RESEARCH ARTICLE – Pharmaceutical Nanotechnology
Protein Nanoparticles for Intracellular Delivery of Therapeutic Enzymes LINA HERRERA ESTRADA, STANLEY CHU, JULIE A. CHAMPION Department of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia Received 22 November 2013; revised 7 March 2014; accepted 25 March 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23974 ABSTRACT: The use of enzymes as therapeutics is very promising because of their catalytic activity and specificity. However, intracellular delivery of active enzymes is challenging due to their low stability and large size. The production of protein-enzyme nanoparticles was investigated with the goal of developing a protein carrier for active enzyme delivery. -Galactosidase (-gal), an enzyme whose deficiency is the cause of some lysosomal storage disorders, was incorporated into enhanced green fluorescent protein nanoparticles prepared via desolvation. Particle size was found to be sensitive to the type of cross-linker, cross-linking time, and the presence of imidazole. The results indicate that -gal activity is highly retained (>70%) after particle fabrication and >85% of protein is incorporated in the particles. Proteinenzyme nanoparticles exhibited higher internalization in multiple cell lines in vitro, compared with the soluble enzyme. Importantly, -gal retained its activity following intracellular delivery. These data demonstrate that protein nanoparticles are a biocompatible, highC 2014 Wiley Periodicals, Inc. and the American Pharmacists efficiency alternative for intracellular delivery of active enzyme therapeutics. Association J Pharm Sci Keywords: therapeutic enzymes; protein delivery; nanoparticles; intracellular delivery; enzyme activity; cross-linking; desolvation; macromolecular drug delivery; particle size
INTRODUCTION Enzyme therapeutics offer multiple advantages over small molecule drugs. Enzymes can bind their targets with high affinity and act with high specificity.1 Most importantly, an enzyme’s catalytic activity allows it to rapidly convert multiple target molecules into products, which significantly amplifies its effect compared with that of small molecules.2,3 These unique properties make enzymes promising therapeutic agents, and an important and growing segment of pharmaceuticals. Although most targets of United States Food and Drug Administration-approved enzymatic drugs are extracellular, there are many diseases for which treatment requires intracellular delivery.4 Effective enzyme replacement for genetic diseases such as lysosomal storage diseases requires intracellular delivery to lysosomal compartments.2,5 More therapeutic applications of intracellular enzymes are expected to emerge, including inhibition or reversal of ubiquitination for cancer6 and phosphorylation of Tau protein for Alzheimer’s disease.7 Enzymes can effectively perform these functions,8–11 but require modification, encapsulation, or immobilization on biocompatible matrices to improve their stability and limited distribution.1,2,12 Hence, there is a critical need to develop delivery systems that will improve accessibility of enzymatic drugs to intracellular compartments.
Abbreviations used: eGFP, enhanced green fluorescent protein; $gal, $-galactosidase; GTA, glutaraldehyde; DTSSP, 3,3 3 dithiobis(sulfosuccinimidylpropionate); BS , bis(sulfosuccinimidyl)suberate; ONPG, ortho-nitrophenyl-D-galactopyranoside. Correspondence to: Julie A. Champion (Telephone: +404-894-2874; Fax: +404385-2713; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com/. Journal of Pharmaceutical Sciences
C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
There are many challenges associated with the delivery and retention of activity, given the large size of enzymes and their complex tertiary or quaternary structure that can be highly sensitive to the environment.2 A delivery vehicle must be produced in conditions gentle enough for activity retention and be small enough to penetrate tissues and be internalized by cells.13,14 The size of delivery vehicles should be in the nanometer range, but the exact size depends on the application. Enzymes packaged in a delivery vehicle possess several advantages over soluble formulations, such as higher stability, lower immune response and targeting capabilities.12,13,15–19 Most delivery systems immobilize enzymes on the surface of another material or encapsulate them in polymeric, lipid, or mesoporous materials.2,4,20–25 Other methods involve fusion of an enzyme to other proteins or peptides.26 However, challenges remain, such as low encapsulation efficiencies, reduction in activity from fabrication conditions, or undesirable degradation products.27 A biodegradable, alternative method is to use protein particles for delivery of enzymes. Protein particles offer multiple advantages over their polymeric, inorganic, and liposomal counterparts including their high loading capacity, ease of production, surrounding protein environment, and amino-acid degradation products.28,29 Most protein particles are studied for intracellular delivery of small molecule drugs,29–35 only a few protein drugs have been investigated.36,37 Little is known about the production or function of enzyme nanoparticles. Some proteins and enzymes have been encapsulated in albumin microspheres38,39 or crystallized and cross-linked40,41 but the large size of these particles hinders efficient delivery and intracellular uptake. Recently, particles made directly with glucose oxidase have been shown to be more efficient as biosensors than immobilized enzyme on the surface of gold nanoparticles.42 In this study, we report the production of protein particles as enzyme carriers that maintain enzymatic activity and increase cellular uptake. For this purpose, we studied Estrada, Chu, and Champion, JOURNAL OF PHARMACEUTICAL SCIENCES
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RESEARCH ARTICLE – Pharmaceutical Nanotechnology
Figure 1. Schematic diagram of the desolvation process. Ethanol is added drop by drop to a solution of carrier protein and enzyme. The protein and enzyme precipitate into nanoparticles that are stabilized by cross-linking.
