Isabel Izquierdo Barba Universidad Complutense de Madrid, Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN).
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This document summarizes research on using mesoporous silica nanoparticles (MSNs) to target and treat bacterial infections. Specifically, it discusses functionalizing MSNs with targeting agents like dendrimers (MSN-G3) to selectively deliver antibiotics to gram-negative bacteria and destroy biofilms. MSN-G3 showed effective targeting of E. coli and biofilm destruction within 1 hour when loaded with the antibiotic levofloxacin. The research aims to develop new nanomedicine approaches for treating drug-resistant infections by using stimuli-responsive MSNs for controlled antibiotic delivery directly to bacteria.
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Isabel Izquierdo Barba Universidad Complutense de Madrid, Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN).
- 1. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Dra. Isabel Izquierdo-Barba Smart Bimaterials Research Group Dpto. Química en Ciencias Farmacéuticas, Universidad Complutense de Madrid, España. ibarba@ucm.es VERDI polyValent mEsopoRous nanosystem for bone DIseases Recent advances in mesoporous materials for management of bone infection
- 2. Infection !! Evolution in modern aseptic techniques International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018
- 3. Current therapies Antibiotics Replacements Amputation Death (7%) International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018
- 4. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Infection Persistence Antimicrobial Resistance (AMR) Development and maduration Detachment Antibiotic Immune system Free-Bacteria (Planktonic state) Substrate Biofilm Formation Adhesion Monolayer Proliferation Microcolony D. Campoccia, L. Montanaro, C.R. Arciola, Biomaterials 27 (2006) 2331–2339. Mature Biofilm Super Bug
- 5. New Post antibiotic era: The next generation challenge? World Health Organization. Antimicrobial Resistance: Global Report on Surveillance 2014 (WHO, 2014). G. Taubes, The bacteria fight back, Science 321 (2008) 356–361
- 6. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Drug resistant deadlier than cancer by 2050 World Health Organization. Antimicrobial Resistance: Global Report on Surveillance 2014 (WHO, 2014). G. Taubes, The bacteria fight back, Science 321 (2008) 356–361
- 7. A ten point plan for beating drug resistant 1. A Global public awareness 2. Improve sanitation and prevent the spread of infection 3. Reduce unnecessary use of antimicrobials in agriculture 4. Improve global surveillance of drug resistance and antimicrobial consumption 5. Promote new rapid diagnostics 6. Promote use of vaccines 7. Improve the number, pay and recognition of people working in infection diseases 8. Increase the supply of new antimicrobial effective against drug-resistant bugs 9. A global innovation fund for early stage and non commercial R&D 10. Better incentives to promote investment for new drugs and improving existing ones The Review on Antimicrobial Resistance Chaired by Jim O’Neill December 2014 8. Increase the supply of new antimicrobial effective against drug-resistant bugs 9. A global innovation fund for early stage and non commercial R&D 10. Better incentives to promote investment for new drugs and improving existing ones
- 8. Now WHAT TO DO? Nanomedicine: The future of infection treatment A.J. Huh, Y.J. Kwon, J. Control. Release 156 (2011) 128–145. L. Zhang, D. Pornpattananangku, C.M.J. Hu, C.M. Huang, Curr. Med. Chem. 17 (2010) 585–594 Drug delivery systems Selectively transport antimicrobial agents (target site) Controlled antimicrobial fashion Improve antimicrobial efficacy Safety and Biocompatibility Repair damaged tissues International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018
- 9. Numberofpublications 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 Publication Year “Mesoporous + silica + drug + delivery” ISI Web of Science (7.107 publications) 1300 2018 International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 M. Vallet-Regí, F. Balas and D. Arcos Angew Chem Int Ed 46 (2007) 7548-7558. M. Vallet-Regí, M. Colilla, I. Izquierdo-Barba, M. Manzano, Molecules, 23 (2018) 47.
- 10. 50 nm International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 MSN Stimuli- responsive drug delivery Targeting Biomolecules delivery Gene therapy Intracelullar labeling Diagnostic imaging Fluorescent molecule MRI agent Drugs Proteins Photosensitizer Photodynamic therapy Nucleid acids Dendritic polymer Responsive blocking caps Mesoporous Silica Materials Stimuli-responsive local drug delivery Local Biomolecules delivery Proteins Bone tissue regeneration 1 cm Local drug delivery Drugs Growth factors, peptides 3D scaffolds Responsive blocking caps Mesoporous Silica Nanoparticles MSM 50 nm M. Manzano, M. Colilla, M. Vallet-Regí Expert Opin Drug Deliv 6 (2009) 1-18. M. Vallet-Regí, L. Ruiz-González, I. Izquierdo-Barba, JM. González-Calbet J Mater Chem 16 (2006) 26-31. M. Vallet-Regí, M. Colilla, I. Izquierdo-Barba, M. Manzano, Molecules, 23 (2018) 47.
