Development of wheat glutenin nanoparticles and their biodistribution in mice Narendra Reddy,1 Zhen Shi,1,2 Helan Xu,1 Yiqi Yang1,2,3,4 1
Department of Textiles, Merchandising, and Fashion Design, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0802 2 Key Laboratory of Eco-textiles, Ministry of Education, College of Textiles and Garments, Jiangnan University, Lihu Road #1800, Wuxi 214122, China 3 Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0802 4 Nebraska Center for Materials and Nanoscience, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0802 Received 6 May 2014; revised 9 July 2014; accepted 31 July 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35302 Abstract: Wheat glutenin nanoparticles intended for targeted drug delivery were biocompatible and were detected in the kidney, liver, and spleen in mice. Protein-based nanoparticles are preferred for therapeutic drug and gene delivery owing to their biocompatibility and ability to load various types of drugs. However, proteins such as a collagen and albumin are unstable in aqueous environments and are not ideal for drug delivery applications. Wheat glutenin has been demonstrated to be biocompatible and have good stability under aqueous conditions. Films and fibers have been made from wheat glutenin for medical applications but there are no reports on developing micro- or nanoparticles. In this research, wheat glutenin nanoparticles (70–140 nm) were prepared and the stability of the nanoparticles under various physiological con-
ditions was investigated. Nanoparticles were fluorescently labeled and later injected into mice and the ability of the nanoparticles to penetrate into the cells in various organs was studied. Strong acidic or alkaline conditions provided glutenin nanoparticles with low diameters and the particles were more stable under the pH 7 rather than pH of 4. Glutenin nanoparticles were predominantly found in the liver in mice. Our in vivo and in vitro studies suggest that glutenin C nanoparticles are suitable for drug delivery applications. V 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000–000, 2014.
Key Words: glutenin, protein nanoparticles, drug delivery, biodistribution, kidney
How to cite this article: Reddy N, Shi Z, Xu H, Yang Y. 2014. Development of wheat glutenin nanoparticles and their biodistribution in mice. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Nanoparticles have unique properties that make them preferable for controlled release and other medical applications. Small size, large surface area, ability to escape detection by the immune system, improve therapeutic index, and reduced side effects are some the features that make nanoparticles desirable for medical applications.1,2 Nanoparticles are also reported to have a tendency to accumulate in tumors owing to their “enhanced permeation and retention effect” which allows drugs to be delivered more effectively at the site of the tumors. Owing to these advantages, nano-drug delivery
systems are being extensively investigated as drug delivery and nonviral gene delivery vehicles.3 Nanoparticles for drug delivery applications have been developed from natural and synthetic polymers and also from metals. Metallic nanoparticles such as gold, copper, iron, and silver have been used for drug delivery and reported to have been able to cross the blood–brain barrier.4–6 Similarly, natural polymers such as cellulose, starch, and chitosan and synthetic polymers such as poly(lactic acid) and poly(ethylene glycol) have also been made into nanoparticles for drug delivery applications.7–10
Correspondence to: Y. Yang; e-mail: [email protected] Contract grant sponsor: The Agricultural Research Division at the University of Nebraska-Lincoln, USDA Hatch Act and Multi-State Project S1054; contract grant number: NEB 37-037 Contract grant sponsor: The John and Louise Skala Fellowship and AATCC Student Research Grant Contract grant sponsor: The Program for Changjiang Scholars and Innovative Research Team in University; contract grant number: IRT1135 at Jiangnan University Contract grant sponsor: Scientific Support Program of Jiangsu Province; contract grant number: BE2011404 Contract grant sponsor: The Graduate Student Innovation Plan of Jiangsu Province; contract grant number: CX10B_222Z Contract grant sponsor: The Doctor Candidate Foundation of Jiangnan University; contract grant number: JUDCF10004
C 2014 WILEY PERIODICALS, INC. V
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Proteins derived from cereals such as soybeans, corn, and wheat commonly referred to as plant proteins or cereal proteins have also been extensively investigated for medical applications.11 Unlike collagen, plant proteins are nonimmunogenic and unlike silk they are much easier to dissolve and easily made into various types of biomaterials. Plant proteins have been made into films, fibers, hydrogels, and micro- and nanoparticles.11–15 Hollow and solid zein nanoparticles were found to enter the cytoplasm of cells during in vitro studies and the hollow zein nanoparticles had considerably higher absorption of drugs and dyes.14,16 Zein nanoparticles were also found to be located in the liver when injected in vivo into mice.17 Wheat gluten consists of two major proteins: glutenin and gliadin. It is generally considered that gliadin in gluten is immunogenic and is responsible