desolvation and size tuning of enhanced green fluorescent protein (eGFP) as an enzyme carrier. eGFP has the added advantage of fluorescence for intracellular delivery tracking and imaging. We incorporated $-galactosidase ($-gal), a hydrolase homo-tetramer, whose deficiency is the cause of lysosomal storage disorders GM1 gangliosidosis and Morquio B disease43 into eGFP nanoparticles and show retention of enzymatic activity. In multiple cell types, we found that uptake of particles was greater than uptake of soluble protein. Finally, we successfully demonstrate intracellular delivery of active enzyme in vitro and the potential of protein nanoparticles as therapeutic enzyme drug carriers.
linking reaction was stopped by centrifugation at 1000g for 1 min and removal of supernatant. Particles were resuspended in phosphate buffered saline (PBS, 10 mM NaH2 PO4 , 137 mM NaCl, 2.7 mM KCl, 2 mM KH2 PO4 ; pH 7.4) and sonicated on ice for 1 s every 15 s at 30% amplitude, for 5 min. To determine the yield, the amount of protein not precipitated during desolvation was measured by BCA protein assay (Pierce). Following centrifugation, the concentration of protein in the supernatant was measured and the yield was estimated to be 100% minus the percentage of protein in the supernatant. Data presented are the arithmetic mean of the yield of three samples. Determination of Particle Size and Zeta Potential
MATERIALS AND METHODS Materials and Cell Lines $-Galactosidase (from Escherichia coli, lyophilized powder >500 U/g) and glutaraldehyde (GTA, 8% solution) were purchased from Sigma–Aldrich (Saint Louis, Missouri). 3,3´-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and bis(sulfosuccinimidyl)suberate (BS3 ) were obtained from Pierce (Rockford, Illinois). eGFP was expressed in E. coli and purified with Ni-NTA agarose (Qiagen, Valencia, California) (Fig. S1, Supporting Information). HeLa, SK-BR-3, and NIH/3T3 cells were purchased from American Type Culture Collection (Manassas, Virginia). HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS). SK-BR-3 cells were grown in McCoy 5A Media and supplemented with 10% FBS. 3T3 cells were cultured in DMEM and supplemented with10% calf serum. All media were supplemented with 1% (v/v) penicillin/streptomycin and cells were incubated at 37◦ C in a 5% CO2 humidified atmosphere. Preparation of Particles Protein particles were prepared by desolvation as described by Weber et al.44 Briefly, 6 mg/mL of protein (pure eGFP or 1:24 enzyme:eGFP) was dissolved in water, NaCl solution or imidazole solution (0–250 mM in a 300 mM NaCl, 50 mM NaH2 PO4 buffer; pH 8). Hundred microliters of protein solution was desolvated by continuous, drop-by-drop addition of 400 :L ethanol or acetone at a rate of 1 mL/min (Fig. 1). After desolvation, cross-linker was added at a ratio of cross-linker to lysines of 1:2.2. After 2 h stirring, unless otherwise stated, the crossEstrada, Chu, and Champion, JOURNAL OF PHARMACEUTICAL SCIENCES
Particle size distribution was measured by dynamic light scattering using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., Westborough, Massachusetts). The samples were measured in PBS at 25◦ C and a scattering angle of 90◦ . Average particle size was calculated as the arithmetic mean of the distribution of at least three batches of particles and the SD was calculated as the variance between average diameters of batches. Zeta potential was determined by measuring the electrophoretic mobility of nanoparticles in PBS and 10 mM HEPES buffer using the same instrument. Determination of -Gal Activity The activity of $-gal was measured by quantification of hydrolysis of ortho-nitrophenyl-D-galactopyranoside (ONPG) using a colorimetric $-Gal Assay Kit (Invitrogen, Carlsbad, California). Activity in particles was measured by diluting the particles to a final concentration of 0.5 ng/mL of $-gal and analyzing according to kit instructions. For quantification of activity of $-gal in cells, HeLa and 3T3 cells were seeded at a density of 1 × 104 per well in a 96-well plate in their growth medium. After 16 h, the cells were incubated with particles (87.5 :g/mL) in growth medium for 6 h. The cells were washed five times with PBS, then lysed and assayed for hydrolysis of ONPG. The number of moles of ONPG that are hydrolyzed are calculated using the following equation:
(OD420 )(final volume) nmoles of ONPG hydrolyzed = nL 4500 nmoles (1 cm) cm DOI 10.1002/jps.23974
