Multilayer Hydrogel Capsules of Interpenetrated Network for

Subscriber access provided by UNIV OF WESTERN ONTARIO

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

Multilayer Hydrogel Capsules of Interpenetrated Network for Encapsulation of Small Molecules Veronika Kozlovskaya, Jun Chen, Oleksandra Zavgorodnya, Mohammad B Hasan, and Eugenia Kharlampieva Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02465 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 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

Multilayer Hydrogel Capsules of Interpenetrated Network for Encapsulation of Small Molecules Veronika Kozlovskaya,1,& Jun Chen,1,& Oleksandra Zavgorodnya,1 Mohammad B. Hasan,1 Eugenia Kharlampieva1,2* 1

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

Alabama at Birmingham, Birmingham, AL, USA Abstract We report on a facile capsule-based platform for efficient encapsulation of a broad spectrum of hydrophilic compounds with molecular weight less than 1,000 g mol-1.

The encapsulated

compounds extend from low-molecular weight anionic Alexa Fluor 532 dye and cationic anticancer drug doxorubicin to FITC-dextrans with Mw ranging from 4,000 to 40,000 g mol-1. The pH-sensitive hydrogel capsules with interpenetrated network shell are synthesized by layerby-layer assembly of poly(methacrylic acid) (PMAA, Mw = 150,000 g mol-1) and poly(Nvinylpyrrolidone) (PVPON, Mw = 1,300,000 g mol-1) on 5 µm silica microparticles followed by chemical crosslinking of the PMAA multilayers. Following core dissolution, the result is a hollow microcapsule with PVPON interpenetrated in the PMAA network. The capsules exhibit reversible change in dimeter with a swelling ratio of 1.5 upon pH variation from 7.5 to 5.5. Capsules crosslinked for 4 hours display high permeability toward molecules with molecular weight under 1,000 g mol-1 at pH = 7.5 but exclude dextran molecules with Mw ≥ 40,000 g mol1

. Encapsulation of small molecules was achieved at pH = 7.5 followed by sealing the capsule

wall with 40,000 g mol-1 dextran at pH = 5.5. This approach results in negatively charged molecules such as Alexa Fluor being entrapped within the capsule cavity, while positively charged molecules such as DOX are encapsulated within the negatively charged capsule shell. Considering the simple post-loading approach, ability to entrap both anionic and cationic small molecules, and the pH-responsiveness of the interpenetrated network in the physiologically relevant range, these capsules offer a versatile method for controlled delivery of multiple hydrophilic compounds.

1 ACS Paragon Plus Environment

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

Keywords: multilayer hydrogel, microcapsules, poly(N-vinylpyrrolidone), poly(methacrylic acid), encapsulation, permeability Introduction Free drug administration is usually associated with many challenges including poor drug solubility, rapid clearance from the blood stream, and suboptimal bio-distribution.1,2 A wide variety of drug delivery carriers made of synthetic and biological macromolecules have therefore been developed to increase drug efficacy via increasing solubility, offering protection from degradation, enhancing bioavailability, and minimizing side effects. 1,3 Layer-by-layer (LbL) microcapsules composed of a nanothin polymer wall (< 50 nm) enclosing a micrometer size cavity have emerged as promising drug-delivery carriers due to their high loading capacity and easily adjustable chemical composition and precisely controlled properties.4,5,6,7,8 These hollow particles are synthesized through alternating assembly of watersoluble polymers on sacrificial templates followed by dissolution of the sacrificial cores.9 In contrast to traditional nanoscale drug delivery vehicles such as liposomes, 10 polymersomes, 11 micelles,12 and nanoparticles,13 multilayer microcapsules have high loading capacity and easily functionizable surfaces.

14

Equally important, the chemical and physical properties of the

microcapsules can be finely tuned by choosing appropriate polymers, deposition conditions, and by the nature of sacrificial cores, which allows for controlled delivery of therapeutics in response to various stimuli.5,4,9,15,16 Typically, therapeutic molecules can be encapsulated into multilayer microcapsules through pre-loading or post-loading of the therapeutic cargo.17 In the pre-loading process, molecules are incorporated into inorganic sacrificial nano- or microparticle templates followed by capsule fabrication,18 while post-loading is based on free diffusion of molecules into preformed capsules by external or internal stimuli-controlled variation of the shell permeability.19,20 The pre-loading method provides high loading capacity, but the cargo can be exposed to harsh conditions during template dissolution including hydrochloric or hydrofluoric acids.21 The use of porous calcium carbonate particles as templates allows milder dissolution conditions in the physiologically relevant pH range using ethylene diamine tetraacetic acid (EDTA).20 The post-loading strategy categorically eliminates exposure of the cargo to the chemical species intrinsic to template dissolution and provides a cleaner route for drug entrapment. Along with temperature, ionic strength and solvent exchange, pH changes offer a fast and efficient way to load cargo via 2 ACS Paragon Plus Environment

Page 2 of 33

Page 3 of 33 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

controlling the permeability of the capsule shell.5,9,22 In contrast to the abovementioned stimuli, pH change is favorable for encapsulating sensitive biomolecules; methods like temperature and solvent exchange can trigger protein denaturation.58 Furthermore, post-loading based on solvent exchange and salt changes is limited to encapsulation of large molecules, and the encapsulation efficiency is low.23 Therefore, developing a simple, versatile post-loading method for multilayer capsules is a crucial step toward realization of their potential as universal carriers. pH-Responsive and biodegradable multilayer hydrogel capsules made of poly(methacrylic acid) (PMAA) were previously reported to have a pKa value around 6 which was shown to be useful for controlled drug delivery.24,25,26,27,28 The pH-triggered swelling of these capsules due to ionization of carboxylic groups was employed to control the permeability of the capsule wall to hydrophilic dextran molecules of various molecular weights via changing the mesh size.29 Although some reports have presented PMAA hydrogel capsules as carriers for macromolecules, the capsules were not applicable for encapsulation of small molecular weight therapeutics due to their high permeability at physiological pH. 7,24 Decreasing the mesh size by chemically

co-crosslinking

poly(N-vinylpyrrolidone)-co-(aminopropyl)methacrylamide

(PVPON-NH2) copolymers within the shell was reported to decrease the permeability of (PMAA-PVPON) hydrogel capsules.29 For instance, while (PMAA)7 hydrogel capsules were found to be impermeable only to FITC-dextran with Mw ≥ 500,000 g mol-1, (PMAA-PVPON)7 hydrogel capsules were able to exclude 70,000 g mol-1 FITC-dextran at pH > 6.524,29 However, that approach requires synthesis of PVPON-NH2 copolymers with specifically tailored amine group molar ratios to precisely regulate the mesh size of the capsule shell and does not support entrapment of small molecular weight species at pH = 7.4.29,30 Alternatively, the permeability of hydrogel capsules to macromolecules can be decreased by sealing the capsule shell through interaction with various macromolecules. For example, deposition of FITC-dextran on poly(glycidyl methacrylate)/poly(allylamine hydrochloride) (PGMA/PAH)4 multilayer capsules decreased the permeability towards 4,000 g mol-1 FITCdextran. 31 , 32 Likewise, complexation of the (PMAA)7 hydrogel capsules with quaternized poly(N-vinylpyridine) resulted in the exclusion of 500,000 g mol-1 FITC-dextran for two weeks, while the unlocked capsules became permeable to the dextran after 40 min.24 Although the aforementioned studies showed a significant decrease in FITC-dextran permeation through the capsule walls, the entrapment of molecules with Mw < 4,000 g mol-1 remains a challenge. 3 ACS Paragon Plus Environment

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 4 of 33

Herein, we report on a facile approach to efficiently encapsulate a broad spectrum of hydrophilic compounds including those with Mw < 1,000 g mol-1 by developing pH-sensitive capsules with interpenetrated hydrogel network walls.

We show encapsulation of

macromolecules including FITC-dextrans ranging from 4,000 to 40,000 g mol-1 and the charged small molecules Alexa Fluor 532 carboxylate and doxorubicin HCl.

The interpenetrated

hydrogel capsules are synthesized via multilayer assembly of PMAA (Mw = 150,000 g mol-1) and PVPON (Mw = 1,300,000 g mol-1) on porous and solid silica microparticles (< 5 µm in diameter), followed by crosslinking of the PMAA multilayers with ethylenediamine (EDA) which results in physical entrapment of PVPON layers within the PMAA network. PVPON is biocompatible33,34 and, similar to poly(ethylene glycol), has been shown to prevent protein adsorption due to its highly hydrophilic nature.35,36 Encapsulation of low molecular weight hydrophilic compounds is studied by combining pH-induced post-loading and dextran-based resealing of the hydrogel capsule shell. Considering the simple post-loading approach, ability to entrap both anionic and cationic small molecules, and the pH-responsiveness of the interpenetrated network in the physiologically relevant range, these capsules offer a versatile method for controlled delivery of multiple hydrophilic compounds.