for causing celiac disease but some researchers have provided evidence which indicates that glutenin could also be immunogenic.18–20 Both gliadin and glutenin are reported to contain homologous repetitive amino acid sequences and such sequences obtained from glutenin have shown to be stimulatory under immune activity assays. Although recent research suggests the immunogenic nature of glutenin, conclusive proof of the involvement of glutenin in immunogenicity is yet to be provided.20 Nevertheless, films, fibers, and hydrogels have been made from both wheat proteins for tissue engineering and controlled release applications.11,21 As gliadin dissolves in aqueous ethanol, it has been made into films, fibers, and nanoparticles for medical applications.21 Gliadin nanoparticles crosslinked with glutaraldehyde used as carriers for all-trans-retionic acid (RA) were found to have a payload limit of 76.4 mg RA/mg of nanoparticles and followed a zero-order diffusion.22 Gliadin nanoparticles (450–475 nm) were also used as drug delivery systems to deliver vitamin E, linalyl acetate, and benzalkonium chloride.23 Unlike gliadin, glutenin was found to have excellent biocompatibility and supported the attachment and proliferation of fibroblasts and osteoblasts better than films made from poly(lactic acid).24 Although films made from wheat glutenin have demonstrated to be suitable for medical applications, there are no reports on developing nano- or microparticles from wheat glutenin. Our hypothesis is that wheat glutenin can be made into biocompatible nanoparticles that have the ability to biodistribute into the various organs in mice. To test this hypothesis, wheat glutenin was made into nanoparticles and the potential of the nanoparticles to enter cells in vitro and penetrate into various organs in mice was investigated. The effects of nanoparticle fabrication conditions on the size of the nanoparticles and the morphology and stability of the nanoparticles were also investigated. MATERIALS AND METHODS
Materials Commercially available wheat gluten (WhetPro 80) was supplied by Archer Daniels Midlands, Decatur, IL. Glutenin was purified from the wheat gluten. First, gluten was treated
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with 70% v/v of ethanol for 24 h to remove gliadin. The gliadin-free-gluten was later treated with enzymes (1% amylase on weight of protein) for 24 h at pH 8 buffer to remove starch. Gliadin and starch-free glutenin were washed using deionized water and later dried and used for the preparation of the nanoparticles. Preparation of nanoparticles Wheat glutenin was treated with sodium bisulfite solution (30% on weight of protein) at a ratio of 20:1 for 4 h at 70 C to reduce the proteins, decrease their molecular weights, and make them soluble. Later, the proteins were hydrolyzed using 0.1M of sodium hydroxide at ratio of 15:1 for 2 h at 80 C. Proteins were precipitated by adding HCl (1 mol/L) and the solution was centrifuged (10,000 rpm) to collect the solid proteins. To prepare the nanoparticles, the hydrolyzed proteins (1–20% w/w) were dissolved in ethylene glycol at room temperature. Water was added into the ethylene glycol solution to form the nanoparticles by phase separation. The value of pH of the solution during phase separation varied and the changes in the size of the nanoparticles were studied. Characterizing nanoparticles Size and f potential. Size of the glutenin nanoparticles in solution was observed in a particle size analyzer (Model: Delsa Nano, Beckman Coulter) to determine the particle size and f potential. At least three measurements from separate samples were carried out for each condition and the average and standard deviation of the data are reported. Transmission electron microscopy Nanoparticles were freeze-dried and later cryosectioned to observe the morphology under a transmission electron microscope (Hitachi H7500 Transmission Electron Microscope, Hitachi, Parlin, NJ). Morphology of the nanoparticles was observed to confirm their size and shape. Stability of the nanoparticles Stability of the nanoparticles was studied in pH 7 and 4 water at 37 and 4 C to simulate physiological conditions and understand the ability of the nanoparticles to be used for medical applications. To study the stability, the dry nanoparticles were dispersed (5%) in pH 7 or pH 4 water and heated in an oven at 37 C or maintained in the refrigerator at 4 C. At the predetermined time interval, samples were drawn and measured for particle size in a particle size analyzer. At least three different readings from three different samples were collected and the average and standard deviations are reported. In vitro cytotoxicity studies Cytotoxicity of the nanoparticles was evaluated by coculturing the nanoparticles with media containing mouse fibroblasts (NIH 3T3). Nanoparticles (10% on weight of the Dulbecco’s Modified Eagle’s media) were added into 48-well culture plate containing 1 3 105 cells/mL and cultured for 5 days. After every 24 h, about 100 mL of DMEM containing 20% MTS assay solution was added and the plate was
DEVELOPMENT OF WHEAT GLUTENIN NANOPARTICLES
ORIGINAL ARTICLE
FIGURE 2. Changes in the size of the nanoparticles with varying pH during precipitation. FIGURE 1. TEM image of the glutenin nanoparticles confirmed that the nanoparticles had diameters in the range of 70–120 nm.
