribution, which negatively affects the echogenicity of this type of UCA.9 The other class of MBs − polymeric − have found a place in the search for UCAs. Lipidic and polymeric MBs display different US behavior and circulation times.10 Both MB types can be loaded with drugs to add UCAs with a therapeutic functionality. Recently, strategies for using lipidshell MBs as a multifunctional device supporting sonography and targeted drug delivery have been comprehensively reviewed by Lentacker et al.11 The targeting and drug-loading of polymeric and lipidic MBs have been reviewed by Klibanov et al.12 Experimental approaches to the development of future UCAs that use polymers dispersed in an oil-in-water medium13−16 or that possess an amphiphilic character have been reported as well.17,18 However, difficulties with timeconsuming production procedures and the use of reactants with different degrees of toxicity need to be overcome. Several polymer MB fabrication methods have been described in the literature: (i) MB synthesis assisted by the presence of surfactants;13−16 (ii) strictly controlled size distribution using microfluidics approach;19,20 and (iii) foaming a polymer solution and stabilizing it with chemical cross-links.21 Polymer-shelled MBs are usually thicker than lipidic monoor multilayer MBs. This dampens the US-driven oscillations of the UCA, resulting in a loss of scattering power.22,23 This is a drawback of polymer MBs. However, if echogenicity can be preserved, then the chemical and physical stability, which is one of the main assets of this type of MBs, becomes a clear advantage as it enables the bubble surface to be functionalized by conjugating molecules that can be used for targeting,24,25 imaging,26,27 and drug delivery.28,29 In recent years, we have developed and characterized a polymeric MB based on poly(vinyl alcohol) (PVA). The structural features,21,30−32 the chemical versatility for the coating and the conjugation with different low- and highmolecular-weight molecules,33,34 the echogenicity,22,35−38 the biocompatibility39 and cytotoxicity,40 and the ability to carry therapeutic gases41 have been investigated. Recently, we explored the potentials that this concept can offer for multimodality imaging. In this work, we address the issue of whether this microdevice can support a simultaneous approach for US and MRI methods, considering handy synthesis and facile shell modification as assets of the investigated MB. In particular, we have investigated two types of microdevices for US-MRI imaging, differing in the coupling of SPIONs42 to the PVA shell. First, we used the chemical association of silanized iron oxide nanoparticles to the polymer MB. As a second option, SPIONs were embedded in the shell during the crosslinking reaction at the water/air interface, resulting in a physical embedding of the nanoparticles in the polymer shell.

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MATERIALS AND METHODS

Materials. Ferric chloride hexahydrate (FeCl3·6H2O, purity >99%), ferrous chloride tetrahydrate (FeCl2·4H2O, purity >99%), rhodamine 1391