EXPERMENTAL SECTION Materials. Poly(methacrylic acid) (PMAA, average Mw = 150,000 g mol-1), poly(Nvinylpyrrolidone) (PVPON; average Mw = 1,300,000 g mol-1), poly(ethylene imine) (PEI, average Mw = 70,000 g mol-1), fluorescein isothiocyanate (FITC)-labeled dextrans (average Mw = 40,000; 20,000; and 4,000 g mol-1), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC), NaH2PO4 and Na2HPO4, ethylenediamine (EDA), 46 wt% hydrofluoric acid (HF), ethylenediaminetetraacetic acid (EDTA) and poly(glycidyl methacrylate) (PGMA) were purchased from Sigma-Aldrich. Alexa Fluor 532 carboxylic acid (MW = 723.8 g mol-1) was obtained from Invitrogen. Porous 5 µm silica microspheres were from Restek Corp. Doxorubicin hydrochloride (DOX) was from LC Laboratories. Solid silica microparticles of 2.0 ± 0.2 µm were from Polysciences Inc.

Fabrication of PMAA/PVPON Hydrogel Films. Prior to fabrication, Si wafers (University Wafer) were cleaned by exposure to UV light (185 and 254 nm) for 6 hours using a Novascan 4 ACS Paragon Plus Environment

Page 5 of 33 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

UV ozone cleaner, followed by thorough rinsing with water and immersion in Nochromix (Godax Laboratories, Inc) with concentrated sulfuric acid according to the manufacture’s protocol. After 12 hours, wafers were thoroughly washed with DI water, dried with a stream of nitrogen (Airgas) and immediately used for coating. The thicknesses of the silicon oxide and polymer coatings on the silicon surface were measured using spectroscopic ellipsometry (M2000U, J.A. Woollam). The layer of PGMA was spin-cast from a 0.1 mg mL−1 chloroform solution onto the Si wafer surfaces (3000 rpm, 1 min) and covalently anchored to the surface by heating at 110 °C for 1 h, followed by chloroform rinses to remove unreacted PGMA. After that, a layer of PEI was spin-cast from a 0.5 mg mL−1 isopropanol solution (3000 rpm, 1 min) and cross-linked to PGMA by heating at 70 °C for 4 h. Uncross-linked PEI was rinsed off in water. The PGMA-PEI primed wafer was used to build up a hydrogen-bonded (PMAA/PVPON) multilayer by alternatingly exposing the wafer to 0.5 mg mL−1 PMAA and PVPON solutions (0.01 M phosphate buffer) at pH = 3.5 for 10 min for each polymer deposition with two rinsing steps (0.01 M phosphate buffer, pH = 3.5) in between. The (PMAA/PVPON)n multilayers with a varied number of (PMAA/PVPON) bilayers (denoted as a subscript ‘n’) were obtained and chemically cross-linked by exposure to a 5 mg mL-1 solution of EDC (pH = 5) for 30 min, followed by the immersion to a 5 mg mL-1 EDA solution (pH = 5.5) for various cross-linking time points. The cross-linked (PMAA/PVPON)n hydrogel coatings were rinsed with a 0.01 M phosphate buffer solution (pH = 4.6) and exposed to a solution with pH = 8 (0.01 M phosphate) for varied time periods for hydrogel stability studies. Before ellipsometry measurements on dry films, all hydrogel coatings were left to shrink in 0.01 M phosphate solution at pH = 4.6 for 15 min, dried with nitrogen, and analyzed.

Preparation of PMAA/PVPON Hydrogel Capsules. For the capsule synthesis, the (PMAA/PVPON)14 multilayer was adsorbed on PEI-coated porous silica cores (35 mg) from 0.5 mg mL−1 polymer solutions at pH = 3.5 (0.01 M phosphate) for 15 min. After each polymer deposition for 10 min, the particles were separated from the polymer solution by centrifugation and rinsed two times with a 0.01 M buffer solution (pH = 3.5) for 5-min rinse for each cycle. The (PMAA/PVPON)14 multilayer was cross-linked for 4 and 24 hours as described for the (PMAA/PVPON) hydrogel on Si wafers. After cross-linking, the core-shells were rinsed 4 times with a solution at pH= 4.6 (0.01 M phosphate) and left in 0.01 M phosphate buffer at pH = 8 for 5 ACS Paragon Plus Environment

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

24 h. To obtain hollow (PMAA/PVPON)14 hydrogel capsules, the silica cores were dissolved in aqueous 8% HF and purified by dialysis in DI water using a 1-mL Float-A-Lyzer (MWCO 20 kDa, Spectrum Laboratories). The capsules cross-linked for 4 or 24 h are denoted as 4H(PMAA/PVPON)14 and 24H-(PMAA/PVPON)14, respectively. For the capsule stability study, the (PMAA/PVPON)14 hydrogel capsules were incubated in a 0.01 M phosphate buffer solution at pH = 8, followed by dialysis in DI water.

The same protocol was used to synthesize

(PMAA/PVPON)14 capsules using non-porous 2-µm spherical silica particles as sacrificial templates.

Ellipsometry. Hydrogel film thickness was measured using a M-2000U spectroscopic ellipsometer (J.A. Woollam) between 400 and 1000 nm at 65, 70, and 75° angles of incidence. 37,38 For data interpretation, the ellipsometric angles, Ψ and ∆, were fitted using a multilayer model composed of silicon, silicon oxide and the multilayer film to obtain the thickness of films. The thickness of SiO2 was measured and determined using known optical constants. The thickness of the films were obtained by fitting data with the Cauchy Film approximation with the refractive index as n(λ) = An + Bn/λ2 + Cn/ λ4, with An = 1.5, Bn = 0.01, and Cn = 1.3 × 10−5. The mean squared error for data fitting was less than 50. Fourier Transform Infrared Spectroscopy (FITR). FTIR was used to analyze the chemical composition of (PMAA/PVPON)14 hydrogel capsules after various crosslinking time points. For that, (PMAA/PVPON)14 hydrogel capsule solutions were freeze-dried (Labconco) and the powder was analyzed using Alpha-FTIR (Bruker).

ζ-Potential Measurements. ζ-Potential measurements on (PMAA/PVPON)14 capsules were done using a Nano Zetasizer (Malvern). Before measurements, the pH value of (PMAA/PVPON)14 hydrogel capsule solution (1x106 capsules mL-1) was adjusted using 0.1 M HCl or NaOH solutions. Average ζ-potential values (±standard deviation) were obtained by averaging three independent measurements.

Confocal laser scanning microscopy (CLSM). The CLSM imaging of the capsule in aqueous solutions was carried out using a Nikon A1R+ confocal laser microscope equipped with a 60× oil 6 ACS Paragon Plus Environment

Page 6 of 33

Page 7 of 33 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

immersion objective using FITC (green) or TRITC (red) filter sets.

For capsule size

measurements, 50 µL of 4H-(PMAA/PVPON)14 or 24H-(PMAA/PVPON)14 capsules were added to an 8-well Lab-Tek chamber (Electron Microscopy Sciences), which was filled with 0.01 M phosphate buffer solutions with varied pH values and capsule images were collected after 15 min of incubation. For pH-dependent permeability studies, 50 µL of 4H-(PMAA/PVPON)14 or 24H(PMAA/PVPON)14 capsule solutions were added to the 8-well Lab-Tek chamberglass, which was filled with 0.01 M phosphate buffer solution at pH = 7.5 or pH = 5.5 (15-min equilibration time) and 150 µL of 1 mg mL-1 FITC-dextran solution (Mw = 4,000; 20,000; or 40,000 g mol-1) was added to a chamber. The CLSM images were collected after 15-min incubation of the capsules with a FITC-dextran solution.

