Nanotechnology and Biomedicine better understanding and treatment of living systems synthesis of new drugs and their targeted delivery regenerative medicine neuromorphic engineering biocompatible materials for sustainable environment improvements in human capabilities, societal outcomes, and the quality of life_ attention to ethical issues and societal needs BIOMATERIALS AND PROCESSES S&T PLATFORMS NANO Nanobiomedicine applies nanoscale principles and techniques to understanding and transforming inert materials and biosystems (nonliving, living or thinking) for medical purposes such as drug synthesis, brain understanding, body part replacement, visualization, and tools for medical interventions better understanding of living systems and for developing new tools for medicine and solutions for health care understanding the processes inside cells and neural systems Molecular medicine nanotechnology provides research tools and technology platforms for biomedicine - examples include working in the subcellular environment - investigating and transforming nanobiosystems (e.g. nervous system) rather than individual nanocomponents - developing new nanobiosensor platforms methods of nanotechnology have made inroads in uncovering fundamental biological processes - self-assembling, subcellular processes, and system biology cell as a highly organized molecular mechanism - abilities of information utilization, self-organization, self-repair, and self-replication single molecule measurements - dynamic and mechanistic properties of molecular biomachines, both in vivo and in vitro - direct investigation of molecular motors, enzyme reactions, protein dynamics, DNA transcription, and cell signaling Nanotechnology in drug delivery releasing, targeting, and controlled membrane transport ideal drug for avoiding side effects: - raise concentration up to the efficient level immediately after the dose is given - hold the level for a constant period to allow the drug to do its work - return to the original level soon after the treatment period (no interference with the subsequent dose) controlled-release technologies: - pulse - a constant amount of drug at a constant time interval - feedback - released on command from a physical signal - constant rate release targeting technologies: - active type utilizes a signal peptide, antigen-antibody, receptor-ligand - passive - enhanced permeation and retention (EPR) effect near a malignant tumor organ membrane transport: - easily membrane-transfering pro-drug is activated to drug after crossing the membrane Nanodelivery of nucleic acids ■ base for the gene therapy ■ DNA should be compacted in the presence of polycations ■ ordered structures - toroids, rods, and spheroids Interact with anionic proteoglycans, transported by endocytosis accumulate in the acidic vesicles and raise the pH of the endosomes, inhibiting the degradation of DNA protein influx destabilizes the endosome and releases DNA translocated to nucleus through pore or with the aid of nuclear localization signals decondenses DNA NPs polyamines spermidine and spermine (3-4-3) and their synthetic analogues are excellent promoters of DNA nanoparticle formation polyamines cationic lipids PEI polyethylene imines linear / branched ( N- s s 1H 2 NH- NH NH NH s Í NH2 ■ induce the condensation of DNA to nanoparticles ■ branched PEI acts as a proton sponge at the endosomal pH ■ buffering capacity of PEI is believed to contribute to its ability to deliver DNA within cells without degradation ■ PEGylation improves the stability of nanoparticles and increases their in vivo circulation time Dend rimers polyamidoamine (PAMAM) and polypropylenimine (PPI) dend rimers - ability to provoke DNA nanoparticle formation and facilitate DNA transport monodispersity and controllable surface functionality are advantages five generations of polypropylene imine dendrimers: DNA with dendrimers (AFM) i G -1 TV" ? 2-\ ^/ J. t ^ G -2 G-3 G-4 -"7 iT v G-5 Peptides ■ native - cationic histones and protamines ■ polylysines Chitosan ^H2 NH2 NH2 Poly-L-lysine (linear) Lys /L*s Lys / Lys - Lys Lys Lys r , I y Lys-t-Lya-Lys-]_ya-Lvs_Lys j|_Lys Single tumor cells invade vascular system Tumor tissues various structures and areas, actual cancer cells can occupy <50%, vasculature 1-10%, remaining structure consists of a collagen-rich matrix tumor blood vessels differ from normal vasculature in being up to 3-10 times more permeable drugs are transported into the tumor cells through interendothelial junctions and vesicular vacuolar organelles and fenestrations pore cutoff size in tumor tissue is between 100 and 780 nm Macromolecules or nano particles Drug delivery to tumors interstitial compartment of a tumor contains a network of collagen and elastic fiber, which is immersed by hyaluronate and proteoglycan-containing fluid interstitial pressure within the tumor tissue is elevated due to the lack of a lymphatic drainage system transport of a drug into the tumor area is dependent on the interstitial pressure as well on its composition, charge, and the characteristics of the drug colloidal particles larger than 50 kDa enter the interstitial compartment through leaky vessels and accumulate in tumor