Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Hyaluronic Acid Microgels as Intracellular Endosomolysis Reagents Kishore Raghupathi,† Matthew Skinner,† Grace Chang, Cristin Crawley, Takako Yoshida-Moriguchi, Peter Pipenhagen, Yunxiang Zhu, Luis Z. Avila, Robert J. Miller, and Pradeep K. Dhal* Sanofi Global R&D, 153 Second Avenue, Waltham, Massachusetts 02451, United States ABSTRACT: Hyaluronic acid (HA) microgels were investigated as biocompatible and biodegradable reagents for facilitating endosomolysis in human cells. Employing inverse emulsion templates, HA microgels were prepared by crosslinking aqueous sodium hyaluronate droplets with divinyl sulfone (DVS). Introduction of ether sulfone cross-links was confirmed by infrared (IR) spectroscopy and elemental analysis. The degree of cross-linking of the microgels was estimated using high performance liquid chromatography (HPLC). The size distribution of the water-dispersible HA microgels was studied by laser diffraction analysis, and the gel morphology was investigated using scanning electron microscopy (SEM). Aqueous microgel suspensions were found to be well-tolerated in human cells at concentrations of up to 100 μg/mL. Endosome-rupturing properties of the HA microgels were evaluated in vitro using calcein internalization and Cre protein delivery assays. The results of this study serve as a proof-of-principle for the utility of cross-linked HA microgels as a new class of biocompatible and biodegradable endosomolytic reagents. KEYWORDS: hyaluronic acid, microgel, endosomal escape, endosomolysis
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INTRODUCTION Trafficking into the cellular cytoplasm is essential for the efficacy of many pharmaceuticals, yet efficient cytosolic distribution remains a significant challenge in the field of intracellular delivery of biopharmaceutical drugs. Intracellular uptake of therapeutic agents typically occurs via endocytosis, a pathway through which molecules cross the cellular membrane by encapsulation in endosomal vesicles. In the cytoplasm, endosomes mature and, if not disrupted, fuse with lysosomes, which metabolize encapsulated components.1,2 In light of the degradative function of lysosomes, endosomal rupture and escape prior to lysosomal fusion is critical for retaining the therapeutic activity of a range of pharmaceuticals, especially proteins and oligonucleotides, which are particularly prone to lysosomal degradation.1 Numerous reagents have been developed for facilitating endosome disruption, including peptides,1,3−7 lipids,3,8,9 viral glycoproteins,10 and synthetic polymers that respond to the acidic microenvironments of endosomes and lysosomes.1,3,11−16 Although effective in vitro, most of these reagents are too cytotoxic and/or immunogenic to be used in vivo.17−21 Amphiphilic anionic polymers, such as poly(2-ethylacrylic acid), poly(2-propylacrylic acid), and poly(2-butylacrylic acid), have been examined as biocompatible reagents to trigger endosome disruption.22−26 These water-soluble polymeric amphiphiles are protonated in acidic endosomal compartments, affording hydrophobic materials which fuse with lipid membranes and lyse endosomes. Despite the viability of these synthetic materials as nontoxic endosomolytic reagents, their nonbiodegradable backbone limits their therapeutic utility. © XXXX American Chemical Society
Thus, there is a need for biodegradable alternatives to such endosome-disrupting materials. Hyaluronic acid (HA) is a carboxylate-rich carbohydrate polymer composed of glucuronic acid and N-acetyl-glucosamine moieties. Constituting major portions of the extracellular matrix, skin, and articular cartilage, HA is nontoxic, nonimmunogenic, and biodegradable.27,28 These attributes make HA an ideal biomaterial for biomedical applications.29 Notable HA-based biomedical products include surgical adhesion barriers,30 viscosupplements,31 and dermal fillers.32 Because HA readily undergoes decomposition through enzymatic and free radical pathways,33,34 cross-linking is typically employed to extend in vivo residence time and afford HA hydrogels with tunable mechanical properties.35 To expand the toolbox of materials suitable for facilitating endosomal escape, we sought to investigate cross-linked micron- and sub-micron-sized HA hydrogels (i.e., microgels) as a new class of biodegradable endosomolytic reagents. Although functionalization of linear HA with polyamines has previously been shown to impart endosome-disrupting properties,36 the very short half-life of HA in the bloodstream precludes the use of soluble HA derivatives for sustained delivery applications.32 Water-dispersible HA microgels, on the other hand, are more robust than soluble HA, allowing their use as matrices for controlled and extended release of a variety of pharmaceuticals.37,38 The presence of a high density of pHresponsive glucuronic acid units on the HA backbone led us to Received: December 8, 2017 Accepted: January 10, 2018
A
DOI: 10.1021/acsbiomaterials.7b00966 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 1. Synthesis of HA microgels by DVS cross-linking of w/o emulsion droplets.
