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Engineering Strategies for Oral Therapeutic Enzymes to Enhance Their Stability and Activity

  • Philipp Lapuhs
  • Gregor FuhrmannEmail author
Chapter
  • 1.1k Downloads
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1148)

Abstract

Oral application of therapeutic enzymes is a promising and non-invasive administration that improves patient compliance. However, the gastrointestinal tract poses several challenges to the oral delivery of proteins, including harsh pH conditions and digestive proteases. A promising way to stabilise enzymes during their gastrointestinal route is by modification with polymers that can provide both steric shielding and selective interaction in different digestive compartments. We give an overview of modification technologies for oral enzymes ranging from functionalisation of native proteins, to site-specific mutation and protein-polymer engineering. We specifically focus on enzymes that are active directly in the gastrointestinal lumen and not systemically absorbed. In addition, we discuss examples of microparticle and nanoparticle encapsulated enzymes for improved oral delivery. The modification of orally administered enzymes offers a broad chemical variability and may be a promising tool for enhancing their gastrointestinal stability.

Keywords

Exogenous enzymes Gastrointestinal tract Oral delivery Enzyme therapy Protein-polymer conjugates Non-invasive imaging Gastro-resistant coating Pharmaceutical formulation Stomach-resistant coatings 

Abbreviations

2-BIBB

2-bromoisobutyryl bromide

AP

Alkaline phosphatase

ATRP

Atom transfer radical polymerization

BCA

Bicinchoninic acid

BTpNA

Benzoyl-l-tyrosine p-nitroanilide

CAP

Cellulose acetate phthalate

CD

Circular dichroism

CLSM

Confocal laser scanning microscopy

CM

Carboxymethyl

CT

α1-antichymotrypsin

DLS

Dynamic light scattering

DSC

Differential scanning calorimetry

EDC

1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide

FDA

Food and drug administration USA

FITC-BSA

Fluorescein isothiocyanate conjugate - bovine serum albumin

FTIR

Fourier-transform infrared spectroscopy

GI

Gastrointestinal

GPC

Gel permeation chromatography

H/D

Hydrogen/deuterium

HA

Hyaluronic acid

HAP

Hydroxyapatite

HPLC

High performance liquid chromatography

HPMCP

Hydroxyl propyl methyl cellulose phthalate

HRP

Horseradish peroxidase

LC-MS

Liquid chromatography–mass spectrometry

LCST

Lower critical solution temperature

MALDI-TOF-MS

Matrix assisted laser desorption ionization time-of-flight mass spectrometry

MP

Microparticle

mPEG2-NHS

Branched PEG N-hydroxysuccinimide

MW

Molecular weight

NCC

Nanoceramic cores

NHS

N-hydroxysuccinimide

NHS-Br

N-Hydroxysuccinimide-bromide

NMR

Nuclear magnetic resonance

o-NP

Ortho-nitrophenol

o-NPG

Ortho-nitrophenyl-β-galactoside

PAMAM

Poly(amidoamine)

PBPE

Polymer-based protein engineering

pCBAm

Poly (carboxybetaine acrylamide)

PDMAEMA

Poly(2-(dimethylamino)ethyl methacrylate)

pDMAPS

Poly[N,N′-dimethyl (methacryloylethyl) ammonium propane sulfonate]

PEG

Polyethylene glycol

PEP

Proline-specific endopeptidase

pNIPAm

Poly (N-isopropylacry-lamide)

pOEGMA

Poly(oligoethylene glycol monomethylether methacry-late)

pQA

Poly-(quarternary ammonium methacrylate

pSMA

Poly-(sulfonate methac-rylate)

