potential applications of nanotechnology in medicine include drug delivery, targeted therapy, and disease progression detection through diagnostics.

potential applications of nanotechnology in medicine include drug delivery, targeted therapy, and disease progression detection through diagnostics.potential nanotechnology applications in medicine include drug delivery, targeted therapy, and disease progression detection through diagnostics.

1. OUTLINE NANOSCIENCE IN BIOTECHNOLOGY/LIFE SCIENCE Nanomedicine Health Imaging Diagnostics Treatment Phytosome ( Nanomedicine in Herbal Medicine) Nanoscience in Agro Food and Nutrition Sustainable Agriculture NanoScience in Water Annual global death toll of TB, HIV/AIDS and Malaria alone approaches 6 million people ( over 85% from Sub Sahara Africa ) Fighting these diseases remains one of the most effective ways to alleviate poverty and promote economic progress in these countries.

2. © CSIR 2006 www.csir.co.za INRODUCTION TO NANOMEDICINE

3. Nanotechnology: Manipulation of matter at atomic/molecular level Nanomedicine : Applications of nanotechnology in health: Mainly for treatment, diagnosis, monitoring of diseases Nanomedicine

4. How small?

5. Ant head - 1mm Human hair - 100um , 100 000 nm Red blood cell - 10um, 10 000 nm DNA - 4nm wide Water molecule - 0.2 nm NANOMEDICINE - 100 nm to 500 nm 0.1% of human hair width or 1% of smallest human cell How small?

6. Nanocarriers Multifunctional, targeted devices, capable of crossing all biological barriers to deliver multiple therapeutic agents directly to diseased cells and disease-associated tissues Size Biological barriers Increased cellular uptake Versatile routes of admin Solubility Large surface area to volume ratio Surface Large and active Tailor ability Targeted Early detection Imaging Diagnosis Treatment Disease monitoring Surface charge McNeil, J Leukocyte Biol. 78:585-594, 2005

7. © CSIR 2006 www.csir.co.za NANOMEDICINE IN IMAGING AND DIGNOSTICS

8. www.csir.co.za Liu et al., Int. J. Cancer: 120:2527–2537, 2007. Schematic illustration of bioconjugated Qdots for in vivo cancer targeting and imaging. Target specific tumour cells, Monitoring and manipulating individual cells Track the movements of cells and individual molecules Detect, locate, treat and monitor Imaging, Diagnosis and Monitoring Diseases

9. Types of nanocarries http://www.pharmatutor.org/articles/review-article-nanoparticle

10. Materials used in nanoparticle synthesis Polymeric nanoparticles (GRAS materials) Chitosan, Alginate, dextran PLA (poly-lactic acid), PLGA (poly- lactide co- glycolide ), PCL ( polycaprolactone ) Inorganic nanoparticles Gold nanoparticles, silver nanoparticles Liposomes Phosphatidylcholine and cholesterol Solid lipid nanoparticles Triglycerides, carnauba wax, beeswax, emulsifying wax Dendrimers polyamidoamines (PAMAM), poly(propylene imine) Nanotubes Carbon

11. © CSIR 2006 www.csir.co.za NANOMEDICINE IN DRUG DELIVERY - TREATMENT

12. Nanomedicine Drugs can be contained in nanoparticles for delivery into the body Nanoparticle drug delivery systems are a platform for delivery of multiple types of therapeutic compounds, e.g. conventional drug compounds, herbal medicine, DNA/genes, proteins, vaccines Various routes of delivery, e.g. oral, intravenous, inhalation, intranasal, occular , topical etc. 13

13. NANOMEDICINE/NANOTECHNOLOGY www.csir.co.za

14. www.csir.co.za Conventional Oral Delivery System Limiting factors for oral delivery Gastric Intestinal Track (GIT): Harsh environment Bioactives degradation Poor permeability Relatively short gastric emptying and intestinal transit time Pre-systemic clearance All these lead to: toxicity Increased length of treatment Increased dose & dose frequency Poor bioavailability Drug interaction

15. www.csir.co.za Nanodrug delivery system in the GIT Structure of the GIT Internal structure of the intestine Para-cellular via M cell Intracellular via epithelial cell-intestine mucosa Peyer's patches