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homogenization with an ultraTurrax equipped with a Teflon-coated head at 8000 rpm, the MBs incorporating the magnetic nanoparticles were washed in a separatory funnel with distilled water. Confocal Laser Scanning Microscopy. MBs Type A and B were fluorescently labeled with RBITC (10 μM). MBs were washed from excess fluorescence dye by resuspending them in Milli-Q quality water until residual fluorescence was absent. A confocal laser-scanning microscope (Nikon Eclipse Ti-E, Japan) with 100× (PlanFluo, Nikon, Japan) or 60× (PlanApo, Nikon, Japan) immersion objectives was used for the equatorial images. MBs were detected exciting the sample at 543 nm with a He−Ne (Spectra Physics) laser. Images were captured and processed using EZ-C1 and NIS software packages (Nikon, Japan). Transmission Electron Microscopy Study of MB Shell. The distribution and morphology of SPIONs coupled to the MBs, both Type A and B, were observed by transmission electron microscopy (TEM). Samples were applied to glow-discharged copper grids coated with a thin carbon film. Imaging was performed using a JEOL JEM2100F electron microscope at an acceleration voltage of 200 kV. Micrographs were recorded on a 4k CCD camera. An aliquot of MBs was transferred to Eppendorf tubes containing preheated 10% gelatin (Merck, Darmstadt, Germany) solution. The tubes were then placed in a stand for 15 min at room temperature, upon which the gelatin started to solidify and MBs were trapped in the gelatin. To stabilize further the gelatin, 3% paraformaldehyde in 0.1 M phosphate buffer was added to the Eppendorf tubes and placed in a refrigerator overnight. The gelatin pellet was then embedded in epoxy resin, LX 112 (Ladd, Burlington, VT) following a standard protocol.48 Sections of ∼50 nm were cut using a Leica Ultracut UCT (Leica, Vienna, Austria) and placed on Formvar-coated 50 mesh copper grids coated with a thin carbon film and imaged in a Tecnai 10 electron microscope (FEI, Utrecht, The Netherlands) at 80 kV. Thereafter, the micrographs were recorded using a Veleta CCD camera (Olympus Soft Imaging Solutions, Münster, Germany). Fourier Transform Infrared Spectroscopy. SPIONs silanization was monitored by Fourier transform infrared spectroscopy (FT-IR) using a SPECTRUM 100 (Perkin-Elmer, France) spectrometer. SPIONs and silanized-SPIONs were probed after dispersing the sample in Nujol using a NaCl cell. The spectrum is recorded from 450 to 4000 cm−1 with a resolution of 2 cm−1. Thermogravimetric Analysis. Determination of the amount of iron oxide magnetic nanoparticles bound to (MBs Type A) or embedded in (MBs Type B) MBs was estimated by TGA combined with differential thermal analysis (DTA). Thermogravimetric analysis (TGA)/DTA analysis model STA 409 (Netzsch, Milan, IT) was deployed to perform TGA. Lyophilized samples (20−30 mg) were placed in the TGA furnace and heated from 20 to 1000 °C at a rate of 10 °C/min under nitrogen flux. Differential Scanning Calorimetry. The crystallinity of the PVA shells in the presence of iron nanoparticles was investigated using a TA Q200 (Waters, Milan, Italy) differential scanning calorimeter (DSC). A known amount, typically 2 to 3 mg, of lyophilized MBs and MBsSPIONs (Types A and B) was sealed in an aluminum pan. The scans were performed from 50 to 250 °C at heating and cooling rates of 10 °C/min under a flux of 50 mL/min of dry nitrogen. Data were collected after the first dummy thermal cycle. Ultrasound Characterization. The acoustic system, which was used to determine the scattering properties of the MB suspension, employed single-crystal focused transducer (Panametrics V311, Waltham, MA) with central frequency of 5 MHz and −20 dB bandwidth ranging from 1.2 to 8.1 MHz. The transducer was driven to oscillation by a pulser-receiver (Panametrics PR 5072) with broadband spike pulses emitted at a pulse repetition frequency of 1 kHz. The receiving part of the system contains a 40 dB amplifier and a 1 MHz high pass filter, which were applied to the detected signal before it was visualized and stored by a digital oscilloscope (Tektronix TDS 5052; Tektronix, Beaverton, OR). MBs were enclosed into a custom-made chamber covered with thin, optical, and acoustic transparent plastic material. The size of the chamber along the direction of the beam propagation was S = 10 mm. The center of the chamber was

positioned at a focal length of the transducer (l = 50 mm), so the front and the back wall of it were equally spaced from the focal point. This choice was made to minimize uncertainties caused by dispersive propagation of the acoustic wave along its path to the transducer. The peak negative pressure detected by PVdF 75 μm needle hydrophone (Precision Acoustics, Dorchester, Dorset, U.K.) within the volume occupied by the chamber was not larger than 100 kPa. Five different concentrations of the MBs were tested, ranging from 1.8 × 106 to 1.1 × 105 MBs/mL for bound MBs (Type A) and from 4 × 106 to 2.5 × 105 MBs/mL for embedded MBs (Type B). All experiments were carried out at room temperature in a plastic bath filled with deionized degassed water. Magnetic Characterization. The magnetization measurements were carried out with a vibrating sample magnetometer (VSM) equipped with a superconducting quantum interference device (SQUID) (MPSM VSM SQUID, Quantum Design, San Diego, CA). Measurements were performed at the temperature of 37 °C between the magnetic field strengths of ±70 kOe. Magnetic characterization of MBs Types A and B loaded with SPIONs, containing 29% (w/w) and 15% (w/w) of Fe3O4, respectively, was investigated. For the most accurate measurements, the samples were lyophilized using a cold trap (Thermo Scientific Heto CT110) operating at −110 °C, equipped with a vacuum pump (Vacuubrand, Wertheim, Germany) operating at 4 × 10−4 mbar. The liquid samples were frozen in a dry ice/ethanol bath at −72 °C and then put on the lyophilizer for 24 h. Freeze-dried solid powder samples were loaded in the magnetometer sample holders with known amounts of MBs Type A or Type B, respectively. Magnetic Resonance Imaging. After animal ethics approval, a rat was imaged at 3 T using a Siemens Trio MR Scanner (Siemens, Erlangen, Germany) and the abdomen coil before and ∼1 h after administration of 0.5 mL of 4 × 107 MBs/mL of Type B. An in-house developed pulse-sequence was used to obtain images with a fixed repetition time of 2000 ms and increasing echo time (TE) from 2 to 22.9 with 1.9 ms increments. The signal intensity of the brain (right frontal lobe), liver (right lobe), right kidney (cortex, apex), spleen, and muscle was measured from 13 to 38 mm2 large circular regions of interest placed in the respective organ. The spin−spin relaxation time, T2*, was calculated as the slope of a semilog plot of the signal intensity versus the echo time (TE) 1 1 1 = + T2 T ′2 T2*