RT

Room temperature

SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SEC

Size exclusion chromatography

SEM

Scanning electron microscopy

SGC

Simulated gastric conditions

SGF

Simulated gastric fluid

SIC

Simulated intestinal tract conditions

SIF

Simulated intestinal fluid

Suc-Ala-Ala-Pro-Phe-pNA

N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyla-lanine4-nitroanilide

TEM

Transmission electron microscopy

Tm

Denaturation midpoint

TM-AvPAL

Triple mutant-Anabaena variabilis phenylalanine ammonia lyase

TNBSA

2,4,6-trinitrobenzene sulfonic acid

UCST

Upper critical solution temperature

UV-vis

Ultraviolet-visible α1-anti

α-CT

α-chymotrypsin

References

  1. Aguado BA, Grim JC, Rosales AM, Watson-Capps JJ, Anseth KS (2018) Engineering precision biomaterials for personalized medicine. Sci Transl Med 10(424)PubMedPubMedCentralCrossRefGoogle Scholar
  2. Aloulou A, Schué M, Puccinelli D, Milano S, Delchambre C, Leblond Y, Laugier R, Carriere F (2015) Yarrowia Lipolytica lipase 2 is stable and highly active in test meals and increases fat absorption in an animal model of pancreatic exocrine insufficiency. Gastroenterology 149(7):1910–1919.e5PubMedCrossRefGoogle Scholar
  3. Bansil R, Turner BS (2018) The biology of mucus: composition, synthesis and organization. Adv Drug Deliv Rev 12:43–15Google Scholar
  4. Bélanger-Quintana A, Burlina A, Harding CO, Muntau AC (2011) Up to date knowledge on different treatment strategies for phenylketonuria. Mol Genet Metab 104(0):S19–S25PubMedPubMedCentralCrossRefGoogle Scholar
  5. Blau N, Van Spronsen FJ, Levy HL (2010) Phenylketonuria. Lancet 376(9750):1417–1427CrossRefGoogle Scholar
  6. Blocher M, Walde P, Dunn IJ (1999) Modeling of enzymatic reactions in vesicles: the case of alpha-chymotrypsin. Biotechnol Bioeng 62(1):36–43PubMedCrossRefGoogle Scholar
  7. Brock A, Aldag I, Edskes S, Hartmann M, Herzog T, Uhl W, Schnekenburger J (2016) Novel ciliate lipases for enzyme replacement during exocrine pancreatic insufficiency. Eur J Gastroenterol Hepatol 28(11):1305–1312PubMedCrossRefGoogle Scholar
  8. Chopra S, Bertrand N, Lim J-M, Wang A, Farokhzad OC, Karnik R (2017) Design of insulin-loaded nanoparticles enabled by multistep control of nanoprecipitation and zinc chelation. ACS Appl Mater Interfaces 9(13):11440–11450PubMedPubMedCentralCrossRefGoogle Scholar
  9. Clardy-James S, Allis DG, Fairchild TJ, Doyle RP (2012) Examining the effects of vitamin B12 conjugation on the biological activity of insulin: a molecular dynamic and in vivo oral uptake investigation. MedChemComm 3(9):1054–1058CrossRefGoogle Scholar
  10. Cook MT, Tzortzis G, Charalampopoulos D, Khutoryanskiy VV (2012) Microencapsulation of probiotics for gastrointestinal delivery. J Control Release 162(1):56–67PubMedCrossRefGoogle Scholar
  11. Couvreur P, Puisieux F (1993) Nano- and microparticles for the delivery of polypeptides and proteins. Adv Drug Deliv Rev 10(2):141–162CrossRefGoogle Scholar
  12. Cummings C, Murata H, Koepsel R, Russell AJ (2013) Tailoring enzyme activity and stability using polymer-based protein engineering. Biomaterials 34(30):7437–7443PubMedCrossRefGoogle Scholar
  13. Cummings CS, Campbell AS, Baker SL, Carmali S, Murata H, Russell AJ (2017) Design of stomach acid-stable and mucin-binding enzyme polymer conjugates. Biomacromolecules 18(2):576–586PubMedCrossRefGoogle Scholar