16. www.csir.co.za Routes and mechanisms of particle transport across epithelia DC UPTAKE via MALT Lymph capillary SEROSAL SIDE MUCOSAL SIDE mucus Epithelial cell Cell junction ENDOCYTOSIS by ordinary enterocytes PARACELLULAR LN Blood circulation blood capillary Modified surface Increase circulation time: PEG Enhance particle uptake: Chitosan

17. www.csir.co.za Blood stream and Lymphatic system

18. © CSIR 2006 www.csir.co.za WHAT HAS BEEN DONE IN NANOMEDICINE

19. Failure of drug in clinical development

20. A lmost 50% of drugs with some at Phase III fail; this drives up costs Reasons for failure half-life toxicity dose solubility d ose frequency FDA Phase III and submission failures, 2007-2010 Potential of Nanomedicine in drug development programs

21. Plasma Levels: tremendous prolonged half-life (Single Dose, 50 mg/m 2 ) Hours After Infusion Doxorubicin (µg/ mL ) 4 8 12 16 20 24 .2 2.5 2 5 . .1 1 . 10.0 PLD (T ½=50-80 hours) Doxorubicin (T ½= 2-3 hours) Gabizon et al., Cancer Res. 1994 Pharmacokinetics of Doxil vs Free Doxorubicin Nanodrug Doxil Free drug

22. 23 Abraxane – nano formulated (dose-adjusted to 175 mg/m 2 ) Taxol – free drug (dose175 mg/m 2 ) Clinical PK Comparison of Total Paclitaxel Study

23. B A B; treated with ITZ – 1mg/animal Daily for 2 weeks A; treated with ITZ-NANO - 100µg/animal every 3 days for 2 weeks Nanoencapsulated Itraconazole for the treatment of lung fungus Improved Bioavailability, Reduced Toxicity (R Bentes – unpublished)

24. www.csir.co.za Bawarski et al., Nanomedicine: Nanotechnology, Biology, and Medicine 4:273–282, 2008 Examples of Therapeutic Nanocarriers in Medical Applications

25. © CSIR 2006 www.csir.co.za NANOMEDICINE POVERTY RELATED DISEASES (PRDs) Malaria and tuberculosis as a case study

26. The Innovation Gap For every hundred drug leads, only one is likely to make it to market 14-25 years later. Funding is available for basic research in PRD drug development, but the support falls away as the research becomes more applied, hence hardly any new drugs get to approval stage The Developed world drug development Neglected Disease drug development

27. TB drug pipeline + TMC207 and SQ109

28. www.thelancet.com Vol 375 June 12, 2010 TB drug pipeline (TB global alliance) Novartis cancer drug pipeline http://www.novartisoncology.com/research-innovation/pipeline.jsp it’s been 40 years since the last new drug, yet only 2 drugs going through phase III clinical trials! Example of PRD Drug Development: TB Drug Pipeline

29. WHO report: 2005 TB leading cause of death in SA, highest infection rate in the world due to: Co-infection of HIV and TB in 80% of cases Patient non compliance; length of treatment ( 6-9 months) Poor bioavailability and toxicity, hence: Multi-drug resistant TB (MDR-TB) Extremely resistant TB (XDR-TB) South Africa TB Statistics Daily intake of antibiotics TB pills burden

30. www.csir.co.za MDR TB 1-2 years treatment 50% die 100X more expensive to treat As infectious Implementation of DOT’s program 53% cure rates; WHO target is 85% Logistics are impractical Expensive program, hospitalization Education Annual expenditure of TB drugs in SA Valued at 21.8M USD SA annual TB treatment expenses (e.g. DOTS, hospitalization) Estimated at 250-300M USD TB treatment: Main challenges in South Africa 14 Tablets for 2 years 1 injection daily for 6 months MDR-TB/HIV pills burden

31. Objectives Improve the bioavailability of anti-TB drugs Nanocapsule : slow release Minimise drug-drug interactions Improve solubility and half-life Reduce dose Size: improve biodistribution Reduce dose frequency Polymer degradation: Sustained release over days Reduce treatment time and cost 6-9 months: potentially 2 months Current drugs cost: 1% of the total treatment management Sustained release

32. www.csir.co.za Nano encapsulated TB drugs Anti-TB drugs Polymeric shell INH-loaded PLGA nanoparticles 1 um CLSM image of macrophage cell taken up 1 um size particles