incubated for 3 h at 37 C. Later, 100 mL of the media was transferred into 96-well plate and the metabolic activity was read on a multiwell plate reader (Thermo Scientific Model: Multiskan) at 490 nm. Six to nine replications were done for each time point and the average and standard deviation of the cytotoxicity readings are reported. Media containing cells without any nanoparticles were used as control. In vivo studies Ability of the nanoparticles to enter the various organs in mice was studied. Nanoparticles (100 mg/mL) were first labeled with fluorescein isothiocyanate which is a far red fluorescent dye and the dyed nanoparticles were intravenously injected into mice for up to 5 days in 24-h intervals. Mice were sequentially sacrificed 24 h after injection for up to 5 days. Mice sacrificed after the first 24 h had one injection of the nanoparticles, mice sacrificed after 48 h had two injections, and so on. At the required time, mice were euthanized using CO2 and the organs (liver, kidney, spleen, heart, and lungs) were surgically removed. All experiments were performed according to the Institutional Animal Care and Use Committee (IACUC) approved protocol and two mice were sacrificed for each time point. Organs collected were stored in RPMI solution at 4 C until further analysis. Flow cytometry analysis The organ tissues were minced using a 70 mm cell strainer (Falcon, Tewksbury, MA) to obtain single-cell suspension in RPMI solution. For each organ, an aliquot of 500 mm of cell suspension was measured on the flow cytometer. A flow cytometer (BD Accuri, Franklin Lakes, NJ) was used to determine the presence of fluorescent nanoparticles in the various organs collected from the mice. Population of single cells was defined in a plot of 90 side scattering versus forward scattering, and the percentage of nanoparticle-loaded
cells was counted by measuring fluorescence signal in the green fluorescence channel (FL1). RESULTS AND DISCUSSION
Morphology of the nanoparticles Glutenin nanoparticles had size in the range of 70–140 nm, confirmed using the particle size analyzer and the transmission electron microscopy (TEM) image shown in Figure 1, which makes them suitable for drug delivery and other medical applications. Proteins such as insulin, lysozyme, and myoglobin with diameters of 3–3.5 nm had blood half-time of 8.8–12 min and renal filtration between 75 and 100%.25 It has been reported that nanoparticles with diameters of 5.5 nm would be completely eliminated from the body.25 Although there has been no information on the size of the nanoparticles preferable for drug delivery, the previous studies have shown that protein nanoparticles with size around 100 nm could be used for targeted drug delivery. Glutenin nanoparticles could be made with diameters required for drug delivery. Effect of fabrication conditions on size of nanoparticles Size of the nanoparticles was considerably affected by the pH during precipitation and varied from 65 to 145 nm as shown in Figure 2. Both low and high pHs were suitable to obtain relatively smaller nanoparticles and the largest size was close to the isoelectric point of glutenin where agglomeration of the nanoparticles and resulting increase in size occurs. Although high pH (9–10) produced smaller particles, alkaline conditions could damage the proteins and it is preferable to choose pH of 2 or between 7 and 8 to produce the nanoparticles. Glutenin nanoparticles had considerably high f potential variation ranging from 2200 to 1 320 mV with the isoelectric point at about pH 6.8 similar to that reported earlier. Changing pH also affected the f potential of the nanoparticles and therefore the ability of the nanoparticles to attract various drugs and payloads as shown in Figure 3. f Potential also represents particle stability and larger (6) the f potential larger is the repulsion and
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FIGURE 3. f Potential of the glutenin nanoparticles at various pHs and the isoelectric point.
FIGURE 5. Stability of the nanoparticles at two different temperatures and pHs.
therefore more stable particles.26 Both f potential and pH suggest that acidic and slightly alkaline pH were more suitable to form the glutenin nanoparticles. f Potential at physiological pH of 7.2 (range, 25 to 215 mV) is suitable for medical applications. Glutenin nanoparticles have strong cationic/anionic charge, beneficial for loading drugs by changing the pH of the solution.27 Changing the ratio of ethylene glycol to glutenin above 4% showed a substantial increase in the size of the nanoparticles as shown in Figure 4. When the concentration of the protein in the solution is increased, the solubility decreased and there were considerably higher number of protein molecules. These protein molecules will easily agglomerate and increase in size. However, high temperatures may be useful to get lower size of nanoparticles even at higher concentrations of the protein solution.
agglomerate with increasing time except for the particles in pH 7 solution at 37 C. The agglomeration was higher when the pH was 2 and when the temperature was 4 versus 37 C. The ability of the nanoparticles to maintain their size at pH 7 and 37 C indicates that the nanoparticles would be suitable for most medical applications. Higher stability at pH 7 should be owing to the pH being close to the isoelectric point (6.8–7.0).28 Nanoparticles are more stable at 37 C than at 4 C because of the higher mobility of the particles and lesser chance to get agglomerated.
Stability of the glutenin nanoparticles Glutenin nanoparticles were more stable at pH 7 than pH 4 and at 37 C compared to the stability of the particles at4 C as shown in Figure 5. However, the nanoparticles tend to
FIGURE 4. Influence of the ratio of protein to ethylene glycol on the size of the nanoparticles.
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In vitro cell assay Addition of glutenin nanoparticles into the cell culture media did not affect cell viability or growth as shown in Figure 6. In fact, wells containing the glutenin nanoparticles showed slightly higher metabolic activity compared to the cells without any nanoparticles. It was confirmed that glutenin nanoparticles did not influence the assay results, suggesting that the increase in optical density was owing to the higher metabolic activity of the cells. Glutenin nanoparticles
FIGURE 6. Comparison of the optical densities of mouse fibroblast cells with and without nanoparticles based on MTS assay. The cells were cultured at 37 C for 5 days.
DEVELOPMENT OF WHEAT GLUTENIN NANOPARTICLES
ORIGINAL ARTICLE
cytotoxic. Other proteins such as silk are difficult to be dissolved and made into nanoparticles and also have excessively long degradation in the body making them not useful for targeted drug delivery. Previously, our group and other researchers have demonstrated that nanoparticles made from corn zein are biocompatible and could be used for drug delivery. However, the stability of the zein nanoparticles under different physiological conditions and their biodistribution are not fully understood. Compared to zein, nanoparticles from glutenin are more stable and also do not require crosslinking. In addition, glutenin nanoparticles would not have the risks associated with using animal proteins such as collagen.