Figure 2. (a) Representative SEM micrograph of HA microgels; (b) particle size distribution of HA microgels suspended in physiological saline.
780 nm. The difference in microgel size determined by SEM and laser diffraction may be attributed to swelling of the hydrophilic HA matrix in water. In comparison, the hydrodynamic size of the native HA in aqueous solution measured using dynamic light scattering (DLS) was found to be approximately 8 nm. Using this facile templated cross-linking strategy, HA microgels were prepared in high yield as waterdispersible spherical particles with a mean size less than 1 μm. Molecular Characterization of Cross-Linked HA Microgels. Chemical cross-linking of the HA microgels with DVS was assessed by infrared (IR) spectroscopy and elemental analysis. IR spectra of native HA and its cross-linked microgel counterpart are shown in Figure 3. In both spectra, character-
consider that HA microgels could lyse endosomes by membrane fusion following uptake and protonation in these acidic cellular compartments. HA microgels were synthesized by optimizing a previously described microemulsion strategy,39,40 with cross-linking achieved at room temperature using divinyl sulfone as the cross-linking agent. The chemical composition of the resulting particles was evaluated by spectroscopy, elemental analysis, and chromatography. The size of the dispersible microgels was estimated by laser diffraction analysis and electron microscopy. The role of HA microgels in facilitating endosomal escape was demonstrated using confocal microscopy and protein delivery assays.
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RESULTS AND DISCUSSION HA Microgel Synthesis. Chemically cross-linked HA microgels were prepared by optimizing an emulsion-based cross-linking process.39,40 As shown in Figure 1, an aqueous solution of sodium hyaluronate was dispersed in a mixture of Brij L4 surfactant and n-heptane, affording a stable water-in-oil (w/o) emulsion that was homogenized by sonication and shear. The resulting HA droplets were cross-linked with divinyl sulfone (DVS) via base-mediated oxa-Michael addition of primary hyaluronate alkoxide moieties to vinyl sulfone groups, a chemical strategy frequently employed for HA hydrogel synthesis.41,42 Unreacted cross-linker and surfactant were subsequently removed by precipitating and triturating the microgels in acetone. After vacuum drying, the HA microgels were obtained as white powders in an overall yield of >50%. Microgel morphology and size were assessed using scanning electron microscopy (SEM) and laser diffraction analysis, as shown in Figure 2. SEM images suggest that the microgels were obtained as spherical particles with a broad size distribution and a mean size of 560 nm. Laser diffraction analysis of microgel suspensions in physiological saline confirmed the broad nature of the particle size distribution and gave a mean particle size of
Figure 3. Representative IR spectra of unmodified HA and HA microgels.