(1)

considering that the spin−spin relaxation process is characterized by an irreversible and a reversible relaxation time, T2 and T′2, respectively. In terms of transverse relaxation rates, eq 1 becomes

R 2* = R 2 + R′2

(2)

Assuming that the effect of R2 on the overall relaxation rate, R2*, is negligible, the influence of injected contrast media on the reversible transverse relaxation rate (ΔR′2) can be calculated as * − R 2,before * = (R 2 + R′2,before ) − (R 2 + R′2,after ) R 2,after = R′2,before − R′2,after

(3)

where subscripts “before” and “after” refer to the relaxation rate values before and after the administration, respectively.

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RESULTS AND DISCUSSION The use of superparamagnetic nanoparticles of iron oxide in biological fluids depends directly on their colloidal stability. PVA is considered to be good stabilizing agent for the dispersion of SPIONs due to the favorable physical interaction of PVA with Fe3O4 nanoparticles.49 This feature can be used for the chemical conjugation of silanized SPIONs on the MB shell (Type A) or for a physical embedding into the PVA-based MB shell (Type B). We explored both shell types to evaluate which 1392

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Figure 1. TGA and DTA analysis of MBs modified with iron oxide nanoparticles. (A) MBs Type A and (B) MBs Type B. Black lines: weight loss; dashed lines: DTA.

Table 1 provides a summary of the iron oxide content for the two MB types.

MB type enabled the strongest effect on the spin−spin relaxation rate R*2 at MRI, while maintaining the highest possible echogenicity. SPIONs were prepared using the method of controlled coprecipitation43,44 and surface-modified by silanizing the iron oxide particles with APTMS. The amine groups contained in the moiety can be readily coupled via reductive amination with the aldehyde groups of the PVA shell exposed to the solvent. The SPIONs-APTMS system is reacted with the polysaccharide chitosan, partially oxidizing the C2 and C3 of the saccharide-repeating unit to aldehydes. The chitosan coating was adopted to promote bioadhesion processes and to function as a chelator of transition metal ions for allowing future imaging methods such as SPECT, a tomography technique that uses 99mTc as a source of γ rays as well as a substrate for further functionalization of the MB surface. Moreover, this coating was effective in the prevention of MB aggregation. These features characterize MB Type A. In the case of MB Type B, SPIONs were physically embedded in the PVA shell during the MB formation, taking advantage of the favorable interaction of iron oxide nanoparticles with PVA. The SPIONs, with an average dimension of ∼10 nm, as determined by TEM, were coupled to APTMS for the chemical decoration to the MB surface. The silanization was monitored by FT-IR recording the Si−O stretching at 809 and at 1095 cm−1 and the absorption band at 1050 cm−1 assigned to the −SI−O−CH2 stretching modes present in the spectra of silanized Fe3O4 nanoparticle.44 The amount of SPIONs linked to MB types A and B was determined by TGA/DTA combined method. The thermograms of SPION-MB assemblies of Types A and B are reported in Figure 1A,B, respectively. After moisture evaporation (endothermic peak at 100 °C, see DTA trace), a massive weight loss occurs in the temperature range from 200 to 400 °C due to the PVA and chitosan decomposition of MB Type A (exothermic peak shown by the DTA trace). From the DTA traces, we can observe a decomposition pattern for MBs Type A with well-resolved peaks in the temperature range 200−500 °C, denoting a decomposition process characterized by sequential steps, whereas MBs Type B show a shallow trend of the DTA curve in the 200−600 °C temperature range. This could be interpreted as a consequence of the more complex structure of MBs Type A compared with Type B.