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Cellular Uptake of Particles Uptake of particles was assessed by flow cytometry and confocal microscopy. HeLa, SK-BR-3, and 3T3 cells were seeded at a density of 2.5 × 105 per well in a 24-well plate in growth medium. After 16 h, the cells were incubated with particles (71 :g/mL eGFP or 87.5 :g/mL eGFP + $-gal) in growth medium for 6 h, unless otherwise stated. The cells were washed twice with PBS, trypsinized, and collected. Cells were washed twice more by centrifugation (25 g for 5 min), resuspended in PBS, and filtered with a 35 :m cell strainer. The cells were analyzed by an Accuri C6 (Becton Dickinson and Company, Franklin Lakes, New Jersey) flow cytometer and relative uptake was quantified as the ratio of fluorescence of the sample population compared with the control population (no particles given). 4 ◦ C controls were performed to ensure the observed fluorescence corresponded to uptake and not surface binding. HeLa cells, seeded at a density of 1 × 104 /cm2 , were seeded on glass bottom culture dishes (MatTek Corporation, Ashland, Massachusetts) and incubated with particles (87.5 :g/mL eGFP + $-gal) for 6 h. Cells were washed five times with PBS, fixed for 10 min in 3.7% paraformaldehyde, washed, and permeabilized with 0.1% Triton-X in PBS. The nuclei were stained with Hoechst 33342 (20 :g/mL) and actin filaments with PhalloidinTRITC (50 :g/mL) for 15 min. Cells were imaged using a Zeiss LSM 700–405 confocal microscope. Statistical Analysis All quantitative experiments were performed in triplicate and are presented as arithmetic mean ± SD. One-way ANOVA was used to determine significance among groups. p Values 50 mg/mL), which are ideal for encapsulation of small molecules.35 However, most therapeutic proteins often have a lower solubility than BSA and therefore, it is desirable to work at lower carrier protein concentrations that can yield higher therapeutic enzyme to carrier ratios. Substitution of albumin by eGFP not only facilitates particle tracking, it also decreases the risks associated with using an animal source and leads to higher yields and smaller particles at low carrier protein concentrations. Using eGFP allowed the reduction of carrier protein concentration to 6 mg/mL and thus increased enzyme to carrier ratios. Protein particle size is critical for delivery and uptake by cells. Langer et al.30 reported that particle production and size can be controlled by varying the amount and rate of addition of desolvation agent, the concentration and pH of the initial protein solution, and the stirring rate during fabrication. Our results prove that the type of cross-linker, cross-linking time, and the use of additives can also be manipulated for further tuning of particle size and size distribution. The presence of NaCl, as an additive, at low protein concentrations ( $-gal) and presence of His-tag (eGFP contains His-tag, $-gal does not). The absence of a histidine tag in $-gal supports the observation that imidazole had little effect on particle size. The effect of adding an enzyme on particle yield is not as significant as it is on particle size distribution, although it is also likely due to the differences between the enzyme and carrier protein. The yields of the desolvation method for protein and enzyme coacervation are quite high compared with those from production of many polymeric or lipid nanoparticle formulations containing proteins.2 Higher yield is not only cost effective but reduces the total dosage of material required to deliver the same amount of active enzyme. Importantly, the desolvation approach is generally applicable to different enzymes, but as with all drug carriers, fine-tuning of the formulation and fabrication parameters is required for different enzymes and delivery applications. In addition to size, particle surface charge is also important for effective particle delivery and internalization. It is well established that neutral particles decrease immune recognition and increase circulation times, but charged particles exhibit higher cellular internalization.36 For protein particles, the surface charge is dictated by the identity of the protein and specifically, which residues are exposed. eGFP and eGFP + $-gal nanoparticles exhibit very similar zeta-potentials as a result of the high content of eGFP in