Shape Recovery of Spherical Hydrogen-Bonded Multilayer Capsules

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Shape recovery of spherical hydrogen-bonded multilayer capsules after osmotically induced deformation Nirzari Gupta, Veronika Kozlovskaya, Maksim Dolmat, and Eugenia Kharlampieva Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b01795 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on July 30, 2019

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Shape recovery of spherical hydrogen-bonded multilayer capsules after osmotically induced deformation Nirzari Gupta, 1,& Veronika Kozlovskaya, 1,& Maksim Dolmat,1 and Eugenia Kharlampieva1,2* 1

Department of Chemistry, 2Center for Nanomaterials and Biointegration, the University of

Alabama at Birmingham, Birmingham, AL, USA, 35294 Abstract The mechanical properties of microparticles intended for in vivo applications as drug delivery vehicles are among important parameters, which influence their circulation in the blood and govern particle biodistribution. We report on synthesis of soft but mechanically robust spherical capsules via hydrogen-bonded multilayer assembly of (poly(N-vinylpyrrolidone), Mw = 10 000 g mol-1) with (poly(methacrylic acid) Mw = 100 000 g mol-1)) (PVPON/PMAA)n in methanol using nonporous silica microparticles of 4 µm as sacrificial templates, where n = 5, 10 and represents bilayer number. The mechanical properties of (PVPON/PMAA)n spherical capsules was assessed using the osmotic pressure difference method and resulted in the elasticity modulus of 97 ± 8 MPa, which is in the range of Young’s modulus for elastomeric networks. We also found that hydrogen-bonded (PVPON/PMAA)10 capsules demonstrated almost a complete recovery from a concave buckled inward shape induced by the osmotic pressure difference from addition of polystyrene sulfonate (PSS) to the capsule solution to their initial spherical shape within 12 hours after the PSS solution was rinsed off. The permeability measurements through the capsule shell using fluorescently labeled dextran molecular probes revealed that the average mesh size of the hydrogen-bonded network assembled in methanol is in the range between 3 and 9 nm and is not permeable to the FITC-dextran with molecular weight above 40 000 g mol-1. Our study shows that physicallycrosslinked polyelectrolyte multilayer capsules are capable of withstanding large deformations, which is essential for the development of adaptable particles for controlled delivery.

Keywords: hydrogen-bonded, multilayer capsules, poly(N-vinylpyrrolidone), poly(methacrylic acid), permeability, osmotic pressure, buckling

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Introduction The mechanical stiffness of microparticles intended for in vivo applications is an important parameter which influences their circulation in the blood and governs particle biodistribution.1,2,3 Rigidity/elasticity is among the main advantages of polymeric hydrogels allowing for facile regulation of their biological activity.3 Thus, for instance, decreasing the elastic modulus of polyethylene glycol (PEG) nanogels from 3000 to 10 kPa increased their in vivo circulation up to 2 hours.4 Lowering the elastic modulus of PEG diacrylate-crosslinked poly(2-hydroxyethyl acrylaye) hydrogel microparticles 8-fold led to a 30-fold increase in the circulation half-life.5 The elasticity can also regulate particle association with cancer cells and improve the accumulation in targeted sites.4,6 For example, softer nanoliposomes with a Young’s modulus of 45 kPa were shown to be 2.6-fold more efficient in accumulating in 4T1 tumors compared to more rigid particles with a Young’s modulus of 19 MPa.7 Therefore, a better understanding of the factors governing the particle elasticity is crucial in developing viable nano- and microparticle drug delivery vehicles. Unlike continuous particulates, hollow microparticles, known as capsules and made by the layer-by-layer (LbL) assembly of water-soluble polyelectrolytes on sacrificial particulate templates (cores), can demonstrate a greater structural flexibility. The multilayer approach offers control over capsule size, geometry, composition and thickness at the nanoscale.8-14 For instance, 7-µm discoidal capsules of poly(allylamine hydrochloride) (PAH) crosslinked with glutaraldehyde demonstrated the elasticity modulus in the order of hundreds MPa which allowed them to squeeze through a 5-μm glass capillary tip and recover their original shape.15 The elasticity modulus of PRINT colloids needed to be much less to ensure that passage.4 This difference is due to the fact that the hollow capsule interior resembles a red blood cell with fluid wrapped by a flexible cell membrane,16 which facilitates better shape deformation and recovery via fluid-like deformation of the capsule membrane compared to continuous hydrogels which cannot facilitate such dramatic displacements.17 After the core dissolution, the capsule wall is subject to the thermodynamic force of mixing which favors capsule shell swelling due to the increased osmotic pressure while the elastic retractive force of the network opposes swelling.18 The critical pressure difference and the corresponding shell stiffness control the capsule deformation and are directly proportional to the shell thickness and inversely proportional to the capsule size.19