33. Distribution Following oral exposure, NPs distributed to several organs Particles taken up from intestine ___ ____ __ __ © CSIR 2009 www.csir.co.za LUNGS LIVER KIDNEY SPLEEN BRAIN HEART MUSCLES

34. In vivo release Increase in drug half-life PK of RIF and INH show slow release © CSIR 2006 www.csir.co.za

35. Effects of the Nanodrug on Mycobacaterium tuberculosis replication Nanodrug once a week vs conventional drug daily Treatment with nanoencapsulated TB drugs once a week, comparable to daily treatment with conventional drugs © CSIR 2006 www.csir.co.za

36. Targeted drug delivery Target either the liver or RBC stage of the parasite Identify unique markers in parasite infected RBC or liver cells Targeting ligands Responsive polymers (micro environment changes) Nanomedicine for Malaria- Prospectives

37. www.csir.co.za Encapsulation of Malaria drugs Possible targeting approaches Nanomedicine for Malaria: Treatment

38. Severe malaria © CSIR 2011 Slide 1 Artesunate Artemether Quinine Quinidine gluconate Requirement for rate controlled infusion (quinine) Poor penetration of DHA into cerebrospinal fluid (~10% compared to plasma) Short plasma half-life (Artemisinins) Erratic absorption ( i.m. Artemether) Cardiotoxicity (quinidine) Nanoformulations to enhance drug penetration into CSF Nanoformulations to extend plasma circulation time of drug (e.g. polymer-drug conjugates). Controlled release Targeted nanoformulations to iRBC in brain (e.g. PfEMP-1 targeted nanoparticles). Can also targeted nanoformulations to iRBC in placenta (e.g. VAR2CSA targeted nanoparticles) Inhibit iRBC sequestration in brain and placenta microvasculature Drugs Potential Nano application Key shortcomings

39. Funding agency: water Nanomedicine for Malaria Issues with malaria drugs: Poor bioavailability of drugs Short residence time of drugs in blood Toxicity Bioavailability of tafenoquine increased from 55% to 99% Plasma half-life of Tafenoquine increased from 38.3 ± 4.1 hours to 44.7 ± 1.3 hours 1 invention disclosure filed Assessing the mitigation of toxicity Looking for pharma partners

40. © CSIR 2006 www.csir.co.za Malaria Results — (still going through patenting) Methods: Drugs were reformulated in nanomedicine drug delivery systems by applying our novel technology. Pharmacokinetic profiles of free and reformulated drugs were evaluated in mice. Results: Significant enhancement in the bioavaialbility of the drug was recorded in nano (81%)and micro formulations (99%) when compared to the free drug (55%). Similarly, eliminattion half life was increased in the formulations.   Conclusions: The results provide evidence that nanomedicine drug delivery systems have the ability to enhance bioavailability.

41. What have we achieved technically? Flexible: Can be used for various active compounds Platform technology Ability to produce multifunctional nano-carriers that allow lesser dose Hydrophilic drug Hydrophobic drug Muco -adhesive ligand Stealth coating Targeting ligand

42. 1. Aptamers Target TB infected macrophages by manipulating inherent mechanism Uptake: target the macrophage mannose receptor 2. Mycolic acids Wax coat of Mtb mainly consists of mycolic acids Attract cholesterol May possibly target cholesterol enriched infected areas © CSIR 2006 www.csir.co.za Targeting- 2 approaches Single stranded RNA Folding Incubation with target protein Molecular complex Dube et al. 2012

43. Conclusion Nanomedicine has opportunities to address PRDs What we can accomplish Burden reduction Cost reduction Training of young South Africans in this state of the art novel technology Having invested in this we can expand the program into rest of Africa www.csir.co.za

44. International Workshop on Nanomedicine for Infectious Diseases of Poverty, 27 – 31 March 2011 Opened by Minister of DST, HE Ms Naledi Pandor First Pan-African Nanomedicine Summer school Nov 2012 Nanomedicine Workshop for Poverty Related Diseases: perspectives and possibilities

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46. Nanomedicine Applications of nanotechnology in health: Mainly for treatment, diagnosis, monitoring of diseases Truly interdisciplinary Theranostics Personalised Medicine Tissue engineering (Tissue regeneration) (Stem cell research )

