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Article Cite This: ACS Omega 2017, 2, 9002−9012

Intracellular Delivery of β‑Galactosidase Enzyme Using ArginaseResponsive Dextran Sulfate/Poly‑L‑arginine Capsule for Lysosomal Storage Disorder Meenakshi Gupta,†,‡ Himanshu Pandey,‡ and Sri Sivakumar*,§ †

Institute of Pharmacy, Chhatrapati Shahu Ji Maharaj University, Kanpur, Uttar Pradesh 208024, India Department of Pharmaceutical Sciences, Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad, Uttar Pradesh 211007, India § Department of Chemical Engineering, Material Science Programme, Centre for Nanoscience and Soft Nanotechnology, Centre for Environmental Science and Engineering, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India ‡

S Supporting Information *

ABSTRACT: β-Galactosidase (β-gal) is one of the important lysosomal enzymes that is involved in the breakdown of glycosphingolipids (e.g., GM1 ganglioside), and its deficiency leads to GM1 Gangliosidosis, a lysosomal storage disorder (LSD). Intracellular delivery of β-gal is one of the preferable methods to treat this kind of LSDs. However, it cannot permeate the cell membrane due to its intricate macromolecular nature, low stability, and degradation by endogenous proteases. To this end, we report efficient intracellular delivery of β-gal via arginase-responsive dextran sulfate/poly-Larginine polymer capsules (DS/PA capsules). The therapeutic activity of β-gal enzyme has been assessed in two genedeficient diseased cell lines, SV (β-galactosidase gene-deficient mouse fibroblast) and R201C (deficient human β-galactosidase gene-introduced mouse fibroblast), and in wild-type mouse fibroblast immortalized cell lines. The activity of β-gal enzyme has been estimated within cells by using fluorescein isothiocyanate-cholera toxin B as a florescent probe that illustrates the level of GM1 ganglioside, the β-gal substrate. We found 1.8-, 3.4-, and 2.8-fold reduction in the substrate level in R201C, SV, and wild-type mouse fibroblast, respectively, which confirms the release and therapeutic activity of β-gal enzyme inside the cells. Moreover, enzyme delivery in gene-deficient diseased cell lines (SV and R201C) via DS/PA capsules reduced the level of enzyme substrate to a normal endogenous level, which is present in untreated wild-type mouse fibroblast cells. We note that loading of β-gal enzyme within DS/PA capsules was estimated to be 3 mU per hundred capsules and more than 77% of β-gal is released within 12 h. Overall, these results highlight the potential of DS/ PA capsules as an efficient delivery carrier for therapeutic enzyme.

1. INTRODUCTION Lysosomes are cell compartments, responsible for catabolism of endogenous and exogenous macromolecules and recycle them. More than 50 digestive enzymes are involved in cellular waste disposal by breakdown of all kinds of biomolecules. βGalactosidase (β-gal) is one of lysosomal enzymes that is involved in the breakdown of glycosphingolipid (e.g., GM1 ganglioside) and its deficiency leads to GM1 gangliosidosis, lysosomal storage disorder (LSD) that results in progressive destruction of the central nervous system (CNS).1−3 This can even lead to the death of the person due to permanent cellular and tissue damage. Several strategies have been used in the past to treat LSDs, such as the substrate reduction therapy that reduces biosynthesis of the substrate for correction of the imbalance between formation and breakdown of the substrate (can affect the cellular substrate balance),4−6 pharmacological chaperones therapy that stabilizes mutant lysosomal proteins © 2017 American Chemical Society

(limited to selective patients with mutant lysosomal enzyme LSDs or chaperone responsive mutations),7−13 bone marrow transplant (challenges in identifying compatible donors, high morbidity, graft failure, and mortality),14gene delivery (challenges in obtaining adequate levels of gene product in specific tissues like CNS, maintaining in vivo expression, random integration, and immune reactions),15−17 cell-penetrating peptides (short circulating half-life in vivo),18 and DNAmediated enzyme delivery (in vivo stability).19 Most of the above approaches are limited due to presence of inherent complexities, which are mentioned above within the parenthesis of the corresponding therapy. Presently, the most promising approach to treat LSDs is enzyme replacement therapy (ERT), Received: August 22, 2017 Accepted: November 23, 2017 Published: December 15, 2017 9002

