AFM Investigation of Liquid-Filled Polymer Microcapsules Elasticity

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AFM Investigation of Liquid-Filled Polymer Microcapsules Elasticity Baptiste Sarrazin, Nicolas Tsapis, Ludivine Mousnier, Nicolas Taulier, Wladimir Urbach, and Patrick Guenoun Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00431 • Publication Date (Web): 08 Apr 2016 Downloaded from http://pubs.acs.org on April 12, 2016

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AFM Investigation of Liquid-Filled Polymer Microcapsules Elasticity Baptiste Sarrazin,∗,† Nicolas Tsapis,‡ Ludivine Mousnier,‡ Nicolas Taulier,¶ Wladimir Urbach,§ and Patrick Guenoun∗,† LIONS, NIMBE, CEA, CNRS, Université Paris-Saclay, CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France, Institut Galien Paris-Sud, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Sorbonne Universités, UPMC Univ Paris 06, CNRS, INSERM, Laboratoire d’Imagerie Biomédicale (LIB), 75006, Paris, France, and Laboratoire de Physique Statistique de l’ENS, UPMC , CNRS UMR 8550, Paris, France E-mail: [email protected]; [email protected]

Abstract Elasticity of polymer microcapsules (MCs) filled with a liquid fluorinated core is studied by atomic force microscopy (AFM). Accurately characterized spherical tips are employed to obtain the Young’s moduli of MCs having four different shell thicknesses. We show that those moduli are effective ones because the samples are composite. The strong decrease of the effective MC elasticity (from 3.0 to 0.1 GPa) as the shell thickness decreases (from 200 to 10 nm) is analyzed using a novel numerical approach. This model describes the evolution of the elasticity of a coated half-space according to the ∗

To whom correspondence should be addressed LIONS, NIMBE, CEA, CNRS, Université Paris-Saclay, CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France ‡ Institut Galien Paris-Sud, CNRS, Univ. Paris-Sud, Université Paris-Saclay ¶ Sorbonne Universités, UPMC Univ Paris 06, CNRS, INSERM, Laboratoire d’Imagerie Biomédicale (LIB), 75006, Paris, France § Laboratoire de Physique Statistique de l’ENS, UPMC , CNRS UMR 8550, Paris, France †

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contact radius, the thickness of the film and the elastic moduli of bulk materials. This numerical model is consistent with the experimental data and allows simulating the elastic behavior of MCs at high-frequencies (5 MHz). While the quasi-static elasticity of the MCs is found to be very dependent on the shell thickness, the high frequency (5 MHz) elastic behavior of the core leads to a stable behavior of the MCs (from 2.5 to 3 GPa according to the shell thickness). Finally, the effect of thermal annealing on the MCs elasticity is investigated. The Young’s modulus is found to decrease because of the reduction of the shell thickness due to the loss of the polymer.

Introduction The mechanical properties of heterogeneous micro and nanometric systems such as cells, viral capsides or polymer capsules are challenging to measure and to quantify. Indeed, the measurement is complicated by the scale and the heterogeneity of those objects leads to heterogeneous mechanical properties. In particular, it has been shown that multiple regimes of elasticity and stiffness can be revealed from AFM force-indentation curves on complex objects. 1,2 Atomic Force Microscopy (AFM) is precisely able to probe such mechanical properties of isolated nanometer scale objects. 3 AFM used as a nano indenter allows characterizing single particle using contact models taking into account geometries of the object and of the AFM tip. During the last twenty years, it has been used to study mechanical properties of various systems such as thin polymer films, 4–7 microbubbles, 8–11 microcapsules, 12–14 hollow colloidal particles, 15,16 nanotubes, 17 thin virus shells, 18 polymer brushes 19,20 and cells. 21–23 The work presented here is dedicated to microcaspules made of a hard polymeric shell enclosing a soft liquid core. Those capsules are particularly interesting in the field of medical applications when the core is a fluorinated liquid. Since perfluorinated liquids are relatively inert and interact poorly with hydrogenated molecules, 24 they have been successfully encapsulated into biodegradable polymers to yield theranostic capsules. 25–27 The obtained 2 ACS Paragon Plus Environment

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capsules with perfluorinated core may act as contrast agents both for ultrasound imaging by backscattering ultrasound waves or for fluorine magnetic resonance imaging given their high fluorine content. 28,29 In addition, polymer capsules may serve as drug carriers by delivering a drug – localized into the shell – to a target tissue thereby enhancing efficacy while reducing deleterious effect on healthy organs. Ultrasounds can be used as a trigger to destroy the capsules locally and deliver the drug. This can be achieved either by inducing cavitation bubbles leading to a thermo-mechanical stress or using mild hyperthermia if capsules are thermosensitive. 30,31 One of the main challenges is to understand and to theoretically describe the capsules mechanical properties and their behavior when subjected to mechanical stresses. Guédra et al. 32 proposed several theoretical models (Church, Rayleigh-Plesset, Kelvin-Voigt, Zener) which take into account the shell elasticity, stiffness, viscosity or surface tension to describe the influence of MCs shell mechanics on the ultrasound waves. Nevertheless, there is a crucial lack of experimental data to depict the elastic behavior of those polymer MCs. The aim of the present study is to better describe the mechanical properties of single MCs at the nanometer scale. Previous studies described the elastic behavior of hollow polymer microspheres 33 or phospholipid microbubbles 8 measured by AFM nanoindentation and using the Reissner analytical solution for thin shell elasticity. 34,35 Nevertheless, this approach is only adapted to thin shells. The present work proposes a new way to study elastic properties of capsules, whatever the shell thickness and including the composite aspect of the sample. A numerical model, introduced by Perriot and Barthel, 36 is used to describe the evolution of the effective elastic modulus of the MCs as a function of the contact radius, the shell thickness and the bulk elastic moduli of the sample compounds. This model (CHIMER : Coated Half-space Indentation Model of Elastic Response) has been improved by Sarrazin et al. 2 and has proven to be effective to describe the elastic behavior of flat bilayer sample. The originality of the present work is to use the CHIMER model in spherical geometry to describe the evolution of the effective elastic modulus during the indentation of a hard microcapsule.