- 11. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Hypothesis Use MSNs with targeting properties to release antibiotics in the environment of the bacteria that cause the infection. G. Domenico. The procession of the trojan horse into Troy, 1760 Targeting bacteria 20 mm Targeting biofilm
- 12. Targeting to Gram-negative bacteria Gram (-) - - - - - - - - - - - - - Antimicrobial agent Levofloxacin Targeting agent Polycationic dendrimer (G3) +δ N N N N N NH2 NH2 NH2 NN N H2N H2N H2N NN N H2N H2N H2N H2N N N N NH2 NH2 NH2 NH2 NH2 N C NSi O O O H H O Nanoplatform MSN Nanoantibiotic +δ +δ +δ +δ +δ +δ +δ +δ +δ International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 B. González, M. Colilla, J. Diez, D. Pedraza, M. Guembe, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 68 (2018) 261-271. Aim #1:
- 13. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 CH2Cl2 , N2 , RT N N N N N NH2 NH2 NH2 NN N H2N H2N H2N NN N H2N H2N H2N H2N N N N NH2 NH2 NH2 NH2 NH2 N C NSi O O O H H O G3 (NH2)15 MSN-G3 Targeting to Gram-negative bacteria Dry toluene N2 , 110 ºC, 16 h 1) 2) NH4NO3 / EtOH , 60 ºC (EtO)3Si N NH2 H Si N NH2 O O O H MSN-DAMO Si N NH2 O O O H Synthesis B. González, M. Colilla, J. Diez, D. Pedraza, M. Guembe, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 68 (2018) 261-271.
- 14. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 50 nm MSN 1 2 3 4 5 6 Intensity(a.u.) 2q (degree) 10 11 20 MSN MSN-DAMO MSN-G3 MSN-DAMO 50 nm MSN-G3 50 nm Targeting to Gram-negative bacteria B. González, M. Colilla, J. Diez, D. Pedraza, M. Guembe, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 68 (2018) 261-271.
- 15. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Targeting to Gram-negative bacteria B. González, M. Colilla, J. Diez, D. Pedraza, M. Guembe, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 68 (2018) 261-271. D. Pedraza, J. Díez, I. Izquierdo, M. Colilla, M. Vallet-Regí, Biomed. Glasses. 4 (2018) 1-12 . z-potential/TGA Sample Potencial ζ (mV) F (mmol/g) MSN -36.4 - MSN-DAMO 37.4 1.86 MSN-G3 31.8 0.106 -20 -60 -100 -140 -180 -111.2 -102.2 Q4 Q3 MSN Q2 -95.4 -58.7 -68.1T2 T3 -94.7 -102.4 -111.8 Q2 Q3 Q4 MSN-DAMO d (ppm) MSN-G3 Q4 -112.4 Q3 -102.1 -92.8 Q2 29Si MAS NMR MSN-G3 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 d (ppm) N N N N N NH2 NH2 NH2 NN N H2N H2N H2N NN N H2N H2N H2N H2N N N N NH2 NH2 NH2 NH2 NH2 N C NSi O O O H H O # # 1H HRMAS NMR
- 16. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 0 100 200 300 400 0.0 0.2 0.4 0.6 0.8 1.0 W/W0 Time (h) MSN-L MSN-DAMO-LMSN-G3-L MSN-L: W/W0 = 0.46(1-e-0.021t)0.56 ; R2 = 0.994 MSN-DAMO-L: W/W0 = 0.98(1-e-0.051t)0.68 ; R2 = 0.9997 MSN-G3-L: W/W0 = 0.99(1-e-0.239t)1.0 ; R2 = 0.995 3 Targeting to Gram-negative bacteria B. González, M. Colilla, J. Diez, D. Pedraza, M. Guembe, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 68 (2018) 261-271. D. Pedraza, J. Díez, I. Izquierdo, M. Colilla, M. Vallet-Regí, Biomed. Glasses. 4 (2018) 1-12 . Levofloxacin Release
- 17. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 20183 Targeting to Gram-negative bacteria B. González, M. Colilla, J. Diez, D. Pedraza, M. Guembe, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 68 (2018) 261-271. D. Pedraza, J. Díez, I. Izquierdo, M. Colilla, M. Vallet-Regí, Biomed. Glasses. 4 (2018) 1-12 . Microbiology assays: E. coli 5 - 50 μg/mL 5-50 μg/mL 90 min Bacteria targeting assays E. coli 90 min Effectiveness biofilm assays CONFOCAL MICROSCOPE
- 18. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 E. coli CONTROL 3 mm MSN 3 mm MSN-G3 3 mm MSN-DAMO 3 mm Targeting to Gram-negative bacteria
- 19. MSN-G3 (5 mg/mL) 3 mm 3 mm MSN-G3 (10 mg/mL) MSN-G3 (20 mg/mL) International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Targeting to Gram-negative bacteria B. González, M. Colilla, J. Diez, D. Pedraza, M. Guembe, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 68 (2018) 261-271.