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As shown earlier, in osmotically buckled capsules, the solvent inside the capsules (buffer solution) diffuses through the capsule shell, which has a selective permeability towards macromolecules due to osmolyte concentration gradient between the capsule interior and exterior causing a decrease in the interior pressure.19 The capsule shell can lose shape stability and transform from a convex shape to a concave inward buckled shape when the work done by the external pressure equals the deformation energy; from which the critical pressure for the onset of capsule buckling can be obtained.19-22 Gao et al. tested the buckling of elastic spherical capsules under osmotic pressure induced by polystyrene sulfonate, sodium salt (PSS), finding that the buckling instability of spherical ionic polyelectrolyte microcapsules is due to the isotropic osmotic pressure difference between inside and outside of the multilayer shell induced by surrounding PSS molecules (the osmotic pressure difference method).19

Thus, spherical ionically-paired

(PSS/PAH) multilayer capsules irreversibly deformed (buckled) in response to isotropic osmotic pressure from added PSS macromolecules.19 The shape loss due to osmotic buckling observed previously is generally irreversible. We also showed earlier that random irreversible buckling of initially spherical hydrogen-bonded (PVPON/tannic acid)n shells can be induced by drying leading to irreversible formation of hemispherical concave shells.23 The capsule deformability and robustness has been recently demonstrated crucial for capsule cell internalization as various cells can generate different mechanical force during particle uptake which can lead to different capsules deformations within the cell interior.24 For example, the strongest capsule deformation occurred in HeLa cells when the (PMAA) hydrogel capsules were highly deformed and lost their initial shape during uptake by the cells, while a large amount of the capsules could maintain their shape after the cell internalization by differentiated human monocyte-derived macrophages.24 Unlike ionically-paired multilayer capsules, mechanical properties of hydrogen-bonded capsules have been rarely explored. Yet, hydrogen-bonded multilayers offer unique properties such as pH- and temperature responsiveness to mild environmental changes, biocompatibility, biodegradability, and including a variety of biologically active molecules.

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In one study,

Elsner et al found that Young’s modulus of hydrogen-bonded (PVPON/PMAA) multilayer capsules deposited from aqueous solutions was 610 ± 70 MPa at acidic pH using small deformation by atomic force microscopy (AFM) colloidal tip.31 However the AFM colloidal tip method does not allow for probing large deformations. 3 ACS Paragon Plus Environment

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In this work, we report a facile approach for synthesis of soft and elastic multilayer capsules capable of recovering their shape from a concave buckled inward shape induced by an osmotic pressure to their initial spherical shape after the pressure is removed. Spherical (PVPON/PMAA)n capsules are synthesized by multilayer assembly of the polymers from methanol solutions onto surfaces of 4-µm non-porous silica particles. The larger micrometer particles are used due to easiness of observation of the capsule shape deformation behavior using confocal microscopy. The reported procedure allows for making the multilayer particles of a smaller sub-micrometer size to be used for biologically relevant applications.32 We have recently demonstrated that 3micrometer size capsules of (tannic acid/PVPON) can be successfully applied in biomedical field for in vivo delivery and imaging applications.33,34 Herein, we adopt osmotic pressure difference approach to investigate mechanical properties of these capsules using PSS sodium salt to induce osmotic pressure difference in capsule solutions at pH = 3. This approach allows to investigate dimensional and shape changes of capsules induced in response to increasing isotropic osmotic pressure (shrinkage and buckling instability). 19,20,22 In addition, utilizing organic solvents for multilayer assembly gives an opportunity to control multilayer thickness and morphology as well as to incorporate water-insoluble polymers in the capsule wall. 35,36,37 Spectroscopic ellipsometry and ATR-FTIR spectroscopy are employed to study the multilayer growth and pH-stability of the capsules. Capsule morphology and dry thickness are explored using AFM. The permeability of the capsules obtained in methanol is studied at pH = 3 using fluorescently labeled FITC-dextrans as molecular probes of varied molecular weights and confocal laser scanning microscopy (CLSM). We demonstrate that these hydrogen-bonded capsules assembled in methanol have sufficient elasticity and softness to recover their spherical shape after large deformations when the stress is removed. To the best of our knowledge, this is a first example of shape recovery observed for multilayer physically crosslinked polyelectrolyte capsules after large deformations.