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In this study, microcapsules (MCs) consist of a polymer shell (poly(lactide-co-glycolide), PLGA) surrounding a perfluorinated liquid core (perfluorooctylbromide, PFOB). PLGA is a biocompatible and biodegradable polymer. 37,38 PFOB is chosen as a fluorinated compound whose absence of toxicity has already been reported. 39,40 The indentations of individual PLGA MCs filled with liquid PFOB are studied for capsule radii (R) between 400 and 600 nm and for shell thicknesses (T) such that T/R = 0.025; 0.15 ; 0.25; 0.35 and 1 for full PLGA microspheres. Capsules, deposited on a hard surface, are imaged and subsequently probed by measuring the force-displacement curves with an AFM tip. An inalterable diamond coated tip whose apex dimension is several tens of nanometers (20 to 50 nm depending on the batch, see Experimental Section for deconvolution process) is used to probe capsules and full spheres whose diameter is in the order of the micron. Hertz 41 and Johnson-KendallRoberts 42 models are employed to analyze the force versus deformation curves and compute compressive effective elastic moduli (Young’s modulus) of each MC. Particular attention is paid to the precise calibration of cantilever stiffness and tip geometry which are critical parameters to interpret force curves. PLGA films and full microspheres are also probed for assessing a precise calibration. A crucial point is to locally deform the shell while limiting the global non-elastic crushing of the capsule. Moreover, frequency and temperature influences have been investigated in order to get closer to real experimental conditions of use for MCs for biomedical application. As a result, it is shown that the Young’s modulus is highly dependent on thicknesses at low frequency. The CHIMER numerical model is used to validate experimental data and describe the evolution of elasticity according to the contact radius and the shell thickness. The results are confronted to the thin shell model interpretation and we show that CHIMER analysis is able to go beyond the Reissner analytical solution for thick shells. Subsequently, the same approach is used to simulate the high frequency mechanical behavior of the MCs. The dependence of the shell elasticity to the thickness is found to be much lower because of the PFOB elastic behavior at high frequency.


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Experimental Section Materials. Poly(D,L-lactide-co-glycolide) (PLGA) Resomer RG502 (intrinsic viscosity 0.160.24 dl.g−1 ; Mn=7000-17000 g.mol−1 ; ester terminated) was provided by Evonik Röhm Pharma GmbH (Germany). Sodium cholate (SC) and Nile Red were purchased from SigmaAldrich (France). Perfluorooctyl bromide (PFOB) was obtained from Fluorochem (United Kingdom). Methylene chloride RPE-ACS 99.5 % was provided from Carlo-Erba Reactifs (France). Water was purified using a RIOS/Milli-Q system (Millipore, France). AFM Sample Preparation. Silica substrates are cleaned by UV-Ozone, plasma and acid technique 43 to get reproducible wetting and surface state. In the case of mica substrate, they are cleaned using glue tape to peel off the top atomic layers and to expose fresh mica surface. Freeze-dried MCs and full polymer microspheres are rehydrated in ultrapure deionized MilliQ water (resistivity 18.2 M Ω.cm). A droplet (100 µL) of the obtained suspension is deposited on bare silica wafer and dried in vacuum chamber during 1 to 5 hours. To homogenize the distribution of MCs on the substrate, the sample is rinsed with pure water and dried again during 30 to 60 min. PLGA films are performed using a spin-coating technique: a droplet of PLGA solution (1 g.L−1 in dichloromethane) is deposited on bare silicon wafer and spun at 500 to 5000 RPM depending on the desired thickness (basically 100 to 1000 nm). Obtained films are very flat (roughness lower than 5 nm on 20 µm). In order to measure film thickness, a medical blade is used to scratch the surface. Atomic Force Microscopy. Nanoindentation has been performed using an AFM Dimension V (Bruker Nano , Santa Barbara, CA, USA) mounted with an optical microscope. AFM probes used to perform force curves are silicon-nitride cantilevers with diamond coated tips (DT-NCHR, NanoWorld and B1-NCHR, Nanotools). This diamond coating reduces tip wear and damage during force measurements, particularly on hard surface (calibration curves). Cantilever stiffness is about 60 to 90 N.m−1 and tip radius is in the range of 20 to 150 nm. This is quite larger than classic sharp silicon nitride AFM tips (about 10 nm radius) which are used for high resolution imaging (RFESPA, Bruker). An AFM Dimension 5 ACS Paragon Plus Environment

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Icon (Bruker Santa Barbara, CA, USA) has been used to probe forces at higher frequency. All the force measurements reported in that article are performed in air. The tip speeds during indentation are about 0.2 to 1 µm.s−1 for quasi-static measurements and 50 to 250 µm.s−1 for Peak Force measurements. Calibration of the Cantilever Spring Constant k. Several procedures are described in literature to measure cantilever stiffness. 44 The most common one is the thermal tune computation. 45 Measurement of mechanical response of the cantilever to thermal noise allows us to compute spring constant (Lorentzian fit of the frequency spectrum). The cantilever stiffness calibration procedure can be described as follow: a force curve is performed on hard surface and the slope of approaching curve is measured to determine the sensitivity of the cantilever. Then thermal tune is made far from the surface and the frequency spectrum is fitted around the peak which is at the resonance frequency. AFM Tip Geometry. Blind deconvolution algorithm 46 is applied to a characterization surface picture and allows to get back to the tip shape. Deconvolution is computed using Gwyddion software (David Neˇcas and Petr Klapetek, Department of Nanotechnology, Czech Metrology Institute). The characterization surface used for blind deconvolution consists of pyramidal hard sharp nanostructures (PA series from Mikromasch, NanoAndMore GmbH).

Results and discussion Polymer Films and Microcapsules In order to measure the mechanical properties of the MC’s shell main compound, nanoindentations are first performed on thin PLGA films deposited on a hard surface of silicon wafers (100 to 1000 nm thick). Indentation depths (5 to 20 nm depth) are kept much lower than film thicknesses to avoid all influences from the substrate 4 , to probe the elastic regime and limit viscoplastic deformation. This can be checked by imaging the surface after indentation. Such AFM pictures of residual deformations are presented in Supporting Information. 6 ACS Paragon Plus Environment

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Figure 1(a) presents typical force curves on PLGA film compared to polystyrene (PS) films (500 nm) and a hard surface (silicon substrate). Mechanical properties of PS are well known and described in literature, 47 providing a relevant reference to check the AFM calibration accuracy as well as to compare values with our samples data. The approaching force curves are used to calculate the Young’s moduli of the samples. First, the indentation δ is calculated from equation 1:

δ = |z − z0 | − |D − D0 |

(1)

where z and D are the piezo-displacement and the cantilever deflection respectively and z0 and D0 are the piezo-displacement and the cantilever deflection at which the tip-surface contact occurs. The loading force F is determined from the Hookean equation 2:

F = k(D − D0 )

(2)

where k is the spring constant of the AFM cantilever. This constant is experimentally measured using the mechanical response to thermal noise. Two different models are used in this study. The first and the simplest one is the Hertz model that describes the purely elastic deformation of two spherical bodies in contact. 41 The second is the Johnson-Kendall-Roberts (JKR) model that describes an elastico-adhesive contact. 42 Hertz theory leads to Young’s modulus as equation 3:

E=

F 3(1 − ν 2 ) √ 4 R∗ (δ − δ0 )3/2

1/R∗ = 1/Rtip + 1/Rsample

(3)