Table 1. Iron Content in Microbubbles of Types A and B MBs SPIONs

iron content % (w/w)

Type A Type B

29 ± 6 15 ± 1

Confocal laser scanning microscopy on rhodaminated MBs could not detect, within the resolution of the method, any difference in the size of Types A and B or unloaded MBs. The diameter, determined by averaging more than 100 equatorial focal planes for each MB types, is 3.8 ± 0.6 μm for all types of MBs. For a more detailed morphology of the magnetic MBs, transmission electron micrographs, shown in Figure 2A,B, confirm the coupling of magnetic iron oxide nanoparticles on the MB shells of Types A and B.

Figure 2. TEM micrographs of MBs coated with SPIONs: (A) chemical conjugation and (B) physical embedding.

Despite the higher amount of magnetite present in Type A (Table 1), TEM shows the presence of a more even distribution in Type B. A more in-depth TEM investigation of MB Type B showed that the SPION penetration in the shell is limited to the center and the outer part of the MB shell; see Figure 3. The apparent change in the shell thickness is explained by considering that the sectioning of MBs takes place at random distance from the MB center. Therefore, some MBs 1393

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enthalpy of melting after the chemical modifications carried out for coupling SPIONs. Similar DSC analysis, carried out on MBs Type B, is reported in Figure 5. Also in Figure 5, the thermogram of the naked PVA

Figure 5. Differential scanning calorimetry of MBs Type B with physically embedded SPIONs (full line) and PVA MB (dashed line).

Figure 3. TEM micrograph of MB Type B. Note the black dots, representing SPIONs embedded in the MB shell. Arrows highlight the SPIONs embedded in the PVA shell.

shells is reported for comparison (dashed line). In this case, the crystallinity of the shell is partially maintained as monitored by the melting and recrystallization peaks at 192.8 and 169.6 °C, respectively. The melting temperatures and the peak areas are lower than those measured in the absence of the embedded SPIONs, denoting a decrease in the stability of the crystalline domains contained in the shell. The plot scales used in the thermograms shown in Figures 4 and 5 of the PVA do not allow us to evidence the glassy transition at 75 °C present for unloaded MBs of Type A and Type B. The calorimetric parameters resulting from these experiments are summarized in Table 2. With an enthalpy of melting of 6.87 kJ/mol of crystalline PVA,52 the unloaded PVA MBs possess a degree of crystallinity of ∼12.5%. This value is smaller than the degree of crystallinity of the PVA powder determined by DSC, ∼30%, used as starting materials for the MB synthesis. The decrease in crystallinity can be attributed to the disordering effect of the chains of PVA acting as a cross-linking agent to form the hydrogels. In MBs Type B, the crystallinity of the shell is about halved with respect to the unloaded PVA MB, and the melting temperature is decreased, denoting reduced stability in the crystalline domain after SPIONs are embedded in the shell. A similar effect has been reported for the hybrid system containing semicrystalline poly(caprolactone fumarate) (PCLF) and hydroxyapatite (HA) nanoparticles and for poly(styrene-b-ethylene oxide), PSEO, and CdS nanoparticles.53,54 The decrease in the melting enthalpy of PCLF and PSEO, observed for increasing amounts of HA and CdS, respectively, was confirmed by wide-angle Xray diffraction (WAXD) measurements. The scattering properties of the suspension of MBs Type A and Type B in a low-pressure field were characterized. The purpose of this study was to assess the extent to which MBs Types A and B with combined functionalities could be used for the most common clinical application, including Doppler signal enhancement. In Doppler mode, commercial UCAs typically produce linear backscattering enhancement about 20 dB above the noise, resulting in improved differentiation of the echo from blood55,56 In fact, the intensity of the US scattered by the small spherical particles (