1. Mechanical and Industrial Engineering University of Massachusetts Amherst, MA, USA Nanomedicine Jonathan P. Rothstein

2. Introduction What is nanomedicine? It is nanotechnology used for the treatment, diagnosis, monitoring and control of biological systems It includes the delivery and targeting of pharmaceutical, therapeutic, and diagnostic agents using nanoparticles to cancer and other cells It includes nanomaterial for bone, cartilage, vascular, bladder and neural applications What isnt nanomedicine? Flesh eating/repairing nanorobots

3. Introduction – Goals of Nanomedicine End goal of nanomedicine is improved diagnostics, treatment and prevention of disease For a great review see http://www.wtec.org/nano2/Nanotechnology_Research_Directions_to_2020/

4. Introduction – Goals of Nanomedicine Ultimate goal is to integrate detection, diagnostics, treatment and prevention of disease into a personalized single platform

5. Introduction Nanotechnology holds key to a number of recent and future breakthroughs in medicine

6. Goal is to develop handheld diagnostic devices for personalized medical testing and treatment BioMEMS for Screening and Diagnostics Biomedical Analysis and Communication System Disposable Diagnostic BioChip

7. BioMEMS – Micro and Nanofluidics

8. Nanoparticles for Pathogen Detection Gold nanoparticles can be functionalized with thiolated oligonucleatides. Bound to the oligonucleatides are fluorophores which are quenched by their proximity to the nanoparticle. When the targeted RNA (H2N2, HIV or a cancer) bindes to the oligonucleatide, the fluorophore is released and becomes fluorescence. The fluorescence can be detected in a BioMEMS device. Challenge is developing oligonucleatides with high selectivity for the target RNA. Nanoparticle ProbeTargeted RNA Fluorophore Release

9. Nanoparticles for Targeted Detection of Cancer As an example, nanoparticle probes were developed by Chad Mirkin at Northwestern Univ. that target the survivin RNA sequence known to exist in a certain breast cancers. Experiments are done ex-vivo. On the left, cancer cells fluoresce. On the right, healthy cells show minimal fluorescence. Breast Cancer Cells Healthy Cells

10. Nanoparticles for In-vivo Detection of Pathogens Fluorescence is not a viable option in-vivo, but magnetic tagging works very well. Harmless virus can used as a building block to produce contrast agents that can be used in Magnetic Resonance Imaging (MRI). Here, magnetic metal ions are bonded to the virus as are molecules that bind to cancer cells. A full body MRI scan detects these contrast agents and even very small tumors throughout the body

11. Nanoparticle Encapsulation for Drug Delivery Nanoparticle shells can be formed around spherical droplets A.D. Dinsmore, et al., Science 298, 1006 (2002), Y. Lin, et al., Science 299, 226 (2003) Shells are porous at lengthscales much smaller than size of nanoparticle. A: Scanning electron microscope of a dried 10- μ m-diameter colloidosome composed of 0.9- μm-diameter polystyrene spheres.

12. P Interfacial Area = A Energy = A O/W + 4 R 2 P/O (Oil) (Water) Energy = (A- R 2 ) O/W + 2 R 2 P/O + 2 R 2 P/W II. Particle sitting astride the interface (half-in, half-out): [Pickering (1907); Pieranski PRL 45, 569 (1980)] I. Particle (P) away from interface: If | P/O – P/W | < O/W /2, then adsorption reduces surface energy. Why Particles Adsorb to Interfaces surface tension

13. Nanoparticles At Interfaces nm m to mm oil-nanoparticle suspension, w/ droplets water droplet: Nanoparticles can be functionalized, cross linked or sintered to make shell permanent, strengthen shell or change shell permeability.

14. Nano-Encapsulation for Drug Delivery By making the holes between nanoparticles approximately the same size as the drug you want to administer you can get a constant release rate – avoids spikes in dosage. Can also allow encapsulation of hydrophobic drugs which are difficult to get into you mostly water body. Drug Concentration in Patient Time Nano-Encapsulated Drug Delivery Standard Diffusion Based Drug Delivery

15. Targeted Delivery to Tumors Goal is to inject treatment far from tumor and have large accumulation in tumor and minimal accumulation in normal cells/organs.

16. Cancer Treatments Tumor penetration is a key issue for successful chemotherapy

17. Nanoparticle use in Cancer Treatments Because of their small size, nanoparticles can pass through interstitial spaces between necrotic and quiescent cells. Tumor cells typically have larger interstitial spaces than healthy cells Particles collect in center bringing therapeutics to kill the tumor from inside out.

18. Making Gold Nanoparticles AuCl4- salts are reduced using NaBH 4 in the presence of thiol capping ligands The core size of the particles formed can be varied from <1 nm to ~ 8 nm The surface functionality can be controlled through the choice of thiols Diffusion speed can be controlled by length of thiols

19. Nanoparticles as Sensors and Therapeutics Glutathione (GSH) provides a selective and tunable release mechanism Once inside cells, fluorophores and drugs selectively dissociate

20. Nanoparticle Success Both cationic and anionic particles penetrate and accumulate in tumors. However, only cationic particles diffuse fully throughout the tumor. Work of Neil Forbes and Vince Rotello at UMASS

21. Nanoparticle Targeting and Accumulation To maximize their effectiveness, the microenvironment of the tumor must be quantified and vectors developed to specifically target the tumor. These treatment approaches have shown great promise in mice. Necrotic Quiescent Proliferating Therapeutic

22. First Successful Nanomedicine - Abraxane

23. Alternatives to Nanoparticles - Surfactants Surfactants are composed of a hydrophilic head and a long hydrophobic tail When dissolved in water above the critical micellar concentration (CMC) surfactants can self-assemble into large aggregate Spherical micelles are around 10nm in size Hydrophobic drugs can be encapsulated and in their core and delivered throughout the body or to a specific target.