(4)

where R∗ is the effective radius of the two bodies in contact, F is the applied force, δ


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is the indentation depth and ν is the Poisson ratio of the sample. The contact point δ0 is determined as a fit parameter. Reduced elastic modulus (E ∗ = E/(1 − ν 2 )) is simply determined from the linear fit of the F vs. δ 3/2 experimental data (measured between 10 to 70 % of the full indentation depth) (Figure 1(b)) In this study, ν is assumed to be 0.33 for the PS as well as for the PLGA and the PLGA/PFOB MC (glassy polymer 48 ). In the case of a polymer film, Rsample is infinite so R∗ = Rtip . It is found that PLGA is slightly harder than PS. Young’s modulus of PLGA is not modified with an increase of frequency. For 1 Hz and 2 kHz (AFM PeakForce QNM, Bruker) we measured 3.0 ± 0.2 GPa and 3.08 ± 0.35 GPa respectively. This value reaches 4.3 GPa for frequencies between 1 to 10 GHz. 49 P S F ilm P L G A F ilm H a rd s u rfa c e (F = k z )

1 1 0 0 1 0 0 0

1 2 0 0

9 0 0 1 0 0 0

8 0 0 Force (µN)

7 0 0

F o rc e (n N )

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P L G A F ilm P S F ilm

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-2 0 0

-2 0

-1 5

-1 0

-5

0

5

1 0

1 5

2 0

2 5

-1 0

3 0

0

z d is p la c e m e n t ( n m )

1 0

2 0

In d e n t a tio n d e p th

(a)

3 0

4 0 3 /2

(n m

3 /2

5 0

6 0

)

(b)

Figure 1: (a) Force vs. displacement curves on PLGA and PS. (b) Hertz plots of PLGA and PS. As adhesion may play a role in the elastic response, an elastico-adhesive model is needed. The Johnson-Kendall-Roberts (JKR) model was the first to incorporate adhesion into Hertzian contact. The traditional equation of JKR model 42 gives the contact radius a as a function of the applied force F , the surface energy γ, the elastic modulus E and the effective radius R∗ :

a3 =

p 3R∗ (1 − ν 2 ) F + 3πγR∗ + 6πγR∗ F + (3πγR∗ )2 4E

(5)

As it has been shown, 50 contact radius is difficult to estimate in the case of AFM nanoin8 ACS Paragon Plus Environment

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dentation experiments. Indeed, even for very definite tip geometry, the nanoindentation area can be hard to estimate because of nanoroughness, localized adhesion or Van der Waals forces. Thus schemes where the elastic modulus is expressed as a function of the cantilever deflection (or loading force) and indentation depth have been derived. Lin et al. 47 and Tsukruk et al. 51 reported the following equation derived from JKR model : " 1/2 #3/2 2 6πR∗ γ 3F1 (1 − ν 2 ) 1− E= √ 3 F1 4 R∗ δ 3/2

(6)

where

F1 = F + 3γπR∗ +

p 6γπR∗ F + (3γπR∗ )2 )

(7)

and the adhesion energy γ is determined by the pull-off force Fpo needed to detach the tip from the surface by : 3 Fpo = − γπR∗ 2

(8)

The adhesion energy γ can be a critical parameter for the interpretation of elasticity at low indentation depth. Actually, the pull-off force and the adhesion energy can be highly influenced by humidity, temperature, maximum applied force, tip and surface properties, leading to a large error in the computed elastic moduli. Figure 2 shows the “instantaneous” Young’s modulus derived from equation 6 (directly calculated from Hertz and JKR relations using instantaneous F and δ extracted from force curves) during the progression of the indentation. JKR model provides higher Young’s moduli than Hertz model at low indentation depth. Adhesion effect is actually significant when the applied force is low, increasing the effective Young’s modulus. However for both models (Hertz and JKR), our results show that Young’s modulus at low indentation depth (< 2-5 nm) is varying with deformation. This is due to the destabilizing attractive force gradient in the vicinity of surfaces and the possible lower molecular mobility at the interface between the two contacting surfaces. 52,53 For larger 9 ACS Paragon Plus Environment

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indentation depth, elastic bulk effects are predominant and adhesion effects become negligible against applied force. Hence, in the range of our experimental indentation depths (5 to 10 nm), the convergence of Hertz and JKR models is observed, showing that adhesion effect can be neglected if the elastic modulus determination does not take into account the first few nanometers. Accordingly the first data points at low indentations are not taken into account for the fit of the data presented about the capsules.

I n s t a n t a n e o u s Y o u n g ’s M o d u lu s ( G P a )

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P L G A I n s t a n t a n e o u s Y o u n g ’s M o d u lu s J K R m o d e l H e rtz m o d e l

1 0 A d h e s io n C o m p r e s s ib ilit y A ttr a c tiv e fo r c e 9 8 7 6

F it t in g D e p t h E la s t ic R e g im e 5

P la s t ic D e fo r m a tio n

4 3 2 1 0 -2

0

2

4

6

8

1 0

1 2

1 4

In d e n t a t io n D e p t h δ( n m )

Figure 2: Instantaneous Young’s modulus of PLGA vs. Indentation depth computed with JKR and Hertz models. The convergence of both models is the fitting range (elastic behavior) which is limited by a more complex behaviors (attractive forces, adhesion, low molecular mobility...) for low indentation and visco-plastic behavior for high indentation. Oscillations in the data are probably due to parasitic optical interference or piling-up of material due to viscous flows in the upper few nanometers of the film. It must also be noted that viscoplastic effects occur even for tiny indentation depth (≈ 5 nm). Therefore, after tip withdrawing from the surface, a circular ridge surrounding the residual hole is observed. Indeed, plastic deformation in soft material is mainly associated with piling up effect which is more important at higher yield stress 54 . Although this behavior seems to be incompatible with the fully elastic approach of the PLGA, we observed that this 10 ACS Paragon Plus Environment

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effect does not critically upset the linearity of the Hertz model (F/δ 3/2 ) mainly because the amount of piled up polymer is tiny when compared to the quantity of displaced material during indentation. This effect can be related to the local mobility close to the surface 55 and the residual deformation is relaxed after several hours at ambient temperature. Finally microparticles made of PLGA (T/R = 1) are probed in order to check the applicability of the above presented measurement procedures to spherical geometries. Figure 3(a) shows PLGA microparticles that appear to be considerably wider than high. This effect is due to the convolution between the tip shape and the particle shape. 56 Nonetheless, AFM topographic images allow determining the top of each particle with an accuracy of 5 to 10 nm. Nanoindentation points are determined on the AFM image and force curves are recorded on the top of each localized particle. Young’s modulus of PLGA microparticles is found to be 2.8 ± 0.4 GPa. The slightly larger dispersion than in planar case is most probably due to the uncontrolled slight lateral motion of the spherical particles during the tip approach. As expected however, PLGA films and microparticles give very similar responses to normal compressive forces.