FIGURE 7. Comparison of the percentage of cells containing nanoparticles detected in various organs in mice using flow cytometer after 1–4 days of injection.
may act as a nutrient or growth factor and promote cell growth thereby increasing the metabolic activity. Biodistribution of nanoparticles in mice Glutenin nanoparticles were predominantly found in the kidneys followed by liver and spleen in mice as shown in Figure 7. Increasing the dosage of the nanoparticles and/or residence time did not significantly affect the amount of nanoparticles found in the different organs. Kidneys are reported to filter (renal clearance) nanoparticles with a hydrodynamic radius of up to about 8 nm. As the glutenin nanoparticles were considerably larger, the nanoparticles accumulated in the kidneys.25,29 Particles that are not cleared by the kidneys are removed through the liver (hepatic clearance) and it has been reported that the livers clear nanoparticles in the range of 10–20 nm.29 Therefore, considerable amounts of nanoparticles were also found in the kidneys. Lower amounts of the glutenin nanoparticles were detected in the spleen and lungs as most of the particles were taken up by the kidneys and liver. There was no significant increase in the amount of nanoparticles in any of the organs after multiple injections, suggesting that the nanoparticles were easily digested in the body or were removed within 24 h before the next injection was made. It appears that glutenin nanoparticles could be used to effectively deliver drugs to kidneys. However, varying the size and surface modifications to the nanoparticles may increase the biodistribution of the nanoparticles in the other organs in mice. Advantages of glutenin nanoparticles The ability to develop nanoparticles from wheat glutenin with the size, stability, and biocompatibility required for medical applications could have significant implications. Common proteins used for medical applications such as collagen and albumin do not have the stability to be made into nanoparticles. Although crosslinking of protein nanoparticles has been done to improve stability, most of the crosslinking agents currently used for protein crosslinking are
CONCLUSIONS
Nanoparticles developed from wheat glutenin were biocompatible and found to be predominantly biodistributed in the kidneys in mice. With diameters as low as 30 nm and having good stability under physiological conditions, glutenin nanoparticles had the qualities required for medical applications. Changing the pH or ratio of solvent during preparation provided nanoparticles with different sizes. Glutenin nanoparticles have had strong anionic/cationic charge that would be helpful to load various types of drugs. In vitro studies indicated that glutenin-containing wells had higher metabolic activity compared to wells without the nanoparticles. Biocompatible and stable glutenin nanoparticles without the need for external crosslinking agents would be unique nanoparticles for drug delivery and other medical applications. REFERENCES 1. Nitta SK, Numata K. Biopolymer-based nanoparticles for drug/ gene delivery and tissue engineering. Int J Mol Sci 2013;14:1629– 1654. 2. Mailander V, Landfester K. Interaction of nanoparticles with cells. Biomacromolecules 2009;10:2379–2400. mos C, Grandemange S, Mograbi B, 3. Eidi H, Joubert O, Ne Foliguet B, Tournebize J, Maincent P, Le Faou A, Aboukhamis I, Rihn BH. Drug delivery by polymeric nanoparticles induces autophagy in macrophages. Int J Pharm 2012;422:495–503. 4. Yokel R, Grulke E, MacPhail R. Metal-based nanoparticle interactions with the nervous system: The challenge of brain entry and the risk of retention in the organism. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2013;5:346–373. 5. Kharissova OV, Dias HVR, Kharisov BI, Perez BO. The greener synthesis of nanoparticles. Cell 2013;31:240–248. 6. Chen P, Kanehira K, Sonezaki S, Taniguchi A. Detection of cellular response to titanium dioxide nanoparticle agglomerates by sensor cells using heat shock protein promoter. Biotechnol Bioeng 2012;109:3112–3118. 7. Lee KY, Yuk SH. Polymeric protein delivery systems. Prog Polym Sci 2007;32:669–697. 8. Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev 2003;55: 329–347. 9. Lopez P, Murdan S. Zein microspheres as drug antigen carriers: A study of their degradation and erosion, in the presence and absence of enzymes. J Microencapsul 2006;23:303–314. 10. Jahanshahi M, Babaei Z. Protein nanoparticle: A unique system as drug delivery vehicles. Afr J Biotechnol 2008;7:4926–4934. 11. Reddy N, Yang Y. Potential of plant proteins for medical application. Trends Biotechnol 2011;29:490–498. 12. Cai S, Xu H, Jiang Q, Yang Y. Novel 3D electrospun scaffolds with fibers oriented randomly and evenly in three dimensions to
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closely mimic the unique architectures of extracellular matrices in soft tissues: Fabrication and mechanism study. Langmuir 2013;29: 2311–2318. Dong J, Sun, Q, Wang J. Basic study of corn protein, zein, as a biomaterial in tissue engineering, surface morphology and biocompatibility. Biomaterials 2004;25:4691–4697. Xu H, Jiang Q, Reddy N, Yang Y. Hollow nanoparticles from zein for potential medical applications. J Mater Chem 2011;21:18227– 18235. Jiang Q, Reddy N, Yang Y, Cytocompatible crosslinking of electrospun zein fibers for the development of water stable tissue engineering scaffolds. Acta Biomaterial 2010;6:4042–4051. Xu H, Zhang Y, Jiang Q, Reddy N, Yang Y. Biodegradable hollow zein nanoparticles for removal of reactive dyes from wastewater. J Environ Manag 2013;125:33–40. Lai LF, Guo HX. Preparation of new 5-fluorouracil-loaded zein nanoparticles for liver targeting. Int J Pharm 2011;404:317–323. Real A, Comino I, Lorenzo L, Merchan F, Gil-humanes J, Gimenez M J, Lopez-Casado MA, Torres IM, Cebolla A, Sousa C, Barro F, Piston F. Molecular and immunological characterization of gluten proteins isolated from oat cultivars that differ in toxicity for celiac disease. PLoS One 2012;7:e48365. Camarca A, Anderson RP, Mamone G, Fierro O, Facchiano A, Costantini S, Zanzi D, Sidney J, Auricchio S, Sette A, Troncone R, Gianfrani C. Intestinal T cell responses to gluten peptides are largely heterogeneous: Implications for a peptide-based therapy in celiac disease. J Immunol 2009;182:4158–4166. Howdle PD. Gliadin, glutenin or both? The search for the Holy Grail in coeliac disease. Eur J Gastroenterol Hepatol 2006;18:703– 706.