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rescein isothiocyanate (FITC), and gelatin were purchased from Sigma-Aldrich (India). Fetal bovine serum (FBS) was purchased from Gibco. Formaldehyde, toluene, liquor ammonia (24%), ammonium fluoride, ethanol, and dimethyl sulfoxide (DMSO) were obtained from Merck Chemicals (India). Hydrofluoric acid (40% w/v) was purchased from SD FineChem Ltd. and tetraethyl orthosilicate from Fluka. HeLa and L929 (wild-type mouse fibroblast) cell lines were purchased from the National Centre for Cell Science, Pune, India. SV (βgalactosidase gene-deficient mouse fibroblast, JCRB1207) and R201C (deficient human β-galactosidase gene-introduced mouse fibroblast, JCRB1199), were purchased from the Japanese Collection of Research Bioresources cell bank. 2.2. Methods. 2.2.1. Synthesis of β-Gal-Loaded DS/PA Polymeric Capsules. The synthesis of β-gal-loaded DS/PA capsules was carried out by sequential layer-by-layer assembly of dextran sulfate and poly-L-arginine on β-gal-loaded calcium carbonate templates, followed by core removal. The synthesis of the calcium carbonate (CaCO3) template was done as previously reported.33,34 Briefly, 1 M calcium chloride dihydrate (CaCl2·2H2O) was added to 1 M sodium carbonate (Na2CO3) under vigorous stirring for 1 min and was left undisturbed for 20 min. The particles were separated by centrifugation at 3000 rpm for 3 min, washed thrice with deionized water and acetone, followed by drying by heating at 80 °C. Further, β-gal loading was done by dispersing calcium carbonate templates (20 mg) in the β-gal enzyme (8 U/mL in Z buffer) overnight at 4 °C, followed by separation at 3000 rpm for 3 min. The enzymeloaded calcium carbonate template was sequentially coated with polyanionic and polycationic polymer solution, as reported by De Geest et al.35,36 Layer-by-layer polymer coating was initiated by addition of dextran sulfate (1 mL, 1 mg/mL in 0.5 M NaCl) polymer to β-gal-loaded calcium carbonate particles (20 mg). This was kept in rotospin for 15 min. The adsorption step was followed by washing of the sample thrice with deionized water to remove unbound polymer. The separation of the sample was done at 3000 rpm for 3 min. Subsequently, seven alternate polymeric layers were made of poly-L-arginine (1 mg/mL in 0.5 M NaCl) and dextran sulfate (1 mg/mL in 0.5 M NaCl) to form eight layers with alternate negative and positive charges. Finally, removal of the calcium carbonate template was carried out by using 0.2 M EGTA (7.5 pH) solution. The pH of EGTA solution was adjusted by using NaOH solution. The prepared capsules were mixed with 0.2 M EGTA (7.5 pH) solution for 5 min. This process was repeated four times; each time fresh EGTA solution was added, followed by three times of washing with deionized water. The prepared β-gal-DS/PA capsules were dispersed in 1 mL of deionized water. Bare DS/PA capsules were prepared without loading β-gal enzyme as the negative control. 2.2.2. Synthesis of β-Gal-Loaded PSS/PAH Polymeric Capsules. The synthesis of β-gal-loaded PSS/PAH capsules was carried out by a sequential layer-by-layer assembly of poly(sodium 4-styrene-sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) on β-gal-loaded 3-(aminopropyl)triethoxysilane (APTES)-functionalized silica templates, followed by removal of the silica core. Silica particles were prepared by Stober’s37 method by hydrolysis of tetraethyl orthosilicate (4.7 mL) using ammonia (54.4 mL) in a solution of water (2 mL) and ethanol (50 mL). The particles were separated by centrifugation, washed with deionized water followed by ethanol to remove the excess of reactants, and dried at 80 °C overnight. Further, for APTES functionaliza-