PLGA/PFOB Microcapsules As microcapsule radii are found to be in between 400 and 600 nm, the shell thicknesses span the following ranges: 140 to 210 nm (for T/R = 0.35), 100 to 150 nm (for T/R = 0.25), 60 to 90 nm (for T/R = 0.15) and 10 to 15 nm (for T/R = 0.025). In the following study, indentation depth is limited to several nanometers to avoid visco-plastic deformation and thus satisfy elastic model condition. AFM pictures are taken on microcapsules monolayer (Figure 3). The surface and morphology of these dried microcapsules differ according to the shell thickness. For T/R = 0.35 and 0.25, the surface exhibits a fairly regular morphology – similar to full PLGA microparticles – though some roughness is observed (Figure 3(b) and 3(c)). In the case of the thinnest capsules (T/R = 0.15 and 0.025), surface seems to be smoother (Figures 3(d) and 3(e)). For 11 ACS Paragon Plus Environment

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(a)

(b)

(c)

(d)

(e)

(f)

Figure 3: (a) PLGA Microparticles. (b) PLGA/PFOB Microcapsules T/R = 0.35 (c) T/R = 0.25. (d) T/R = 0.15. (e) and (f) T/R = 0.025. T/R = 0.025, shells become very fragile and broken capsules are sometimes observed (Figure 3(e) and 3(f)).


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Normal compression is applied at the top of each microcapsule using a trigger force to limit the deformation. It has to be noted that the results obtained on MCs monolayer do not differ from results obtained on isolated MC. Thus, this feature of the sample enables to systematically perform a lot of force curves in order to get statistically reliable results. As a first analysis, the Reissner analytical solution is used to fit the F − δ curves. This model is a reliable approximation of the elastic behavior of a thin spherical empty shell. 34 Reissner predicts the following relationship for the shell stiffness k:

k=

4 T2 F =p E δ 3(1 − ν 2 ) Rs

(9)

where ν and E are the Poisson ratio and the Young’s modulus of the shell material respectively, Rs is the radius of the spherical shell and T is the shell thickness. Figure 4(a) shows the double logarithmic F − δ plot for different shell thicknesses. A decrease of the exponent is observed when T/R decreases (remember that the first data points at low indentation depth are not taken into account). Thus, for thick capsules (T /R ≥ 0.25), the value of the exponent is in between 1.35 and 1.55, which is consistent with a Hertzian behavior. By contrast, for thinnest shells (T /R ≤ 0.15) the exponent is close to one, suggesting a linear behavior of the F −δ curve and the applicability of the Reissner analytical solution. Figure 4(b) shows the fit of the shell thickness (T/R = 0.15) using Reissner model. Imposing the PLGA bulk elasticity in the formula provides a correct thickness T as a fit result within the expected error bars. Although our capsules cannot be described as simple empty shells, Reissner theory works rather well as previously shown by Taber. 57 For larger indentations, deviations are expected to occur due to shear exerted on the shell but our capsules are probably too fragile to reach this range. For thicker shells, PLGA/PFOB capsues cannot be describe as simple empty shells which leads to a deviation from Reissner analytical solution. Moreover, the Reissner model does not take into account the composite aspect of the sample. CHIMER model is able to describe the evolution of the effective elasticity, whatever the shell thickness. Actually, this numerical 13 ACS Paragon Plus Environment

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F o rc e (n N )

= 1 , α= = 0 .3 5 , = 0 .2 5 , = 0 .0 2 5

T /R T /R T /R T /R

1 0 0 0

1 .5 α= 1 .5 α= 1 .3 , α= 1 .0

1 0 0

1 0

1 1

1 0 In d e n ta tio n ( n m ) (a)

1 0 0 0

E x p e r im e n ta l d a ta M C P L G A /P F O B ( T /R = 0 .1 5 ) R e is s n e r t h e o r y ( T = 6 5 n m , E = 3 G P a )

8 0 0 6 0 0 F o rc e (n N )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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4 0 0 2 0 0 0 -5

0

5

1 0

1 5

2 0

2 5

In d e n ta tio n ( n m ) (b)

Figure 4: (a) Double logarithmic F − δ plot for different shell thicknesses with power law fits. α is the exponent of the power law. (b) F − δ plot of a thin PLGA/PFOB MC (T/R = 0.15) fitted with the Reissner analytical solution. The expected value of T is 72 nm and the fitted value is 65 nm.


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approach describes the transition between the Young’s modulus of bulk shell compound at low indentation depth to the bulk elastic modulus of the core at high indentation depth. For this purpose, Hertz model was used to obtain effective elastic moduli Eef f and such values are implemented in CHIMER algorithm (see Supporting Information and Perriot et al. 36 Figure 5(a) shows that Hertz model fairly describes the mechanical behavior of the thickest microcapsules. An additional feature for improving the fit is to determine the best contact point δ0 which has the advantage to take into account residual adhesive effects. The typical deviations to zero of this parameter are small and of the order a few nanometers. For the thinnest ones (T/R = 0.15 and 0.025), ruptures and plastic deformations of the shells are observed which explain the discrepancy of the experimental force curves with the Hertzian model at high indentation depth. For T/R = 0.35 capsules, the Young’s modulus (E0.35 = 2.27 ± 0.56 GPa) is close to the elastic moduli of microparticles (T/R = 1) and PLGA film. Microcapsules with thinner shells present significantly smaller Young’s moduli: E0.25 = 1.25 ± 0.23 GPa, E0.15 = 0.57 ± 0.22 GPa, E0.025 = 0.1 ± 0.05 for T/R = 0.25, 0.15, and 0.025 respectively (Figure 5(b)). For the two thinnest capsules, effective Young’s modulus cannot be evaluated with accuracy due to the fragility of the shells and the force resolution of the used cantilever. We observe breaks in several force curve slopes showing the sudden rupture of the shell. The tip perforates the capsule and penetrates into the liquid core. This can be checked after hand on AFM pictures (see Supporting Information). This effect is not reproducible in term of indentation depth. Fully described in literature, the behavior of pure elastic shells under flat compression presents a first-order transition between the flatten deformation and the buckling deformation with the inversion of the curvature. Pauchard et al. 58 reported that such transition occurs at a deformation close to twice the thickness of the shell. In our case, force curves do not present this behavior because of the sharp shape of the indenter, the brittle hallmark of the PLGA and the incompressible PFOB core. Indeed, local deformation is observed preceding the sudden ruptures of the MCs.