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21. Reddy N, Yang Y. Self-crosslinked gliadin fibers with high strength and water stability for potential medical applications. J Mater Sci Mater Med 2008;19:2055–2061. 22. Ezpeleta I, Irache JM, Stainmesse S, Chabenat C, Gueguen J, Popineau Y, Orrecchioni A. Gliadin nanoparticles for the controlled release of all-trans-retinoic acid. Int J Pharm 1996;131:191– 200. 23. Duclairoir C, Orecchioni AM, Depraetere P, Osterstock F, Nakache E. Evaluation of gliadins nanoparticles as drug delivery systems: A study of three different drugs. Int J Pharm 2003;253:133–144. 24. Reddy N, Jiang Q, Yang Y. Novel wheat protein films as substrates for tissue engineering. J Biomater Sci Polym Ed 2011;22: 2063–2077. 25. Choi HS, Lui W, Misra P, Tananka E, Zimmer JP, Ipe, BI, Bawendi MG, Frangioni JV. Renal clearance of nanoparticles. Nat Biotechnol 2007;25:1165–1170. 26. Hans ML, Lowman AM. Biodegradable nanoparticles for drug delivery and targeting. Curr Opin Solid State Mater Sci 2002;6: 319–327. 27. Clogston JD, Patri AK. Zeta potential measurement. In: McNeil SE, editor. Characterization of Nanoparticles Intended for Drug Delivery. New York: Humana Press; 2011. p 63–70. 28. Rombouts I, Lamberts L, Celus I, Lagrain B, Brijs K, Delcour JA. Wheat gluten amino acid composition analysis by highperformance anion-exchange chromatography with integrated pulsed amperometric detection. J Chromatogr A 2009;1216:5557– 5562. 29. Longmire M, Choyke PL, Kobayashi H. Clearance properties of nano-sized particles and molecules as imaging agents: Considerations and caveats. Nanomedicine 2008;3:703–717.
DEVELOPMENT OF WHEAT GLUTENIN NANOPARTICLES
Department of Textiles, Merchandising, and Fashion Design, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0802 2 Key Laboratory of Eco-textiles, Ministry of Education, College of Textiles and Garments, Jiangnan University, Lihu Road #1800, Wuxi 214122, China 3 Department of Biological Systems Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0802 4 Nebraska Center for Materials and Nanoscience, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0802 Received 6 May 2014; revised 9 July 2014; accepted 31 July 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35302 Abstract: Wheat glutenin nanoparticles intended for targeted drug delivery were biocompatible and were detected in the kidney, liver, and spleen in mice. Protein-based nanoparticles are preferred for therapeutic drug and gene delivery owing to their biocompatibility and ability to load various types of drugs. However, proteins such as a collagen and albumin are unstable in aqueous environments and are not ideal for drug delivery applications. Wheat glutenin has been demonstrated to be biocompatible and have good stability under aqueous conditions. Films and fibers have been made from wheat glutenin for medical applications but there are no reports on developing micro- or nanoparticles. In this research, wheat glutenin nanoparticles (70–140 nm) were prepared and the stability of the nanoparticles under various physiological con-
ditions was investigated. Nanoparticles were fluorescently labeled and later injected into mice and the ability of the nanoparticles to penetrate into the cells in various organs was studied. Strong acidic or alkaline conditions provided glutenin nanoparticles with low diameters and the particles were more stable under the pH 7 rather than pH of 4. Glutenin nanoparticles were predominantly found in the liver in mice. Our in vivo and in vitro studies suggest that glutenin C nanoparticles are suitable for drug delivery applications. V 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000–000, 2014.
Key Words: glutenin, protein nanoparticles, drug delivery, biodistribution, kidney
How to cite this article: Reddy N, Shi Z, Xu H, Yang Y. 2014. Development of wheat glutenin nanoparticles and their biodistribution in mice. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Nanoparticles have unique properties that make them preferable for controlled release and other medical applications. Small size, large surface area, ability to escape detection by the immune system, improve therapeutic index, and reduced side effects are some the features that make nanoparticles desirable for medical applications.1,2 Nanoparticles are also reported to have a tendency to accumulate in tumors owing to their “enhanced permeation and retention effect” which allows drugs to be delivered more effectively at the site of the tumors. Owing to these advantages, nano-drug delivery
systems are being extensively investigated as drug delivery and nonviral gene delivery vehicles.3 Nanoparticles for drug delivery applications have been developed from natural and synthetic polymers and also from metals. Metallic nanoparticles such as gold, copper, iron, and silver have been used for drug delivery and reported to have been able to cross the blood–brain barrier.4–6 Similarly, natural polymers such as cellulose, starch, and chitosan and synthetic polymers such as poly(lactic acid) and poly(ethylene glycol) have also been made into nanoparticles for drug delivery applications.7–10
Correspondence to: Y. Yang; e-mail: [email protected] Contract grant sponsor: The Agricultural Research Division at the University of Nebraska-Lincoln, USDA Hatch Act and Multi-State Project S1054; contract grant number: NEB 37-037 Contract grant sponsor: The John and Louise Skala Fellowship and AATCC Student Research Grant Contract grant sponsor: The Program for Changjiang Scholars and Innovative Research Team in University; contract grant number: IRT1135 at Jiangnan University Contract grant sponsor: Scientific Support Program of Jiangsu Province; contract grant number: BE2011404 Contract grant sponsor: The Graduate Student Innovation Plan of Jiangsu Province; contract grant number: CX10B_222Z Contract grant sponsor: The Doctor Candidate Foundation of Jiangnan University; contract grant number: JUDCF10004
C 2014 WILEY PERIODICALS, INC. V
1
