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SAXS Analysis of Shell Formation During Nanocapsule Synthesis via Inverse Miniemulsion Periphery RAFT Polymerization Robert H. Utama, Martin Dulle, Stephan Förster,* Martina H. Stenzel,* Per B. Zetterlund* Currently available methods for synthesis of polymeric nanocapsules only offer limited control over the shell thickness, even though it is an important parameter for various applications. Furthermore, suitable methods to critically measure this parameter in a facile way are still nonexistent. Here, lab-scale small-angle X-ray scattering (SAXS) is utilized to in situ measure the evolution of shell thickness during nanocapsule synthesis via inverse miniemulsion periphery reversible addition–fragmentation chain transfer (RAFT) polymerization (IMEPP). The measured shell thickness is consistent with estimates from the commonly used transmission electron microscopy (TEM) technique. Moreover, the individual thicknesses of two concentric shells comprising different polymeric materials (the outer shell formed via IMEPP chain extension of the inner shell) can be determined, thus further demonstrating the versatility of this approach.
1. Introduction The administration of water-soluble therapeutic agents such as proteins to treat various ailments is inhibited by R. H. Utama, Prof. P. B. Zetterlund Centre for Advanced Macromolecular Design School of Chemical Engineering The University of New South Wales Sydney, NSW 2052, Australia E-mail: [email protected] Dr. M. Dulle, Prof. S. Förster Physikalische Chemie I Universität Bayreuth 95447 Bayreuth, Germany E-mail: [email protected] Prof. M. H. Stenzel Centre for Advanced Macromolecular Design School of Chemistry The University of New South Wales Sydney, NSW 2052, Australia E-mail: [email protected] Macromol. Rapid Commun. 2015, 36, 1267−1271 © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
their apparent cytotoxicity and low bioavailability.[1,2] Hollow polymeric nanocapsules, where the drugs can be protected in a water pool, are considered to be suitable candidates to circumvent these issues and therefore the development of loaded nanocapsules has grown very rapidly in recent years. The synthetic pathways to nanocapsules can be divided into three categories: i) self-assembly,[3,4] ii) sacrificial template,[5,6] and iii) miniemulsion polymerization.[7] Significant efforts have been directed towards analysis of various nanoparticle parameters such as size, shape, and surface properties, which affect cellular interactions and penetrations.[8–11] However, one aspect that is frequently overlooked is the shell thickness, despite important characteristics such as shell degradation, diffusion, and release rate of encapsulated molecules being critically controlled by this parameter.[12–14] Only few synthetic pathways allow control of shell thickness. The sacrificial template/layer by layer (LbL) method enables control over the shell thickness by varying the number of layers added.[15,16] Polymerization within the interfacial region of miniemulsion droplets
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has also been reported to be suitable for controlling the shell thickness. For example, interfacial polyaddition was claimed to enable control of the shell thickness by adjustment of the stoichiometry.[17,18] However, the inherent limitations of these techniques lie in the relatively poor precision in obtaining the desired shell thickness and/or the complexity of the process. Controlled/living radical polymerization (CLRP), such as reversible addition–fragmentation chain transfer (RAFT) polymerization, has been used extensively to control the size and morphology of linear and branched polymers.[19,20] It has also been used, in conjunction with miniemulsions, to create nanoparticles suitable for drug delivery.[21–23] We have recently reported a new method, namely inverse miniemulsion periphery polymerization (IMEPP) via RAFT, for synthesis of nanocapsules in one-pot, which readily allows control of the shell thickness.[24–26] The mechanism of shell formation in IMEPP is controlled by RAFT polymerization as described previously.[26] The inverse miniemulsion comprises sodium chloride (lipophobe) in the aqueous dispersed phase and monomer, cross-linker, and initiator in the toluene continuous phase and is stabilized by amphiphilic RAFT block copolymers. This confines the subsequent RAFT polymerization on the periphery of the droplet, the chains growing out towards the continuous phase. There are to date no reported techniques suitable for measuring the thickness of the polymeric shell of nanocapsules in their native state. The most commonly used technique is transmission electron microscopy (TEM), but the results are greatly affected by issues related to the behavior of the polymeric chains as well as collapsing of the nanocapsules upon drying. Although cryo-TEM may overcome such limitations, its application is relatively tedious and time consuming. Here, we report the utilization of smallangle X-ray scattering (SAXS) to perform online investigations on the evolution of the polymeric shells of nanocapsules in their native state, synthesized via RAFT IMEPP.