We believe that our study brings new

knowledge about controlling mechanical stiffness of hydrogen-bonded multilayers and polyelectrolyte capsules. This work might be essential for the development of soft and adaptable particles that can be tailored for various intended purposes.

EXPERIMENTAL SECTION

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Materials. Poly(styrene sulfonate) sodium salt (PSS, Mw = 70 000 g mol-1), poly(Nvinylpyrrolidone) (PVPON, Mw = 10 000 g mol-1), poly(methacrylic acid) (PMAA, Mw = 100 000 g mol-1), Na2HPO4 and NaH2PO4 phosphate buffers, and fluorescein isothiocyanate (FITC)-labeled dextrans with average molecular weights of 4 000, 40 000, 150 000, and 500 000 g mol-1 were purchased from Sigma-Aldrich. Non-porous silica microspheres of 3.9 ± 0.2 µm were purchased from Cospheric LLC. Alexa Fluor 568 carboxylic acid succinimidyl ester was purchased from Invitrogen. Ultrapure deionized (DI) water (18.2 Ω cm) was used in all experiments (Evoqua). Methanol was purchased from Fisher Scientific and used as received. Multilayer assembly of hydrogen-bonded (PVPON/PMAA) capsules. Spherical hydrogenbonded capsules of capsules of (PVPON/PMAA) were assembled on the surfaces of 4-µm nonporous silica microspheres by alternate exposure of the silica cores to 0.5 mg mL-1 methanol solutions of PVPON and PMAA for 10 minutes each with two rinses with methanol after each deposited polymer layer to remove excess of non-adsorbed polymer using centrifugation. For that, the particle suspension was centrifuged at 7000 rpm for 45 s followed by supernatant removal using a single use plastic pipette (Fisher Scientific) and particle re-suspension in methanol using a sonication bath (Branson Ultrasonic bath; 2 min). After a (PVPON/PMAA)n multilayer coating (n = 5, 10) was assembled, hollow (PVPON/PMAA)n capsules were obtained via silica core dissolution in 8%wt hydrofluoric acid for 12 hours. The capsules were purified by dialysis in DI water (pH = 3, adjusted by 0.1 M HCl) for 3 days using a Float-A-lyzer (MWCO = 20 kDa; Spectrum Labs). Growth of (PMAA/PVPON)n multilayers. The growth of (PVPON/PMAA)n multilayers on Si wafers (University Wafer) in methanol was assessed with spectroscopic ellipsometry (J.A. Woollam M2000U). The wafers were cleaned as described previously38 and the PVPON and PMAA polymers were adsorbed onto the wafer surfaces similarly as described for the (PVPON/PMAA)n capsule assembly. The dry thickness measurements were performed in the range of 400-1000 nm at 65°, 70°, and 75° angles of incidence. For data fitting, a multilayer model composed of silicon, silicon oxide, and the Cauchy film was used, and the ellipsometric angles, Ψ and Δ, were fitted using the Cauchy approximation with a wavelength-dependent refractive index as n (λ) = An + Bn/λ2 + Cn/λ4, with An = 1.5, and Bn and Cn as fitted parameters (Figure S1). The SiO2 thickness was measured first and the thickness of the dry (PVPON/PMAA)n film was obtained by fitting the data. The mean-squared error for data fitting was less than 30. The film 5 ACS Paragon Plus Environment