Proteins derived from cereals such as soybeans, corn, and wheat commonly referred to as plant proteins or cereal proteins have also been extensively investigated for medical applications.11 Unlike collagen, plant proteins are nonimmunogenic and unlike silk they are much easier to dissolve and easily made into various types of biomaterials. Plant proteins have been made into films, fibers, hydrogels, and micro- and nanoparticles.11–15 Hollow and solid zein nanoparticles were found to enter the cytoplasm of cells during in vitro studies and the hollow zein nanoparticles had considerably higher absorption of drugs and dyes.14,16 Zein nanoparticles were also found to be located in the liver when injected in vivo into mice.17 Wheat gluten consists of two major proteins: glutenin and gliadin. It is generally considered that gliadin in gluten is immunogenic and is responsible for causing celiac disease but some researchers have provided evidence which indicates that glutenin could also be immunogenic.18–20 Both gliadin and glutenin are reported to contain homologous repetitive amino acid sequences and such sequences obtained from glutenin have shown to be stimulatory under immune activity assays. Although recent research suggests the immunogenic nature of glutenin, conclusive proof of the involvement of glutenin in immunogenicity is yet to be provided.20 Nevertheless, films, fibers, and hydrogels have been made from both wheat proteins for tissue engineering and controlled release applications.11,21 As gliadin dissolves in aqueous ethanol, it has been made into films, fibers, and nanoparticles for medical applications.21 Gliadin nanoparticles crosslinked with glutaraldehyde used as carriers for all-trans-retionic acid (RA) were found to have a payload limit of 76.4 mg RA/mg of nanoparticles and followed a zero-order diffusion.22 Gliadin nanoparticles (450–475 nm) were also used as drug delivery systems to deliver vitamin E, linalyl acetate, and benzalkonium chloride.23 Unlike gliadin, glutenin was found to have excellent biocompatibility and supported the attachment and proliferation of fibroblasts and osteoblasts better than films made from poly(lactic acid).24 Although films made from wheat glutenin have demonstrated to be suitable for medical applications, there are no reports on developing nano- or microparticles from wheat glutenin. Our hypothesis is that wheat glutenin can be made into biocompatible nanoparticles that have the ability to biodistribute into the various organs in mice. To test this hypothesis, wheat glutenin was made into nanoparticles and the potential of the nanoparticles to enter cells in vitro and penetrate into various organs in mice was investigated. The effects of nanoparticle fabrication conditions on the size of the nanoparticles and the morphology and stability of the nanoparticles were also investigated. MATERIALS AND METHODS
Materials Commercially available wheat gluten (WhetPro 80) was supplied by Archer Daniels Midlands, Decatur, IL. Glutenin was purified from the wheat gluten. First, gluten was treated
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with 70% v/v of ethanol for 24 h to remove gliadin. The gliadin-free-gluten was later treated with enzymes (1% amylase on weight of protein) for 24 h at pH 8 buffer to remove starch. Gliadin and starch-free glutenin were washed using deionized water and later dried and used for the preparation of the nanoparticles. Preparation of nanoparticles Wheat glutenin was treated with sodium bisulfite solution (30% on weight of protein) at a ratio of 20:1 for 4 h at 70 C to reduce the proteins, decrease their molecular weights, and make them soluble. Later, the proteins were hydrolyzed using 0.1M of sodium hydroxide at ratio of 15:1 for 2 h at 80 C. Proteins were precipitated by adding HCl (1 mol/L) and the solution was centrifuged (10,000 rpm) to collect the solid proteins. To prepare the nanoparticles, the hydrolyzed proteins (1–20% w/w) were dissolved in ethylene glycol at room temperature. Water was added into the ethylene glycol solution to form the nanoparticles by phase separation. The value of pH of the solution during phase separation varied and the changes in the size of the nanoparticles were studied. Characterizing nanoparticles Size and f potential. Size of the glutenin nanoparticles in solution was observed in a particle size analyzer (Model: Delsa Nano, Beckman Coulter) to determine the particle size and f potential. At least three measurements from separate samples were carried out for each condition and the average and standard deviation of the data are reported. Transmission electron microscopy Nanoparticles were freeze-dried and later cryosectioned to observe the morphology under a transmission electron microscope (Hitachi H7500 Transmission Electron Microscope, Hitachi, Parlin, NJ). Morphology of the nanoparticles was observed to confirm their size and shape. Stability of the nanoparticles Stability of the nanoparticles was studied in pH 7 and 4 water at 37 and 4 C to simulate physiological conditions and understand the ability of the nanoparticles to be used for medical applications. To study the stability, the dry nanoparticles were dispersed (5%) in pH 7 or pH 4 water and heated in an oven at 37 C or maintained in the refrigerator at 4 C. At the predetermined time interval, samples were drawn and measured for particle size in a particle size analyzer. At least three different readings from three different samples were collected and the average and standard deviations are reported. In vitro cytotoxicity studies Cytotoxicity of the nanoparticles was evaluated by coculturing the nanoparticles with media containing mouse fibroblasts (NIH 3T3). Nanoparticles (10% on weight of the Dulbecco’s Modified Eagle’s media) were added into 48-well culture plate containing 1 3 105 cells/mL and cultured for 5 days. After every 24 h, about 100 mL of DMEM containing 20% MTS assay solution was added and the plate was
DEVELOPMENT OF WHEAT GLUTENIN NANOPARTICLES
ORIGINAL ARTICLE
FIGURE 2. Changes in the size of the nanoparticles with varying pH during precipitation. FIGURE 1. TEM image of the glutenin nanoparticles confirmed that the nanoparticles had diameters in the range of 70–120 nm.