in which the missing enzyme is replaced by intravenous infusion. Although ERT is commercially available currently, it is limited to six lysosomal enzyme therapies.20,21 Thus, it is essential to design suitable delivery vehicles that can translocate lysosmal enzymes inside the cell. In this regard, few attempts have been made to deliver β-gal enzyme using different vehicles, such as lipid vesicles(liposome),22 polymeric nanoparticles,23 protein nanoparticles,24 cyclodextrin,25 functionalized gold nanoparticles,26 and polymersome.27 However, these approaches suffer from the following challenges, such as low encapsulation efficiency, reduction in activity, formation of undesirable degradation products, etc. Herein, we report efficient intracellular delivery of β-gal enzyme via dextran sulfate and poly-L-arginine polymeric capsules prepared by layer-by-layer assembly for GM1 gangliosidosis management. The current approach has the following advantages: (1) good enzyme loading (3 mU/100 capsules),28 (2) retention of enzyme activity due to mild capsule synthesis conditions,29 (3) enhanced cellular uptake of enzyme as compared to that of the free enzyme, (4) protection of the encapsulated enzyme by the LbL polymeric shell from the endogenous proteases inactivation and immunological reactions due to shielding,30,31 (5) biodegradability due to arginase enzyme response capability,32 and (6) reduced cytotoxicity. Enzyme-loaded polymeric capsules were synthesized by the sequential adsorption of oppositely charged polymers, dextran sulfate (anionic polyelectrolyte), and poly-L-arginine (cationic polyelectrolyte) on β-gal enzyme-preloaded calcium carbonate, followed by the removal of the sacrificial calcium carbonate template by using ethylene glycol-bis(2-aminoethylether) NNN′N′-tetraacetic acid (EGTA). The therapeutic activity of β-gal has been evaluated in SV (β-galactosidase gene-deficient mouse fibroblast), R201C (deficient human β-galactosidase gene-introduced mouse fibroblast), and wild-type mouse fibroblast. To mark curative activity of the β-gal enzyme, fluorescein isothiocyanate (FITC)-cholera toxin, a florescent probe, has been used to illustrate the level of the substrate. The decrease in the substrate level within gene-deficient fibroblast (SV and R201C) as well as wild-type mouse fibroblast confirms the release (77%) and therapeutic activity of the β-gal enzyme inside the cells after delivery of β-gal-DS/PA capsules. Our studies reveal that the bare DS/PA capsules showed no in vitro cytotoxicity and were efficiently taken up by SV, R201C, wildtype mouse fibroblast, and HeLa cells.

2. MATERIALS AND METHODS 2.1. Materials. Dextran sulfate sodium salt from Leuconostos spp., poly-L-arginine hydrochloride (mol. wt. 15 000− 70 000), calcium chloride dihydrate, sodium carbonate, disodium ethylenediaminetetraacetate (EDTA), ethylene glycol-bis(2-aminoethylether)-NNN′N′-tetraacetic acid (EGTA), poly(sodium 4-styrene-sulfonate) (PSS) Mw 70 000, poly(allylamine hydrochloride) (PAH) Mw 56 000, β-gal enzyme, β-galactosidase reporter gene activity detection kit (GAL-A), rhodamine isothiocyanate (RITC), cholera toxin B conjugated with fluorescein isothiocyanate (FITC), trypsin-EDTA, low glucose Dulbecco’s modified Eagle’s medium (DMEM), medium-to-high glucose Dulbecco’s modified Eagle’s medium (DMEM), 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), penicillin−streptomycin antibiotic, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fluorescein isothiocyanate-phalloidin (FITC-phalloidin), fluo9003

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solution at 4 °C for RITC labeling. Nearly, 104 cells were seeded in a 24-well plate containing 13 mm gelatin (0.2% w/v)coated coverslips. RITC-labeled DS/PA capsules (50 capsules/ cell) were added to each well and incubated for 12 h in an incubator. After incubation, the medium was discarded and cells were washed thrice with phosphate-buffered saline (PBS) to remove free uninternalized capsules. Cells were fixed with 4% formaldehyde solution, and nuclei were stained using DAPI (10 μg/mL) for 10 min. Staining protocols were used as provided by the manufacturer. These samples were mounted on glass slides and observed under a confocal laser scanning microscope (Zeiss LSM 710). 2.3.2. In Vitro Enzyme Release Studies. First, to observe βgal release from DS/PA capsules within HeLa cells, approximately 104 HeLa cells were seeded in a 24 well plate containing 13 mm gelatin coated coverslip and incubate for 6 h. After 6 h incubation, β-gal-loaded DS/PA capsules (3 mU) were dispersed in media and added to a cell culture plate. Cells were incubated at 37 °C under 5% CO2 for different time periods (0, 6, 12, and 24 h). Thereafter, the medium was removed and cells were washed thrice with PBS to remove free uninternalized capsules. Cells were incubated with ONPG buffer for 1 h. Subsequent to this, cells were fixed with 4% formaldehyde solution for 20 min, mounted on glass slides, and observed under a confocal laser scanning microscope. Cells were quantified for yellow fluorescence produced by onitrophenol (ONP) using Image J software. In the second set of experiments, in vitro β-gal release from β-gal-loaded DS/PA capsules was analyzed in HeLa cell lysate after 4, 6, 12, and 24 h time point. Around 104 HeLa cells were seeded in a 96-well plate and incubated for 6 h. After 6 h incubation, β-gal-loaded DS/PA capsules (3 mU) were dispersed in media and added into a 96-well plate and allowed to incubate at different time points in the CO2 incubator. Thereafter, the medium was removed and cells were washed thrice with PBS to remove free uninternalized capsules. Cells in the cell culture plate were trypsinized and washed with PBS. Cell lysis was done by incubating these cells in ice-cold lysis buffer for 30 min, and the cell lysate was collected by centrifugation at 14 000 rpm for 20 min at 4 °C. Quantification of β-gal in cell lysate was done by β-galactosidase detection kit using protocol provided by the manufacturers. Further, similar release studies (12 h time point) were done with nondegradable PSS/PAH capsules as the control experiment. 2.3.3. Enzyme Activity Studies. Therapeutic activity of the β-gal enzyme, after delivering β-gal-loaded DS/PA capsules inside the cells, was evaluated in SV, R201C, and wild-type mouse fibroblast cell lines. For enzyme activity studies, about 104 cells were seeded in a 24-well plate containing 13 mm gelatin (0.2% w/v)-coated coverslips and were rested for 6 h. After 6 h of incubation, cells were treated with two different concentrations of β-gal-loaded DS/PA capsules (50 capsules/ cell, 1.5 mU and 100 capsules/cell, 3 mU). In a separate set of experiments, cells were treated with free β-gal enzyme (1.5 and 3 mU) equivalent to the β-gal-loaded in DS/PA capsules as a control. A negative control was also taken in which cells were treated with bare DS/PA capsules (50 and 100 capsules/cell). Cells were incubated at 37 °C under 5% CO2 for different time points (0, 6, 12, and 24 h). Afterward, the medium was removed and cells were washed thrice with PBS to remove free uninternalized capsules. FITC-cholera toxin B (0.75 μg/mL) in DMEM was added to cell culture plates and incubated for 2 h followed by washing thrice with PBS. After this, cells were fixed