3. Results and Discussion Two systems with different types of shells were investigated: methyl methacrylate (MMA) homopolymer and diblock copolymer of methyl methacrylate/tert-butyl methacrylate (MMA/tBMA), both utilizing ethylene glycol dimethacrylate (EGDMA) as the cross-linker. The MMA system represents a “Core/1 Shell” morphology whereby the initial stabilizer hydrophobic block (Shell 1, MMA) was chain extended with MMA (Shell 1 + polymerized MMA via IMEPP, Scheme 1). In contrast, the MMA/tBMA system represents a “Core/2 Shells” morphology (Scheme 1) whereby the initial Shell 1 was chain extended to yield a second shell (“Shell 2”) comprising tBMA. SAXS was then used to analyze in situ the growth in shell thickness during the RAFT controlled chain extension polymerization. The SAXS data were fitted to a model of a sphere with two concentric shells of different but spatially uniform contrast. The first step was to determine the size of the aqueous core by fitting the first scattering profile (before polymerization) with a Core/1 Shell model with a very thin shell, the main scattering contrast thus originating from the core. The radius and contrast (with respect to toluene) of the core was then kept constant for determination of the shell thickness (“Shell 1” in Scheme 1) by varying its value and contrast to give the best fitting model for the scattering curve. Once the radius of the aqueous core, the thickness of Shell 1 and their respective contrasts had been determined, these parameters were kept constant for the subsequent fitting to obtain the thickness of the shell after IMEPP (Core/1 Shell and Core/2 Shells; Scheme 1), using the shell thickness of Shell 2 and its contrast as adjustable parameters. As an example, the equation for the case of a single shell is given below: I ( q) =
[sin( qrc ) − qrc cos( qrc )] scale ⎡ ⎢3Vc ( ρ c − ρ s ) ( qrc )3 Vs ⎣ 2
[sin( qrs ) − qrs cos( qrs )] ⎤ + 3Vs ( ρs − ρsolv ) ⎥ + bckg ( qrs )3 ⎦
2. Experimental Section
O
S
O
S O
IMEPP n
o
Ph
O
O
Stabilizer hydrophobic block Shell 1
O
O
MMA EGDMA
Ph
S
Aq. Core
o
O
Polymeric shell
where scale is a scale factor, Vs is the volume of shell, Vc is the volume of the core, rs is the radius of the shell, rc is the radius of the core, ρc is the scattering length density of the
O
O
S
O
Shell 1 IMEPP tBMA EGDMA
Core/1 Shell
O
Shell 2
O
O
S n
O
o
Detailed experimental on the synthesis of the block-copolymer, SAXS data analysis, and all analytical techniques are available on the Supporting Information.
(1)
O
O
O
S
Ph
Core/2 Shells
Scheme 1. Schematic representation of the “Core/1 Shell” and “Core/2 Shells” nanocapsules.
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SAXS Analysis of Shell Formation During Nanocapsule Synthesis via Inverse Miniemulsion Periphery RAFT Polymerization
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core, ρs is the scattering length density of the shell, ρsolv is the scattering length density of the solvent, and bckg is the background scattering. The suitability of SAXS to measure the shell growth was initially examined for a Core/1 Shell-type nanocapsule using the previously established IMEPP system based on poly(N-(2-hydroxypropyl)methacrylamide)b-poly(methyl methacrylate) (PHPMA19-b-PMMA79) as macro-RAFT stabilizer with MMA and EGDMA (Table S1, Supporting Information). A dispersed phase of 10% (weight fraction with respect to the continuous phase) was confirmed to be optimum as it confines any structural factor effects to only the lower q-range, and eliminates any oscillation from interparticle cross-linking. Such a relatively low weight fraction of water was also found to not affect the q-region of interest whereby the shell growth is visible. Normalized scattering curves over the period of polymerization are shown in Figure S4 (Supporting Information). Fitting of a Core/1 Shell model to the scattering curves corresponding to t = 0 and during polymerization resulted in excellent fits (Figure 1A), confirming nanocapsule morphology. Analysis of the scattering curve at t = 0 (Figure S3, Supporting Information) revealed a distinct minimum, which was used to determine the aqueous core radius of 92 nm. The thickness of Shell 1 was successfully determined to be 7.5 nm despite a small difference in contrast between the shell and the toluene background (Table S3, Supporting Information). This gives a total initial droplet diameter of 199 nm (92 + 92 + 7.5 + 7.5), comparable to the DLS values (dn = 169 nm, dv = 253 nm, Table S2, Supporting Information). As the shell progressively grows, the distinctive minimum point was overshadowed by the feature of the growing shell. Subsequent fitting of the normalized scattering curve every 30 min provided information on the shell growth over time. The total integrated scattering intensity (area under the scattering curve) increased and the shape of the scattering curve changed with time due to shell growth. The shell growth was quite significant