istic peaks corresponding to pendent carboxylate and N-acetyl moieties were observed at 1614, 1558, and 1405 cm−1, along with a peak at 1036 cm−1 corresponding to glycosidic backbone linkages.43,44 In the IR spectrum of the microgel, unique signals were observed at 1284 and 1310 cm−1 which were attributed to C−O and SO stretching modes, respectively, of ether sulfone bonds formed as a result of the reaction between pendent hydroxyl groups of HA and vinyl sulfone groups.45,46 Results of the elemental analysis of three different microgel samples are summarized in Table 1. The results showed that the mean B
DOI: 10.1021/acsbiomaterials.7b00966 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering Table 1. Elemental Analysis Results for Three Batches of DVS Cross-Linked HA Microgels HA microgel batch
carbon content (wt %)
hydrogen content (wt %)
nitrogen content (wt %)
sulfur content (wt %)
N:S molar ratio
1 2 3 avg
39.5 34.9 35.5 36.6
5.9 5.3 5.0 5.4
3.0 2.4 2.5 2.6
1.2 1.0 1.3 1.2
5.7:1 5.5:1 4.4:1 5.2:1
sulfur incorporation in the microgel matrix was 1.2 wt %, corresponding to an average nitrogen to sulfur (N:S) molar ratio of 5.2:1. This data further confirmed vinyl sulfone mediated cross-linking and suggests that vinyl sulfone moieties were introduced at every fifth HA repeat unit. The batch-tobatch variation in sulfur content was minimal, demonstrating that microgel synthesis using the emulsion cross-linking strategy is a reproducible process. The degree of cross-linking of the HA microgels was estimated by hyaluronidase enzymolysis of the gels followed by high performance liquid chromatography (HPLC) characterization. Owing to differences in charge density and molecular weight, oligomers formed from hyaluronidase digestion of HAbased materials can be separated chromatographically and detected by the UV−vis absorbance of unsaturated chain-end functionality. As shown in Figure 4a, complete digestion of soluble HA by Streptomyces-derived hyaluronidase enzyme affords a binary mixture of tetrameric and hexameric oligosaccharides bearing δ-4,5-alkene moieties.47 Enzymolysis of DVS-cross-linked HA, on the other hand, yields a more complex mixture of tetramers, hexamers, and fragments bearing pendent vinyl sulfone groups and ether sulfone-linked oligosaccharides (Figure 5). Using a calibration standard generated from incompletely digested HA, the identities of
Figure 5. Chemical structure of DVS-modified HA fragments: (a) single point modification, and (b) cross-linking modification.
these higher molecular weight fragments were readily determined. As shown in the chromatogram of the HA microgel digestion product (Figure 4b), the tetrameric and hexameric species were observed at characteristic retention times of 6.1 and 14.2 min, respectively. Species eluting between 6.1 and 14.2 and 14.2−18.5 min were attributed to single point vinyl sulfone-modified tetramers and hexamers, respectively. Peaks eluting at retention times longer than that of the octameric species (21.9 min) were identified as DVS-crosslinked fragments. By comparing the peak areas of modified and cross-linked species, the degrees of monofunctional modification and cross-linking by DVS were estimated to be approximately 2.8 and 5.0%, respectively. These findings suggest that hyaluronidase digestion followed by HPLC analysis is a valuable tool for analyzing the composition and crosslinking densities of HA microgels. Furthermore, these results suggest that the inverse emulsion synthesis affords lightly crosslinked HA microgels. Assessment of the Biocompatibility of HA Microgels. The biocompatibility of these HA microgels was assessed using
Figure 4. Representative HPLC chromatograms of (a) soluble HA after complete (left) and incomplete (right) hyaluronidase digestion, and (b) HA microgels after hyaluronidase digestion. C
DOI: 10.1021/acsbiomaterials.7b00966 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering human embryonic kidney cells (HEK 293F). The cells were exposed to microgel suspensions at concentrations of 25, 50, and 100 Åg/mL. After 48 h of incubation, viability of the cells was assessed using a CellTiter-Glo luminescence assay. Cells exposed to native growth media were used as a negative control. At all three microgel concentrations, viability was comparable to that of cells incubated with the growth media control, as shown in Figure 6, suggesting that the gels are
Scheme 1. Proposed mechanism of action for the endosomolytic activity of HA microgels
Figure 6. Viability of human embryonic kidney cells incubated with suspensions of HA microgels.