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P L G A F ilm

1 6 0 0

T /R = 1 T /R = 0 .3 5

1 4 0 0 1 2 0 0

F o rc e (n N )

1 0 0 0 8 0 0 T /R = 0 .2 5

6 0 0 4 0 0

T /R = 0 .1 5

2 0 0

T /R = 0 .0 2 5 0 0

1 0

2 0 3 0 4 0 5 0 3 /2 3 /2 In d e n ta tio n D e p th δ ( n m )

6 0

7 0

(a)

N o r m a liz e d F r e q u e n c y

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1 ,0

T / R T / R T / R T / R

0 ,8 0 ,6

= 0 .1 = 0 .2 = 0 .3 = 1 .0

0 5 5 0

0 ,4 0 ,2 0 ,0 0 ,0

0 ,5

1 ,0

1 ,5

2 ,0

2 ,5

3 ,0

3 ,5

4 ,0

4 ,5

A p p a r e n t Y o u n g ’s M o d u lu s ( G P a )

(b)

Figure 5: (a) Hertzian fitting (straight lines) of PLGA film, full PLGA microparticles and PLGA / PFOB microcapsules experimental force curves (scatter) . (b) Statistical histograms of Young’s moduli depending on shell thickness. Solid lines are Gaussian fits. The decreasing measured elastic modulus with the shell thickness is directly related to the composite nature of MCs which are made of a polymer film coated on a liquid core. The measured elastic moduli indeed characterize the elastic behavior of the MCs through an


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effective modulus (Eef f ) which describes the local elasticity (i.e. at a given indentation) of the shell/core system. A. Perriot and E. Barthel modeled this elastic contact in the case of a flat coated infinite half-space. 36 They proposed a numerical analysis describing the change of the effective modulus according to the a/T (or δ/T ) ratio. This numerical approach can also describe the evolution of the contact radius a according to the indentation depth δ. This evolution is particularly interesting in the case of coated material because the contact radius √ a is no longer proportional to δ (Hertzian contact radius) owing to the compliance of the lower part (liquid core). We previously showed that low and high indentation depths are not relevant to describe the mechanical behavior with Hertz model (adhesion and compressive effects below 2-5 nm and visco-plasticity above 10 to 15 nm). In the relevant range of indentation depth (5-15 nm), the numerical analysis of Perriot and Barthel shows that the contact radius does not dramatically change with δ/T and remains at about 7 to 11 nm (Figure 6(a)). Hence, this justifies to assume a constant effective elasticity for one AFM nanoindentation experiment. Moreover, the slope in this range of δ/T is 0.39 to 0.45 which is close to the Hertzian dependency a ∝ δ 1/2 . Thus, the Hertzian approximation remains relevant despite the composite aspect of the sample. In a heuristic approach, Perriot and Barthel proposed a simple expression of the coated half-space indentation model which can be written as follows:

Eef f

a T

= E1 +

(E0 − E1 ) n 1 + Tax0

(10)

where E1 is the elastic modulus of the coating and E0 is the elastic modulus of the lower part. In our case, E1 and E0 are considered to be EP LGA and Ecore respectively. x0 and n are adjustable parameters. Approximating the shells as a flat surfaces (Rtip >> Rcapsule ) and considering EP LGA as the bulk PLGA elastic modulus and Ecore ≈ 0 (PFOB liquid core), experimental data are well fitted to equation 9 (x0 =0.05 and n=1.3). Figure 6(b) shows the transition of Eef f according to the shell thickness. The effective elastic modulus Eef f is close 17 ACS Paragon Plus Environment

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to the bulk PLGA modulus EP LGA for thick shells and tends towards Ecore for thin shells.

C o n t a c t r a d iu s a ( n m )

This simplified model is a good approximation of the shell behavior.

T = 2 T = 1 T = 5 T = 1

1 0

5

1 0

4

1 0

3

1 0

2

1 0

1

1 0

0

1 0

-1

1 0

-5

1 0

-4

1 0

-3

1 0

0 0 n m 2 5 n m 0 n m 5 n m

-2

1 0

-1

1 0

0

1 0

1

1 0

2

1 0

3

4

1 0

1 0

5

1 0

6

1 0

7

δ/ T

(a)

3 0 0 0 E f f e c t iv e Y o u n g ’s m o d u lu s ( M P a )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 P L G A /P F O B P e r r io t a n d B H e u r is tic a p p H ig h fr e q u e n

5 0 0 0 1 E -4

1 E -3

M a r ro c y

ic r o c a p s u le s th e l m o d e l a c h e x p e c ta tio n

0 ,0 1

0 ,1

1

a /T

(b)

Figure 6: (a) Simulation of the evolution of the contact radius a according to the shell thickness T and the indentation depth δ using the complete numerical algorithm. The range of experimental indentation depth induces slight variation of the contact radius during the measure (7 to 11 nm). (b) Effective Young’s Modulus versus a/T. Unbroken red line is the fit from heuristic model (x0 = 0.05 and n = 1.3) and considering EP F OB >> EP LGA . Squares are the fitting of MCs experimental value using the complete numerical algorithm from Perriot and Barthel. 36 Dashed line is the prediction of the high-frequency (5MHz) effective elastic modulus of the MCs (EP F OB /EP LGA ≈ 0.73) and the grey area is the Young’s modulus of bulk PLGA.


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Using the complete numerical algorithm (see supporting information and Sarrazin et al. 2 ) enables to fit Ecore knowing EP LGA and Eef f . Thus, Ecore is found to be a finite value of 100 MPa. By contrast with the heuristic approach, E0 is no longer considered to be zero. We interpret this elastic contribution as a consequence of the spherical shape of the particle. Indeed, for thin enough capsules, the deformation of the liquid is able to induce a longrange stress on the polymer shell, thus invalidating the model. Nevertheless, this ”effective” spherical shape contribution remains small, less than 3 % of the PLGA elasticity so the flat model is reasonable, especially for the thickest capsules. Considering quasi-static mechanical behavior, PFOB is a liquid which is considered as incompressible. In contrast, PFOB exhibits an elastic behavior at high-frequency (EP F OB =2.18 GPa at 5 MHz 59 ). Concerning PLGA at high frequency, we showed that the elasticity is really close to the quasi-static one at 2 kHz and reaches 4.3 GPa for frequencies of about 1 to 10 GHz. 49 It is probable that the gap between experimental values of PLGA elasticity is mostly due to the measurement technique and PLGA Young’s modulus is almost not influenced by frequency. Consequently, in the case of ultrasound waves in the MHz range, an increase of the shell elasticity is expected because of the increasing of EP F OB whereas EP LGA is almost unchanged. Using the coated elastic half-space model, the capsule elasticity at high frequency can be estimated (Figure 6(b)). The conclusion of this simulation is that the mechanical behavior of the capsules depends less on the shell thickness – contrary to the quasi-static case – because of the elastic property of the PFOB at high frequency. The effect of thermal annealing on MCs mechanical behavior is also investigated (Figure 7). Indeed, MCs should be injected several hours in advance before reaching the targeted organ and the modifications of the shell thickness T and its mechanical properties have to be predicted. After heating MCs in water, a droplet of the solution is deposited and dried on mica substrate. Force measurements are then performed in air at room temperature. Effective Young’s moduli of annealed MCs are found significantly lower than non-annealed