for the first few hours before reaching a plateau value (Figure 1B). The overall thickness of the polymeric shell was ≈17 nm after 7 h. The calculated shell thickness value was very similar to the previously published results of ≈20 nm after 7 h of polymerization, measured from the TEM data.[26] The developed SAXS method was further applied to a system with more than one shell material. Analysis of the MMA/tBMA system (EGDMA cross-linker) was conducted to investigate the capability of the developed approach in differentiating concentric shells of polymers having relatively similar electron densities. To illustrate the versatility of the technique, a macro-RAFT diblock copolymer different from that of the Core/1 Shell model above and to the previously reported tBMA polymeric nanocapsules, synthesized via IMEPP,[24] was employed. Here, methyl methacrylamide (MAAm) was used instead of HPMA as the hydrophilic block and MMA as the hydrophobic block (PMAAm16-b-PMMA64). Initially, the normalized scattering curves (Figure S3, Supporting Information) were fitted to the Core/1 Shell model used above for the MMA system, giving an aqueous core radius and Shell 1 thickness of 79.5 and 4.8 nm, respectively, and thus an overall droplet diameter of ≈170 nm in close agreement with DLS data (dn = 174 nm, dv = 243 nm, Table S2, Supporting Information). As the polymerization proceeded, changes in the scattering curves conformed to a Core/2 Shell model (Figure 2A and S5, Supporting Information). Determination of the polydispersity of the growing shell was not attempted, thus leading to the small discrepancy observed on the fitted curve (Figure 2A, square). However, this did not change the position of the form factor maximum, but would only flatten the undulation of the overall form factor. The form factor maximum is a distinctive feature of a Core/ 2 Shell model and its shift into lower q-values as the polymerization progress represents the increase in shell thickness. Therefore, the shell growth can be determined correctly from the fitting.
Figure 1. A) Form factor fitting of the scattering curve with a core–shell model and B) evolution of polymeric shell (the initial value of 7.5 nm corresponds to “Shell 1” (Scheme 1)) thickness over time of the MMA system.
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Figure 2. A) Normalized Core/2 Shell model fits for the nanocapsules for IMEPP of tBMA/EGDMA, curves have been shifted along y-axis for better visibility), B) evolution of shell thickness (Shell 2) over time.
The subsequent determination of the thickness of Shell 2 (and its contrast) as a function of time was conducted using the Core/2 Shell model by keeping constant the values of the core radius, the Shell 1 thickness as well as their respective contrasts (Table S4, Supporting Information). The Shell 2 thickness versus time (Figure 2B) revealed steady growth in the first 4.5 h, reaching a plateau value at 30.6 nm. The initial linear increase of shell thickness with time is in agreement with classical seeded growth kinetics (Figure 2B).[27] Now, if the shell was growing from a flat surface, one would anticipate a linear increase in shell thickness with conversion if the density of the crosslinked shell (Shell 2) is constant throughout the shell region and does not depend on conversion. Figure 3 shows the Shell 2 thickness plotted versus conversion, revealing a close to linear relationship except for the highest con-
version range. Given that the shell growth occurs from a sphere, the surface area gradually increases with conversion (unlike in case of a flat surface). As a consequence, a given conversion increment would lead to progressively smaller increases in shell thickness assuming the shell density is constant. To correct for the effect of increasing surface area on shell growth, the experimental data were “corrected” so as to correspond to the initial droplet surface area before polymerization as a flat surface (see Supporting Information for details). The resulting plot deviates significantly upwards from linearity, indicating that as the shell thickness increases, a given conversion increment leads to a progressively greater increase in thickness for the corresponding hypothetical flat surface. This is consistent with the density of the polymer network decreasing with increasing conversion, well in agreement with spherical polymer brush models where a decreasing segment density with increasing radius is expected.[28] The shell contrasts with respect to the continuous phase (Table S5, Supporting Information) decreased with time for Shell 2, eventually reaching a value lower than for Shell 1, consistent with the density of Shell 2 decreasing with increasing shell growth.