exposed to media containing Hoechst stain or a mixture of Hoechst stain and 0.25 mM calcein. As shown in Figure 7, cells incubated with the Hoechst stain showed a characteristic blue signal corresponding to labeled nuclei, while cells incubated with the calcein fluorophore exhibited green punctate spots attributed to calcein localized in endosomal vesicles. On the other hand, cells incubated with the Cy5-functionalized HA microgels (pink signal) showed colocalized microgel and calcein fluorescence diffused throughout the cellular cytosol, an observation attributed to endosomal escape and cytosolic release of calcein. These results suggest that the HA microgels readily undergo endocytosis and facilitate endosomal disruption in these mammalian cells. Although, the calcein release assay provides evidence supporting the endosomolytic characteristics of HA microgels, confocal microscopy is limited to a qualitative assessment of such phenomenon. In order to quantitatively evaluate the endosomolytic activity of HA microgels, we set out to perform an in vitro Cre protein delivery assay. In this assay, human embryonic kidney cells (HEK 293F) expressing green fluorescence protein (GFP) were transformed to incorporate a genomic Cre recombination response. In the absence of the Cre protein, the cells stably express GFP. On the other hand, nuclear introduction of the Cre enzyme results in site-specific lox-P cleavage, a scission event which causes expression of red fluorescence protein (RFP). As this change in protein expression inherently depends on endocytosis of Cre and release into the cellular cytosol, endosomolytic activity can be quantified using flow cytometry by measuring the percentage of cells expressing RFP. To assess the endosomolytic activity of HA microgels, GFP-expressing HEK 293F cells were incubated in media containing 50 nM Cre protein and microgels at concentrations of 25, 50, and 100 mg/mL. A negative control experiment was performed by incubating cells in the presence of Cre protein alone. After a two-day incubation, the cells were analyzed by flow cytometry to quantify the percentage of cells expressing RFP. While only one percent of cells incubated with the Cre protein alone expressed RFP (Figure 8), cells incubated in the presence of the HA microgels showed a significant increase in RFP expression, with 17−23% of the cells exhibiting
biocompatible. Furthermore, these results suggest that the purification procedure employed for microgel isolation effectively removed residual DVS and surfactant from the reaction mixture. In light of this promising biocompatibility, we subsequently set out to evaluate the endosomolytic characteristics of the HA microgels using in vitro cellular uptake and protein delivery assays. In Vitro Endosomolytic Activity of HA Microgels. Protonatable glucuronate functionality imparts pH-responsive characteristics to the HA microgels. This feature led us to hypothesize that HA microgels could potentially be utilized as endosomolytic reagents. At physiological pH, pendent carboxylate moieties of HA are essentially deprotonated, affording water-dispersible hydrophilic microgels. Following endocytosis, the glucuronate groups can be protonated in the acidic environment of endosomes, leading to an increase in the hydrophobicity of the microgels. As observed with poly(alkyl acrylic acid)s,22−26 increased hydrophobicity would impart HA microgels with sufficient amphiphilicity to facilitate endosomal membrane fusion and disruption, as shown in Scheme 1. The endosomolytic activity of the HA microgels was evaluated using fluorescence confocal laser scanning microscopy (CLSM) and an in vitro calcein endosomolysis assay.48 For this purpose, HA microgels bearing covalently bound cyanine5 (Cy5) fluorophores were prepared as shown in Scheme 2 by amidation of glucuronate moieties with an aminofunctionalized Cy5 dye. The microgel functionalization was mediated using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), an acylating agent which has been found to be superior to traditional water-soluble carbodiimide reagents.49 Upon completion of the dye coupling reaction, the Cy5-labeled gels were extensively dialyzed in saline to remove any unconjugated dye and were isolated by lyophilization as a fluffy blue powder. The Cy5-functionalized HA microgels were incubated with human umbilical vein endothelial cells at a concentration of 400 μg/mL in the presence of nuclear Hoechst stain and 0.25 mM calcein, a water-soluble fluorophore that undergoes fluid-phase endocytosis. After 18 h of incubation, the cells were visualized using fluorescence CLSM and compared to control cells D
DOI: 10.1021/acsbiomaterials.7b00966 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering Scheme 2. Synthesis of Fluorescently Labeled HA Microgels
Figure 7. Fluorescence CLSM images of human umbilical vein endothelial cells incubated in media containing (a) Hoechst stain; (b) Hoechst stain and 0.25 mM calcein; and (c) Hoechst stain, 0.25 mM calcein, and 400 mg/mL fluorescent HA microgels.