Encapsulation of Molecules in (PMAA/PVPON) Hydrogel Capsules. For encapsulation of 20,000 or 4,000 g mol-1 FITC-dextran, 50 µL of 4H-(PMAA/PVPON)14 capsules (0.01 M phosphate buffer, pH = 7.5) were soaked with 150 µL of 1 mg mL-1 solutions of the corresponding FITC-dextran solution for 15 min at pH = 7.5. After that, the solution pH was decreased to pH = 5.5 for 15-min, followed by addition of 150 µL of 1 mg mL-1 solution of 40,000 g mol-1 FITC-dextran (15-min incubation, pH = 5.5). Finally, the capsules were rinsed with 0.01 M buffer solution at pH = 7.5 three times with 5-min cycle for each rinse. Encapsulation of anionic Alexa Fluor 532 carboxylic acid fluorescent dye was carried out in a similar manner by exposing the capsules first to the dye solution (1 mg mL-1, pH = 7.5, 15 min), followed by rinsing with the 0.01 M phosphate buffer solution at pH = 5.5 (15-min incubation) and addition of 150 µL of 40,000 g mol-1 FITC-dextran solution (pH = 5.5). Finally, the capsules were rinsed with 0.01 M buffer solution at pH = 7.5. The CLSM imaging was performed after each incubation step. For encapsulation of cationic DOX, the 4H-(PMAA/PVPON)14 hydrogel capsules (300 µL of 4H-(PMAA/PVPON)14 capsule solution with 1 x 106 capsules per mL, pH = 7.5, 0.01 M phosphate buffer) were loaded with 4,000 g mol-1 FITC-dextran as described above and sealed with 40,000 g mol-1 FITC-dextran (150 µL, 1 mg mL-1, pH = 5.5; 24-h incubation). After 24 hincubation, the solution pH was increased to pH = 7.5, 200 µL of 1 mg mL-1 DOX was added and the solution was gently shaken for 24 h (500 rpm). Finally, the capsules were rinsed with 0.01 M phosphate buffer at pH = 7.5. The capsules were stable for the time of the permeability 7 ACS Paragon Plus Environment

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 8 of 33

experiment, specifically, for at least 4 days (including 24-h exposure to DOX at pH= 7.5 after 24-h sealing with 40,000 g mol-1 FITC-dextran described above, and a 3-day dialysis at pH = 7.5 to remove the DOX excess.

RESULTS AND DISCUSSION PMAA/PVPON Interpenetrated Hydrogel Films Hydrogen-bonded multilayers of PMAA (Mw = 150,000 g mol-1) and high molecular weight PVPON (Mw = 1,300,000 g mol-1), denoted as PMAA-150 and PVPON-1300, respectively, were constructed on planar surfaces of silicon wafers or on sacrificial silica microparticle templates as shown in Figure 1 via layer-by-layer assembly of polymers from acidic solutions (see Experimental).

After the desired number of (PMAA-150/PVPON-1300) bilayers (n) was

adsorbed, the (PMAA/PVPON)n coating, either on planar surface or on inorganic cores, was subjected to carbodiimide-assisted chemical crosslinking with ethylenediamine (EDA) for 2, 4, 8, 24 or 48 hours. Importantly, the high molecular weight PVPON was used to prevent release from the network upon PMAA crosslinking, as PVPON with Mw ≤ 60,000 g mol-1 can completely release from the crosslinked (PMAA)n hydrogels. 7,24,30 The growth of the PMAA-150/PVPON-1300 hydrogen-bonded film at pH = 3.5 is linear as measured by ellipsometry of dry films for the 12-layer (PMAA/PVPON)6 coating. The average thickness of PMAA and PVPON per (PMAA/PVPON) bilayer are 2.6 ± 0.8 and 2.4 ± 0.5 nm, respectively (Fig. 2a). This result is in good agreement with our previous study on hydrogenbonded (PMAA-100/PVPON-1300) films built using spin-assisted multilayer assembly at pH = 2.5 with the (PMAA/PVPON) bilayer thickness of 4.4 ± 0.5 nm.39 Next, we studied the effect of crosslinking time on the (PMAA/PVPON) hydrogel thickness above the pKa of PMAA. We hypothesized that increasing the crosslinking time would enable PVPON to be physically entrapped within the EDA-crosslinked PMAA layers rendering a mechanically robust and less permeable interpenetrated network at pH = 8, despite the disruption of hydrogen bonds between PMAA and PVPON at the higher pH. The initial hydrogen-bonded (PMAA/PVPON)4 films of 31 ± 2 nm were crosslinked for 2, 8, 24, and 48 hours at pH = 5.5. The pH value being less than the pKa of PMAA (~6)

allowed the hydrogen-bonded

(PMAA/PVPON)4 film to be stable at pH = 5.5 for longer than 140 hours (Fig. 2b). There was no significant thickness change in all crosslinked hydrogel films either before or after their 8 ACS Paragon Plus Environment

Page 9 of 33 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

exposure to pH = 8 for 24 h, and the multilayer hydrogel remained stable for at least 45 days indicating that (PMAA/PVPON)4 can be successfully obtained by even 2-hour crosslinking (Fig. 2). Our results also show that the hydrogels obtained via dipped multilayer assembly allow the PVPON-1300 chains to be highly entangled within the network unlike our previously reported spin-assisted (PMAA-100/PVPON-1300) films that showed 18% release of PVPON-1300 from 16-h crosslinked hydrogels.39 Another important observation is that the high molecular weight of PVPON is crucial for its retention in the ‘dipped’ hydrogels. Indeed, the PVPON with Mw = 1,300,000 g mol-1 cannot diffuse out from these (PMAA-150/PVPON-1300) networks unlike shorter PVPON chains (55,000 g mol-1 and 360,000 g mol-1) that were completely released after crosslinking giving one component (PMAA) hydrogels.24,40

The enhanced degree of chain

entanglement and interdiffusion with increasing polymer molecular weight correlates well with prior studies on other hydrogen-bonded and ionically paired multilayers which exhibited slower pH-triggered disintegration when composed of higher Mw polymers.41,42,43

(PMAA/PVPON) Interpenetrating Hydrogel Capsules The fact that the bilayer thickness of the (PMAA-150/PVPON-1300) hydrogel (~5 nm) is greater than that of other PMAA-containing two-component systems such as (PMAA/PVPON-NH2-20) having bilayer thicknesses of 3 ± 1 nm,30 implies that the (PMAA/PVPON) system is a good candidate for encapsulation of small molecules.

As shown previously, 8-bilayer tannic

acid/PVPON (TA/PVPON-1300) capsules had a bilayer thickness of ~ 5 nm and successfully entrapped the anticancer drug doxorubicin (DOX, MW = 543.5 g mol-1) at pH = 7.4. 34, 38,44, 45 To synthesize 14-bilayer PMAA/PVPON capsules, 5-µm porous silica particles were used as sacrificial templates to give ~70-nm thick capsule wall based on the ellipsometry data for thin films. A 14-bilayer system was selected as the high thickness should decrease permeation of small molecules as shown in ionic capsules.4 The (PMAA-150/PVPON-1300)14 capsules were crosslinked for 4 and 24 hours and are denoted as 4H-(PMAA/PVPON)14 and 24H(PMAA/PVPON)14. FTIR analyses of these capsules dried from solution at pH = 3.5 before and after crosslinking as well as spectra of PMAA and PVPON homopolymers are shown in Figure 3. The hydrogenbonded non-crosslinked (PMAA/PVPON)14 capsules showed two absorbance bands at 1704 cm-1 and 1635 cm-1, corresponding to the stretching vibrations of protonated carboxylic groups of 9 ACS Paragon Plus Environment

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

PMAA and PVPON carbonyls, respectively.

24,46

Page 10 of 33

These spectral features correlate well with

protonated PMAA and PVPON homopolymers that have the corresponding bands of their carboxylic and carbonyl groups centered at 1694 cm-1 and 1651 cm-1, respectively (Fig. 3a). After crosslinking, both 24H-(PMAA/PVPON)14 capsules and 4H-(PMAA/PVPON)14 capsules were dialyzed at pH = 7.5 for one week. The FTIR spectra for both 4 h- and 24 h-crosslinked capsules show the presence of the PVPON carbonyl signals around 1657 cm-1 while the carboxylate signals appear around 1545 cm-1 because of the ionization of COOH groups in PMAA at pH = 8 (Fig. 3c).24 These results confirm the successful formation of two-component PMAA/PVPON interpenetrated hydrogel shells after 4- and 24-hour crosslinking. Interestingly, the PVPON was irreversibly trapped within the capsule wall even though swelling and chain rearrangements should be more pronounced at high pH within free-standing hydrogels compared to surface-constrained networks.47,48 Since the entrapment of PVPON within the capsule wall along with the higher density of the crosslinks could affect the pH-responsive properties including permeability toward encapsulated molecules, we investigated the pH-dependent size change of these capsules. Optical microscopy images of the capsule solutions were analyzed at pH = 5.5 and pH = 7.5 (Fig. 4). At pH = 5.5, average diameters of 4H-(PMAA/PVPON)14 and 24H-(PMAA/PVPON)14 capsules are 6.0 ± 0.4 µm and 5.8 ± 0.7 µm, respectively (Fig. 4a, b). At pH = 7.5, the average capsule diameters increased to 9.5 ± 0.5 and 8.9 ± 0.8 µm for 4H-(PMAA/PVPON)14 and 24H-(PMAA/PVPON)14, respectively (Fig. 4c, d). The swelling ratios, calculated as the ratio of capsule diameters at pH = 7.5 to those at pH = 5.5, are similar for both types of capsules with 1.58- and 1.53-fold for the 4H-(PMAA/PVPON)14 and 24H-(PMAA/PVPON)14, respectively. This ~1.5-fold swelling ratio of the interpenetrated (PMAA/PVPON) capsules is similar to that of 22-hour crosslinked (PMAA)7 and thiolated PMAA capsules,24,