  14. Dean SN, Turner KB, Medintz IL, Walper SA (2017) Targeting and delivery of therapeutic enzymes. Ther Deliv 8(7):577–595PubMedCrossRefGoogle Scholar
  15. Donaldson GP, Lee SM, Mazmanian SK (2015) Gut biogeography of the bacterial microbiota. Nat Rev Microbiol 14:20PubMedPubMedCentralCrossRefGoogle Scholar
  16. Eek D, Krohe M, Mazar I, Horsfield A, Pompilus F, Friebe R, Shields AL (2016) Patient-reported preferences for oral versus intravenous administration for the treatment of cancer: a review of the literature. Patient Prefer Adherence 10:1609–1621PubMedPubMedCentralCrossRefGoogle Scholar
  17. Ehren J, Govindarajan S, Moron B, Minshull J, Khosla C (2008) Protein engineering of improved prolyl endopeptidases for celiac sprue therapy. Protein Eng Des Sel 21(12):699–707PubMedPubMedCentralCrossRefGoogle Scholar
  18. Frizzell H, Ohlsen TJ, Woodrow KA (2017) Protein-loaded emulsion electrospun fibers optimized for bioactivity retention and Ph-controlled release for peroral delivery of biologic therapeutics. Int J Pharm 533(1):99–110PubMedPubMedCentralCrossRefGoogle Scholar
  19. Fuhrmann G (2018) Luminal coating of the intestine. Nat Mater 17:754–755PubMedCrossRefGoogle Scholar
  20. Fuhrmann K, Fuhrmann G (2017) Recent advances in oral delivery of macromolecular drugs and benefits of polymer conjugation. Curr Opin Colloid Interface Sci 31:67–74CrossRefGoogle Scholar
  21. Fuhrmann G, Leroux J-C (2011) In vivo fluorescence imaging of exogenous enzyme activity in the gastrointestinal tract. Proc Natl Acad Sci U S A 108(22):9032–9037PubMedPubMedCentralCrossRefGoogle Scholar
  22. Fuhrmann G, Leroux J-C (2014) Improving the stability and activity of oral therapeutic enzymes – recent advances and perspectives. Pharm Res 31(5):1099–1105PubMedCrossRefGoogle Scholar
  23. Fuhrmann G, Grotzky A, Lukic R, Matoori S, Luciani P, Yu H, Walde P, Schlüter AD, Gauthier MA, Leroux J-C (2013) Sustained gastrointestinal activity of dendronized polymer-enzyme conjugates. Nat Chem 5:582–589PubMedCrossRefGoogle Scholar
  24. Fuhrmann G, Chandrawati R, Pamar PA, Keane TJ, Maynard SA, Bertazzo S, Stevens MM (2018) Engineering extracellular vesicles with the tools of enzyme prodrug therapy. Adv Mater 30:1706616CrossRefGoogle Scholar
  25. Gauthier MA, Klok H-A (2010) Polymer-protein conjugates: an enzymatic activity perspective. Polym Chem 1(9):1352–1373CrossRefGoogle Scholar
  26. Graham ML (2003) Pegaspargase: a review of clinical studies. Adv Drug Deliv Rev 55(10):1293–1302PubMedCrossRefGoogle Scholar
  27. Grotzky A, Nauser T, Erdogan H, Schlüter AD, Walde P (2012) A fluorescently-labeled dendronized polymer-enzyme conjugate carrying multiple copies of two different types of active enzymes. J Am Chem Soc 134:11392–11395PubMedCrossRefGoogle Scholar
  28. Gupta V, Hwang BH, Doshi N, Mitragotri S (2013) A permeation enhancer for increasing transport of therapeutic macromolecules across the intestine. J Control Release 172(2):541–549PubMedCrossRefGoogle Scholar
  29. Hafner A, Lovrić J, Lakoš GP, Pepić I (2014) Nanotherapeutics in the Eu: an overview on current state and future directions. Int J Nanomed 9:1005–1023Google Scholar
  30. Hamuro Y, Coales SJ, Molnar KS, Tuske SJ, Morrow JA (2008) Specificity of immobilized porcine pepsin in H/D exchange compatible conditions. Rapid Commun Mass Spectrom 22(7):1041–1046PubMedCrossRefGoogle Scholar
  31. Ishida T, Maeda R, Ichihara M, Irimura K, Kiwada H (2003) Accelerated clearance of pegylated liposomes in rats after repeated injections. J Control Release 88(1):35–42PubMedCrossRefGoogle Scholar