tion,38 silica nanoparticles (600 mg) were sonicated in toluene (106.5 mL) for 20 min and kept for reflux for 24 h after adding 460 μL of 3-(aminopropyl)triethoxysilane (APTES) at 130 °C. APTES-functionalized silica nanoparticles were separated at 3900 rpm for 3 min and washed twice with toluene and with methanol. Drying was done by heating at 80 °C for 16 h. β-Gal enzyme loading was done using the same protocol as that in the case of DS/PA capsules (Section 2.2.1). A layer-by-layer polymer assembly was started with the addition of poly(sodium 4-styrene-sulfonate) (PSS) (1 mg/mL in 0.5 M NaCl) to enzyme-loaded APTES-functionalized silica nanoparticles (10 mg), and the mixture was kept on rotospin for 15 min. This adsorption step was followed by washing thrice the sample with 0.5 M NaCl to remove unbound polymer. Separation of the sample was done at 3900 rpm for 3 min. Subsequently, seven alternate polymeric layers were made of poly(allylamine hydrochloride) (PAH) (1 mg/mL in 0.5 M NaCl) and PSS (1 mg/mL in 0.5 M NaCl) to form eight layers with alternate negative and positive charges. Finally, removal of the silica template was done using HF buffer (0.75 M HF/4 M NH4F). (Caution: HF is very toxic and should be handled with all precautions mentioned in Material Safety Data Sheet.) Centrifugation was performed at 8000 rpm for 3 min, followed by five times washing with deionized water. The prepared β-galPSS/PAH capsules were dispersed in 1 mL of deionized water. 2.2.3. Estimation of β-Gal Loading in Polymeric Capsules. To estimate β-gal enzyme entrapment within capsules, first, the percent enzyme retained on the template was analyzed. After a layer-by-layer synthesis, β-gal enzyme entrapment in polymeric capsules was quantified by mixing an equal volume of polymeric capsules and double strength o-nitrophenyl β-D-galactopyranoside (ONPG) assay buffer using β-galactosidase detection kit (GAL-A) for an hour. The mixture was ultasonicated for 15 min, followed by centrifugation at 14 000 rpm for 10 min at 4 °C. Quantification of β-gal was done as per the protocol provided by the manufacturer. 2.3. Cell Culture Studies. Cell culture studies were done on SV (β-galactosidase gene-deficient mouse fibroblast) and R201C (deficient human β-gal introduced fibroblast, KO mouse-derived SV cells). L929 cell lines were used as wildtype mouse fibroblast as negative control. SV and R201C cells were cultured in low glucose DMEM medium, whereas HeLa and wild-type mouse fibroblast were cultured in medium-tohigh glucose DMEM medium supplemented with FBS (10% v/ v) and penicillin−streptomycin (1% v/v). All of the cells were grown in a CO2 (5%) incubator at 37 °C. 2.3.1. Cytotoxicity and Capsule Uptake Studies of DS/PA Polymeric Capsules. The cytotoxicity of DS/PA capsules was studied on four cell lines (SV, R201C, wild-type mouse fibroblast, and HeLa) using MTT assay. For MTT assay, approximately 104 cells were seeded in each well of a 96-well plate in DMEM (100 μL) and rested for 6 h. After 6 h, different concentrations of DS/PA capsules (0, 25, 50, and 100 capsules/ cell) were added to wells of a 96-well plate. After incubation for 18 h, the medium was removed and MTT dye (0.5 mg/mL) was added followed by incubation of 4 h. After 4 h, MTT dye was removed followed by addition of DMSO (200 μL). The optical density of each sample was analyzed at 570 nm using a UV−vis spectrophotometer. All samples were taken in triplicates. For capsule uptake studies, post-labeled DS/PA capsules with RITC were used to observe their localization inside the cells. DS/PA capsules were incubated overnight with RITC dye 9004