4. Conclusion
Figure 3. Shell (Shell 2) thickness versus conversion obtained by SAXS (solid squares; full line is guide to the eye only) for the tBMA/MMA system. The broken line shows predicted shell thickness from a flat surface corresponding to the initial droplet surface area (see text and Supporting Information for details).
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By using an appropriate and simple SAXS model to fit the data, we have presented an efficient way to monitor the controlled creation of polymeric shells of nanocapsules using SAXS. The results confirm the suitability of IMEPP to synthesize nanocapsules with excellent control over the shell and the suitability of lab-scale SAXS as a tool to monitor the shell growth. This capability to accurately control the shell thickness is extremely useful in designing polymeric nanocapsules whereby the release of the loads is controlled by the degradation of the shell overtime and/ or the porosity of the shells.
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Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: R.H.U. and M.D. contributed equally. R.H.U thank the UNSW student exchange office for a Student Exchange Scholarship. Note: Stephan Förster and Martina H. Stenzel were added as corresponding authors on April 22, 2015. Received: February 12, 2015; Revised: March 18, 2015; Published online: April 17, 2015; DOI: 10.1002/marc.201500096 Keywords: IMEPP; inverse miniemulsion; nanocapsules; RAFT polymerization; small-angle X-ray scattering [1] K. Cho, X. Wang, S. Nie, Z. Chen, D. M. Shin, Clin. Cancer Res. 2008, 14, 1310. [2] D. J. Stewart, Crit. Rev. Oncol./ Hematol. 2007, 63, 12. [3] A. Blanazs, S. P. Armes, A. J. Ryan, Macromol. Rapid Commun. 2009, 30, 267. [4] A. Rösler, G. W. M. Vandermeulen, H.-A. Klok, Adv. Drug Delivery Rev. 2012, 64, 270. [5] A. P. R. Johnston, C. Cortez, A. S. Angelatos, F. Caruso, Curr. Opin. Colloid Interface Sci. 2006, 11, 203. [6] Y. Wang, V. Bansal, A. N. Zelikin, F. Caruso, Nano Lett. 2008, 8, 1741. [7] K. Landfester, A. Musyanovych, V. Mailänder, J. Polym. Sci., Polym. Chem. 2010, 48, 493. [8] C. E. Ashley, E. C. Carnes, G. K. Phillips, D. Padilla, P. N. Durfee, P. A. Brown, T. N. Hanna, J. Liu, B. Phillips, M. B. Carter, N. J. Carroll, X. Jiang, D. R. Dunphy, C. L. Willman, D. N. Petsev, D. G. Evans, A. N. Parikh, B. Chackerian, W. Wharton, D. S. Peabody, C. J. Brinker, Nat. Mater. 2011, 10, 389.
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[9] M. C. Garnett, P. Kallinteri, Occup. Med. 2006, 56, 307. [10] W. Jiang, B. Y. S. Kim, J. T. Rutka, W. C. W. Chan, Nat Nano 2008, 3, 145. [11] R. A. Petros, J. M. DeSimone, Nat. Rev. Drug Discovery 2010, 9, 615. [12] H. Ai, S. A. Jones, M. M. de Villiers, Y. M. Lvov, J. Controlled Release 2003, 86, 59. [13] Y. Gao, Y. Chen, X. Ji, X. He, Q. Yin, Z. Zhang, J. Shi, Y. Li, ACS Nano 2011, 5, 9788. [14] Y. Kim, M. H. Pourgholami, D. L. Morris, M. H. Stenzel, Biomacromolecules 2012, 13, 814. [15] T. Wu, Z. Ge, S. Liu, Chem. Mater. 2011, 23, 2370. [16] S. Ye, C. Wang, X. Liu, Z. Tong, J. Biomater. Sci., Polym. Ed. 2005, 16, 909. [17] G. Baier, A. Musyanovych, M. Dass, S. Theisinger, K. Landfester, Biomacromolecules 2010, 11, 960. [18] E.-M. Rosenbauer, K. Landfester, A. Musyanovych, Langmuir 2009, 25, 12084. [19] A. Gregory, M. H. Stenzel, Prog. Polym. Sci. 2012, 37, 38. [20] C. Boyer, M. H. Stenzel, T. P. Davis, J. Polym. Sci., Part A: Polym. Chem. 2011, 49, 551. [21] J. K. Oh, S. A. Bencherif, K. Matyjaszewski, Polymer 2009, 50, 4407. [22] M. A. M. Oliveira, C. Boyer, M. Nele, J. C. Pinto, P. B. Zetterlund, T. P. Davis, Macromolecules 2011, 44, 7167. [23] P. B. Zetterlund, Y. Kagawa, M. Okubo, Chem. Rev. 2008, 108, 3747. [24] R. H. Utama, M. Drechsler, S. Förster, P. B. Zetterlund, M. H. Stenzel, ACS Macro Lett. 2014, 3, 935. [25] R. H. Utama, Y. Guo, P. B. Zetterlund, M. H. Stenzel, Chem. Commun. 2012, 48, 11103. [26] R. H. Utama, M. H. Stenzel, P. B. Zetterlund, Macromolecules 2013, 46, 2118. [27] A. P. Philipse, Colloid Polym. Sci. 1988, 266, 1174. [28] M. Daoud, J. P. Cotton, J. Phys. France 1982, 43, 531