all formulations. The results shown here prove that the produced protein particles are inherently charged, as indicated by the strongly negative zeta-potentials measured in HEPES buffer. However, in the presence of ions, such as those observed in physiological conditions, the measured zeta-potential value decreases drastically. This indicates that ions adsorb to the surface of the particles and shield its charge, which causes the particles to seem only slightly charged.51 Despite this, carrier-enzyme particles are effectively internalized. The low particle uptake reported at 4◦ C, compared with 37◦ C, indicates that particles are internalized by active uptake mechanisms, such as endocytic processes.52,53 The high internalization reported here is ideal for drug delivery. In addition, the inherent charge of the carrier-enzyme particles will lead to repulsive forces between particles that, in low ionic strength buffers, will improve particle physical stability. In moderate to high ionic strength buffers, the low apparent charges will lead to lack of repulsive forces between particles, which causes to aggregation over time. These issues were overcome by lyophilization prior to particle storage. Upon resuspension, lyophilized particles showed high enzymatic activity retention even though their size decreases considerably. The observed decrease in size is most likely the result of particle drying, as the water trapped inside the particles is removed they collapse into smaller particles, which can be advantageous for drug delivery. Estrada, Chu, and Champion, JOURNAL OF PHARMACEUTICAL SCIENCES
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Enzymatic activity retention was observed to depend on the type of cross-linker and cross-linking time. The activity of $-gal in 20 min GTA cross-linked particles was significantly higher than the activity of enzyme in particles cross-linked with BS3 and DTSSP for 2 h, although the particle sizes were the same. These differences were seen for particles in solution; however, the activity of cell-internalized GTA cross-linked particles (20 min) was not significantly different than that of internalized particles cross-linked with BS3 and DTSSP in cell lysates. Studies performed with the three types of particles in HeLa cell lysates indicate that DTSSP and BS3 particles are broken apart at similar rates, possibly by the reducing agents and proteases found in cells; however, particles cross-linked for 2 h with GTA are not degraded (Fig. S13, Supporting Information). Additional studies indicate that that BS3 and DTSSP particles break apart while inside of the cells, releasing the enzyme and achieving the same activity levels of their higher activity counterpart, 20 min GTA cross-linked particles, which do not break apart (Fig. S14, Supporting Information). Intact GTA particles may have increased mass transfer limitations for substrate access compared with the DTSSP and BS3 cross-linked particles that are broken up inside the cells, which cancels the increased activity seen for the GTA particles when all are evaluated intact in solution. Thus, the use of reducible or degradable cross-linkers may prove advantageous for enzyme or protein intracellular delivery. The relative importance of each particle property must now be evaluated in specific disease models both in vitro and in vivo.
CONCLUSIONS The results presented here illustrate the feasibility of producing protein nanoparticles for intracellular delivery of therapeutic enzymes. Particle size can be tuned by adjusting a variety of parameters, including NaCl and imidazole concentration, type of cross-linker and cross-linking time. The effect of each parameter is dependent on the presence of the enzyme. We demonstrate high enzymatic activity after desolvation and crosslinking of enzymes with carrier proteins. Multiple cell lines exhibited greater internalization of enzyme-protein nanoparticles than soluble protein. Most significantly, this protein particle delivery system effectively delivers active enzyme inside cells. These results are very promising for therapeutic enzyme delivery and provide a new application for protein nanoparticles drug carriers.
ACKNOWLEDGMENTS This work was supported by the National Science Foundation (NSF 1105248) and the Kenneth Rainin Foundation. The authors thank Dr. Andreas S. Bommarius for the generous donation of the eGFP gene.