- 20. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 MSN-G3-L 20 mm MSN-DAMO-L 20 mm E. coli CONTROL 20 mm MSN-L 20 mm Targeting to Gram-negative bacteria Antimicrobial efefcts (biofilm):
- 21. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Targeting to Gram-negative bacteria levofloxacin 20 mm20 mm3 mm Effective biofilm destruction with levofloxacin loaded dendrimer-decorated MSNs E. coli E. coli biofilm MSN-G3-L 1 h Targeting to Gram-negative bacteria N N N N N NH2 NH2 NH2 NN N H2N H2N H2N NN N H2N H2N H2N H2N N N N NH2 NH2 NH2 NH2 NH2 N C N H H O Si O O O MSN-G3
- 22. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Targeting to bacterial biofilm 10 mm Ti6Al4V Nano-Ti6Al4V extracellular matrix S. Wagner, D.Hauck, M. Hoffmann, R. Sommer, I. Joachim, R.Mgller, A.Imberty, A. Varrot, A. Titz. Angew.Chem. Int. Ed. 2017, 56, 16559 –16564 M. Mlouka, T. Cousseau, P. Di Martino, AIMS Molecular Science, 3 (2016) 338-356. Lectins (Glycoconjugates) High Affinitty Polysaccharides DAPI (germs) FITC-Lentins TRITC-Lentins Cacofluor (matrix) SYTO (alive cells) IP (dead cells) Biofilm Specific stainned
- 23. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 M. Martínez-Carmona, D. Lozano, M. Colilla, M. Vallet-Regí, Acta Biomater. 2018, 65, 393–404. MC3T3-E1 HOS Lectin-conjugated MSNs for targeted bone cancer treatment Targeting to bacterial biofilm
- 24. Targeting to bacterial biofilm Antimicrobial agent Levofloxacin Targeting agent Lectin ConA Nanoplatform MSN Nanoantibiotic International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 MSN-ConA M. Martínez-Carmona, I. Izquierdo-Barba, M. Colilla, M. Vallet-Regí (under preparatiom) Aim #2:
- 25. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Targeting to bacteria biofilm SATES Toluen Anhydre conditions ConA EDC/ Sulfo-NHS Surfactant removal NH4NO3 (10 mg/mL) EtOH/H2O (95%) Levofloxacin Loading EtOH M. Martínez-Carmona, I. Izquierdo-Barba, M. Colilla, M. Vallet-Regí (under preparatiom)
- 26. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 50 nm 50 nm50 nm MSN MSNConAMSNSATES Targeting to bacterial biofilm 0 10 20 30 40 50 60 70 80 90 100 0 50 100 150 200 250 300 350 Levofloxacinrelease(%) T (h) MSN MSNConA 5001000150020002500300035004000 MSN MSNConA MSNSATES NHbend COOH Amide cm-1
- 27. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 20183 Targeting to bacterial biofilm Microbiology assays: CONFOCAL MICROSCOPE 5-20 μg/mL 90 min Biofilm targeting assays E. coli MSN MSN-ConA 5-10 μg/mL E. coli, S.aureus 90 min Effectiveness biofilm assays MSN@Levo MSN-ConA@Levo
- 28. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Targeting to bacterial biofilm MSN-ConA 50ug/mL MSN 10ug/mL MSN 50ug/mL 20µm Biofilm polysaccharide matrix Nanoparticles (MSN and MSNConA) Alived bacteria 20µm 20µm MSN-ConA 10ug/mL 20µm
- 29. Antimicrobial effect of MSN-ConA MSN-ConA 10ug/mLMSN 10ug/mLBiofilm International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Biofilm polysaccharide matrix Alived bacteria Dead bacteria Targeting to bacterial biofilm 20mm20mm20mm Antimicrobial effect of MSN-ConA@Levo MSN-ConA Levo 10ug/mLMSN-Levo 10ug/mLBiofilm 20mm20mm20mm
- 30. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Targeting to bacterial biofilm levofloxacin 20 mm20 mm Effective biofilm destruction with levofloxacin loaded Lectin decorated-MSN E. coli E. coli biofilm MSNConA-L 1 h Targeting to the bacterial biofilm OH OHSiO2 OH OH MSN-ConA-
- 31. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Multifunctional 3D implants Use mesoporous materials able to inhibit the bone infection and to regenerate new bone Aim #3: Robocasting
- 32. Atomic-scale 5 nm 5 nm SiO2-P2O5-CaO OH- OH-OH- OH-OH- OH- OH- OH- OH- OH- OH- OH- OH-OH-OH- OH- Si P Ca O Sol-gel glass Ordered mesoporous glass Atomic-scale 50nm Meso-scale Multifunctional 3D implants International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 M. Vallet-Regí, Chem. Eur. J. 12 (2006) 5934–5943. M. Vallet-Regí, I. Izquierdo-Barba, M. Colilla. Phil. Trans. R. Soc. A 370 (2012) 1400–142.