26

but only a half of that shown for 5-hour

crosslinked one-component (PMAA)7 capsules (~3-fold).24

These results indicate that the

(PMAA/PVPON) capsules studied here maintain their pH-triggered swelling due to ionization of the PMAA chains and osmotic flux of ions, which is typical for multilayer (PMAA) hydrogel capsules.27,24 However, the incorporation of long PVPON chains within the capsule shell results in suppressed mobility of PMAA segments between the crosslinks. At the same time, the pHtriggered size change in (PMAA/PVPON) capsules is not affected by their crosslinking time


Page 11 of 33 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

suggesting that the swelling is mostly controlled by the presence of the PVPON, unlike the previously reported (PMAA) capsules that swelled much less if more tightly crosslinked.24 We also found that the porosity of the sacrificial silica particles used for synthesis of the capsules did not affect swelling. The 24-hour crosslinked (PMAA/PVPON)14 capsules obtained on non-porous 4-µm silica particles swelled 1.5-fold, changing from 3.0 ± 0.1 µm to 4.5 ± 0.1 µm when pH was varied from pH = 5.5 to 7.5 (Fig. 4e, f), which is similar to the swelling ratio of the 24H-(PMAA/PVPON)14 capsules obtained on porous silica as discussed above.

In

addition, both 4H-(PMAA/PVPON)14 and 24H-(PMAA//PVPON)14 capsules obtained on porous silica templates displayed similar surface charge properties with negative zeta-potentials at pH = 7.5 of -14.1 ± 0.3 mV and -13.7 ± 0.2 mV, respectively. Similarly, the 24H-(PMAA//PVPON)14 capsules synthesized using non-porous silica microparticles had negative surface charge at pH = 7.5 with the average zeta potential of -8.3 ± 0.4 mV (Fig. 5). The zeta potential values reversed from negative at pH = 7.5 to neutral at pH ~5.5 and to positive at pH < 5 which is due to the formation of ionic pairs between carboxylates of PMAA and the NH3+ from singly reacted EDA crosslinker at pH < 7 and their dissociation at pH < 5.7

pH-Dependent Permeability of (PMAA/PVPON) Hydrogel Capsules and Their Interaction with Dextrans It has been previously shown that single-component (PMAA) capsules have pH-controlled permeability as molecules can easily permeate through the swollen capsule wall at pH = 7.5 but experience suppressed diffusion at pH = 5 due to the decreased mesh size of the network7,24,29 Herein, we explored the permeability of (PMAA/PVPON)14 hydrogel capsules toward dextran molecules with molecular weights from 4,000 to 40,000 g mol-1 at pH = 5.5 (the minimal average size of the capsules, Fig. 4a, b, e) and at pH = 7.5 (the maximum average capsule size, Fig. 4c, d, f). The 4H-(PMAA/PVPON)14 and 24H-(PMAA/PVPON)14 capsule solutions were incubated with fluorescently FITC-labeled dextran molecules for 15 min and images were collected using confocal microscopy. Using the CLSM images, we calculated the ratio of the fluorescence intensities from the capsule interior (Ii) and the exterior dextran solution (Ie) and considered the capsules impermeable when Ii/Ie = r was less than 0.5, and permeable when r > 0.5.49,50,51


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 12 of 33

CLSM images in Figure 6 and the analysis of the intensity ratios in Table 1 demonstrate that 4H-(PMAA/PVPON)14 capsules were impermeable to both 20,000 and 40,000 g mol-1 dextran molecules at both pH = 5.5 and pH = 7.5 (Fig. 6a, b, g, h). The r values were 0.18 ± 0.02 and 0.41 ± 0.03 for 40,000 and 20,000 g mol-1 FITC-dextran, respectively, at pH = 7.5, while at pH = 5.5, the intensity ratios were 0.13 ± 0.01 and 0.23 ± 0.03 for 40,000 and 20,000 g mol-1 dextrans, respectively (Table 1). Since the capsules were permeable to 4,000 g mol-1 FITC-dextran at pH = 7.5 (r value of 1.2 ± 0.1; Fig. 6c, Table 1), the mesh size of the swollen hydrogel shell of 4H(PMAA/PVPON)14 at pH = 7.5 must be larger than that of the 4,000 g mol-1 FITC-dextran (3 nm).52 Conversely, as the capsules were impermeable to the larger dextrans, the mesh size must be less than the hydrodynamic radii of 40,000 and 20,000 g mol-1 FITC-dextrans which have been reported as 6.6 ± 0.2 and 5.2 ± 0.5 nm, respectively.52

The 4-h crosslinked

(PMAA/PVPON)14 capsules were still permeable to 4,000 g mol-1 dextran at pH = 5.5 when the hydrogel shell was in its collapsed state, giving the r value of 0.7 ± 0.3 (Fig. 6i, Table 1).

Table 1. The ratio of fluorescence intensities r = Ii/Ie with fluorescence intensities from the capsule interior (Ii) and the exterior FITC-dextran solution (Ie) for 4H-(PMAA/PVPON)14 and 24H-(PMAA/PVPON)14 hydrogel capsules (labeled as 4H and 24H, respectively) probed with FITC-dextrans of varied molecular weight at pH = 7.5 and pH = 5.5 using CLSM. r Mw (FITC-dextran), g mol

-1

pH = 7.5

pH = 5.5

4H

24H

4H

24H

40 000

0.18±0.02

0.06±0.01

0.13±0.01

0.11±0.01

20 000

0.41±0.03

0.12±0.02

0.23±0.03

0.09±0.02

4 000

1.2±0.1

1.12±0.02

0.7±0.3

0.3±0.2

In contrast to the 4H crosslinked capsules, the 24H crosslinked (PMAA/PVPON)14 capsules demonstrated pH-controlled selective permeability to the low molecular weight dextran molecules (Mw = 4,000 g mol-1) by excluding them from the capsule at pH = 5.5 (intensity ratio of 0.3 ± 0.2) but being permeable at pH = 7.5 (Fig. 6l) (r of 1.12 ± 0.02 (Table 1)). These 24H(PMAA/PVPON)14 capsules completely excluded 40,000 and 20,000 g mol-1 FITC-dextran at


Page 13 of 33 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

both pH values (Fig. 6d,e, j, k and Table 1) due to the decreased mesh size of the hydrogel shell at a higher crosslink density. 24 In contrast to the previously reported (PMAA)7 and (PMAA/PVPON-NH2)7 capsules that were only impermeable to FITC-dextran with Mw ≥ 70,000 g mol-1 (hydrodynamic radius of 7.2 ± 0.3 nm52) at pH = 7.4, 24,29 both 4H-(PMAA/PVPON)14 and 24H-(PMAA/PVPON)14 capsules in the current study showed less permeability toward the dextrans. These capsules also showed better exclusion of the dextrans compared to hydrogen-bonded (silk fibroin/poly(Nvinylcaprolactam)) microcapsules that were found to be permeable to 150,000 g mol-1 FITCdextran at pH = 7.5.49 Our (PMAA/PVPON) capsules showed similar permeability to that of the thiolated PMAA capsules which could retain macromolecules with Mw = 15,000 g mol-1. 53 Our results on capsule permeability indicate that 4H-(PMAA/PVPON)14 and 24H-(PMAA/PVPON)14 capsules can be used for encapsulation of hydrophilic molecules with molecular weights as low as 4,000 g mol-1 even in their swollen state at pH = 7.5 (Fig. 6c, f). As previously reported, resealing the capsule wall and therefore lowering the effective mesh size could be achieved through ionic or non-ionic interactions of the capsule shell with macromolecular chains. 24,31,54,55 In this study, we found that large molecular weight dextrans can interact with the (PMAA/PVPON) shell and decrease its permeability. We found that while 4H-(PMAA/PVPON)14 capsules were permeable to the dextrans with Mw < 20,000 g mol-1 even at pH = 5.5 where the hydrogel shell is in its collapsed state (Fig.6c, i), the capsules excluded the dextran after exposure to a solution of 40,000 g mol-1 dextran at pH = 5.5 for 15 min and subsequent rinsing with the same buffer (Fig. 7). These capsules showed decreased permeability toward 20,000 g mol-1 FITC-dextran with r = 0.41 ± 0.03 for the untreated capsules, while those resealed with 40,000 g mol-1 FITC-dextran (Fig. 7a, b) showed r = 0.13 ± 0.02. A similar decrease in permeability of the 4H-(PMAA/PVPON)14 capsules was observed toward 4,000 g mol-1 FITC-dextran after interaction with 40,000 g mol-1 FITC-dextran as measured by CLSM (Fig. 7c, d). In this case, the intensity ratio decreased from r = 1.2 ± 0.1 for the untreated capsules to r = 0.48 ± 0.06 for the dextran-treated capsules. Shell resealing can be attributed to strong entanglements of long dextran molecules with the highly interpenetrated network of the (PMAA/PVPON) hydrogel at pH = 5.5. When the dextran chains interdiffuse inside the hydrogel, their hydroxyl groups can form hydrogen bonds with the carbonyls of PVPON56 and amide carboxyl groups57 from EDA crosslinks with PMAA at pH < 13 ACS Paragon Plus Environment