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41. Roy JJ, Abraham TE. 2004. Strategies in making cross-linked enzyme crystals. Chem Rev 104(9):3705–3721. 42. Sharma S, Shrivastav A, Gupta N, Srivastava S. 2011. Amperometric biosensor: Increased sensitivity using enzyme nanoparticles int conf nanotech biosens. IACSIT Press, Singapore, 24–27. 43. Hoogeven AT, Graham-Kawashima H, d’Azzo A, Galjaard H. 1984. Processing of human beta-galactosidas in GM1-gangliosidosis and Morquio B syndrome. J Biol Chem 259:1974–1977. 44. Weber C, Coester C, Kreuter J, Langer K. 2000. Desolvation process and surface characterization of protein nanoparticles. Int J Pharm 194:91–102. 45. Mehravar R, Jahanshahi M, Saghatoleslami N. 2009. Production of biological nanoparticles from lactalbumin for drug delivery and food science application. Afr J Biotech 8(24):6822–6827. 46. Munishkina LA, Ahmad A, Fink AL, Uversky VN. 2008. Guiding protein aggregation with macromolecular crowding. Biochemistry 47(34):8993–9006. 47. Migneault I, Dartiguenave C, Bertrand MJ, Waldron K. 2004. Glutaraldehyde: Behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques 37:790–802. 48. Dooley C, Dore TM, Hanson GT, Jackson WC, Remington SJ, Tsien RY. 2004. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem 279(21):22284– 22293. 49. Hamilton S, Odili J, Pacifico MD, Wilson GD, Kupsch JM. 2003. Effect of imidazole on the solubility of a his-tagged antibody fragment. Hybrid Hybrid 22(6):347–355. 50. Langer K, Anhorn MG, Steinhauser I, Dreis S, Celebi D, Schrickel N. 2008. Human serum albumin (HSA) nanoparticles: Reproducibility of preparation process and kinetics of enzymatic degradation. Int J Pharm 347:109–117. 51. Lieleg O, Baumgartel RM, Bausch AR. 2009. Selective filtering of particles by the extracellular matrix: An electrostatic bandpass. Biophys J 97:1569–1577. 52. Weigel PH, Oka JA. 1981. Temperature dependence of endocytosis mediated by the asialoglycoprotein receptor in isolated rat hepatocytes. J Biol Chem 256:2615–2617. 53. Silverstein SC, Steinman RM, Cohn ZA. 1977. Endocytosis. Ann Rev Biochem 46:669–722.
Estrada, Chu, and Champion, JOURNAL OF PHARMACEUTICAL SCIENCES
Protein Nanoparticles for Intracellular Delivery of Therapeutic Enzymes LINA HERRERA ESTRADA, STANLEY CHU, JULIE A. CHAMPION Department of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia Received 22 November 2013; revised 7 March 2014; accepted 25 March 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23974 ABSTRACT: The use of enzymes as therapeutics is very promising because of their catalytic activity and specificity. However, intracellular delivery of active enzymes is challenging due to their low stability and large size. The production of protein-enzyme nanoparticles was investigated with the goal of developing a protein carrier for active enzyme delivery. -Galactosidase (-gal), an enzyme whose deficiency is the cause of some lysosomal storage disorders, was incorporated into enhanced green fluorescent protein nanoparticles prepared via desolvation. Particle size was found to be sensitive to the type of cross-linker, cross-linking time, and the presence of imidazole. The results indicate that -gal activity is highly retained (>70%) after particle fabrication and >85% of protein is incorporated in the particles. Proteinenzyme nanoparticles exhibited higher internalization in multiple cell lines in vitro, compared with the soluble enzyme. Importantly, -gal retained its activity following intracellular delivery. These data demonstrate that protein nanoparticles are a biocompatible, highC 2014 Wiley Periodicals, Inc. and the American Pharmacists efficiency alternative for intracellular delivery of active enzyme therapeutics. Association J Pharm Sci Keywords: therapeutic enzymes; protein delivery; nanoparticles; intracellular delivery; enzyme activity; cross-linking; desolvation; macromolecular drug delivery; particle size
INTRODUCTION Enzyme therapeutics offer multiple advantages over small molecule drugs. Enzymes can bind their targets with high affinity and act with high specificity.1 Most importantly, an enzyme’s catalytic activity allows it to rapidly convert multiple target molecules into products, which significantly amplifies its effect compared with that of small molecules.2,3 These unique properties make enzymes promising therapeutic agents, and an important and growing segment of pharmaceuticals. Although most targets of United States Food and Drug Administration-approved enzymatic drugs are extracellular, there are many diseases for which treatment requires intracellular delivery.4 Effective enzyme replacement for genetic diseases such as lysosomal storage diseases requires intracellular delivery to lysosomal compartments.2,5 More therapeutic applications of intracellular enzymes are expected to emerge, including inhibition or reversal of ubiquitination for cancer6 and phosphorylation of Tau protein for Alzheimer’s disease.7 Enzymes can effectively perform these functions,8–11 but require modification, encapsulation, or immobilization on biocompatible matrices to improve their stability and limited distribution.1,2,12 Hence, there is a critical need to develop delivery systems that will improve accessibility of enzymatic drugs to intracellular compartments.