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Figure 1. (a) SEM image of DS/PA capsules and (b) TEM image of DS/PA capsules.

Scheme 2. Schematic Illustration for Reaction of βGalactosidase Enzyme on o-Nitrophenyl β-DGalactopyranoside (ONPG) Artificial Substrate

with 4% formaldehyde solution for 20 min, mounted on glass slides, and observed under a confocal laser scanning microscope. 2.4. Characterization of Polymeric Capsule. The morphology of the samples was acquired from a scanning electron microscope and transmission electron microscope. Layer-by-layer (LBL) polymeric capsules were analyzed for zeta potential values and particle size using dynamic light scattering (DLS) by Malvern zeta sizer. Confirmation of β-gal enzyme loading and quantification of the enzyme in β-gal-DS/PA and β-gal-PSS/PAH were carried out by using ONPG, an artificial β-gal substrate, provided by Sigma GAL-A, β-Galactosidase detection kit. Thermo Scientific Multiskan UV−vis spectrophotometer was used for measuring absorbance at 570 nm for MTT assay. For localization of capsules in cells and to mark a substrate level inside the cells, imaging was done by confocal laser scanning microscopy (Carl Zeiss LSM 710).

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization. Scheme 1 demonstrates the synthesis of β-gal-loaded DS/PA polymeric capsules prepared by the sacrificial template method35,36 that consists of

the following steps: (a) synthesis of CaCO3 template, (b) loading of β-gal enzyme on CaCO3 template, (c) sequential 9005

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Figure 2. Cytotoxicity studies of bare DS/PA capsules with (a) R201C, (b) SV, (c) HeLa, and (d) wild-type mouse fibroblast cell lines.

Figure 3. Uptake studies of DS/PA capsules with (a) SV, (b) R201C, (c) wild-type mouse fibroblast, and (d) HeLa cells after 12 h. (Note: Inset figures show Z-stack images of corresponding cells.)

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Figure 4. (a) Confocal microscopy images of β-gal enzyme release from β-gal DS/PA capsule in HeLa cells at different time points, (b) level of βgalactosidase in HeLa cells at different time points, (c) % release of β-galactosidase from DS/PA capsules at different time points in HeLa Cells, and (d) release of β-gal from DS/PA and PSS/PAH capsule (control sample) at 12 h time point.

charged polymer on CaCO3 template, the LBL assembly was further characterized by zeta potential measurements. Alteration in the surface potential of the CaCO3 template (Figure S4) from positive to negative after each consecutive layer formation verifies the sequential adsorption of oppositely charged polymer. SEM (Figure 1a) and TEM (Figure 1b) images of DS/PA capsules also confirm the formation of capsules with size ranging from 1 to 3 μm. To quantify the loading of β-gal enzyme within DS/PA capsules, we have used ONPG as the assay buffer. Scheme 2 illustrates the reaction of β-galactosidase enzyme over ONPG, an artificial substrate. A yellow color compound, o-nitrophenol (ONP), is formed after hydrolysis of o-nitrophenyl β-Dgalactopyranoside (ONPG) in presence of β-gal enzyme. Confirmation of β-gal loading was done by mixing an equal volume of polymeric capsules (β-gal-DS/PA) and double strength ONPG assay buffer provided in β-galactosidase detection kit (GAL-A) for an hour. Confocal images of treated capsules (β-gal DS/PA, Figure S5A and β-gal PSS/PAH, Figure S5B) demonstrate the formation of a yellow color compound, o-nitrophenol, after hydrolysis of o-nitrophenyl β-D-galactopyranoside (ONPG) and hence confirm the β-gal enzyme loading in β-gal-DS/PA and β-gal-PSS/PAH capsules. The loading of βgal enzyme within DS/PA capsules is found to be 3 mU/100 capsules. 3.2. Cell Culture Studies. To access the delivery competence, polymeric capsules have been analyzed for cytotoxicity, internalization by the cells, and release of enzyme and therapeutic activity at an in vitro level. SV (β-galactosidase gene-deficient mouse fibroblast) and R201C (deficient human