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

5.8. When the pH is increased to pH > 6, dextran chains entangled within the network can be further locked in place by ionic pairs between the carboxylates of PMAA (-COO-) and NH3+ groups from single-end attached EDA in the hydrogel matrix,7 which will retain the dextran molecules within the matrix at higher pH values.

Encapsulation of Large and Small Molecular Weight Molecules in (PMAA/PVPON) Hydrogel Capsules Herein, we explore the encapsulation of large and small molecular weight molecules into the capsule cavity using a post-loading approach followed by shell resealing with 40,000 g mol-1 dextran. To encapsulate 20,000 and 4,000 g mol-1 FITC-dextrans inside 4H-(PMAA/PVPON)14 capsules, the hydrogel shells were soaked in the 20,000 g mol-1 or 4,000 g mol-1 FITC-dextran solutions at pH = 7.5 for 15 min to allow the FITC-dextran to diffuse through the swollen capsule shell into the cavity. After rinsing off the FITC-dextran solutions using pH = 5.5 buffer, the capsules were exposed to a solution of 40,000 g mol-1 FITC-dextran for 15 min at pH = 5.5 and rinsed several times with pH = 7.5 buffer.

We found that 15-min exposure time of the

-1

40,000 g mol dextran to the capsule is enough to completely seal the capsule shell. The CLSM imaging carried out at every stage of the encapsulation revealed that while fluorescence can be observed from both inside and outside the capsules at the loading step at pH = 7.5 (Fig. 8a, e), the green fluorescent signal is seen exclusively in the capsule interiors after changing the solution pH to 5.5 and sealing the shell with the 40,000 g mol-1 FITC-dextran (Fig. 8b, f). Importantly, after the dextran-loaded capsule solution was brought to pH = 7.5, the green fluorescence from the encapsulated 20,000 and 4,000 g mol-1 dextrans (Fig. 8c, g) was still present in the capsule interiors for at least 1 hour of measurement as evidenced by the fluorescence profiles (Fig. 8d, h). In contrast to previously reported encapsulation of 10,000 g mol-1 FITC-dextran into poly(diallyldimethylammonium chloride)/poly(styrene sulfonate) (PDADMAC/PSS)4 capsules by heating to 55 °C, 58 our approach is performed at room temperature. Also, compared to PMAA capsules prepared by copper-catalyzed azide-alkyne cycloaddition, which could retain 45 base-pair double stranded 13,000 g mol-1 DNA upon disulfide crosslinking, 59 our pH-induced post-loading approach with dextran resealing allows for encapsulation of molecules with molecular weights down to 4,000 g mol-1. 14 ACS Paragon Plus Environment

Page 14 of 33

Page 15 of 33 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

Next, we applied the dextran-based resealing approach to encapsulate model anionic small molecule drugs within the 4H-(PMAA/PVPON)14 capsules. For that, the capsules were exposed to a solution of Alexa Fluor 532 carboxylate, a hydrophilic fluorescent dye with molecular weight of 723.8 g mol-1, at pH = 7.5 for 15 min. The CLSM images in Figure 9 demonstrate that the red fluorescence from the dye can be observed both in the capsule interiors and in the bulk solution (Fig. 9a). The fluorescence intensity profile of the capsules indicates that the shell is highly permeable toward the dye molecule under this condition (Fig. 9b). After the capsule shell was allowed to collapse at pH = 5.5 and interact with 40,000 g mol-1 FITC-dextran, the dye was entrapped inside the capsule as demonstrated by CLSM (Fig. 9c) and the fluorescence intensity profile (Fig. 9d). In contrast to the untreated capsule shell, the dextran-treated capsule shell showed significantly decreased permeability toward the dye at pH = 7.5 as evidenced from the CLSM imaging in Figure 9e and the fluorescence intensity profile of the capsule in Figure 9f. Although the small molecule 6-carboxyfluorescein was previously reported to be successfully encapsulated inside (PDADMAC/PSS)4 polyelectrolyte capsules using heat to induce shell thickening, the required temperature was 55 °C,58 which can be a limitation for temperaturesensitive molecules such as proteins or DNA. Since the fluorescence of the dye is not pH-dependent at pH > 5, the analysis of the intensity profiles of the capsules at pH = 5.5 and pH = 7.5 revealed that ~80% of the dye was entrapped inside the capsules after dextran resealing at pH = 5.5 (Fig. 9b, d), while ~60% of the dye was retained inside the capsules after the increasing pH from 5.5 to 7.5 (Fig. 9d, f). The 20% release of the dye at pH = 7.5 can be attributed to the dye trapped within the capsule shell at pH = 5.5 compared to the dye portion encapsulated in the capsule interior. The shell-trapped dye could be easily rinsed off from the anionic PMAA/PVPON capsule wall during buffer rinses at pH = 7.5. Finally, we investigated the ability of (PMAA/PVPON) capsules to encapsulate cationic small molecules by using the anticancer drug doxorubicin (DOX, MW = 543.5 g mol-1) as a functional load. The DOX has a molecular weight smaller than that of the Alexa Fluor dye discussed above and could not be entrapped into pristine 4H-(PMAA/PVPON)14 hydrogel capsules by collapsing the shell network at pH = 5.5 (data not shown). To facilitate DOX entrapment inside the capsules, we prepared the 4H-(PMAA/PVPON)14 hydrogel capsules with 4,000 g mol-1 FITCdextran entrapped in the capsule interior and sealed the capsule shell with 40,000 g mol-1 FITCdextran (Fig. 8g). The DOX solution (1 mg mL-1, pH = 7.5) was added to the dextran-loaded 15 ACS Paragon Plus Environment

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 16 of 33

capsules and incubated for 24 hours at pH = 7.5. The CLSM images in Figure 10a show that DOX was encapsulated exclusively in the capsule wall (the red fluorescence signal), while the capsule interior was completely occupied by the FITC-dextran molecules (the green fluorescence signal). This result can be explained by the fact that the shell network has a negative charge at pH = 7.5, while DOX is cationic (pKa of DOX is 8.3),60 which results in strong electrostatic interaction. This ionic pairing can result in the collapse of the PMAA/PVPON network and prevent further diffusion of DOX inside the capsule interior. The ionic nature of DOX interaction with the dextran-loaded capsule wall was further confirmed by decreasing the pH of the capsule solution to pH = 5.5. As observed with confocal microscopy, the capsule walls showed a decreased red fluorescence signal, implying a release of some DOX from the capsule shell into the solution (Fig. 10b). The observed release of DOX is due to mutual repulsions of cationic DOX molecules whose charge became unshielded by the neutral PMAA/PVPON hydrogel matrix at pH = 5.5 (Fig. 5). Similar DOX release triggered by lowering the pH value to 5 or 3 was previously reported for PMAA multilayer hydrogel cubes explored as drug delivery microcarriers. 60,61 Caruso et al., reported the use of polymer-stabilized CaCO3 sacrificial microparticles with capping polymers for loading of small hydrophilic molecules into multilayer PMAA capsules.

62

Also, encapsulation of DOX into

PSS(PDADMAC/PSS)5 capsules via spontaneous deposition of DOX into PSS-doped CaCO3 sacrificial template was demonstrated by Gao et al., where a moderately high temperature of 55 °C was used to trigger shell thickening to prevent leakage of the small molecule load from the capsules. 63 DOX was also reported to be encapsulated into the PMAA capsule shell by conjugating DOX with PMAA, 64 however, the loading efficiency was low.

Unlike those

previously reported approaches, the post-loading approach presented in this work relies on template-free hollow capsules whose loading mechanism is triggered by a simple increase in solution pH followed by sealing the capsule wall with non-toxic and biocompatible dextran molecules.