Abbreviations used: eGFP, enhanced green fluorescent protein; $gal, $-galactosidase; GTA, glutaraldehyde; DTSSP, 3,3 3 dithiobis(sulfosuccinimidylpropionate); BS , bis(sulfosuccinimidyl)suberate; ONPG, ortho-nitrophenyl-D-galactopyranoside. Correspondence to: Julie A. Champion (Telephone: +404-894-2874; Fax: +404385-2713; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com/. Journal of Pharmaceutical Sciences
C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
There are many challenges associated with the delivery and retention of activity, given the large size of enzymes and their complex tertiary or quaternary structure that can be highly sensitive to the environment.2 A delivery vehicle must be produced in conditions gentle enough for activity retention and be small enough to penetrate tissues and be internalized by cells.13,14 The size of delivery vehicles should be in the nanometer range, but the exact size depends on the application. Enzymes packaged in a delivery vehicle possess several advantages over soluble formulations, such as higher stability, lower immune response and targeting capabilities.12,13,15–19 Most delivery systems immobilize enzymes on the surface of another material or encapsulate them in polymeric, lipid, or mesoporous materials.2,4,20–25 Other methods involve fusion of an enzyme to other proteins or peptides.26 However, challenges remain, such as low encapsulation efficiencies, reduction in activity from fabrication conditions, or undesirable degradation products.27 A biodegradable, alternative method is to use protein particles for delivery of enzymes. Protein particles offer multiple advantages over their polymeric, inorganic, and liposomal counterparts including their high loading capacity, ease of production, surrounding protein environment, and amino-acid degradation products.28,29 Most protein particles are studied for intracellular delivery of small molecule drugs,29–35 only a few protein drugs have been investigated.36,37 Little is known about the production or function of enzyme nanoparticles. Some proteins and enzymes have been encapsulated in albumin microspheres38,39 or crystallized and cross-linked40,41 but the large size of these particles hinders efficient delivery and intracellular uptake. Recently, particles made directly with glucose oxidase have been shown to be more efficient as biosensors than immobilized enzyme on the surface of gold nanoparticles.42 In this study, we report the production of protein particles as enzyme carriers that maintain enzymatic activity and increase cellular uptake. For this purpose, we studied Estrada, Chu, and Champion, JOURNAL OF PHARMACEUTICAL SCIENCES
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Figure 1. Schematic diagram of the desolvation process. Ethanol is added drop by drop to a solution of carrier protein and enzyme. The protein and enzyme precipitate into nanoparticles that are stabilized by cross-linking.
desolvation and size tuning of enhanced green fluorescent protein (eGFP) as an enzyme carrier. eGFP has the added advantage of fluorescence for intracellular delivery tracking and imaging. We incorporated $-galactosidase ($-gal), a hydrolase homo-tetramer, whose deficiency is the cause of lysosomal storage disorders GM1 gangliosidosis and Morquio B disease43 into eGFP nanoparticles and show retention of enzymatic activity. In multiple cell types, we found that uptake of particles was greater than uptake of soluble protein. Finally, we successfully demonstrate intracellular delivery of active enzyme in vitro and the potential of protein nanoparticles as therapeutic enzyme drug carriers.
linking reaction was stopped by centrifugation at 1000g for 1 min and removal of supernatant. Particles were resuspended in phosphate buffered saline (PBS, 10 mM NaH2 PO4 , 137 mM NaCl, 2.7 mM KCl, 2 mM KH2 PO4 ; pH 7.4) and sonicated on ice for 1 s every 15 s at 30% amplitude, for 5 min. To determine the yield, the amount of protein not precipitated during desolvation was measured by BCA protein assay (Pierce). Following centrifugation, the concentration of protein in the supernatant was measured and the yield was estimated to be 100% minus the percentage of protein in the supernatant. Data presented are the arithmetic mean of the yield of three samples. Determination of Particle Size and Zeta Potential
MATERIALS AND METHODS Materials and Cell Lines $-Galactosidase (from Escherichia coli, lyophilized powder >500 U/g) and glutaraldehyde (GTA, 8% solution) were purchased from Sigma–Aldrich (Saint Louis, Missouri). 3,3´-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and bis(sulfosuccinimidyl)suberate (BS3 ) were obtained from Pierce (Rockford, Illinois). eGFP was expressed in E. coli and purified with Ni-NTA agarose (Qiagen, Valencia, California) (Fig. S1, Supporting Information). HeLa, SK-BR-3, and NIH/3T3 cells were purchased from American Type Culture Collection (Manassas, Virginia). HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS). SK-BR-3 cells were grown in McCoy 5A Media and supplemented with 10% FBS. 3T3 cells were cultured in DMEM and supplemented with10% calf serum. All media were supplemented with 1% (v/v) penicillin/streptomycin and cells were incubated at 37◦ C in a 5% CO2 humidified atmosphere. Preparation of Particles Protein particles were prepared by desolvation as described by Weber et al.44 Briefly, 6 mg/mL of protein (pure eGFP or 1:24 enzyme:eGFP) was dissolved in water, NaCl solution or imidazole solution (0–250 mM in a 300 mM NaCl, 50 mM NaH2 PO4 buffer; pH 8). Hundred microliters of protein solution was desolvated by continuous, drop-by-drop addition of 400 :L ethanol or acetone at a rate of 1 mL/min (Fig. 1). After desolvation, cross-linker was added at a ratio of cross-linker to lysines of 1:2.2. After 2 h stirring, unless otherwise stated, the crossEstrada, Chu, and Champion, JOURNAL OF PHARMACEUTICAL SCIENCES