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Communication
SAXS Analysis of Shell Formation During Nanocapsule Synthesis via Inverse Miniemulsion Periphery RAFT Polymerization Robert H. Utama, Martin Dulle, Stephan Förster,* Martina H. Stenzel,* Per B. Zetterlund* Currently available methods for synthesis of polymeric nanocapsules only offer limited control over the shell thickness, even though it is an important parameter for various applications. Furthermore, suitable methods to critically measure this parameter in a facile way are still nonexistent. Here, lab-scale small-angle X-ray scattering (SAXS) is utilized to in situ measure the evolution of shell thickness during nanocapsule synthesis via inverse miniemulsion periphery reversible addition–fragmentation chain transfer (RAFT) polymerization (IMEPP). The measured shell thickness is consistent with estimates from the commonly used transmission electron microscopy (TEM) technique. Moreover, the individual thicknesses of two concentric shells comprising different polymeric materials (the outer shell formed via IMEPP chain extension of the inner shell) can be determined, thus further demonstrating the versatility of this approach.
1. Introduction The administration of water-soluble therapeutic agents such as proteins to treat various ailments is inhibited by R. H. Utama, Prof. P. B. Zetterlund Centre for Advanced Macromolecular Design School of Chemical Engineering The University of New South Wales Sydney, NSW 2052, Australia E-mail: [email protected] Dr. M. Dulle, Prof. S. Förster Physikalische Chemie I Universität Bayreuth 95447 Bayreuth, Germany E-mail: [email protected] Prof. M. H. Stenzel Centre for Advanced Macromolecular Design School of Chemistry The University of New South Wales Sydney, NSW 2052, Australia E-mail: [email protected] Macromol. Rapid Commun. 2015, 36, 1267−1271 © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
their apparent cytotoxicity and low bioavailability.[1,2] Hollow polymeric nanocapsules, where the drugs can be protected in a water pool, are considered to be suitable candidates to circumvent these issues and therefore the development of loaded nanocapsules has grown very rapidly in recent years. The synthetic pathways to nanocapsules can be divided into three categories: i) self-assembly,[3,4] ii) sacrificial template,[5,6] and iii) miniemulsion polymerization.[7] Significant efforts have been directed towards analysis of various nanoparticle parameters such as size, shape, and surface properties, which affect cellular interactions and penetrations.[8–11] However, one aspect that is frequently overlooked is the shell thickness, despite important characteristics such as shell degradation, diffusion, and release rate of encapsulated molecules being critically controlled by this parameter.[12–14] Only few synthetic pathways allow control of shell thickness. The sacrificial template/layer by layer (LbL) method enables control over the shell thickness by varying the number of layers added.[15,16] Polymerization within the interfacial region of miniemulsion droplets
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has also been reported to be suitable for controlling the shell thickness. For example, interfacial polyaddition was claimed to enable control of the shell thickness by adjustment of the stoichiometry.[17,18] However, the inherent limitations of these techniques lie in the relatively poor precision in obtaining the desired shell thickness and/or the complexity of the process. Controlled/living radical polymerization (CLRP), such as reversible addition–fragmentation chain transfer (RAFT) polymerization, has been used extensively to control the size and morphology of linear and branched polymers.[19,20] It has also been used, in conjunction with miniemulsions, to create nanoparticles suitable for drug delivery.