- 33. Multifunctional 3D implants International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 GRIFMGLEVPVAVAN MGLEVPVAVAN 5 mm LEV-contained MG VAN-contained PVA solution + Gelatin-Glu solution with RIF 5 mm Slurry Robocasting 3D scaffold Dip-coated 50nm50nm -CH2-CH-CH2-CH- OH - OH - Polyvinyl alcohol (PVA) Vancomycin (VAN) 50 nm Polymer PVA part Mesostructured bioceramic part + Nanocomposite MGHA material Levofloxacin (LEV) + Antibiofilm agent: Rifampicin (RIF) External Gelatin-Glu coating Drug compartmented 3D scaffold R. García-Alvarez, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 49 (2017) 113–126
- 34. Multifunctional 3D implants International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 50nm50nm 50nm R. García-Alvarez, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 49 (2017) 113–126
- 35. Multifunctional 3D implants International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018
- 36. Multifunctional 3D implants International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 1 h In vitro assays in SBF: Degradability and Bioactivity R. García-Alvarez, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 49 (2017) 113–126
- 37. Multifunctional 3D implants International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Rifampin 0 30 60 90 120 150 180 0.0 0.2 0.4 0.6 0.8 1.0 Levofloxacin 0 10 20 30 0.0 0.1 0.2 0.3 Time (hours) Vancomycin Multi-therapy: Drug release R. García-Alvarez, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 49 (2017) 113–126 M.Cicuéndez, J.C. Doadrio, A. Hernández, M.T. Portolés, I. Izquierdo-Barba, M. Vallet-Regí. Acta Biomaterialia 65 (2018) 450–461 0 5 10 15 Rifampicin Levofloxacin Vancomycin 1 6 24 t / h µg·mg-1 Time (hours) pH 5.5 pH 6.7 pH 7.4 0 50 100 150 200 250 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Qt/Q0 pH-sensitive release
- 38. Multifunctional 3D implants International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 VL 1h 1h 24h 24h GRIFMGLEVPVAVAN MGLEVPVAVAN SA Bacteria biofilm (initial) Alived bacteria Extracellular polysaccharide matrix biofilm Dead bacteria 10 mm 10 mm 10 mm 10 mm 20 mm R. García-Alvarez, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 49 (2017) 113–126 Antimicrobial effect: importance of multitherapy
- 39. Multifunctional 3D implants International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 10 mm 20 mm Osteoregeneration processes (preosteoblast assays) R. García-Alvarez, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 49 (2017) 113–126 0 2 4 GRIFMGLEVPVAVAN U/LLDH LDH 1d Control 0.0 0.1 0.2 0.3 0.4 0.5 MitochondrialActivity MTT 7d 0 2 4 6 U/LALP ALP 7d GRIFMGLEVPVAVANControl GRIFMGLEVPVAVANControl *
- 40. Multifunctional 3D implants International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Alived bacteria Surface Dead bacteria Biofilm matrix Surface Surface Surface Multidrug 3D scaffold Vancomycin Osteoblast Bioactive layer Bone Tissue regeneration Levofloxacin Rifampin
- 41. Preliminary results Hyperthermia in biofilm treatment International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Preliminary assays: NIR radiation AuNR AuNR@MSN 50nm 0.5 0.6 0.7 1.0 0 5 10 15 20 25 30 50 mg/mL 75 mg/mL T(ºC) Nominal Power (W/cm2 ) T Hyperthermia 20 mm 20 mm
- 42. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 New nanoantibiotics based on mesoporous silica nanoparticles (MSNs) with targeting agents and loaded with antibiotics have been developed. New 3D multifunctional implants which combine the merits of osseous regeneration and local multidrug have been developed. These nanodevices are envisioned as a promising alternative to conventional infection treatments by improving the antimicrobial efficacy and reducing side effects. Conclusions
- 43. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Funding: European Research Council (ERC-2015-AdG). Advanced Grant Verdi-694160 Ministerio de Economía y Competitividad (MINECO), Spain, through projects MAT2012-35556, MAT2015-64831-R Organizing committee: Acknowledgements
- 44. Thank you very much Grupo de Investigación en Biomateriales Inteligentes (GIBI) Smart Biomaterials Research Group https://www.ucm.es/valletregigroup/ polyValent mEsopoRous nanosystem for bone DIseases VERDI