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hydration measurements were done using a 5-mL liquid cell. The cell was filled with 0.01 M phosphate buffer solution at pH = 3 values and measurements were taken at a 70° angle after 20 minutes of equilibration. The thickness of the hydrated multilayer film was obtained by fitting An, Bn, and Cn (Figure S1 in Supporting Information). The pH-dependent stability of the coating was studied by exposure of the film to 0.01 M phosphate buffer solution with a certain pH value for 15 minutes, followed by film drying with a gentle stream of nitrogen (Airgas) and dry thickness measurement. Characterization. For scanning electron microscopy (SEM) analysis, 5 µL of silica particle or capsule suspension was dropped onto a clean chip of Si wafer, dried in Petri dish, sputter-coated with a thin 2-nm layer of silver (Denton Vacuum) and imaged at 10 kV. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) spectra of (PVPON/PMAA)5 hydrogen-bonded capsules freeze-dried from aqueous solution at pH = 3 were collected using a Bruker Alpha ATR-FTIR spectrometer. Surface charge measurements of (PVPON/PMAA)5 capsules were carried out using Nano Zetasizer (Malvern). For that, the capsule suspension was centrifuged and the solution was exchanged for 0.01 M phosphate buffer at pH = 3, followed by capsule re-dispersion by vortexing. An average ζ-potential value ± standard deviation was obtained by averaging three independent zeta-potential measurements. Permeability of hydrogen-bonded (PVPON/PMAA) capsules to FITC-dextrans and osmotic pressure-induced deformation (buckling) of the capsules were visualized using Nikon A1R multiphoton confocal laser scanning microscope (CLSM) equipped with a 60x oil immersion objective. The capsules (30 µL; 1.8 x 108 capsules mL-1) were mixed with 450 µL of 2 mg mL-1 FITC-dextran solution (0.01 M pH = 3) in a well of chambered coverglass (Lab-Tek) for 15 minutes and then imaged. The capsules were considered non-permeable when the ratio r = Ii/Ie where Ii and Ie are the fluorescence intensities from the capsule interior and the exterior dextran solution, respectively, was r < 0.5. A percentage of non-permeable capsules (% NP) was calculated as %NP = [(NP)/(NP + P)] x 100, where NP and P are the numbers of non-permeable and permeable capsules, respectively. In total, 200-300 microcapsules were analyzed using ImageJ at two separate areas from CLSM images for each FITC dextran. For CLSM visualization of capsule buckling in the presence of PSS, the fluorescently labelled Alexa-Fluor 568 PMAA39 (Mw = 100 000 g mol-1) was used to prepare (PVPON/PMAA)5 or (PVPON/PMAA)10 capsules. The capsules (25 µL; 1.8 x 108 capsules mL-1) were mixed with a 6 ACS Paragon Plus Environment

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450 µL of PSS solution at a desired PSS concentration (wt%) and imaged. The number of deformed and intact (PVPON/PMAA) capsules was determined by multi-point analysis of the CLSM images using the ImageJ software. In total, 100 capsules at two separate areas in two CLSM images were counted for each PSS concentration. Any indentation on the capsules was considered as deformation and the percentage of deformed capsules (% D) was calculated as % D = [(D)/(D + I)] x 100, where D and I are the numbers of deformed and intact capsules, respectively. Shape recovery of capsule buckling was studied after the PSS solution was removed and replaced with 0.01 M pH = 3 phosphate buffer solution six times in the CLSM chambered coverglass. CLSM images of the capsules were collected 12 hours after the solution replacement and analyzed as described above. Atomic Force Microscopy. The capsule shell thickness was obtained from the topography atomic force microscopy (AFM) images of capsules dried on Si wafer chips (0.5 cm x 0.5 cm) using AFM Multimode 8 (Bruker). The half of the height of the collapsed flat regions of capsules was measured using capsule topography section profiles40 using NanoScope Analysis v150 software (Figure S2 in Supporting Information). The AFM silicon cantilevers with the tip radius of