[21–23] We have recently reported a new method, namely inverse miniemulsion periphery polymerization (IMEPP) via RAFT, for synthesis of nanocapsules in one-pot, which readily allows control of the shell thickness.[24–26] The mechanism of shell formation in IMEPP is controlled by RAFT polymerization as described previously.[26] The inverse miniemulsion comprises sodium chloride (lipophobe) in the aqueous dispersed phase and monomer, cross-linker, and initiator in the toluene continuous phase and is stabilized by amphiphilic RAFT block copolymers. This confines the subsequent RAFT polymerization on the periphery of the droplet, the chains growing out towards the continuous phase. There are to date no reported techniques suitable for measuring the thickness of the polymeric shell of nanocapsules in their native state. The most commonly used technique is transmission electron microscopy (TEM), but the results are greatly affected by issues related to the behavior of the polymeric chains as well as collapsing of the nanocapsules upon drying. Although cryo-TEM may overcome such limitations, its application is relatively tedious and time consuming. Here, we report the utilization of smallangle X-ray scattering (SAXS) to perform online investigations on the evolution of the polymeric shells of nanocapsules in their native state, synthesized via RAFT IMEPP.
3. Results and Discussion Two systems with different types of shells were investigated: methyl methacrylate (MMA) homopolymer and diblock copolymer of methyl methacrylate/tert-butyl methacrylate (MMA/tBMA), both utilizing ethylene glycol dimethacrylate (EGDMA) as the cross-linker. The MMA system represents a “Core/1 Shell” morphology whereby the initial stabilizer hydrophobic block (Shell 1, MMA) was chain extended with MMA (Shell 1 + polymerized MMA via IMEPP, Scheme 1). In contrast, the MMA/tBMA system represents a “Core/2 Shells” morphology (Scheme 1) whereby the initial Shell 1 was chain extended to yield a second shell (“Shell 2”) comprising tBMA. SAXS was then used to analyze in situ the growth in shell thickness during the RAFT controlled chain extension polymerization. The SAXS data were fitted to a model of a sphere with two concentric shells of different but spatially uniform contrast. The first step was to determine the size of the aqueous core by fitting the first scattering profile (before polymerization) with a Core/1 Shell model with a very thin shell, the main scattering contrast thus originating from the core. The radius and contrast (with respect to toluene) of the core was then kept constant for determination of the shell thickness (“Shell 1” in Scheme 1) by varying its value and contrast to give the best fitting model for the scattering curve. Once the radius of the aqueous core, the thickness of Shell 1 and their respective contrasts had been determined, these parameters were kept constant for the subsequent fitting to obtain the thickness of the shell after IMEPP (Core/1 Shell and Core/2 Shells; Scheme 1), using the shell thickness of Shell 2 and its contrast as adjustable parameters. As an example, the equation for the case of a single shell is given below: I ( q) =
[sin( qrc ) − qrc cos( qrc )] scale ⎡ ⎢3Vc ( ρ c − ρ s ) ( qrc )3 Vs ⎣ 2
[sin( qrs ) − qrs cos( qrs )] ⎤ + 3Vs ( ρs − ρsolv ) ⎥ + bckg ( qrs )3 ⎦
2. Experimental Section
O
S
O
S O
IMEPP n
o
Ph
O
O
Stabilizer hydrophobic block Shell 1
O
O
MMA EGDMA
Ph
S
Aq. Core
o
O
Polymeric shell
where scale is a scale factor, Vs is the volume of shell, Vc is the volume of the core, rs is the radius of the shell, rc is the radius of the core, ρc is the scattering length density of the
O
O
S
O
Shell 1 IMEPP tBMA EGDMA
Core/1 Shell
O
Shell 2
O
O
S n
O
o
Detailed experimental on the synthesis of the block-copolymer, SAXS data analysis, and all analytical techniques are available on the Supporting Information.
(1)
O
O
O
S
Ph
Core/2 Shells
Scheme 1. Schematic representation of the “Core/1 Shell” and “Core/2 Shells” nanocapsules.