- 46. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Targeting to Gram-negative bacteria B. González, M. Colilla, J. Diez, D. Pedraza, M. Guembe, I. Izquierdo-Barba, M. Vallet-Regí, Acta Biomaterialia 68 (2018) 261-271. 52.5 C4,C5 21.2 C2 * # MSN-DAMO 10.7 C1 38.6 C3 47.1 C4,C5 10090 80 70 60 50 40 30 20 10 0 -10 MSN # * d (ppm) * C 1 C 2 C 3 C 4 C 5 Si N NH3 H O O O # # # * 13C CP MAS NMR
- 47. International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Hypothesis Use MSNs with targeting properties to release antibiotics in the environment of the bacteria that cause the infection. G. Domenico. The procession of the trojan horse into Troy, 1760 Targeting bacteria 20 mm Targeting biofilm
- 48. Source: National Healthcare Safety Network (NHSN) data base (March od 2018) 2015 2,400,000 Risk 0.5—2% Infected 96,000 Infection of orthopedic implants Cost 1 50,000 $ Total Cost 4,8 B$ Risk Groups Diabetes mellitus Previous skin infection Rheumatoid arthritis Obesity/Malnutrition Urinary infection 5% 2020 4,800,000 Hip prosthesis International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018
- 49. MSN-ConA 50ug/mL MSN-ConA 10ug/mL MSN-ConA 5ug/mL Targeting to bacterial biofilm International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 Biofilm polysaccharide matrix Nanoparticles (MSN and MSNConA) Alived bacteria M. Martínez-Carmona, I. Izquierdo-Barba, M. Colilla, M. Vallet-Regí (under preparatiom)
- 50. Multifunctional 3D implants International Symposium: Mesoporous Materials: From 1991 to 2018 Madrid, April 10 and 11, 2018 GRIFMGLEVPVAVAN Extracellular polysaccharide matrix biofilm Dead bacteriaLive bacteria EC Bacteria biofilm (initial) 1h 3h 24h 20 mm 20 mm 20 mm 20 mm 1h 24h GRIFMGLEVPVAVANMGLEVPVAVAN SA biofilm log[CFU] 0 5 10 15 20 25 0 5 10 15 20 25 6h 1h 24h6h * GRIFMGLEVPVAVANMGLEVPVAVAN EC biofilm * log[CFU] Gram-positive Gram-negative 1h 24h 6h 1h 24h6h
Editor's Notes
- Good afternoon Ladies and Gentleman. First of all, I would like to thank Areces Fundation and especially Professor Vallet-Regí to invite me to give this talking. I am delighted for this opportunity. It is a honor for me, as well, to be here and share panel with all these prestigious Scientifics in the mesoporous materials field. Today I will talking about our recent researches concerning the infection treatment by mesoporous materials.
- Despite of modern standard asepsis protocols and peri-operative antibiotics prophylaxis …. The infection associated to orthopedic implants still represents one of the most serious and devastating complications.
- In general, after infection diagnosis; it is necessary prolonged systemic treatment with different combinations of antibiotics and long periods of hospitalization. This kind of treatment provoke high side effect and antibiotic resistances. In most cases it is necessary a second replacement. If the bacteria infection is persistent it is necessary an amputation of the member and the in even in the 7% the dead is produced.
- This infection persistance is due to two main factors: The formation of biofilm. It is very well know that once bacteria attach to the implant surface, rapidly the biofilm is formed and the antimicrobial and antibiotics therapies are not very effective. The antimicrobial resistance, and apparition of denoted superbugs where an antibiotic is ineffective.
- Even it is talking about a new emergence post antibiotic era in which the antimicrobial treatment will be useless. This a very serious problem with important social and economical implications.
- Jim O Neel, one of the best world's macroeconomists warns governments that the drug resistant will be deadlier than cancer by 2050.
- And he recommends ten point plan for beating drug resistant, in which the last three points are addressed to increase and foment the research in the development of new antimicrobial agent more effective that current therapies.
- Now, what to do ??? Probably the answer it is in the Nanomedice which offers the possibility for designing drug delivery systems ….
- Mesoporous silica materials have received great attention by the biomedical scientific community, since they were proposed for the first time as drug delivery systems by Prof. Vallet-Regí in 2001.