24. Nanotechnology in Tissue Engineering – Cartilage Replacement Samuel Stupp at Northwestern has shown that nanotechnology can be use to regenerate severed spinal cords. Two polypeptides amphiphiles are used that when mixed in an aqueous solution self assemble into a nanotube As seen on right, these nanotubes display peptide growth factors. In mice, these systems have been shown to promote axonal outgrowth and bridging of injured areas (bottom right).

25. Nanotechnology in Tissue Engineering – Cartilage Replacement Over 15 million people worldwide suffer from knee-joint failure each year due to cartilage deterioration and 1 million spinal surgeries are needed every year When cartilage breaks down, the resulting contact of bones causes pain, swelling, and loss of movement. As observed over the past 250 years, normal (hyaline-type) cartilage is not known to repair itself. Mechanism not fully understood, but cartilage cells, chondrocytes, are sparsely distributed in tissue with poor vasculature, and actually continue to deteriorate after a traumatic incident osteoarthritis. www.allaboutarthritis.com www.healthsolutionsgroup.com

26. Nanotechnology in Tissue Engineering – Cartilage Replacement Because cartilage doesnt have vasculature and cannot repair itself, accepted treatments have been mostly mechanical in their approach. Joint lubricants: Simple and effective at short-term pain relief but do not address cause of the problem or repair any damage. Debridement/lavage/microfracture: Small lesions are repaired by shaving or shaping contour of cartilage. Microfracture penetrates subchondral plate (bone) and actually causes growth of fibrocartilage – a lesser form, not desirable. Total joint replacement: Addresses problem and generally allows full repair, but Very invasive procedure, native tissue removed Prostheses do not last a lifetime in active patients. Nanotechnology approach Regrow patients own cartillage in-vivo to repair damage www.hughston.com/hha/

27. ACT Methods A popular tissue engineering approach has been to introduce new cells, via autologous chondrocyte transplantation/implantation (ACT/ACI). Some of the earliest work by Benya and Shaffer (1982) showed it was possible to isolate and culture chondrocytes. More interesting result was that when cultured in vitro, the cells differentiated and changed their phenotype to produce a lesser quality collagen. Need better tissue scaffolds – nanotech. biomed.brown.edu Important to tissue engineering: Cells will differentiate purely based on mechanical stimulus. Genzyme ACT method: FDA approved 1997

28. Hydrogels – Self Assembly Hydrogels have applications in drug delivery and tissue engineering Regenerating cartilage and other tissue requires scaffolds with similar modulus and other mechanical properties Need to develop stiffer, tunable hydrogels We are currently looking at Polylactide-Polyethylene Oxide-Polylactide triblock copolymers. Systems are biocompatible with a hydrophobic ends (PLA) and a hydrophilic center (PEO) which self-assembles in water and can form a gel under the right conditions CMC Gelation Triblock Copolymer Micelle Gel Reinforced Through Addition of Nanoparticles [kPa]1-10100100010,000 hyaline cartilage ??? amorphous hydrogel crystalline Hydrogel

29. Hydrogels – Self Assembly Hydrogels have applications in drug delivery and tissue engineering Regenerating cartilage and other tissue requires scaffolds with similar modulus and other mechanical properties Need to develop stiffer, tunable hydrogels We are currently looking at Polylactide-Polyethylene Oxide-Polylactide triblock copolymers. Systems are biocompatible with a hydrophobic ends (PLA) and a hydrophilic center (PEO) which self-assembles in water and can form a gel under the right conditions CMC Gelation Triblock Copolymer Micelle Gel Reinforced Through Addition of Nanoparticles

30. Rheology of Hydrogels The hydrogels formed are very stiff with elastic modulus on the order of 1-10 kPa. Within range of moduli of several human tissues including cartilage. Gels formed from polymers with higher degree of polymerization maintain a high storage modulus even at physiological temperatures (37 0 C). In-vivo applications feasible. Rheological response of these polymers can be easily tuned by varying the crystallinity or block length of PLA or through addition of nanoparticles. R-Lactide Amorphous Core L-Lactide Crystalline Core Khaled et al. Biomaterials (2003)

31. Effect of Nanoparticle Addition on Rheology of Hydrogel PEO adsorbs very strongly to laponite Result is an additional, stronger network junction that increases modulus Only a very small amount of laponite (1%) is required to gel the neat polymer Dramatic modulus enhancement is observed shows great promise However, laponite is non-ideal because it is not FDA approved for in-vitro use Currently looking for the right nanoparticle Laponite Clay Nanoparticles

32. Hydroxyapatite (HAP) Nanoparticles Hydroxyapatite (a type of Calcium phosphate) is a mineral found in bone and enamel Bioactive material capable of bonding to living tissue HAP nanowhiskers are 20-80 nm in width but up to 100s of nm in length, and they have a high tendency to aggregate Can HAP serve as a new junction point? Initial results are promising, but still a work in progress