In addition, our approach is versatile and allows for encapsulation of a broad

spectrum of compounds including positively and negatively charged molecules of various molecular weights.

CONCLUSIONS


Page 17 of 33 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

We developed multilayer hydrogel capsules with ultrathin, interpenetrated hydrogel shells for entrapment of low molecular weight molecules. The hydrogel capsules were synthesized by multilayer assembly of PMAA (Mw = 150,000 g mol-1) and PVPON (Mw = 1,300,000 g mol-1) on porous and solid silica microparticles followed by chemical crosslinking of PMAA.

The

PVPON of high molecular weight was physically entrapped within the PMAA network and did not diffuse out from the network at pH = 8 for at least 45 days of measurement.

The

interpenetrated (PMAA/PVPON) capsules were able to encapsulate various hydrophilic molecules including low molecular weight dextran (with Mw ranging from 4,000 to 40,000 g mol-1), anionic Alexa Fluor carboxylate, and the cationic hydrophilic anticancer drug doxorubicin. The 14-bilayer (PMAA/PVPON)14 capsule crosslinked for 4 hours swelled 1.5-fold when the solution pH was changed from 5.5 to 7.5. The capsule wall became highly permeable toward molecules with molecular weight lower than 1000 g mol-1 at pH = 7.5 but non-permeable to dextran molecules with Mw ≥ 40,000 g mol-1. Encapsulation of small molecules was carried out by permeation inside the capsules at pH = 7.5 followed by sealing the capsule shell with 40,000 g mol-1 dextran at pH = 5.5. We found that negatively charged Alexa Fluor carboxylate dye was entrapped within the capsule cavity, while DOX was encapsulated within the capsule shell because of the anionic shell network.

Our study demonstrates that interpenetrated

(PMAA/PVPON) hydrogel capsules are a versatile platform for delivery of a broad spectrum of hydrophilic molecules, including co-delivery of multiple compounds, which is essential for efficient drug therapy.

ACKNOWLEDGEMENTS This work was supported by NSF Career Award # 1350370. The UAB High Resolution Imaging Facility is acknowledged for the use of CLSM. Aaron Alford is acknowledged for technical assistance.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: 205-934-0974. Department of Chemistry, University of Alabama at Birmingham, 901 14th Street South, Birmingham, Alabama 35294, United States 17 ACS Paragon Plus Environment

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

&

Authors (V.K.) and (J.C.) equally contributed to this work.

Notes The authors declare no competing financial interest.


Page 18 of 33

Page 19 of 33 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

FUGURES

Figure 1. Multilayer assembly of PMAA (150,000 g mol-1) and PVPON (1,300,000 g mol-1) polymer layers on sacrificial inorganic microparticles, followed by chemical crosslinking of PMAA layers with EDA and dissolution of the core to obtain hollow (PMAA-PVPON)n interpenetrating multilayer hydrogel capsules with PVPON physically entrapped within the network ( subscript ‘n’ denotes the number of PMAA/PVPON bilayers).


Langmuir

40 Dry thickness, nm

(a)

(PMAA/PVPON)6

30 20 10 0 0

2

4 6 8 10 Number of layers

12

40

Dry thickness, nm

(b)

30 20 10 0 50

Dry thickness, nm

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 20 of 33

0

48 96 Time, hours

144

H-bonded (PVPON/PMAA)4

(c)

hydrogel, pH=4.6 hydrogel; 24 h at pH=8 hydrogel, 45 days at pH=8

40 30 20 10 0

2

24 48 8 Cross-linking time, h

Figure 2. (a) Multilayer growth of (PMAA/PVPON)6 hydrogen-bonded film from 0.5 mg mL-1 polymer solutions (0.01 M phosphate buffer, pH = 3.5) on PGMA-PEI-coated Si wafers; (b) Time-dependent thickness stability of (PMAA/PVPON)6 hydrogen-bonded film in 0.01 M phosphate buffer solution at pH = 5.5 as measured by ellipsometry. (c) Dry thicknesses of hydrogen-bonded (PVPON/PMAA)4 multilayer before (striped blue), and after crosslinking for 2, 8, 24 and 48 h. Film thicknesses were measured right after crosslinking after exposure to pH=4.6 (light grey); followed by measurements after exposure of the film to pH=8 for 24 h (grey) and 45 days (dark grey). 20 ACS Paragon Plus Environment

Page 21 of 33

Transmittance, a.u.

PVPON

1651 PMAA

1694

(a)

Transmittance, a.u.

(b) H-bonded (PMAA/PVPON)14 capsules

1704 1635

24H-(PMAA/PVPON)14 capsules

Transmittance, a.u.

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

4H-(PMAA/PVPON)14 capsules

1545 (c)

1657 1800

1700

1600

1500 Wavenumber,cm-1

1400

Figure 3. (a) FTIR spectra of individual PMAA and PVPON polymers freeze-dried from pH = 3.5 buffer solutions (0.01 M phosphate). (b) FTIR spectrum of (PMAA/PVPON)14 hydrogen-bonded capsules freeze-dried from solutions at pH = 3.5. (c) FTIR spectra of 4H-(PMAA/PVPON)14 and 24H-(PMAA/PVPON)14 capsules freeze-dried from pH = 7.5 buffer solution (0.01 M phosphate).


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

Figure 4. Optical microscopy images of (a, c) 4H-(PMAA/PVPON)14 and (b, d) 24H(PMAA/PVPON)14 hydrogel capsules synthesized using porous silica microparticles and imaged at (a, b) pH = 5.5 and (c, d) pH = 7.5. Scale bar is 10 µm. (e, f) Images of 24H(PMAA/PVPON)14 capsules synthesized using non-porous silica particles of 2 µm and imaged at (e) pH = 5.5 and (f) pH = 7.5.


Page 22 of 33

Page 23 of 33

15 10 ζ -potential, mV

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

5 0 -5

-10 2

4

6

8

pH Figure 5. pH-dependent ζ-potential values of 24H-(PMAA/PVPON)14 capsules measured from 0.01 M phosphate buffer solutions of capsules (1 x 106 capsules mL-1).


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

Figure 6. CLSM images of (a-c, g-i) 4H-(PMAA/PVPON)14 and (d-f, j-l) 24H(PMAA/PVPON)14 hydrogel capsules after exposure to 1 mg mL-1 FITC-dextran solutions with FITC-dextran Mw of 4,000 g mol-1 (a,d,g,j), 20,000 g mol-1 (b,e,h,k), and 40,000 g mol-1 (c,f,i,l) at pH = 7.5 (a-f) and pH= 5.5 (g-l). The scale bar is 5 µm in all images.


Page 24 of 33

Page 25 of 33 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

Figure 7. CLSM images of (a, c) 4H-(PMAA/PVPON)14 capsules after exposure to 1 mg mL-1 FITC-dextran solutions with (a, b) Mw = 20,000 g mol-1 and (c, d) Mw = 4,000 g mol-1 before (a, c) and after (b, d) resealing with 40,000 g mol-1 dextran. The scale bar is 10 µm in all images.


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

Figure 8. CLSM images of 4H-(PMAA/PVPON)14 hydrogel capsules after exposure to FITC-dextran with Mw of (a) 20,000 g mol-1 and (e) 4,000 g mol-1 in solutions at pH=7.5, followed by their rinsing with 0.01 M phosphate buffer at pH= 5.5 and interaction with 40,000 g mol-1 FITC-dextran (b, f), followed by the exposure to 0.01 M phosphate buffer at pH = 7.5 (c, g). (d, h) The corresponding fluorescence intensity profiles from (d) 20,000 g mol-1 and (h) 4,000 g mol-1 FITC-dextran-loaded capsules, respectively. The scale bar is 5 µm in all images.


Page 26 of 33

(b)

240

220 0.0

0.2

0.4 0.6 Distance, a.u.

0.8

280

Fluorescence Intensity, a.u.

260


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


Page 27 of 33

(d)

210 140 70 0

0.2

0.4 0.6 0.8 Distance, a.u.

1.0

280

(f)

210 140 70 0

0.2

0.4 0.6 Distance, a.u.

0.8

Figure 9. Encapsulation of Alexa Fluor 532 carboxylic acid fluorescent dye inside the 4H-(PMAA/PVPON)14 hydrogel capsules: CLSM images of the capsules (a) in the presence of the dye at pH = 7.5, (c) after exposure to 40 000 g mol-1 FITC-dextran at pH = 5.5 and rinsing with the buffer solution at pH = 5.5, and (e) after rinsing with pH = 7.5 buffer for 15 min; (b, d, f) the fluorescence intensity profiles of a capsule in (a), (c), and (e), respectively.