- In this scenario we have two actors: Mesoporous materials. That Initially, they were purposed as bioceramics for local drug delivery and bone tissue regenerations due to their surface characteristics, such as biocompatibility and bioactivity, and their capability to load and release in a controlled fashion different therapeutic cargoes for the treatment of diverse pathologies. Recently, it is possible also to fabricate 3D macroporous scaffolds based on these materials which offers new possibility in regeneration proposed. Mesoporous silica nanoparticles offers new possibility in Nanomedicine and they has been proposed as promising alternatives in cancers therapies.
- As a Trojan horse…. We will design a new nanosystems based on Mesoporous Silica Nanoparticles with targeting to the bacteria surface and biofilm and able to release antibiotics in the environment of the bacteria that cause the infection.
- The first aim constitutes the design of a new nanoantibiotic Taking into account the negatively charged bacteria wall. We have designed a mesoporus silica nanoparticles by grafting a polycationics dendrimer, which confer positive density surfaces and also loading with levofloxacin as antimicrobial agent. This nanosystem will be able to internalize into gram-negative bacteria and release the levofloxacin inside and destroy the superbug.
- The synthesis of this nanosystems is very simple. For covalent anchorage of the dendrimers onto the external surface of the MSN sample, first we obtained the precursor silylated G3-PPI dendrimer, G3-Si(EtO)3, and immediately employed in the post-grafting reaction with the MSN material. For comparative purposes, we also functionalized the surface of MSN with small alcoxysilane contained two amine groups.
- Concerning the structural characterization XRD confirms the 2D hexagonal mesoporous arrangement, showing three well-defined reflections that can be indexed as 10, 11 and 20 of a p6mm plain group for all the materials. These results agree with TEM studies, which show spherical shaped nanoparticles with an average diameter of ca. 120 nm. Regarding the mesoporous structure, TEM images evidence the presence of typical of MCM-41 structure. These results reveal that neither the morphology nor the mesoporous order are affected by the used functionalization procedures.
- A deep physicochemical characterization has been performed. Concerning, the zetha-potential we can observe that after functionalization it is produces a drastic change on the MSN material from negative to positive charge. From 36.4 mV in bare MSN to ca. +35 mV in the hybrid materials The functionalization rate was determined by TGA an elemental analisis and we can observe that this value is higher in the case of DAMO group compared with G3 due to the the steric hindrance that occurs when macromolecules are employed to functionalize the silica surface, resulting in a more effective incorporation of the smaller alkoxysilane. . Both functionalized materials possess amine groups susceptible to protonation; however, the incorporation of the polycationic dendrimer does not exceed the positive value reached with the DAMO molecule. steric hindrance
- The next slide represent the release profiles of levofloxacin from different matrices. The obtained results evidence more accused release from the MSN-G3, followed of MSN-DAMO with respect to unmodificated MSN. All these profiles can be fitted Chapman's equation, where w (t) is the amount of LEVO released at each moment, w0 is the initially charged amount, A is the ratio between total LEVO released and total LEVO charged, k is the release kinetic constant and δ is a non-ideality parameter.
- Concerning the microbiology assays. We have performed two different assays. Bacteria targeting assay on plancktonic bacteria by adition of MSNs without antibiotic. Antimicrobial effect of completed nanoantibiotic, which was carried onto on previosly preformed biofilm. An d then the visulization by confocal microscopy
- Concerning to targeting effects. All images show the bacillus morphology of E. coli with the bacterial wall labeled in red, in good agreement with the control. In the case of pristine MSN, green scattered dots not close to the bacteria walls are observed. On the contrary, MSN-DAMO images display small domains of green aggregates corresponding to nanoparticles in the proximity of the bacterial wall obeying certain interactions between positive charges of amine groups and the negative bacterial wall. However, a complete internalization and uniform localization of MSN-G3 nanoparticles of the bacteria cells is appreciated. Difference in the successful internalization of both hybrid materials may be attributed to the G3 dendritic skeleton, which provides MSN-G3 of high surface flexibility. Consequently, there are more interaction points in the positively charged amine functional groups of MSN-G3 that are exposed to the negatively charged bacterial wall.
- This internalization is dosage dependent …. As you can see in the figure
- Concerning the antimicrobial effects microbiology assays was performed on preformed biofilm and after 90 min of incubation with the completed nanoantibiotic. Initially, the preformed biofilm displays a typical structure composed of colony live bacteria (green) covered by a protective mucopolysaccharide matrix (blue). After 90 min of incubation. MSN-L simple is not able to destroy the biofilm, appearing live bacteria colonies coated with its protective layer. However, small amounts of scattered dead bacteria are present onto the outermost surface probably due to the action of the LEVO released from this material. On the other hand, MSN-DAMO triggers the biofilm disruption, with the presence of small unfilled domains within the colony, showing vastly live cells (green), dispersed mucopolysaccharide (blue) fragments and scarcely dead cells (red). Finally, the best results are derived from MSN-G3-L assays, which is able to totally destroy the biofilm since no blue staining and almost negligible live cells are observed.