(biological temperature and lower than the glass tran-

MCs ones. Thus, after 24h at 38


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sition temperature of the PLGA), Young’s modulus of MC with T/R = 0.25 (0.89 ± 0.24

(> T ),

GPa) is about 30 % less than not annealed MCs. Similarly, after 60 min at 50

g

Young’s modulus of MCs with T/R = 0.35 is 1.02 ± 0.15 GPa (Figure 7(a) and 7(b)). AFM imaging evidences polymer on the substrate between the microcapsules. This may be due to polymer hydration and partial degradation of the shell by hydrolysis even under the PLGA

glass transition temperature (Tg = 42.6 ± 0.5

60

). Some polymer is then deposited on the

substrate during the drying process. Thus, a thinning of MCs during heating is highly probable. The loss of polymer can then explain the softening of the MC and the much better homogeneity of the Young’s moduli.


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T/R = 0.25 T/R = 0.25, T = 50°C / 60 min T/R = 0.25, T = 38°C / 24h

N o r m a liz e d F r e q u e n c y

1 ,0 0 ,8 0 ,6 0 ,4 0 ,2 0 ,0 0 ,6

0 ,8

1 ,0 1 ,2 1 ,4 1 ,6 1 ,8 2 ,0 Apparent Young's Modulus (GPa)

2 ,2

2 ,4

(a)

T/R = 0.35 T/R = 0.35, T° = 50°C / 60 min

1 ,0 N o r m a liz e d F r e q u e n c y

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 ,8 0 ,6 0 ,4 0 ,2 0 ,0 0 ,0

0 ,5

1 ,0

1 ,5 2 ,0 2 ,5 3 ,0 Apparent Young's Modulus (GPa)

3 ,5

4 ,0

4 ,5

(b)

Figure 7: Statistical histograms of Young’s moduli depending on annealing temperature : (a) MC T/R = 0.25 and (b) MC T/R = 0.35. Force measurements are performed at room temperature.


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Conclusion Polymeric microcapsules (≈ 500 nm radius) have been locally probed using an AFM tip (≈ 20-50 nm radius). The comparison between Hertz and JKR models showed that adhesive effect can be neglected if the first few nanometers are not taken into account. As expected, the force curves exhibit a power law behavior (at large enough indentation depth) with a decreasing exponent from 1.5 for thick shells to 1.0 for the thinnest ones. Reissner analytical model has been used to describe the elasticity of shells with T /R ≤ 0.15. Nevertheless, this approach is not suitable for thicker shells and does not provide any information about the effective elasticity of the MCs. Using both JKR and Hertz models , effective elastic moduli of MCs are found to decrease with the shell thickness (200 to 10 nm thick give elastic moduli from 3.0 to 0.1 GPa). Indeed, the effective elastic moduli reflect the combination of the bulk polymer modulus and of the core softness. Since the radius of the tip is much smaller than the radius of the MCs, a semi-analytical model (CHIMER) – describing the effective Youngs modulus of a flat infinite coated half-space – was used to interpret the observed decrease of the MCs effective elasticity. Simulations show that the evolution of the contact radius a is close to the Hertzian dependency (a ∝ δ 1/2 ) and varies very little in the experimental range of indentation. Hence, it is justified to consider a constant effective elasticity during each AFM nanoindentation experiment. Independently obtained effective elastic moduli are thus introduced into the complete numerical algorithm and we argue that the correction due to the spherical shape is represented by an effective core elasticity. The latter is found to be about 100 MPa (≈ 3 % of the bulk PLGA elasticity), showing that the flat assumption is reasonable for the thickest capsules. Nevertheless, the development of a spherical coated model will be needed in the future for refining the analysis. In the context of using MCs for ultrasound examination and drug delivery, this numerical model has been used to simulate the local compression at high frequency, knowing the elastic modulus of each component of the microcapsule. Thus, the coated half-space model predicts that the PLGA/PFOB microcapsule elasticity depends much less on the shell thickness than at low 22 ACS Paragon Plus Environment

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frequency because of the comparable elasticity of PLGA and PFOB at high frequency (2.5 to 3 GPa). Finally, the effect of thermal annealing on MCs elasticity (below and above glass transition of the polymer) has been investigated. Annealed MCs are found to be softer after ten hours at 38

and one hour at 50. This is due to the hydration of the polymer which

probably makes the shell thinner and promotes a decrease of the effective elasticity. This work shows that a semi-analytical model can describe the elastic behavior of a complex nano object. Such a method is a promising way to describe, simulate and interpret the effective elasticity of composite materials.

Acknowledgement We acknowledge the Plan Cancer 2009-2013 (NABUCCO), National Institute for Health and Medical Research (INSERM) and the RTRA Triangle de la Physique (Université ParisSaclay) for funding. This work was also supported by the French National Research Agency under the Program Investing in the Future (Investissements d’Avenir) Grant no ANR-11IDEX-0003-02 and Grant no ANR-10-NANO-06-04. Institut Galien Paris-Sud is a member of the Laboratory of Excellence LERMIT supported by a grant from ANR (ANR-10-LABX33). The authors thank Cindy Rountree from the SPHYNX laboratory (CEA-Saclay) for support during Peak Force experiments, Etienne Barthel (ESPCI, Paris) for his kind help about the numerical model and Rémy Brossard (PhD Student, LIONS, CEA-Saclay) for programming it.

Supporting Information Available This material is available free of charge via the Internet at http://pubs.acs.org/.