  32. Kang TS, Wang L, Sarkissian CN, Gámez A, Scriver CR, Stevens RC (2010) Converting an injectable protein therapeutic into an oral form: phenylalanine ammonia lyase for phenylketonuria. Mol Genet Metab 99(1):4–9PubMedPubMedCentralCrossRefGoogle Scholar
  33. Kim W, Erlandsen H, Surendran S, Stevens RC, Gamez A, Michols-Matalon K, Tyring SK, Matalon R (2004) Trends in enzyme therapy for phenylketonuria. Mol Ther 10(2):220–224PubMedCrossRefGoogle Scholar
  34. Klinger D, Landfester K (2011) Dual stimuli-responsive poly(2-hydroxyethyl methacrylate-co-methacrylic acid) microgels based on photo-cleavable cross-linkers: Ph-dependent swelling and light-induced degradation. Macromolecules 44(24):9758–9772CrossRefGoogle Scholar
  35. Kumar A, Montemagno C, Choi H-J (2017) Smart microparticles with a Ph-responsive macropore for targeted oral drug delivery. Sci Rep 7(1):3059PubMedPubMedCentralCrossRefGoogle Scholar
  36. Lee Y, Deelman TE, Chen K, Lin DSY, Tavakkoli A, Karp JM (2018) Therapeutic luminal coating of the intestine. Nat Mater 17:834–842PubMedCrossRefGoogle Scholar
  37. Leeds JS, Oppong K, Sanders DS (2011) The role of fecal elastase-1 in detecting exocrine pancreatic disease. Nat Rev Gastroenterol Hepatol 8(7):405–415PubMedCrossRefGoogle Scholar
  38. Leonard F, Ali H, Collnot EM, Crielaard BJ, Lammers T, Storm G, Lehr CM (2012) Screening of budesonide nanoformulations for treatment of inflammatory bowel disease in an inflamed 3d cell-culture model. Altex 29(3):275–285PubMedCrossRefGoogle Scholar
  39. Liu M, Tirino P, Radivojevic M, Phillips D, Gibson M, Leroux J-C, Gauthier MA (2012) Molecular sieving on the surface of a protein provides protection without loss of activity. Adv Funct Mater 23(16):2007–2015CrossRefGoogle Scholar
  40. Liu Y, Lee J, Mansfield KM, Ko JH, Sallam S, Wesdemiotis C, Maynard HD (2017) Trehalose glycopolymer enhances both solution stability and pharmacokinetics of a therapeutic protein. Bioconjug Chem 28(3):836–845PubMedPubMedCentralCrossRefGoogle Scholar
  41. Lomer MCE, Parkes GC, Sanderson JD (2008) Review article: lactose intolerance in clinical practice – myths and realities. Aliment Pharmacol Ther 27(2):93–103PubMedCrossRefGoogle Scholar
  42. Lundh G (1957) Determination of trypsin and chymotrypsin in human intestinal content. Scand J Clin Lab Invest 9(3):229–232PubMedCrossRefGoogle Scholar
  43. Mitragotri S, Burke PA, Langer R (2014) Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat Rev Drug Discov 13(9):655–672PubMedPubMedCentralCrossRefGoogle Scholar
  44. Moroz E, Matoori S, Leroux J-C (2016) Oral delivery of macromolecular drugs: where we are after almost 100 years of attempts. Adv Drug Deliv Rev 101:108–121PubMedCrossRefGoogle Scholar
  45. Murata H, Cummings CS, Koepsel RR, Russell AJ (2013) Polymer-based protein engineering can rationally tune enzyme activity, Ph-dependence, and stability. Biomacromolecules 14(6):1919–1926PubMedCrossRefGoogle Scholar
  46. Murgia X, Loretz B, Hartwig O, Hittinger M, Lehr C-M (2018) The role of mucus on drug transport and its potential to affect therapeutic outcomes. Adv Drug Deliv Rev 124:82–97PubMedCrossRefGoogle Scholar
  47. Naikwade SR, Meshram RN, Bajaj AN (2009) Preparation and in vivo efficacy study of pancreatin microparticles as an enzyme replacement therapy for pancreatitis. Drug Dev Ind Pharm 35(4):417–432PubMedCrossRefGoogle Scholar