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

Figure 10. CLSM images of DOX (the red fluorescence signal) encapsulated into the capsule wall of 4H-(PMAA/PVPON)14 hydrogel capsules with the capsule interiors loaded with 4,000 g mol-1 FITC-dextran (the green fluorescence signal) at (a) pH = 7.5 and (b) pH = 5.5.


Page 28 of 33

Page 29 of 33 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

REFERENCES (1) Allen, T. M.; Cullis, P. R. Drug delivery systems: entering the mainstream. Science 2004, 303, 1818-1822. (2) Vrignaud, S.; Benoit, J.-P.; Saulnier, P. Strategies for the nanoencapsulation of hydrophilic molecules in polymer-based nanoparticles. Biomaterials 2011, 32, 8593-8604. (3) Rout, G. K.; Shin, H.-S.; Gouda, S.; Sahoo, S.; Das, G.; Fraceto, L. F.; Patra, J. K. Current advances in nanocarriers for biomedical research and their applications, Artif. Cells, Nanomed. Biotechnol. 2018, DOI: 10.1080/21691401.2018.1478843. (4) Antipov, A. A.; Sukhorukov, G. B. Polyelectrolyte multilayer capsules as vehicles with tunable permeability. Adv. Colloid Interface Sci. 2004, 111, 49-61. (5) De Villiers, M. M.; Otto, D. P.; Strydom, S. J.; Lvov, Y. M. Introduction to nanocoatings produced by layer-by-layer (LbL) self-assembly. Adv. Drug Deliv. Rev. 2011, 63, 701-715. (6) Cui, J.; Richardson, J. J.; Björnmalm, M.; Faria, M.; Caruso, F. Nanoengineered templated polymer particles: Navigating the biological realm. Acc. Chem. Res. 2016, 49, 1139-1148. (7) Kozlovskaya, V.; Sukhishvili, S. A. Amphoteric hydrogel capsules: Multiple encapsulation and release routes. Macromolecules 2006, 39, 6191-6199. (8) Cui, J.; van Koeverden, M. P.; Müllner, M.; Kempe, K.; Caruso, F. Emerging methods for the fabrication of polymer capsules. Adv. Colloid Interface Sci 2014, 207, 14-31. (9) Delcea, M.; Möhwald, H.; Skirtach, A. G. Stimuli-responsive LbL capsules and nanoshells for drug delivery. Adv. Drug Deliv. Rev. 2011, 63, 730-747. (10) Allen, T. M.; Cullis, P. R. Liposomal drug delivery systems: from concept to clinical applications. Adv. Drug Deliv. Rev. 2013, 65, 36-48. (11) Lee, J. S.; Feijen, J. Polymersomes for drug delivery: design, formation and characterization. J. Control. Release 2012, 161, 473-483. (12) Li, W.; Shi, J.; Zhang, C.; Li, M.; Gan, L.; Xu, H.; Yang, X. Co-delivery of thioredoxin 1 shRNA and doxorubicin by folate-targeted gemini surfactant-based cationic liposomes to sensitize hepatocellular carcinoma cells. J. Mater. Chem. B 2014, 2, 4901-4910. (13) Eloy, J. O.; De Souza, M. C.; Petrilli, R.; Barcellos, J. P. A.; Lee, R. J.; Marchetti, J. M. Liposomes as carriers of hydrophilic small molecule drugs: strategies to enhance encapsulation and delivery. Colloids Surf., B 2014, 123, 345-363. (14) Del Mercato, L. L.; Ferraro, M. M.; Baldassarre, F.; Mancarella, S.; Greco, V.; Rinaldi, R.; Leporatti, S. Biological applications of LbL multilayer capsules: From drug delivery to sensing. Adv. Colloid Interface Sci. 2014, 207, 139-154. (15)Driver, K.; Baco, S.; Khutoryanskiy, V. V. Hollow capsules formed in a single stage via interfacial hydrogen-bonded complexation of methylcellulose with poly (acrylic acid) and tannic acid. European Polymer Journal, 2013, 49, 4249-4256. (16) Kozlovskaya, V.; Xue, B;. Kharlampieva, E. Shape-adaptable polymeric particles for controlled delivery. Macromolecules 2016, 49, 8373-8386. (17) Guzmán, E.; Mateos-Maroto, A.; Ruano, M.; Ortega, F.; Rubio, R. G. Layer-by-layer polyelectrolyte assemblies for encapsulation and release of active compounds. Adv. Colloid and Interface Sci. 2017, 249, 290-307.


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) Wang, Y.; Yan, Y.; Cui, J.; Hosta-Rigau, L.; Heath, J. K.; Nice, E. C.; Caruso, F. Encapsulation of water-insoluble drugs in polymer capsules prepared using mesoporous silica templates for intracellular drug delivery. Adv. Mater. 2010, 22, 4293-4297. (19) De Koker, S.; De Cock, L. J.; Rivera-Gil, P.; Parak, W. J.; Velty, R. A.; Vervaet, C.; Remon, J. P.; Grooten, J.; De Geest, B. G. Polymeric multilayer capsules delivering biotherapeutics. Adv. Drug Deliv. Rev. 2011, 63, 748-761. (20) Skirtach, A. G.; Yashchenok, A. M.; Möhwald, H., Encapsulation, release and applications of LbL polyelectrolyte multilayer capsules. Chem. Commun. 2011, 47, 12736-12746. (21) Richardson, J. J.; Choy, M. Y.; Guo, J.; Liang, K.; Alt, K.; Ping, Y.; Cui, J.; Law, L. S.; Hagemeyer, C. E.; Caruso, F. Polymer capsules for plaque‐targeted in vivo delivery. Adv. Mater. 2016, 28, 7703-7707. (22) Dai, X.; Tan, C. Combination of microRNA therapeutics with small-molecule anticancer drugs: mechanism of action and co-delivery nanocarriers. Adv. Drug Deliv. Rev.2015, 81, 184197. (23) Köhler, K.; Sukhorukov, G. B. Heat treatment of polyelectrolyte multilayer capsules: A versatile method for encapsulation. Adv. Funct. Mater. 2007, 17, 2053-2061. (24) Kozlovskaya, V.; Kharlampieva, E.; Mansfield, M. L.; Sukhishvili, S. A. Poly(methacrylic acid) hydrogel films and capsules: Response to pH and ionic strength, and encapsulation of macromolecules. Chem. Mater. 2006, 18, 328-336. (25) Zelikin, A. N.; Quinn, J. F.; Caruso, F. Disulfide cross-linked polymer capsules: En route to biodeconstructible systems. Biomacromolecules 2006, 7, 27-30. (26) Zelikin, A. N.; Li, Q.; Caruso, F. Disulfide-stabilized poly(methacrylic acid) capsules: Formation, cross-linking, and degradation behavior. Chem. Mater. 2008, 20, 2655-2661. (27) Kozlovskaya, V.; Alexander, J. F.; Wang, Y.; Kuncewicz, T.; Liu, X.; Godin, B.; Kharlampieva, E. Internalization of red blood cell-mimicking hydrogel capsules with pHtriggered shape responses. ACS Nano 2014, 8, 5725-5737. (28) De Koker, S.; Cui, J.; Vanparijs, N.; Albertazzi, L.; Grooten, J.; Caruso, F.; De Geest, B. G. Engineering polymer hydrogel nanoparticles for lymph node‐targeted delivery. Angew. Chem. Int. Ed. 2016, 55, 1334-1339. (29 ) Kozlovskaya, V.; Sukhishvili, S. A., pH-controlled permeability of layered hydrogenbonded polymer capsules. Macromolecules 2006, 39, 5569-5572. (30) Kozlovskaya, V.; Shamaev, A.; Sukhishvili, S. A. Tuning swelling pH and permeability of hydrogel multilayer capsules. Soft Matter 2008, 4, 1499-1507. (31) Feng, Z.; Gao, C.; Shen, J. Spontaneous deposition of FITC‐labeled dextran into covalently assembled (PGMA/PAH)4 microcapsules. Macromol. Chem. Phys 2009, 210, 1387-1393. (32) Ibarz, G.; Dähne, L.; Donath, E.; Möhwald, H. Resealing of polyelectrolyte capsules after core removal. Macromol. Rapid Commun. 2002, 23, 474-478. (33) Pham-Hua, D.; Padgett, L. E.; Xue, B.; Anderson, B.; Zeiger, M.; Barra, J. M.; Bethea, M.; Hunter, C. S.; Kozlovskaya, V.; Kharlampieva, E.; Tse, H. M. Islet encapsulation with polyphenol coatings decreases proinflammatory chemokine synthesis and T cell trafficking. Biomaterials 2017, 128, 19-32. (34) Alford, A.; Rich, M.; Kozlovskaya, V.; Chen, J.; Sherwood, J.; Bolding, M.; Warram, J.; Bao, Y.; Kharlampieva, E. Ultrasound-triggered delivery of anticancer therapuetics from MRIvisible multilayer microcapsules. Adv. Therapeutics 2018, DOI: 10.1002/adtp.201800051.