adsorption of oppositely charged dextran sulfate and poly-Larginine polymer on CaCO3 template to form a layer-by-layer assembly, and (d) removal of the template by EGTA solution. We note that the porous and rough surface of CaCO3 template facilitates the loading of β-gal (240 mU/mg). Moreover, the positive surface potential of CaCO3 template (zeta potential +7 mV, in water at pH 7) facilitates the electrostatic deposition of dextran sulfate, a negatively charged polymer, as the first layer, followed by deposition of poly-Larginine as the second layer. This process was repeated to attain eight layers, which makes the capsules structurally stable to prevent the leakage of the enzyme from the capsules. We also note that EGTA is explicitly used as a chelating agent instead of disodium ethylenediaminetetraacetate (EDTA) to dissolve the calcium carbonate template. For the activity of β-gal enzyme over ONPG, an artificial substrate, the presence of Mg++ and Na+ ions is essential, as these ions act as enzyme activators. EGTA preferentially binds with calcium ions (Ca++) and at the same time does not sequester Mg++ ions. Hence, it does not cause inactivation of β-gal enzyme, whereas EDTA sequesters Mg++ ions along with Ca++ ions and causes β-gal enzyme inactivation.39,40 The size and surface morphology of DS/PA capsules were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The SEM image of the CaCO3 template (Figure S1a) clearly shows that the particles are porous with a rough surface. The size of the CaCO3 templates and DS/PA capsules is in the range of 1−3 μm which is further supported by DLS measurements (Figures S2 and S3). To substantiate the sequential adsorption of oppositely 9007

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Figure 5. GM1 ganglioside levels in R201C cells (deficient human β-gal-gene-introduced mouse fibroblast) treated with free β-gal enzyme equivalent to the enzyme loaded in the capsules (top panel, a, c) and 50 β-gal-DS/PA capsules/cells (bottom panel, a) and 100 β-gal-DS/PA capsules/cells (bottom panel, c) at different time points (6, 12, and 24 h). FITC-cholera toxin B was used as a detection probe for GM1 ganglioside accumulation in culture fibroblasts. (b) and (d) represent the corresponding fluorescence intensity. Note: Control is untreated cells, and negative control is cells treated with bare DS/PA capsules (50 and 100 capsules/cell).

Scheme 3. Schematic Illustration of β-Galactosidase Enzyme Activity on GM1 Ganglioside Substrate

capsules possess no cytotoxicity with R201C (a), SV (b), HeLa (c), and wild-type mouse fibroblast (d) cells. To substantiate the capsule internalization inside the cells, RITC-labeled DS/ PA capsules were incubated with cells for 12 h. In Figure 3, red fluorescence exhibited by the RITC-labeled DS/PA capsules, after cellular internalization in SV (a), R201C (b), wild-type mouse fibroblast (c), and HeLa (d), demonstrates that most of the cells have internalized capsules inside. Z-stack images (Figure 3, top inset) state that the red fluorescence due to internalized RITC-labeled capsule also confirms the efficient uptake of capsules inside the cells. Nuclei were stained by DAPI. From the cytotoxicity and capsule uptake studies, it can be concluded that DS/PA capsules can be employed as intracellular delivery cargos in biological systems. 3.2.2. In Vitro Enzyme Release Studies. Figure 4a demonstrates the confocal microscopy images of β-gal enzyme release from DS/PA capsules inside HeLa cells at 6, 12, and 24 h time point (Figure S6). The yellow fluorescence observed in the confocal microscopy images is due to the formation of onitrophenol (ONP), suggesting the release of β-gal from the capsules. The quantification of the enzyme release is given in Figure 4b, and it clearly suggests that the release of enzyme is at the maximum at 12 h time point compared to that at 24 h time point. The reduction in the enzyme release at 24 h time point can be attributed to the consumption of enzyme by the GM1 ganglioside substrates (see Figures 5−7). The release of the enzyme is further confirmed by the quantification of enzyme in cell lysate at 4, 6, 12, and 24 h time point (Figure 4c). We observed the maximum release (77%) at 12 h time point, which