- A biofilm is a bacterial community in which bacteria are embedded into a extracellular matrix, which is mainly composed of polysaccharides. It constitutes a natural mechanism of defense of the microorganisms against external aggressions, including antibiotics and immune system. Lectins such as ConA are glycoproteins that are present in a variety of organisms, and most of them are isolated from plant components. They own the ability to weakly bind glycans with high specificity forming glycoconjugates.
- Actually, ConA has been successfully used to design drug-loaded tumour nanoparticles able to selectively bind and internalize in cancer cells overexpressing membrane glycans. Since glycan-type polysaccharides are also present in the bacterial biofilm, we hypothesize that ConA could be used to target MSNs towards extracellular biofilm matrix.
- Herein, we report the design of a new nanoantibiotic consisting of MSNs loaded with an antimicrobial agent (LEVO) and grafted in their outermost surface with ConA, which has been proved to selectively recognize and bind to certain glycans.
- The nanoantibiotic, denoted as MSNConA@LEVO, was synthesized using several steps. Briefly, pure silica MSNs were synthetized by the well-known modified Stöber method and externally functionalized by grafting an alkoxysilane bearing carboxylic acid groups, which will allow the final anchorage of ConA by reaction with the amine groups present in the protein. LEVO loading was carried out by impregnation method in ethanol, and always before ConA grafting to prevent protein denaturation.
- A deep physical-chemical characterization of the nanosystem was performed. Structural characterization by TEM shows spherical nanoparticles with an average size of ca. 150 nm with 2D-hexagonal structure with a honeycomb mesoporous arrangement, in good agreement with XRD. FTIR studies demostrated the presence of the functional groups corresponding to the molecules anchoared. An in vitro levofloxacin reléase show a gradual antibiotic release from the matrices, the reléase is more acusate in te case of functionalized MSN.
- Concerning the microbiological assays we carried out three different experiments in order to study the effectiveness of the nanosystem. We performed bacteria targeting assays by soaking the different samples without antimicrobial with a suspension of E. coli. We also performed biofilm targeting assays by soaking the different materials (without levofloxacin) in a preformed biofilm. Finally in order to perform the effectiveness onto the biofilm in E. coli and S. aureus of complete systems, we carried out the experiment by soaking the different samples in a preformed biofilm with both strain.
- The 3rd aim is concerning the design of multifunctional 3D implants by using mesoporous materials able to inhibit the bone infection at the same to regenerate new bone.
- To design this type of 3D multifunctional materials it is necessary to fabricate bioactive surface in order to enhance the bone tissue regeneration capability. The synthesis of these mesoporous materials leads the incorporation of different elements as calcium and phosphorous into the silica network in order to prepare mesoporous glasses with similar composition to the conventional sol-gel glasses. In this case, this mesoporous glasses have a added value that it is the ordered mesoporous arrangement which provokes outstanding characteristics.
- In the image it is represented a Schematic design of these therapeutic multidrug 3D scaffolds for bone infection. These 3D scaffolds containing rifampin, levofloxacin and vancomycin, is formed by a mixture of MGHA and PVA processed rapid prototyping technique (MGLEVPVAVAN) and posterior coating with Gel-Glu layer (GRIFMGLEVPVAVAN). In the right, we can see the localization of the different antibiotics in order to make sequential and sustained release.
- Morphology study of the different 3D scaffolds was performed by SEM. Before dip-coating process, the MGLEVPVAVAN scaffolds exhibit a very high porosity in both, surface and inner structure. Giant macropores of about 1 mm can be observed as well as a regular and high porosity all over and in the scaffold. High magnification shows the presence of mesopores of around 50 um with high rate of interconnectivity. A detail of the surface shows a smooth polymeric surface with incrustations of MG ceramic material. In addition, TEM image and FT diagram (Fig. 3E) display a mesoporous arrangement in the 2D hexagonal structure, These results evidence the 3D hierarchical structure of these scaffolds containing Levofloxacin and Vancomicin.
- In order to obtain an early release of RIF antibiofilm, these scaffolds were coated by dip-coating method with a gelatin-glutaraldehyde mixture containing a RIF. SEM studies the 3D scaffolds after coating containing RIF are shown in Fig. The obtained results show that after coating the macropores corresponding to 1 mm are blocked by a layer of thickness of 5 um.