Page 30 of 33

Page 31 of 33 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

(35) Kinnane, C. R.; Such, G. K.; Antequera-García, G.; Yan, Y.; Dodds, S. J.; Liz-Marzan, L. M.; Caruso, F. Low-fouling poly(N-vinyl pyrrolidone) capsules with engineered degradable properties. Biomacromolecules 2009, 10, 2839-2846. (36) Andersen, T. E.; Palarasah, Y.; Skjodt, M. O.; Ogaki, R.; Benter, M.; Alei, M.; Kolmos, H. J.; Koch, C.; Kingshott, P. Decreased material-activation of the complement system using lowenergy plasma polymerized poly(vinyl pyrrolidone) coatings. Biomaterials 2011, 32, 4481-4488. (37) Liang, X.; Kozlovskaya, V.; Chen, Y.; Zavgorodnya, O.; Kharlampieva, E. Thermosensitive multilayer hydrogels of poly (N-vinylcaprolactam) as nanothin films and shaped capsules. Chem. Mater. 2012, 24, 3707-3719. (38) Chen, J.; Kozlovskaya, V.; Goins, A.; Campos-Gomez, J.; Saeed, M.; Kharlampieva, E. Biocompatible shaped particles from dried multilayer polymer capsules. Biomacromolecules 2013, 14, 3830-3841. (39) Kozlovskaya, V.; Zavgorodnya, O.; Ankner, J. F.; Kharlampieva, E. Controlling internal organization of multilayer poly(methacrylic acid) hydrogels with polymer molecular weight. Macromolecules 2015, 48, 8585-8593. (40) Kharlampieva, E.; Erel-Unal, I.; Sukhishvili, S. A. Amphoteric surface hydrogels derived from hydrogen-bonded multilayers: Reversible loading of dyes and macromolecules, Langmuir 2007, 23, 175-181. (41) Kharlampieva, E.; Ankner, J. F.; Rubinstein, M.; Sukhishvili, S. A. pH-Induced release of polyanions from multilayer films. Phys. Rev. Lett. 2008, 100, 128303/1-128303/4. (42) Jang, Y.; Seo, J.; Akgun, B.; Satija, S.; Char, K. Molecular weight dependence on the disintegration of spin-assisted weak polyelectrolyte multilayer films. Macromolecules 2013, 46, 4580-4588. (43) Nestler, P.; Passvogel, M.; Helm, C. A. Influence of polymer molecular weight on the parabolic and linear growth regime of PDADMAC/PSS multilayers. Macromolecules 2013, 46, 5622-5629. (44) Chen, J.; Ratnayaka, S.; Alford, A.; Kozlovskaya, V.; Liu, F.; Xue, B.; Hoyt, K.; Kharlampieva, E. Theranostic multilayer capsules for ultrasound imaging and guided drug delivery. ACS Nano 2017, 11, 3135-3146. (45) Liu, F.; Kozlovskaya, V.; Zavgorodnya, O.; Martinez-Lopez, C.; Catledge, S.; Kharlampieva, E. Encapsulation of anticancer drug with hydrogen-bonded multilayers of tannic acid. Soft Matter 2014, 10, 9237-9247. (46) Kozlovskaya, V.; Yakovlev, S.; Libera, M.; Sukhishvili, S. A. Surface priming and the selfassembly of hydrogen-bonded multilayer capsules and films. Macromolecules 2005, 38, 48284836. (47) Toomey, R.; Freidank, D.; Rühe, J. Swelling behavior of thin, surface-attached polymer networks. Macromolecules 2004, 37, 882-887. (48) Marcombe, R.; Cai, S.; Hong, W.; Zhao, X.; Lapusta, Y.; Suo. Z. A theory of constrained swelling of a pH-sensitive hydrogel. Soft Matter 2010, 6, 784-793. (49) Espinosa-Dzib, A.; Chen, J.; Zavgorodnya, O.; Kozlovskaya, V.; Liang, X.; Kharlampieva, E. Tuning assembly and enzymatic degradation of silk/poly (N-vinylcaprolactam) multilayers via molecular weight and hydrophobicity. Soft Matter 2015, 11, 5133-5145. (50) Shutava, T.; Prouty, M.; Kommireddy, D.; Lvov, Y. pH-Responsive decomposable layerby-layer nanofilms and capsules on the basis of tannic acid. Macromolecules 2005, 38, 28502858. 31 ACS Paragon Plus Environment

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

(51) Shutava, T. G.; Lvov, Y. M. Nano-engineered Microcapsules of Tannic Acid and Chitosan for Protein Encapsulation. J. Nanosci. Nanotechnol. 2006, 6, 1655-1661. (52) Wen, H.; Hao, J.; Li, S. K. Characterization of human sclera barrier properties for transscleral delivery of bevacizumab and ranibizumab. J. Pharm. Sci. 2013, 102, 892-903. (53) Chong, S.-F.; Lee, J. H.; Zelikin, A. N.; Caruso, F. Tuning the permeability of polymer hydrogel capsules: An investigation of cross-linking density, membrane thickness, and crosslinkers. Langmuir 2011, 27, 1724-1730. (54) Kozlovskaya, V.; Liu, F.; Xue, B.; Ahmad, F.; Alford, A.; Saeed, M.; Kharlampieva, E. Polyphenolic polymersomes of temperature-sensitive poly(N-vinylcaprolactam)-block-(poly(Nvinylpyrrolidone) for anticancer therapy. Biomacromolecules 2017, 18, 2552-2563. (55) Li, Y.; Lokitz, B. S.; McCormick, C. L. Thermally responsive vesicles and their structural “locking” through polyelectrolyte complex formation. Angew. Chem. Int. Ed. 2006, 118, 59245927. (56) Huang, M.; Hou, Y.; Li, Y.; Wang, D.; Zhang, L. High performances of dual network PVA hydrogel modified by PVP using borax as the structure-forming accelerator, Des. Monomers Polym. 2017, 20, 505-513. (57) Wang, A.; Tao, C.; Cui, Y.; Duan, L; Yang, Y.; Li, J. Assembly of environmental sensitive microcapsules of PNIPAAm and alginate acid and their application in drug release. J. Colloid Interface Sci. 2009, 332, 271-279. (58) Song, W.; He, Q.; Möhwald, H.; Yang, Y.; Li, J. Smart polyelectrolyte microcapsules as carriers for water-soluble small molecular drug. J. Control. Release 2009, 139, 160-166. (59) Kempe, K.; Noi, K. F.; Ng, S. L.; Müllner, M.; Caruso, F. Multilayered polymer capsules with switchable permeability. Polymer 2014, 55, 6451-6459. (60) Kozlovskaya, V.; Chen, J.; Tedjo, C.; Liang, X.; Campos-Gomez, J.; Oh, J.; Saeed, M.; Lungu, C. T.; Kharlampieva, E. pH-Responsive hydrogel cubes for release of doxorubicin in cancer cells. J. Mater. Chem. B 2014, 2, 2494-2507. (61) Xue, B.; Kozlovskaya, V.; Liu, F.; Chen, J.; Williams, J.; Campos-Gomez, J.; Saeed, M.; Kharlampieva, E. Intracellular degradable hydrogel cubes and spheres for anticancer drug delivery. ACS Applied Mater. & Interfaces 2015, 7, 13633-13644. (62) Richardson, J. J.; Maina, J. W.; Ejima, H.; Hu, M.; Guo, J.; Choy, M. Y.; Gunawan, S. T.; Lybaert, L.; Hagemeyer, C. E.; De Geest, B. G.; Caruso, F. Versatile loading of diverse cargo into functional polymer capsules. Adv. Sci. 2015, 2, 1-2. (63) Tong, W.; She, S.; Xie, L.; Gao, C. High efficient loading and controlled release of lowmolecular-weight drugs by combination of spontaneous deposition and heat-induced shrinkage of multilayer capsules. Soft Matter 2011, 7, 8258-8265. (64) Cui, J.; Yan, Y.; Wang, Y.; Caruso, F. Templated assembly of pH‐labile polymer‐drug particles for intracellular drug delivery. Adv. Funct. Mater. 2012, 22, 4718-4723.

For Table of Contents use only


Page 32 of 33

Page 33 of 33 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


Multilayer Hydrogel Capsules of Interpenetrated Network for

Recommend Documents