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References (1) Bonilla, M. R.; Stokes, J. R.; Gidley, M. J.; Yakubov, G. E. Interpreting atomic force microscopy nanoindentation of hierarchical biological materials using multi-regime analysis. Soft Matter 2015, 11, 1281–1292. (2) Sarrazin, B.; Brossard, R.; Guenoun, P.; Malloggi, F. Investigation of PDMS based bi-layer elasticity via interpretation of apparent Young’s modulus. Soft Matter 2016, (3) Butt, H.-J.; Cappella, B.; Kappl, M. Force measurements with the atomic force microscope: Technique, interpretation and applications. Surface Science Reports 2005, 59, 1–152. (4) Domke, J.; Radmacher, M. Measuring the Elastic Properties of Thin Polymer Films with the Atomic Force Microscope. Langmuir 1998, 14, 3320–3325. (5) O’Connell, P. A. Rheological Measurements of the Thermoviscoelastic Response of Ultrathin Polymer Films. Science 2005, 307, 1760–1763. (6) Du, B.; Tsui, O. K. C.; Zhang, Q.; He, T. Study of Elastic Modulus and Yield Strength of Polymer Thin Films Using Atomic Force Microscopy. Langmuir 2001, 17, 3286–3291. (7) Sun, Y.; Akhremitchev, B.; Walker, G. C. Using the Adhesive Interaction between Atomic Force Microscopy Tips and Polymer Surfaces to Measure the Elastic Modulus of Compliant Samples. Langmuir 2004, 20, 5837–5845. (8) Buchner Santos, E.; Morris, J. K.; Glynos, E.; Sboros, V.; Koutsos, V. Nanomechanical Properties of Phospholipid Microbubbles. Langmuir 2012, 28, 5753–5760. (9) Abou-Saleh, R. H.; Peyman, S. A.; Critchley, K.; Evans, S. D.; Thomson, N. H. Nanomechanics of Lipid Encapsulated Microbubbles with Functional Coatings. Langmuir 2013, 29, 4096–4103.


Page 24 of 31

Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(10) Poehlmann, M.; Grishenkov, D.; Kothapalli, S. V. V. N.; Härmark, J.; Hebert, H.; Philipp, A.; Hoeller, R.; Seuss, M.; Kuttner, C.; Margheritelli, S.; Paradossi, G.; Fery, A. On the interplay of shell structure with low- and high-frequency mechanics of multifunctional magnetic microbubbles. Soft Matter 2014, 10, 214. (11) Grant, C. A.; McKendry, J. E.; Evans, S. D. Temperature dependent stiffness and visco-elastic behaviour of lipid coated microbubbles using atomic force microscopy. Soft Matter 2012, 8, 1321. (12) Ghorbanzadeh Ahangari, M.; Fereidoon, A.; Jahanshahi, M.; Sharifi, N. Effect of nanoparticles on the micromechanical and surface properties of poly(ureaformaldehyde) composite microcapsules. Composites Part B: Engineering 2014, 56, 450–455. (13) De Loubens, C.; Deschamps, J.; Georgelin, M.; Charrier, A.; Edwards-Levy, F.; Leonetti, M. Mechanical characterization of cross-linked serum albumin microcapsules. Soft Matter 2014, 10, 4561. (14) Ye, C.; Drachuk, I.; Calabrese, R.; Dai, H.; Kaplan, D. L.; Tsukruk, V. V. Permeability and Micromechanical Properties of Silk Ionomer Microcapsules. Langmuir 2012, 28, 12235–12244. (15) Zoldesi, C.; Ivanovska, I.; Quilliet, C.; Wuite, G.; Imhof, A. Elastic properties of hollow colloidal particles. Physical Review E 2008, 78 . (16) Yin, J.; Retsch, M.; Lee, J.-H.; Thomas, E. L.; Boyce, M. C. Mechanics of Nanoindentation on a Monolayer of Colloidal Hollow Nanoparticles. Langmuir 2011, 27, 10492– 10500. (17) Tan, E. P. S.; Lim, C. T. Nanoindentation study of nanofibers. Applied Physics Letters 2005, 87, 123106.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(18) Gibbons, M.; Klug, W. Nonlinear finite-element analysis of nanoindentation of viral capsids. Physical Review E 2007, 75 . (19) Sui, X.; Chen, Q.; Hempenius, M. A.; Vancso, G. J. Probing the Collapse Dynamics of Poly(N-isopropylacrylamide) Brushes by AFM: Effects of Co-nonsolvency and Grafting Densities. Small 2011, 7, 1440–1447. (20) Zhang, Z.; Morse, A. J.; Armes, S. P.; Lewis, A. L.; Geoghegan, M.; Leggett, G. J. Nanoscale Contact Mechanics of Biocompatible Polyzwitterionic Brushes. Langmuir 2013, 29, 10684–10692. (21) Horimizu, M.; Kawase, T.; Tanaka, T.; Okuda, K.; Nagata, M.; Burns, D. M.; Yoshie, H. Biomechanical evaluation by AFM of cultured human cell-multilayered periosteal sheets. Micron 2013, 48, 1–10. (22) Carl, P.; Schillers, H. Elasticity measurement of living cells with an atomic force microscope: data acquisition and processing. Pfl¨ ugers Archiv - European Journal of Physiology 2008, 457, 551–559. (23) Vella, D.; Ajdari, A.; Vaziri, A.; Boudaoud, A. The indentation of pressurized elastic shells: from polymeric capsules to yeast cells. Journal of The Royal Society Interface 2011, 9, 448–455. (24) Riess, J. G. Blood substitutes and other potential biomedical applications of fluorinated colloids. Journal of Fluorine Chemistry 2002, 114, 119–126. (25) Pisani, E.; Tsapis, N.; Paris, J.; Nicolas, V.; Cattel, L.; Fattal, E. Polymeric Nano/Microcapsules of Liquid Perfluorocarbons for Ultrasonic Imaging: Physical Characterization. Langmuir 2006, 22, 4397–4402. (26) Diou, O.; Tsapis, N.; Giraudeau, C.; Valette, J.; Gueutin, C.; Bourasset, F.; Zanna, S.;


Page 26 of 31

Page 27 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Vauthier, C.; Fattal, E. Long-circulating perfluorooctyl bromide nanocapsules for tumor imaging by 19FMRI. Biomaterials 2012, 33, 5593–5602. (27) Mousnier, L.; Huang, N.; Morvan, E.; Fattal, E.; Tsapis, N. Influence of polymer endchemistry on the morphology of perfluorohexane polymeric microcapsules intended as ultrasound contrast agents. International Journal of Pharmaceutics 2014, 471, 10–17. (28) Cosco, D.; Fattal, E.; Fresta, M.; Tsapis, N. Perfluorocarbon-loaded micro and nanosystems for medical imaging: A state of the art. Journal of Fluorine Chemistry 2015, 171, 18–26. (29) Srinivas, M.; Morel, P. A.; Ernst, L. A.; Laidlaw, D. H.; Ahrens, E. T. Fluorine-19 MRI for visualization and quantification of cell migration in a diabetes model. Magnetic Resonance in Medicine 2007, 58, 725–734. (30) Somaglino, L.; Bouchoux, G.; Mestas, J.-L.; Lafon, C. Validation of an acoustic cavitation dose with hydroxyl radical production generated by inertial cavitation in pulsed mode: Application to in vitro drug release from liposomes. Ultrasonics Sonochemistry 2011, 18, 577–588. (31) Coussios, C. C.; Roy, R. A. Applications of Acoustics and Cavitation to Noninvasive Therapy and Drug Delivery. Annual Review of Fluid Mechanics 2008, 40, 395–420. (32) Guédra, M.; Valier-Brasier, T.; Conoir, J.-M.; Coulouvrat, F.; Astafyeva, K.; Thomas, J.-L. Influence of shell compressibility on the ultrasonic properties of polydispersed suspensions of nanometric encapsulated droplets. The Journal of the Acoustical Society of America 2014, 135, 1044–1055. (33) Glynos, E.; Koutsos, V.; McDicken, W. N.; Moran, C. M.; Pye, S. D.; Ross, J. A.; Sboros, V. Nanomechanics of Biocompatible Hollow Thin-Shell Polymer Microspheres. Langmuir 2009, 25, 7514–7522.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 31