- In vitro degradability assay in SBF of these 3D multitherapy scaffolds was performed at different key times. After 1 h of incubation, SEM image displays the total dissolution of the RIF coating leaving empty pores 1 mm. In addition, the in vitro degradability assays in SBF were performed during long periods of incubation at 15 and 30 days. The obtained results show a partial degradation of scaffold. Higher magnification micrographs show also a typical layer of hydroxyapatite formed, which is identified by the needle-like particles observed all over the surface of the scaffolds indicating a high level of bioactivity.
- Concerning the In vitro release tests of these 3D scaffolds, in the case of VAN and LEV, the release profiles are more sustained and prolonged in time in comparison with the RIF release, which is characterized by a fast release in just 1 h, according to in vitro degradability tests. The Fig. displays the dosages corresponding to GRIFMGLEVPVAVAN sample after 1, 6 and 24 h of incubation. It is important to remark than the matrix an initial effective dosage formed by three antibiotics followed of an effective and sustained dosage for long periods of time (>10 days) for LEV and VAN. It is important also remark that Levofloxacin released from silica mesoporous matrix has a pH sensitive release according with different type of interaction between the matrix and levofloxacin molecules as function of pH. We observe a higher release at acidid pHs which are characteristics of a infection environment.
- Once determined release profiles, antimicrobial assays have been carried out to determine the effectiveness of these multidrug systems against S. aureus biofilms. These antimicrobial studies have been carried out for all scaffold containing one, or two or three drugs in order to determine the success of the proposed combined therapy. Initially, the preformed biofilm shows a typical structure formed by colony live bacteria (green) covered by protective mucopolysaccharide matrix (blue). After 1 h of incubation with the different samples, notable differences are observed in presence or not of RIF. MGLEVPVAVAN containing both LEV and VAN is not able to destroy completely the biofilm, appearing even live bacteria colonies coated with its protective layer as it can observe. However, GRIFMGLEVPVAVAN scaffolds totally destroy the biofilm, observing colonial killed bacteria without the presence of protective layer of mucopolysaccharides. After 24 h, the GRIFMGLEVPVAVAN scaffold shows complete destruction of biofilm appearing isolated fragments, while MGLEVPVAVAN scaffolds still exhibits bacteria colonies with protective covered, indicating no efficacy against biofilm. It is important to remark that after long time of exposure all scaffold containing one or two drugs showed a total destruction of studied biofilm. These results show that our multidrug systems formed by the combination of RIF, LEV and VAN together with the designed strategy designed are very effective for the total destruction of biofilm in the first 24 h of incubation, which is indicative of their antimicrobial efficient.
- Biocompatibility assays were performed using the MC3T3 preosteoblasts cell culture. The obtained results evidence their biocompatibility, exhibitting an adequate preosteoblast adhesion and spreading on the entire surface of 3D scaffold. The biochemistry studies, showing non cytotoxic effect of these scaffolds only a a slight decrease of ALP activity, which, could be attributed to the presence of antibiotics in the media.
- In this case, we have designed a 3D scaffolds containing 3 types of antimicrobial agents in different compartments into the scaffolds which display a sequential release of the different antimicrobials, first they release rifampicin, which is a antibiofilm agent , followed with a Levofloxacin and Vancomicin with a sustained release. At the same time, the surface of these scaffolds exhibits bioactivity and its able to attach osteoblast for bone tissue regeneration purposes.
- Relationship between the nominal power of the laser (emission at 808 nm) and the temperature increase achieved with the Au @ MSN sample.
- This has been develop in Smart Biomaterials Research Group of Complutense of Madrid
- Now, we will describe the design of new nanoantibiotics based on targeting to biofilm
- Orthopedic infections represent a serious problem due to the total number of hip and knee arthroplasties that are performed worldwide. In USA, only in hip implants, it was in 2015 a total of 2.4 millons and it is forecast that in 2020 will be 4.8 millons. According to the National Health-care Safety Network, if the rate of infection is around 0.5-2% percent, then the number of infected implant will be 96 thousand and taking into account the estimated cost of the medical costs are more than $ 50,000 per patient The total cost is too high, being a serious economic problem. These results are even more worrisome in risk group such as diabetes and so on, where rate of infection is increased up to 5%.
- The effectiveness of these multidrug systems has been also determined on gram-negative E. coli biofilms. In this case, the effectiveness is against Gram-negative biofilm is lower with respect to Gram-positive, due to after 1 h of incubation still appear small colonies of 20 lm, which are formed by live bacteria (green) coated with a protective matrix (blue). After 6 and 24 h of incubation, the scenery is very similar to S. aureus a few scattering formed by small fragments of protective biolayer (blue), dead bacteria (red) and small amount of lived bacteria (green). In order to quantify the number of live bacteria present in each biofilm after of different treatments, CFU was determined. The obtained results shows notable differences in both 3D scaffolds, containing and not RIF, showing the efficacy of combined therapy .