β-gal gene-introduced mouse fibroblast, KO mouse-derived SV cells) were selected as disease model cell lines for cell culture studies. Further, same studies were also done on wild-type mouse fibroblast and HeLa (human cervical adenocarcinoma cells, as a negative control). 3.2.1. Cytotoxicity and Capsule Uptake Studies. The cytotoxicity of DS/PA capsules was studied in all four cell lines: SV, R201C, wild-type mouse fibroblast, and HeLa cells, by using MTT assay to observe the effect of internalized capsules on cell viability. Cells were incubated with DS/PA capsules up to 100 capsules per cell for a period of 18 h, and cell viability was measured. Figure 2 demonstrates that the 9008

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Figure 6. GM1 ganglioside levels in SV cells (β-gal gene-deficient mouse fibroblasts) treated with free β-gal enzyme equivalent to the enzyme loaded in the capsules (top panel, a, c) and 50 β-gal-DS/PA capsules/cells (bottom panel, a) and 100 β-gal-DS/PA capsules/cells (bottom panel, c) at different time points (6, 12, and 24 h). FITC-cholera toxin B was used as a detection probe for GM1 ganglioside accumulation in culture fibroblasts. (b) and (d) represent the corresponding fluorescence intensity. Note: Control is untreated cells, and negative control is cells treated with bare DS/ PA capsules (50 and 100 capsules/cell).

Figure 7. GM1 ganglioside levels in wild-type mouse fibroblast cell line treated with free β-gal enzyme equivalent to the enzyme loaded in the capsules (top panel, a, c) and 50 β-gal-DS/PA capsules/cells (bottom panel, a) and 100 β-gal-DS/PA capsules/cells (bottom panel, c) at different time points (6, 12, and 24 h). FITC-cholera toxin B was used as a detection probe for GM1 ganglioside accumulation in culture fibroblasts. (b) and (d) represent the corresponding fluorescence intensity. Note: Control is untreated cells, and negative control is cells treated with bare DS/PA capsules (50 and 100 capsules/cell). 9009

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Figure 8. GM1 ganglioside levels in treated (50 capsules/cell) and untreated R201C, SV, and wild-type mouse fibroblast cell line.

using Image J software (Figure 5b,d). Figure 5b (50 capsules/ cell) and Figure 5d (100 capsules/cell) clearly suggest that the reduction in GM1 ganglioside is 1.8- and 2.2-fold, respectively, after 24 h β-gal-DS/PA capsules delivery. This result also supports the activity of β-gal enzyme over the substrate. In contrast, Figure 5a (top panel) and Figure 5c (top panel) demonstrate no change in green fluorescence when samples are treated with free β-gal enzyme equivalent to 50 and 100 capsules concentrations, respectively, at time intervals which support the need of capsules as the delivery vehicle. This is further supported by the graph (black bar) in Figure 5b,d. From these results, it can be articulated that reduction in both green fluorescence and intensity per cell shows decline in GM1 ganglioside substrate level in R201C cells when treated with β-gal-DS/PA capsules at different time intervals. R201C cell line, being deficient human β-galactosidase gene-introduced β-galactosidase KO mouse fibroblast, is not able to express βgal enzyme on its own because the gene responsible for expressing β-gal enzyme is lacking. Here, it could be speculated that this enzyme activity is due to the β-gal enzyme that is being released by β-gal-DS/PA capsules inside the cells. β-Gal enzyme in its free form has not been taken up by the cells. This might be due to poor membrane permeability and deprived uptake of β-gal by cells; hence, no cutback is seen in GM1 ganglioside substrate level. Similar results were observed when SV β-gal gene-deficient mouse fibroblast (Figure 6a−d) and wild-type mouse fibroblast (Figure 7a−d) were treated in the same manner. No reduction in the GM1 ganglioside substrate level was seen when the enzyme was delivered in free form. In contrast, β-gal delivered by DS/PA capsules show 3.4- and 1.9-fold reduction in GM1 Ganglioside at 50 and 100 capsules per cell in SV cells. Similarly, 2.8- and 3.1-fold reduction in β-gal substrate was observed at 50 and 100 capsules per cell in wild-type mouse fibroblast cells. Herein, it can be concluded that β-gal enzyme delivered by DS/PA capsules in all three cell lines (R201C, SV, and wild-type mouse fibroblast) shows a significant reduction in the substrate level after a time interval. This shows that enzyme cargos in the form of polymeric capsule are first efficiently taken up by the cells and enzyme is being released from DS/PA capsules by inherent biodegradation and also same results were observed at two different concentrations (50 and 100 capsules