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SAXS Analysis of Shell Formation During Nanocapsule Synthesis via Inverse Miniemulsion Periphery RAFT Polymerization
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core, ρs is the scattering length density of the shell, ρsolv is the scattering length density of the solvent, and bckg is the background scattering. The suitability of SAXS to measure the shell growth was initially examined for a Core/1 Shell-type nanocapsule using the previously established IMEPP system based on poly(N-(2-hydroxypropyl)methacrylamide)b-poly(methyl methacrylate) (PHPMA19-b-PMMA79) as macro-RAFT stabilizer with MMA and EGDMA (Table S1, Supporting Information). A dispersed phase of 10% (weight fraction with respect to the continuous phase) was confirmed to be optimum as it confines any structural factor effects to only the lower q-range, and eliminates any oscillation from interparticle cross-linking. Such a relatively low weight fraction of water was also found to not affect the q-region of interest whereby the shell growth is visible. Normalized scattering curves over the period of polymerization are shown in Figure S4 (Supporting Information). Fitting of a Core/1 Shell model to the scattering curves corresponding to t = 0 and during polymerization resulted in excellent fits (Figure 1A), confirming nanocapsule morphology. Analysis of the scattering curve at t = 0 (Figure S3, Supporting Information) revealed a distinct minimum, which was used to determine the aqueous core radius of 92 nm. The thickness of Shell 1 was successfully determined to be 7.5 nm despite a small difference in contrast between the shell and the toluene background (Table S3, Supporting Information). This gives a total initial droplet diameter of 199 nm (92 + 92 + 7.5 + 7.5), comparable to the DLS values (dn = 169 nm, dv = 253 nm, Table S2, Supporting Information). As the shell progressively grows, the distinctive minimum point was overshadowed by the feature of the growing shell. Subsequent fitting of the normalized scattering curve every 30 min provided information on the shell growth over time. The total integrated scattering intensity (area under the scattering curve) increased and the shape of the scattering curve changed with time due to shell growth. The shell growth was quite significant
for the first few hours before reaching a plateau value (Figure 1B). The overall thickness of the polymeric shell was ≈17 nm after 7 h. The calculated shell thickness value was very similar to the previously published results of ≈20 nm after 7 h of polymerization, measured from the TEM data.[26] The developed SAXS method was further applied to a system with more than one shell material. Analysis of the MMA/tBMA system (EGDMA cross-linker) was conducted to investigate the capability of the developed approach in differentiating concentric shells of polymers having relatively similar electron densities. To illustrate the versatility of the technique, a macro-RAFT diblock copolymer different from that of the Core/1 Shell model above and to the previously reported tBMA polymeric nanocapsules, synthesized via IMEPP,[24] was employed. Here, methyl methacrylamide (MAAm) was used instead of HPMA as the hydrophilic block and MMA as the hydrophobic block (PMAAm16-b-PMMA64). Initially, the normalized scattering curves (Figure S3, Supporting Information) were fitted to the Core/1 Shell model used above for the MMA system, giving an aqueous core radius and Shell 1 thickness of 79.5 and 4.8 nm, respectively, and thus an overall droplet diameter of ≈170 nm in close agreement with DLS data (dn = 174 nm, dv = 243 nm, Table S2, Supporting Information). As the polymerization proceeded, changes in the scattering curves conformed to a Core/2 Shell model (Figure 2A and S5, Supporting Information). Determination of the polydispersity of the growing shell was not attempted, thus leading to the small discrepancy observed on the fitted curve (Figure 2A, square). However, this did not change the position of the form factor maximum, but would only flatten the undulation of the overall form factor. The form factor maximum is a distinctive feature of a Core/ 2 Shell model and its shift into lower q-values as the polymerization progress represents the increase in shell thickness. Therefore, the shell growth can be determined correctly from the fitting.
Figure 1. A) Form factor fitting of the scattering curve with a core–shell model and B) evolution of polymeric shell (the initial value of 7.5 nm corresponds to “Shell 1” (Scheme 1)) thickness over time of the MMA system.
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Figure 2. A) Normalized Core/2 Shell model fits for the nanocapsules for IMEPP of tBMA/EGDMA, curves have been shifted along y-axis for better visibility), B) evolution of shell thickness (Shell 2) over time.