(34) Reissner, E. Stresses and Small Displacements of Shallow Spherical Shells. II. Journal of Mathematics and Physics 1946, 25, 279–300. (35) Fery, A.; Weinkamer, R. Mechanical properties of micro- and nanocapsules: Singlecapsule measurements. Polymer 2007, 48, 7221 – 7235. (36) Perriot, A.; Barthel, E. Elastic contact to a coated half-space - Effective elastic modulus and real penetration. Journal of Material Research 2004, 600–608. (37) Yamaguchi, K.; Anderson, J. M. In vivo biocompatibility studies of medisorb 65/35 D,L-lactide/glycolide copolymer microspheres. Journal of Controlled Release 1993, 24, 81–93. (38) Reed,

A.;

Gilding,

D.

Biodegradable

polymers

poly(glycolic)/poly(Iactic acid) homo and copolymers:

for

use

in

surgery

2. In vitro degradation.

Polymer 1981, 22, 494–498. (39) Leese, P. T.; Noveck, R. J.; Shorr, J. S.; Woods, C. M.; Flaim, K. E.; Keipert, P. E. Randomized Safety Studies of Intravenous Perflubron Emulsion. I. Effects on Coagulation Function in Healthy Volunteers:. Anesthesia & Analgesia 2000, 91, 804–811. (40) Noveck, R. J.; Shannon, E. J.; Leese, P. T.; Shorr, J. S.; Flaim, K. E.; Keipert, P. E.; Woods, C. M. Randomized Safety Studies of Intravenous Perflubron Emulsion. II. Effects on Immune Function in Healthy Volunteers:. Anesthesia & Analgesia 2000, 91, 812–822. (41) Hertz, H. R. Ueber die Beruehrung elastischer Koerper (On Contact Between Elastic Bodies); Gesammelte Werke (Collected Works); 1895; Vol. 1. (42) Johnson, K.; Kendall, K.; Roberts, A. Surface energy and the contact of elastic solids. Proveeding of The Royal Society of London. Series A, Mathematical and Physical 1971, 324, 301–313. 28 ACS Paragon Plus Environment

Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(43) Kern, W. The Evolution of Silicon Wafer Cleaning Technology. Journal of The Electrochemical Society 1990, 137, 1887. (44) Burnham, N. A.; Chen, X.; Hodges, C. S.; Matei, G. A.; Thoreson, E. J.; Roberts, C. J.; Davies, M. C.; Tendler, S. J. B. Comparison of calibration methods for atomic-force microscopy cantilevers. Nanotechnology 2003, 14, 1–6. (45) Butt, H. J.; Jaschke, M. Calculation of thermal noise in atomic force microscopy. Nanotechnology 1995, 6, 1–7. (46) Villarrubia, J. Morphological estimation of tip geometry for scanned probe microscopy. Surface Science 1994, 321, 287–300. (47) Lin, L.-Y.; Kim, D.-E. Measurement of the elastic modulus of polymeric films using an AFM with a steel micro-spherical probe tip. Polymer Testing 2012, 31, 926–930. (48) Lee, M. H.; Lee, D. Elastic instability of polymer-shelled bubbles formed from air-inoil-in-water compound bubbles. Soft Matter 2010, 6, 4326. (49) Astafyeva, K. Thesis : Physical and Acoustical Properties of Fluorocarbon Nanoparticles. 2014. (50) Kopycinska-Mller, M.; Geiss, R. H.; Hurley, D. C. Contact mechanics and tip shape in AFM-based nanomechanical measurements. Ultramicroscopy 2006, 106, 466–474. (51) Tsukruk, V. V.; Huang, Z.; Chizhik, S. A.; Gorbunov, V. V. Probing of micromechanical properties of compliant polymeric materials. Journal of Materials Science 1998, 33, 4905–4909. (52) Grierson, D. S.; Flater, E. E.; Carpick, R. W. Accounting for the JKRDMT transition in adhesion and friction measurements with atomic force microscopy. Journal of Adhesion Science and Technology 2005, 19, 291–311.


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(53) Salez, T.; Benzaquen, M.; Raphal, l. From adhesion to wetting of a soft particle. Soft Matter 2013, 9, 10699. (54) Rodr´ıguez, J.; Maneiro, M. G. A procedure to prevent pile up effects on the analysis of spherical indentation data in elasticplastic materials. Mechanics of Materials 2007, 39, 987 – 997. (55) Forrest, J.; Dalnoki-Veress, K.; Stevens, J.; Dutcher, J. Effect of Free Surfaces on the Glass Transition Temperature of Thin Polymer Films. Physical Review Letters 1996, 77, 4108–4108. (56) H¨ ubner, U.; Morgenroth, W.; Meyer, H.; Sulzbach, T.; Brendel, B.; Mirandé, W. Downwards to metrology in nanoscale: determination of the AFM tip shape with well-known sharp-edged calibration structures. Applied Physics A: Materials Science & Processing 2003, 76, 913–917. (57) Taber, L. A. Large Deflection of a Fluid-Filled Spherical Shell Under a Point Load. Journal of Applied Mechanics 1982, 49, 121 – 128. (58) Pauchard, L.; Rica, S. Contact and compression of elastic spherical shells: the physics of a ’ping-pong’ ball. Philosophical Magazine B 1998, 78, 225–233. (59) André, M.; Nelson, T.; Mattrey, R. Physical and acoustical properties of perfluorooctylbromide, an ultrasound contrast agent. Invest Radiol 1990, 25, 983–987. (60) Passerini, N.; Craig, D. Q. An investigation into the effects of residual water on the glass transition temperature of polylactide microspheres using modulated temperature DSC. J Control Release 2001, 73, 111–115.


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Page 31 of 31

Graphical TOC Entry 3000

AFM Tip PLGA Shell T PFOB Core R

Effective Young’s modulus )MPaC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Bulk PLGA

2500 2000 1500 1000 500

PLGA/PFOB Microcapsules Elastic coated half-space model

0 1E-4

1E-3

0,01

0,1

Contact radius / Capsule thickness


1

AFM Investigation of Liquid-Filled Polymer Microcapsules Elasticity

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