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CONCLUSION This manuscript describes the role of HA microgels as biocompatible endosomal escape reagents. Water-dispersible HA microgels with submicron hydrodynamic diameters were prepared using an microemulsion cross-linking strategy and were systematically characterized by IR spectroscopy, elemental analysis, SEM, and laser light scattering. Furthermore, the degree of cross-linking was estimated by diagnostic HPLC characterization of enzymatically degraded gels. In human umbilical vein endothelial cells, the HA microgels were found to lyse endosomal compartments and mediate cytosolic calcein release. Endosome disruption was further demonstrated and quantified using an in vitro Cre protein delivery assay. The underlying mechanism behind the endosomolytic properties of these anionic microgels has been attributed to fusion of HA microgels with endosomal membranes followed by protonation of the glucuronate groups within acidic endosomal microenvironments. Though additional work is warranted to fully elucidate the endosomolytic mechanism, these results nevertheless reveal the utility of HA microgels as potential biocompatible and biodegradable endosome disrupting reagents. Additionally, these findings demonstrate the versatility of HA-based materials as promising vectors for intracellular delivery of biopharmaceuticals.36,50−52
Figure 8. Percentage of RFP-expressing human embryonic kidney cells following incubation with 50 nM Cre protein and polyhistidine or HA microgels.
a change in protein expression. Interestingly, the endosomolytic activity of the HA microgels decreased with increasing microgel concentration. We attribute this result to possible extracellular electrostatic complexation between cationic Cre proteins and anionic HA microgels in the culture media, a phenomenon which would reduce overall Cre uptake. The results of this protein delivery assay further demonstrate the endosomolytic characteristics of HA microgels in mammalian cells and suggest that endosomolysis can be achieved effectively in cells at lower microgel concentrations.
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EXPERIMENTAL SECTION
Materials. Hyaluronic acid (HA) with an average molecular weight of 200 000 g/mol was produced by bacterial fermentation, followed by controlled radiation53 and obtained as a powder. Sodium hydroxide (NaOH) pellets, disodium succinate hexahydrate, and sodium chloride (NaCl) were purchased from EMD Millipore. HPLC grade water was purchased from Fischer Scientific. Propylene glycol was obtained from Alfa Aesar. Brij L4, n-heptane, divinyl sulfone (DVS), N-methyl morpholine, acetone, ethanol (EtOH), and Tween 20 were purchased E
DOI: 10.1021/acsbiomaterials.7b00966 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering from Sigma-Aldrich. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was purchased from TCI America. Cyanine5 amine (Cy5-amine) was obtained from Lumiprobe Corporation. Streptomyces-derived hyaluronidase enzymes were obtained from CalBiochem. High glucose Dulbecco’s modified Eagle’s Medium (DMEM) [5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 1x glutaMAX, and 100 μg/mL penicillin-streptomycin], high glucose DMEM containing 10% FBS, serum-free high glucose DMEM, trypsin-EDTA solution, Luria−Bertani (LB) broth, kanamycin, heparin, One Shot BL21 Star (DE3) chemically competent E. coli, and FreeStyle 293F cells were obtained from ThermoFisher Scientific. A CellTiter-Glo luminescent cell viability assay kit was purchased from Promega and LoxP Green Fluorescence Protein (GFP)/Red Fluorescence Protein (RFP) ColorSwitch lentivirus (Puro) was obtained from Amsbio. Unless otherwise noted, all chemicals were used as received without further purification. Purification of +36GFPCre protein was performed using a HisTrap FF purchased from GE Healthcare Life Sciences. Instrumentation. Fourier-transform infrared spectroscopy measurements were carried out using a ThermoFisher Scientific Nicolet spectrophotometer equipped with an attenuated total reflectance (ATR) accessory. High performance liquid chromatography (HPLC) was performed using an Agilent system, fitted with an AS3000 auto sampler, Spectra 200 programmable wavelength detector (λ = 232 nm), a CarboPac PA100 guard column (4 × 50 mm), and a CarboPac PA100 LC column (4 × 250 mm). Analytes were eluted in water/0.4 M sodium phosphate buffer (pH 5.8) using a 50 min step-linear gradient at a flow rate of 0.8 mL/min. Dynamic light scattering (DLS) measurements were performed using a Malvern Zetasizer NanoZS. Laser diffraction analysis of HA microgel suspensions was performed using a Malvern Mastersizer 3000 equipped with a Hydro MV dispersion unit operating at 2000 rpm. SEM measurements were performed using an AMRAY 3300FE Field Emission SEM. For SEM analysis, HA microgels were fixed on aluminum stubs and sputter coated with gold. Water-in-oil (w/o) emulsions were homogenized using an IKA T10 Basic Ultra-Turrax hand-held dispersion unit, and probe sonication was performed using a Qsonica Q125 ultrasonic homogenizer operated at 100% intensity. Elemental analyses were performed at Intertek Pharmaceutical Services (Whitehouse, NJ). Synthesis of HA Microgels. Water-dispersible HA microgels were synthesized using a modified literature procedure.39,40 Using a positive displacement pipet, 0.5 mL of an HA solution in aqueous 0.2 M NaOH (at a concentration of 200 mg/mL) was slowly added to nheptane (7.36 mL) containing Brij L4 (0.64 mL). This mixture was vortexed, and the resulting w/o emulsion was probe sonicated for one minute at room temperature. The emulsion was subsequently cooled to 0 °C and homogenized for 15 min using an Ultra-Turrax dispersion unit operating at 11,000 rpm. DVS (44 μL) was added to the emulsion and homogenization was continued at 0 °C. After 2 h, the emulsion was precipitated into acetone (30 mL), and the resulting solid was triturated twice with acetone to remove residual surfactant and DVS. The microgels were dried under reduced pressure and were obtained as fine white powders in yields greater than 50%. Microgels used for cell culture were suspended in deionized water (20 mL), dispersed by probe sonication (30 s, room temperature), mixed gently at room temperature for an additional 30 min, and isolated by centrifugation. This washing procedure was repeated twice and the microgels were isolated by lyophilization as fluffy white powders. Fluorescent Labeling of HA Microgels. HA microgels (46.2 mg) were added to 5 mL of aqueous NaCl (0.9% w/w) solution and dispersed by probe sonication (two minutes, room temperature). To this suspension was added 2.5 mL of ethanol, and the pH of the resulting suspension was adjusted to a value of 6.2−6.5 using 1 M HCl (aq). A solution of DMT-MM (1.9 mg) dissolved in 1.5 mL of ethanol was added to the microgel suspension and the reaction mixture was agitated at room temperature for one hour. Cy5-amine (1.9 mg) dissolved in 1 mL of ethanol was added to the reaction mixture and the pH was adjusted to a value of 6.2−6.5 using solutions of 10% ethanolic N-methylmorpholine and 1 M HCl (aq). The resulting reaction mixture was agitated in dark at room temperature for 16 h. At
the end of this time, the pH of the reaction mixture was adjusted to a value