  48. Parihar AKS, Srivastava S, Patel S, Singh MR, Singh D (2017) Novel catalase loaded nanocores for the treatment of inflammatory bowel diseases. Artif Cells Nanomed Biotechnol 45(5):981–989PubMedCrossRefGoogle Scholar
  49. Parmar PA, Chow LW, St-Pierre J-P, Horejs C-M, Peng YY, Werkmeister JA, Ramshaw JAM, Stevens MM (2015) Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration. Biomaterials 54:213–225PubMedPubMedCentralCrossRefGoogle Scholar
  50. Pascucci T, Rossi L, Colamartino M, Gabucci C, Carducci C, Valzania A, Sasso V, Bigini N, Pierigè F, Viscomi MT, Ventura R, Cabib S, Magnani M, Puglisi-Allegra S, Leuzzi V (2018) A new therapy prevents intellectual disability in mouse with phenylketonuria. Mol Genet Metab 124(1):39–49PubMedCrossRefGoogle Scholar
  51. Pashuck ET, Stevens MM (2012) Designing regenerative biomaterial therapies for the clinic. Sci Transl Med 4(160):160sr4PubMedCrossRefGoogle Scholar
  52. Pinier M, Fuhrmann G, Verdu E, Leroux J-C (2010) Prevention measures and exploratory pharmacological treatments of celiac disease. Am J Gastroenterol 105:2551–2561PubMedCrossRefGoogle Scholar
  53. Regan PT, Malagelada J-R, Dimagno EP, Glanzman SL, Go VLW (1977) Comparative effects of antacids, cimetidine and enteric coating on the therapeutic response to oral enzymes in severe pancreatic insufficiency. N Engl J Med 297(16):854–858PubMedCrossRefGoogle Scholar
  54. Robic S (2007) Pegylated glutenase polypeptides. USA patent application WO/2007/047303. 26.04.2007Google Scholar
  55. Rodríguez-Martínez JA, Solá RJ, Castillo B, Cintrón-Colón HR, Rivera-Rivera I, Barletta G, Griebenow K (2008) Stabilization of Α-chymotrypsin upon pegylation correlates with reduced structural dynamics. Biotechnol Bioeng 101(6):1142–1149PubMedPubMedCentralCrossRefGoogle Scholar
  56. Rodríguez-Martínez JA, Rivera-Rivera I, Solá RJ, Griebenow K (2009) Enzymatic activity and thermal stability of Peg-Α-chymotrypsin conjugates. Biotechnol Lett 31(6):883–887PubMedPubMedCentralCrossRefGoogle Scholar
  57. Sarkissian CN, Shao Z, Blain F, Peevers R, Su H, Heft R, Chang TMS, Scriver CR (1999) A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase. Proc Natl Acad Sci U S A 96(5):2339–2344PubMedPubMedCentralCrossRefGoogle Scholar
  58. Sarkissian CN, Gámez A, Wang L, Charbonneau M, Fitzpatrick P, Lemontt JF, Zhao B, Vellard M, Bell SM, Henschell C, Lambert A, Tsuruda L, Stevens RC, Scriver CR (2008) Preclinical evaluation of multiple species of pegylated recombinant phenylalanine ammonia lyase for the treatment of phenylketonuria. Proc Natl Acad Sci U S A 105(52):20894–20899PubMedPubMedCentralCrossRefGoogle Scholar
  59. Schulz JD, Gauthier MA, Leroux J-C (2015a) Improving oral drug bioavailability with polycations? Eur J Pharm Biopharm 97(Part B):427–437PubMedCrossRefGoogle Scholar
  60. Schulz JD, Patt M, Basler S, Kries H, Hilvert D, Gauthier MA, Leroux J-C (2015b) Site-specific polymer conjugation stabilizes therapeutic enzymes in the gastrointestinal tract. Adv Mater 28(7):1455–1460PubMedCrossRefGoogle Scholar
  61. Shan L, Marti T, Sollid LM, Gray GM, Khosla C (2004) Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue. Biochem J 383(2):311–318PubMedPubMedCentralCrossRefGoogle Scholar
  62. Tack GJ, Verbeek WHM, Schreurs MWJ, Mulder CJJ (2010) The spectrum of celiac disease: epidemiology, clinical aspects and treatment. Nat Rev Gastroenterol Hepatol 7:204–213PubMedCrossRefGoogle Scholar