incubated for 3 h at 37 C. Later, 100 mL of the media was transferred into 96-well plate and the metabolic activity was read on a multiwell plate reader (Thermo Scientific Model: Multiskan) at 490 nm. Six to nine replications were done for each time point and the average and standard deviation of the cytotoxicity readings are reported. Media containing cells without any nanoparticles were used as control. In vivo studies Ability of the nanoparticles to enter the various organs in mice was studied. Nanoparticles (100 mg/mL) were first labeled with fluorescein isothiocyanate which is a far red fluorescent dye and the dyed nanoparticles were intravenously injected into mice for up to 5 days in 24-h intervals. Mice were sequentially sacrificed 24 h after injection for up to 5 days. Mice sacrificed after the first 24 h had one injection of the nanoparticles, mice sacrificed after 48 h had two injections, and so on. At the required time, mice were euthanized using CO2 and the organs (liver, kidney, spleen, heart, and lungs) were surgically removed. All experiments were performed according to the Institutional Animal Care and Use Committee (IACUC) approved protocol and two mice were sacrificed for each time point. Organs collected were stored in RPMI solution at 4 C until further analysis. Flow cytometry analysis The organ tissues were minced using a 70 mm cell strainer (Falcon, Tewksbury, MA) to obtain single-cell suspension in RPMI solution. For each organ, an aliquot of 500 mm of cell suspension was measured on the flow cytometer. A flow cytometer (BD Accuri, Franklin Lakes, NJ) was used to determine the presence of fluorescent nanoparticles in the various organs collected from the mice. Population of single cells was defined in a plot of 90 side scattering versus forward scattering, and the percentage of nanoparticle-loaded
cells was counted by measuring fluorescence signal in the green fluorescence channel (FL1). RESULTS AND DISCUSSION
Morphology of the nanoparticles Glutenin nanoparticles had size in the range of 70–140 nm, confirmed using the particle size analyzer and the transmission electron microscopy (TEM) image shown in Figure 1, which makes them suitable for drug delivery and other medical applications. Proteins such as insulin, lysozyme, and myoglobin with diameters of 3–3.5 nm had blood half-time of 8.8–12 min and renal filtration between 75 and 100%.25 It has been reported that nanoparticles with diameters of 5.5 nm would be completely eliminated from the body.25 Although there has been no information on the size of the nanoparticles preferable for drug delivery, the previous studies have shown that protein nanoparticles with size around 100 nm could be used for targeted drug delivery. Glutenin nanoparticles could be made with diameters required for drug delivery. Effect of fabrication conditions on size of nanoparticles Size of the nanoparticles was considerably affected by the pH during precipitation and varied from 65 to 145 nm as shown in Figure 2. Both low and high pHs were suitable to obtain relatively smaller nanoparticles and the largest size was close to the isoelectric point of glutenin where agglomeration of the nanoparticles and resulting increase in size occurs. Although high pH (9–10) produced smaller particles, alkaline conditions could damage the proteins and it is preferable to choose pH of 2 or between 7 and 8 to produce the nanoparticles. Glutenin nanoparticles had considerably high f potential variation ranging from 2200 to 1 320 mV with the isoelectric point at about pH 6.8 similar to that reported earlier. Changing pH also affected the f potential of the nanoparticles and therefore the ability of the nanoparticles to attract various drugs and payloads as shown in Figure 3. f Potential also represents particle stability and larger (6) the f potential larger is the repulsion and
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FIGURE 3. f Potential of the glutenin nanoparticles at various pHs and the isoelectric point.
FIGURE 5. Stability of the nanoparticles at two different temperatures and pHs.
therefore more stable particles.26 Both f potential and pH suggest that acidic and slightly alkaline pH were more suitable to form the glutenin nanoparticles. f Potential at physiological pH of 7.2 (range, 25 to 215 mV) is suitable for medical applications. Glutenin nanoparticles have strong cationic/anionic charge, beneficial for loading drugs by changing the pH of the solution.27 Changing the ratio of ethylene glycol to glutenin above 4% showed a substantial increase in the size of the nanoparticles as shown in Figure 4. When the concentration of the protein in the solution is increased, the solubility decreased and there were considerably higher number of protein molecules. These protein molecules will easily agglomerate and increase in size. However, high temperatures may be useful to get lower size of nanoparticles even at higher concentrations of the protein solution.
agglomerate with increasing time except for the particles in pH 7 solution at 37 C. The agglomeration was higher when the pH was 2 and when the temperature was 4 versus 37 C. The ability of the nanoparticles to maintain their size at pH 7 and 37 C indicates that the nanoparticles would be suitable for most medical applications. Higher stability at pH 7 should be owing to the pH being close to the isoelectric point (6.8–7.0).28 Nanoparticles are more stable at 37 C than at 4 C because of the higher mobility of the particles and lesser chance to get agglomerated.
Stability of the glutenin nanoparticles Glutenin nanoparticles were more stable at pH 7 than pH 4 and at 37 C compared to the stability of the particles at4 C as shown in Figure 5. However, the nanoparticles tend to
FIGURE 4. Influence of the ratio of protein to ethylene glycol on the size of the nanoparticles.