Particle size distribution was measured by dynamic light scattering using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., Westborough, Massachusetts). The samples were measured in PBS at 25◦ C and a scattering angle of 90◦ . Average particle size was calculated as the arithmetic mean of the distribution of at least three batches of particles and the SD was calculated as the variance between average diameters of batches. Zeta potential was determined by measuring the electrophoretic mobility of nanoparticles in PBS and 10 mM HEPES buffer using the same instrument. Determination of -Gal Activity The activity of $-gal was measured by quantification of hydrolysis of ortho-nitrophenyl-D-galactopyranoside (ONPG) using a colorimetric $-Gal Assay Kit (Invitrogen, Carlsbad, California). Activity in particles was measured by diluting the particles to a final concentration of 0.5 ng/mL of $-gal and analyzing according to kit instructions. For quantification of activity of $-gal in cells, HeLa and 3T3 cells were seeded at a density of 1 × 104 per well in a 96-well plate in their growth medium. After 16 h, the cells were incubated with particles (87.5 :g/mL) in growth medium for 6 h. The cells were washed five times with PBS, then lysed and assayed for hydrolysis of ONPG. The number of moles of ONPG that are hydrolyzed are calculated using the following equation:
(OD420 )(final volume) nmoles of ONPG hydrolyzed = nL 4500 nmoles (1 cm) cm DOI 10.1002/jps.23974
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Cellular Uptake of Particles Uptake of particles was assessed by flow cytometry and confocal microscopy. HeLa, SK-BR-3, and 3T3 cells were seeded at a density of 2.5 × 105 per well in a 24-well plate in growth medium. After 16 h, the cells were incubated with particles (71 :g/mL eGFP or 87.5 :g/mL eGFP + $-gal) in growth medium for 6 h, unless otherwise stated. The cells were washed twice with PBS, trypsinized, and collected. Cells were washed twice more by centrifugation (25 g for 5 min), resuspended in PBS, and filtered with a 35 :m cell strainer. The cells were analyzed by an Accuri C6 (Becton Dickinson and Company, Franklin Lakes, New Jersey) flow cytometer and relative uptake was quantified as the ratio of fluorescence of the sample population compared with the control population (no particles given). 4 ◦ C controls were performed to ensure the observed fluorescence corresponded to uptake and not surface binding. HeLa cells, seeded at a density of 1 × 104 /cm2 , were seeded on glass bottom culture dishes (MatTek Corporation, Ashland, Massachusetts) and incubated with particles (87.5 :g/mL eGFP + $-gal) for 6 h. Cells were washed five times with PBS, fixed for 10 min in 3.7% paraformaldehyde, washed, and permeabilized with 0.1% Triton-X in PBS. The nuclei were stained with Hoechst 33342 (20 :g/mL) and actin filaments with PhalloidinTRITC (50 :g/mL) for 15 min. Cells were imaged using a Zeiss LSM 700–405 confocal microscope. Statistical Analysis All quantitative experiments were performed in triplicate and are presented as arithmetic mean ± SD. One-way ANOVA was used to determine significance among groups. p Values 50 mg/mL), which are ideal for encapsulation of small molecules.35 However, most therapeutic proteins often have a lower solubility than BSA and therefore, it is desirable to work at lower carrier protein concentrations that can yield higher therapeutic enzyme to carrier ratios. Substitution of albumin by eGFP not only facilitates particle tracking, it also decreases the risks associated with using an animal source and leads to higher yields and smaller particles at low carrier protein concentrations. Using eGFP allowed the reduction of carrier protein concentration to 6 mg/mL and thus increased enzyme to carrier ratios. Protein particle size is critical for delivery and uptake by cells. Langer et al.30 reported that particle production and size can be controlled by varying the amount and rate of addition of desolvation agent, the concentration and pH of the initial protein solution, and the stirring rate during fabrication. Our results prove that the type of cross-linker, cross-linking time, and the use of additives can also be manipulated for further tuning of particle size and size distribution. The presence of NaCl, as an additive, at low protein concentrations ( $-gal) and presence of His-tag (eGFP contains His-tag, $-gal does not). The absence of a histidine tag in $-gal supports the observation that imidazole had little effect on particle size. The effect of adding an enzyme on particle yield is not as significant as it is on particle size distribution, although it is also likely due to the differences between the enzyme and carrier protein. The yields of the desolvation method for protein and enzyme coacervation are quite high compared with those from production of many polymeric or lipid