47. First-pass metabolism contributes to low oral bioavailability due to GIT harsh environment Poor permeability Enzymes and transporters At intestine Efflux: P-glycoprotein (Pgp) pumps drug back to intestinal lumen for elimination in faeces Cytochrome P450 enzymes (CYPs) metabolise drug so that only a fraction reaches systemic circulation unchanged At liver Pgp pumps drug into bile CYPs further metabolise unchanged drug Bailey D.G., and Dresser G.K.; Natural products and adverse drug interactions; Canadian Medical Association Journal ; 2004: 170(10): 1531-1532 . Bioavailability problems and toxicity - a summary

48. How to prepare a phytosome Phytosome are formulated by the processes in which the standardized extract of active ingredients of herb are bound to phospholipid like phosphatidylcholine (PC), phosphatidylethanolamin or phosphatidylserine through a polar end. Phytosome are prepared by reacting 3-2 moles of a natural or synthetic phospholipid with one mole of herbal extract The reaction is carried out in aprotic solvent such as dioxane or acetone from which the complex can be isolated by precipitation with non solvent such as aliphatic hydrocarbons or lyophilization or by spray drying Assignment presentation - Nanomedicine 49

49. Introduction The term ‘ Phyto ’ means plant, and ‘Some’ means cell-like. So, phytosome is a delivery system for herbal drugs. phytosome structure is a unit of a few molecules bonded together Shows better absorption which produces better bioavailability and improved pharmacological and pharmacokinetic parameters than conventional herbal extract. Assignment presentation - Nanomedicine 50

50. Flow diagram of phytosome preparation Assignment presentation - Nanomedicine 51

51. Nanoparticles

52. © CSIR 2006 www.csir.co.za ACE11— AFRICAN CENTER OF EXCELLENCE --FUNDED BY THE WORLD BANK

53. Materials used in nanoparticle synthesis Polymeric nanoparticles (GRAS materials) Chitosan, Alginate, dextran PLA (poly-lactic acid), PLGA (poly- lactide co- glycolide ), PCL ( polycaprolactone ) Inorganic nanoparticles Gold nanoparticles, silver nanoparticles Liposomes Phosphatidylcholine and cholesterol Solid lipid nanoparticles Triglycerides, carnauba wax, beeswax, emulsifying wax Dendrimers polyamidoamines (PAMAM), poly(propylene imine) Nanotubes Carbon

54. 55 PRITCHARD STREET , Nanomedicine in Health Cancer Aging Infectious diseases Addiction Obesity Genetic Disorders

55. © CSIR 2006 www.csir.co.za PYHTOSOME (NANOMEDICINE IN HERBAL MEDICINE )

56. Overview of the CSIR nanomedicine CoE Problem statement Death tolI (HIV, Malaria, TB) alone - 6 million people per year Therapeutics available but treatment failure due to Poor bioavailability high dose & dose frequency / long treatment duration Toxic / unpleasant side effects Patient non-compliance  resistance Most of the PRD drugs are over 50 -100 years old Revolutionized other therapies e.g. cancer Great promise to transform IDP therapies Reformulation of drugs for enhanced performance (bioavailability, structure ) Old, new, current drugs Traditional actives Disqualified drugs and drug leads Targeted drug delivery Summary of Pathogen /Disease Characteristics Disease/ pathogen Burden/ annum Population at risk Problems with treatment Malaria: 1 mil deaths 300-500 Mil cases *34.7 M 40% globally 86% in Africa Children & pregnant women Resistance Tuberculosis (TB): 2 mil deaths 9 mil cases (active) *46.4 M Pandemic 2 B latent TB >80% in SS Africa Poor immunity Resistance and long treatment times Human African Trypanoso-miasis (HAT) 50,000 deaths 300,000 cases *1.5M 60 mil (in SS Africa) Safety, efficacy resistance, long treatment time, administration Chagas disease 4,000 deaths 75,000 cases *0.7M 25 mil (Latin America & Caribbean) No treatments for chronic form Leishma-niasis >50,000 deaths 1.5-2 mil cases *2.1M 12 mil infected by ~20 Leishmania sp Safety, long treatment time administration Sources: WHO/TDR and Hotez , P.J., et al., N Engl J Med, 2007. 357(10): *One disability adjusted life year (DALY) equivalent to 1 year of healthy life lost.

57. Uptake of MA-NPs into U937 macrophage cells Particles are inside the cells, not on the surface