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In vitro cell assay Addition of glutenin nanoparticles into the cell culture media did not affect cell viability or growth as shown in Figure 6. In fact, wells containing the glutenin nanoparticles showed slightly higher metabolic activity compared to the cells without any nanoparticles. It was confirmed that glutenin nanoparticles did not influence the assay results, suggesting that the increase in optical density was owing to the higher metabolic activity of the cells. Glutenin nanoparticles
FIGURE 6. Comparison of the optical densities of mouse fibroblast cells with and without nanoparticles based on MTS assay. The cells were cultured at 37 C for 5 days.
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cytotoxic. Other proteins such as silk are difficult to be dissolved and made into nanoparticles and also have excessively long degradation in the body making them not useful for targeted drug delivery. Previously, our group and other researchers have demonstrated that nanoparticles made from corn zein are biocompatible and could be used for drug delivery. However, the stability of the zein nanoparticles under different physiological conditions and their biodistribution are not fully understood. Compared to zein, nanoparticles from glutenin are more stable and also do not require crosslinking. In addition, glutenin nanoparticles would not have the risks associated with using animal proteins such as collagen.
FIGURE 7. Comparison of the percentage of cells containing nanoparticles detected in various organs in mice using flow cytometer after 1–4 days of injection.
may act as a nutrient or growth factor and promote cell growth thereby increasing the metabolic activity. Biodistribution of nanoparticles in mice Glutenin nanoparticles were predominantly found in the kidneys followed by liver and spleen in mice as shown in Figure 7. Increasing the dosage of the nanoparticles and/or residence time did not significantly affect the amount of nanoparticles found in the different organs. Kidneys are reported to filter (renal clearance) nanoparticles with a hydrodynamic radius of up to about 8 nm. As the glutenin nanoparticles were considerably larger, the nanoparticles accumulated in the kidneys.25,29 Particles that are not cleared by the kidneys are removed through the liver (hepatic clearance) and it has been reported that the livers clear nanoparticles in the range of 10–20 nm.29 Therefore, considerable amounts of nanoparticles were also found in the kidneys. Lower amounts of the glutenin nanoparticles were detected in the spleen and lungs as most of the particles were taken up by the kidneys and liver. There was no significant increase in the amount of nanoparticles in any of the organs after multiple injections, suggesting that the nanoparticles were easily digested in the body or were removed within 24 h before the next injection was made. It appears that glutenin nanoparticles could be used to effectively deliver drugs to kidneys. However, varying the size and surface modifications to the nanoparticles may increase the biodistribution of the nanoparticles in the other organs in mice. Advantages of glutenin nanoparticles The ability to develop nanoparticles from wheat glutenin with the size, stability, and biocompatibility required for medical applications could have significant implications. Common proteins used for medical applications such as collagen and albumin do not have the stability to be made into nanoparticles. Although crosslinking of protein nanoparticles has been done to improve stability, most of the crosslinking agents currently used for protein crosslinking are
CONCLUSIONS
Nanoparticles developed from wheat glutenin were biocompatible and found to be predominantly biodistributed in the kidneys in mice. With diameters as low as 30 nm and having good stability under physiological conditions, glutenin nanoparticles had the qualities required for medical applications. Changing the pH or ratio of solvent during preparation provided nanoparticles with different sizes. Glutenin nanoparticles have had strong anionic/cationic charge that would be helpful to load various types of drugs. In vitro studies indicated that glutenin-containing wells had higher metabolic activity compared to wells without the nanoparticles. Biocompatible and stable glutenin nanoparticles without the need for external crosslinking agents would be unique nanoparticles for drug delivery and other medical applications. REFERENCES 1. Nitta SK, Numata K. Biopolymer-based nanoparticles for drug/ gene delivery and tissue engineering. Int J Mol Sci 2013;14:1629– 1654. 2. Mailander V, Landfester K. Interaction of nanoparticles with cells. Biomacromolecules 2009;10:2379–2400. mos C, Grandemange S, Mograbi B, 3. Eidi H, Joubert O, Ne Foliguet B, Tournebize J, Maincent P, Le Faou A, Aboukhamis I, Rihn BH. Drug delivery by polymeric nanoparticles induces autophagy in macrophages. Int J Pharm 2012;422:495–503. 4. Yokel R, Grulke E, MacPhail R. Metal-based nanoparticle interactions with the nervous system: The challenge of brain entry and the risk of retention in the organism. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2013;5:346–373. 5. Kharissova OV, Dias HVR, Kharisov BI, Perez BO. The greener synthesis of nanoparticles. Cell 2013;31:240–248. 6. Chen P, Kanehira K, Sonezaki S, Taniguchi A. Detection of cellular response to titanium dioxide nanoparticle agglomerates by sensor cells using heat shock protein promoter. Biotechnol Bioeng 2012;109:3112–3118. 7. Lee KY, Yuk SH. Polymeric protein delivery systems. Prog Polym Sci 2007;32:669–697. 8. Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev 2003;55: 329–347. 9. Lopez P, Murdan S. Zein microspheres as drug antigen carriers: A study of their degradation and erosion, in the presence and absence of enzymes. J Microencapsul 2006;23:303–314. 10. Jahanshahi M, Babaei Z. Protein nanoparticle: A unique system as drug delivery vehicles. Afr J Biotechnol 2008;7:4926–4934. 11. Reddy N, Yang Y. Potential of plant proteins for medical application. Trends Biotechnol 2011;29:490–498. 12. Cai S, Xu H, Jiang Q, Yang Y. Novel 3D electrospun scaffolds with fibers oriented randomly and evenly in three dimensions to
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