nanoparticle formulations containing proteins.2 Higher yield is not only cost effective but reduces the total dosage of material required to deliver the same amount of active enzyme. Importantly, the desolvation approach is generally applicable to different enzymes, but as with all drug carriers, fine-tuning of the formulation and fabrication parameters is required for different enzymes and delivery applications. In addition to size, particle surface charge is also important for effective particle delivery and internalization. It is well established that neutral particles decrease immune recognition and increase circulation times, but charged particles exhibit higher cellular internalization.36 For protein particles, the surface charge is dictated by the identity of the protein and specifically, which residues are exposed. eGFP and eGFP + $-gal nanoparticles exhibit very similar zeta-potentials as a result of the high content of eGFP in all formulations. The results shown here prove that the produced protein particles are inherently charged, as indicated by the strongly negative zeta-potentials measured in HEPES buffer. However, in the presence of ions, such as those observed in physiological conditions, the measured zeta-potential value decreases drastically. This indicates that ions adsorb to the surface of the particles and shield its charge, which causes the particles to seem only slightly charged.51 Despite this, carrier-enzyme particles are effectively internalized. The low particle uptake reported at 4◦ C, compared with 37◦ C, indicates that particles are internalized by active uptake mechanisms, such as endocytic processes.52,53 The high internalization reported here is ideal for drug delivery. In addition, the inherent charge of the carrier-enzyme particles will lead to repulsive forces between particles that, in low ionic strength buffers, will improve particle physical stability. In moderate to high ionic strength buffers, the low apparent charges will lead to lack of repulsive forces between particles, which causes to aggregation over time. These issues were overcome by lyophilization prior to particle storage. Upon resuspension, lyophilized particles showed high enzymatic activity retention even though their size decreases considerably. The observed decrease in size is most likely the result of particle drying, as the water trapped inside the particles is removed they collapse into smaller particles, which can be advantageous for drug delivery. Estrada, Chu, and Champion, JOURNAL OF PHARMACEUTICAL SCIENCES
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Enzymatic activity retention was observed to depend on the type of cross-linker and cross-linking time. The activity of $-gal in 20 min GTA cross-linked particles was significantly higher than the activity of enzyme in particles cross-linked with BS3 and DTSSP for 2 h, although the particle sizes were the same. These differences were seen for particles in solution; however, the activity of cell-internalized GTA cross-linked particles (20 min) was not significantly different than that of internalized particles cross-linked with BS3 and DTSSP in cell lysates. Studies performed with the three types of particles in HeLa cell lysates indicate that DTSSP and BS3 particles are broken apart at similar rates, possibly by the reducing agents and proteases found in cells; however, particles cross-linked for 2 h with GTA are not degraded (Fig. S13, Supporting Information). Additional studies indicate that that BS3 and DTSSP particles break apart while inside of the cells, releasing the enzyme and achieving the same activity levels of their higher activity counterpart, 20 min GTA cross-linked particles, which do not break apart (Fig. S14, Supporting Information). Intact GTA particles may have increased mass transfer limitations for substrate access compared with the DTSSP and BS3 cross-linked particles that are broken up inside the cells, which cancels the increased activity seen for the GTA particles when all are evaluated intact in solution. Thus, the use of reducible or degradable cross-linkers may prove advantageous for enzyme or protein intracellular delivery. The relative importance of each particle property must now be evaluated in specific disease models both in vitro and in vivo.
CONCLUSIONS The results presented here illustrate the feasibility of producing protein nanoparticles for intracellular delivery of therapeutic enzymes. Particle size can be tuned by adjusting a variety of parameters, including NaCl and imidazole concentration, type of cross-linker and cross-linking time. The effect of each parameter is dependent on the presence of the enzyme. We demonstrate high enzymatic activity after desolvation and crosslinking of enzymes with carrier proteins. Multiple cell lines exhibited greater internalization of enzyme-protein nanoparticles than soluble protein. Most significantly, this protein particle delivery system effectively delivers active enzyme inside cells. These results are very promising for therapeutic enzyme delivery and provide a new application for protein nanoparticles drug carriers.
ACKNOWLEDGMENTS This work was supported by the National Science Foundation (NSF 1105248) and the Kenneth Rainin Foundation. The authors thank Dr. Andreas S. Bommarius for the generous donation of the eGFP gene.
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