further confirms the above observation. To emphasize the releasing capability of DS/PA capsules, we have done the similar release studies (12 h time point) with nondegradable PSS/PAH capsules as the control experiment (Figure 4d), in which PSS/PAH capsules show very minimal release (18%). The control capsules have been prepared using silica as the template, which has also been characterized by scanning electron microscopy (Figure S1B,C) and DLS measurement (Figures S7 and S8). 3.3. Enzyme Activity Studies. Enzyme is a biocatalyst that binds with specific substrate molecules and undergoes a chemical reaction to convert it into a new product. Scheme 3 illustrates the reaction of β-galactosidase enzyme over GM1 ganglioside. β-Gal, being a glycoside hydrolase enzyme, catalyzes the hydrolysis of β-galactoside into monosaccharide by breaking of the glycosidic bond. Figures 5−7 demonstrate the enzyme activity studies of β-gal-DS/PA capsules on R201C, SV, and wild-type mouse fibroblast, respectively. We note that FITC-cholera toxin B specifically binds to GM1 ganglioside and this has been used as track tracer41−46 to mark the level of GM1 ganglioside inside the cell. The binding event is monitored by the observation of green fluorescence from the cells using a confocal microscope. Two hundred cells were quantified for green fluorescence produced by FITC-labeled cholera toxin B using Image J software. Figure 5a,c shows the confocal images and Figure 5b,d shows the fluorescence intensity per cell of R201C cells when treated with β-gal-DS/PA capsules at two different concentrations, 50 capsules (Figure 5a) and 100 capsules (Figure 5c) at 0, 6, 12, and 24 h time intervals. R201C cells were also treated with the equivalent free β-gal enzyme as a control experiment and bare DS/PA capsules in equivalent capsule concentration as a negative control experiment. Confocal images of R201C cells treated with β-gal-DS/PA capsules (50 capsules/cell (Figure 5a) and 100 capsules/cell (Figure 5c)) demonstrate the significant reduction in green fluorescence at 6, 12, and 24 h interval. The decrease in green fluorescence suggests the reduction in GM1 ganglioside level inside cells, which is attributed to the activity of β-gal enzyme over the GM1 ganglioside substrate. It was also found that the reduction in the GM1 ganglioside level increases with time. To roughly estimate the reduction in the GM1 ganglioside level, we have quantified the green fluorescence intensity per cell 9010

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concentration). Figure 8 reveals the level of GM1 ganglioside in treated as well as untreated cells of R201C (black bars), SV (red bars), and wild-type mouse fibroblast (blue bars). The fluorescence intensity per cell of untreated cells is higher in the case of positive control (SV and R201C) than in negative control (wild-type mouse fibroblast), which states the higher level of GM1 ganglioside in SV and R201C as compared to that in wild-type mouse fibroblast. We observed 1.8-, 3.4-, and 2.8fold reduction in GM1 ganglioside in treated R201C, SV, and wild-type mouse fibroblast cells, respectively, at 50 capsules per cell concentration. This confirms the release and therapeutic activity of β-gal enzyme inside the cells through DS/PA capsules. Moreover, the level of GM1 ganglioside substrate in gene-deficient cell lines is reduced to a normal endogenous level that is present in untreated wild-type mouse fibroblast cells.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.7b01230. Zeta potential measurement, DLS, SEM images of calcium carbonate, silica, and PSS/PAH capsules, and confocal images of β-gal enzyme-loaded DS/PA and PSS/PAH capsules (PDF)

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REFERENCES

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4. CONCLUSIONS We report an efficient intracellular delivery of β-gal enzyme by arginase-responsive dextran sulfate/poly-L-arginine layer-bylayer polymeric capsules (DS/PA capsules) for the therapy of GM1 gangliosidosis. The in vitro therapeutic activity of β-gal has been evaluated in SV (β-galactosidase gene-deficient mouse fibroblast), R201C (deficient human β-galactosidase geneintroduced mouse fibroblast), and wild-type mouse fibroblast. Significant reduction in the GM1 ganglioside substrate level present in all of the three cell lines (R201C, SV, and wild-type mouse fibroblast) is observed in the case of β-gal enzyme delivered through DS/PA capsules, whereas the free form showed no reduction. Furthermore, the level of the GM1 ganglioside substrate in gene-deficient cell lines is reduced to a normal endogenous level that is present in untreated wild-type mouse fibroblast cells.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +91-512-2597697. Fax: 91-5122590104. ORCID

Sri Sivakumar: 0000-0002-6472-2702 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was conducted in Indian Institute of Technology, Kanpur (India). We acknowledge Swati Singh (IIT Kanpur) and Neetu Dey (IIT Kanpur) for their help in imaging. We acknowledge Nanomission, Department of Sciences and Technology, Department of Biotechnology (DBT) for providing grant for this work. We also thank Dr. Jayandharan G. Rao, Associate Professor (BSBE, IIT Kanpur), for valuable suggestions and providing backup for cell storage. 9011

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