The subsequent determination of the thickness of Shell 2 (and its contrast) as a function of time was conducted using the Core/2 Shell model by keeping constant the values of the core radius, the Shell 1 thickness as well as their respective contrasts (Table S4, Supporting Information). The Shell 2 thickness versus time (Figure 2B) revealed steady growth in the first 4.5 h, reaching a plateau value at 30.6 nm. The initial linear increase of shell thickness with time is in agreement with classical seeded growth kinetics (Figure 2B).[27] Now, if the shell was growing from a flat surface, one would anticipate a linear increase in shell thickness with conversion if the density of the crosslinked shell (Shell 2) is constant throughout the shell region and does not depend on conversion. Figure 3 shows the Shell 2 thickness plotted versus conversion, revealing a close to linear relationship except for the highest con-
version range. Given that the shell growth occurs from a sphere, the surface area gradually increases with conversion (unlike in case of a flat surface). As a consequence, a given conversion increment would lead to progressively smaller increases in shell thickness assuming the shell density is constant. To correct for the effect of increasing surface area on shell growth, the experimental data were “corrected” so as to correspond to the initial droplet surface area before polymerization as a flat surface (see Supporting Information for details). The resulting plot deviates significantly upwards from linearity, indicating that as the shell thickness increases, a given conversion increment leads to a progressively greater increase in thickness for the corresponding hypothetical flat surface. This is consistent with the density of the polymer network decreasing with increasing conversion, well in agreement with spherical polymer brush models where a decreasing segment density with increasing radius is expected.[28] The shell contrasts with respect to the continuous phase (Table S5, Supporting Information) decreased with time for Shell 2, eventually reaching a value lower than for Shell 1, consistent with the density of Shell 2 decreasing with increasing shell growth.
4. Conclusion
Figure 3. Shell (Shell 2) thickness versus conversion obtained by SAXS (solid squares; full line is guide to the eye only) for the tBMA/MMA system. The broken line shows predicted shell thickness from a flat surface corresponding to the initial droplet surface area (see text and Supporting Information for details).
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By using an appropriate and simple SAXS model to fit the data, we have presented an efficient way to monitor the controlled creation of polymeric shells of nanocapsules using SAXS. The results confirm the suitability of IMEPP to synthesize nanocapsules with excellent control over the shell and the suitability of lab-scale SAXS as a tool to monitor the shell growth. This capability to accurately control the shell thickness is extremely useful in designing polymeric nanocapsules whereby the release of the loads is controlled by the degradation of the shell overtime and/ or the porosity of the shells.
Macromol. Rapid Commun. 2015, 36, 1267−1271 © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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SAXS Analysis of Shell Formation During Nanocapsule Synthesis via Inverse Miniemulsion Periphery RAFT Polymerization
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Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: R.H.U. and M.D. contributed equally. R.H.U thank the UNSW student exchange office for a Student Exchange Scholarship. Note: Stephan Förster and Martina H. Stenzel were added as corresponding authors on April 22, 2015. Received: February 12, 2015; Revised: March 18, 2015; Published online: April 17, 2015; DOI: 10.1002/marc.201500096 Keywords: IMEPP; inverse miniemulsion; nanocapsules; RAFT polymerization; small-angle X-ray scattering [1] K. Cho, X. Wang, S. Nie, Z. Chen, D. M. Shin, Clin. Cancer Res. 2008, 14, 1310. [2] D. J. Stewart, Crit. Rev. Oncol./ Hematol. 2007, 63, 12. [3] A. Blanazs, S. P. Armes, A. J. Ryan, Macromol. Rapid Commun. 2009, 30, 267. [4] A. Rösler, G. W. M. Vandermeulen, H.-A. Klok, Adv. Drug Delivery Rev. 2012, 64, 270. [5] A. P. R. Johnston, C. Cortez, A. S. Angelatos, F. Caruso, Curr. Opin. Colloid Interface Sci. 2006, 11, 203. [6] Y. Wang, V. Bansal, A. N. Zelikin, F. Caruso, Nano Lett. 2008, 8, 1741. [7] K. Landfester, A. Musyanovych, V. Mailänder, J. Polym. Sci., Polym. Chem. 2010, 48, 493. [8] C. E. Ashley, E. C. Carnes, G. K. Phillips, D. Padilla, P. N. Durfee, P. A. Brown, T. N. Hanna, J. Liu, B. Phillips, M. B. Carter, N. J. Carroll, X. Jiang, D. R. Dunphy, C. L. Willman, D. N. Petsev, D. G. Evans, A. N. Parikh, B. Chackerian, W. Wharton, D. S. Peabody, C. J. Brinker, Nat. Mater. 2011, 10, 389.
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