  63. Tack GJ, Van De Water JMW, Bruins MJ, Kooy-Winkelaar EMC, Van Bergen J, Bonnet P, Vreugdenhil ACE, Korponay-Szabo I, Edens L, Von Blomberg BME, Schreurs MWJ, Mulder CJ, Koning F (2013) Consumption of gluten with gluten-degrading enzyme by celiac patients: a pilot-study. World J Gastroenterol 19(35):5837–5847PubMedPubMedCentralCrossRefGoogle Scholar
  64. Tang BC, Dawson M, Lai SK, Wang Y-Y, Suk JS, Yang M, Zeitlin P, Boyle MP, Fu J, Hanes J (2009) Biodegradable polymer nanoparticles that rapidly penetrate the human mucus barrier. Proc Natl Acad Sci 106(46):19268–19273PubMedCrossRefGoogle Scholar
  65. Tang D-W, Yu S-H, Wu W-S, Hsieh H-Y, Tsai Y-C, Mi F-L (2014) Hydrogel microspheres for stabilization of an antioxidant enzyme: effect of emulsion cross-linking of a dual polysaccharide system on the protection of enzyme activity. Colloids Surf B: Biointerfaces 113:59–68PubMedCrossRefGoogle Scholar
  66. Turner JR (2009) Intestinal mucosal barrier function in health and disease. Nat Rev Immunol 9(11):799–809PubMedCrossRefGoogle Scholar
  67. Turner KM, Pasut G, Veronese FM, Boyce A, Walsh G (2011) Stabilization of a supplemental digestive enzyme by post-translational engineering using chemically-activated polyethylene glycol. Biotechnol Lett 33(3):617–621PubMedCrossRefGoogle Scholar
  68. Veronese FM, Pasut G (2005) Pegylation, successful approach to drug delivery. Drug Discov Today 10(21):1451–1458PubMedCrossRefGoogle Scholar
  69. Vllasaliu D, Thanou M, Stolnik S, Fowler R (2018) Recent advances in oral delivery of biologics: nanomedicine and physical modes of delivery. Expert Opin Drug Deliv 15(8):759–770PubMedCrossRefGoogle Scholar
  70. Vorselen D, Van Dommelen SM, Sorkin R, Piontek MC, Schiller J, Döpp ST, Kooijmans SAA, Van Oirschot BA, Versluijs BA, Bierings MB, Van Wijk R, Schiffelers RM, Wuite GJL, Roos WH (2018) The fluid membrane determines mechanics of erythrocyte extracellular vesicles and is softened in hereditary spherocytosis. Nat Commun 9(1):4960PubMedPubMedCentralCrossRefGoogle Scholar
  71. Zelikin AN, Ehrhardt C, Healy AM (2016) Materials and methods for delivery of biological drugs. Nat Chem 8(11):997–1007PubMedCrossRefGoogle Scholar
  72. Zhang S, Bellinger AM, Glettig DL, Barman R, Lee Y-AL, Zhu J, Cleveland C, Montgomery VA, Gu L, Nash LD, Maitland DJ, Langer R, Traverso G (2015) A Ph-responsive supramolecular polymer gel as an enteric elastomer for use in gastric devices. Nat Mater 14:1065–1071. advance online publicationPubMedPubMedCentralCrossRefGoogle Scholar
  73. Zhang Z, Zhang R, Mcclements DJ (2017) Lactase (Β-galactosidase) encapsulation in hydrogel beads with controlled internal Ph microenvironments: impact of bead characteristics on enzyme activity. Food Hydrocoll 67:85–93CrossRefGoogle Scholar
  74. Zhao Y, Wang C, Wang L, Yang Q, Tang W, She Z, Deng Y (2012) A frustrating problem: accelerated blood clearance of pegylated solid lipid nanoparticles following subcutaneous injection in rats. Eur J Pharm Biopharm 81(3):506–513PubMedCrossRefGoogle Scholar

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© Springer Nature Singapore Pte Ltd. 2019

Authors and Affiliations

  1. 1.Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Biogenic Nanotherapeutics Group (BION)SaarbrückenGermany
  2. 2.Department of PharmacySaarland UniversitySaarbrückenGermany

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