tissue enhanced permeability and retention (EPR) effect Help of NPs ■ deliver the pharmacologically required concentration of the drug ■ increase drug concentration at the target site through extended or controlled release ■ overcome multidrug resistance ■ eradicate side effects to vital organs by reducing systemic exposure ■ avoid immune response and hematopoietic toxicity ■ destroy malignant cells specifically, sparing normal cells ■ kill primary tumors inaccessible to surgery ■ destroy seeded cancer and dormant cells and metastases ■ protect the active drug from alteration and inactivation ■ detect cancers at a early stage Application of nanotech Pathways of nanoparticles ■ distribution and routes of NPs after injection ■ coating with plasma proteins - opsonization ■ macrophages internalize the opsonized nanoparticles through phagocytosis and deliver them to the liver, spleen, kidney, lymph node, and bone marrow ■ this clearance can occur within 0.5-5 min ■ prevention - coating, size under 100 nm Cellular uptake pinocytosis, endocytosis, receptor-mediated endocytosis some NPs can escape lysosomal degradation - Important for DNA and macromolecules O Drug |f\(| Receptor^ Ligand Surface treatment or coating of NPs ■ coating with biodegradable matrices - to become "invisible" to macrophages. ■ choice of hydrophilic or hydrophobic matrices for coating determines the fate of NPs ■ hydrophilic coating prevents interaction with macrophages of the RES, reduces their removal from the circulation, and increases their circulation half-life - dextran, PEG, polyethylene oxide (PEO), poloxamers and poloxamines, silicones ■ hydrophobic coatings are applied to increase opsonization, leading to copious interaction with macrophages, and the nanoparticles are therefore rapidly removed from the circulation - this approach is applied for targeted delivery of nanoparticles to the RES of liver and spleen Polymers for encapsulation two different approaches 1) as a drug reservoir, which consists of an oily core as vehicle, which carries the drug, and a polymeric outer core layer with a coating 2) particles are nanospheres in which the drug is dispersed in a polymeric matrix synthetic biodegradable polymers such polyvinylpyrrolidone (PVP), chitosan, polyalkylcyanoacrylates and polylactides such as polyisohexylcyanoacrylate (PIHCA), polyethylcyanoacrylate, and polyisobutylcyanoacrylate (PIBCA) PLGA (FDA approved) degrades slowly, releasing the drug, and is therefore used for controlled release Site-specific delivery passive targeting includes manipulation of the size and/or hydrophobicity or other physicochemical characteristics and can be applied to target the RES active targeting involves the direction of magnetic particles by using an external magnetic field or by using ligand-conjugated nanoparticles folate-coated NPs specifically target folate receptor Release sustained release through polymer degradation enzymatically controlled release controlled release through use of thermosensitive polymers photochemically controlled release pH-responsive release systems laser-induced breakdown (LIB) ultrasound-mediated release. Implants, prostheses increasing life expectancy - growing number of synthetic devices to overcome the problems associated with deteriorating or failing body parts examples of implants are orthopedic joint prostheses, cardiovascular devices, dental implants, for aesthetic reasons from biomaterials that have a common property: biocompatibility this might not be universal for particular type of material -significant effect of location in body and intended function of the device - resistance to adhesion of biomolecules and cells = - is biocompatible for the production of cardiovascular devices - completely different if used to manufacture artificial joints - anyway, small side effects due to "symbiosis" might always occur Self-Assembly and Self-Organization ■ SA - product grows layer by layer with a high degree of equilibrium ■ SO - product is made all at once from the start instead of being assembled one layer at a time - end product made with the desired functional structure by this method does not have a minimum of free energy, but has a minimum loss of entropy a small change in building Self- Self- units can have significant Order , i assembly organization effects f Disorder, J -► Equilibrium Non- equilibrium Synthetic and natural materials in reparative medicine Tissue Material Application Response Titanium and its alloys Joint prostheses, oral implants, fixation plates, Inert pacemakers, heart valves CaP ceramic Joint prostheses, oral implants, bone Bioactive replacement, middle ear replacement Alumina Joint prostheses, oral implants Inert Carbon Heart valves Inert PTFE Joint prostheses, tendon and ligament Inert replacement, artificial blood vessels, heart valves Poly (methylm ethacry late) Eye lenses, bone cement Tolerant Poly(dimethylsiloxane) Breast prostheses, catheters, facial Unknown reconstruction, tympanic tubes Poly(urethane) Breast prostheses, artificial blood vessels, skin Inert replacements PLA Bone fixation plates, bone screws Inert PGA Sutures, tissue membranes Inert PTFE = poly(tetrafluoroethylene). PLA = poly(lactic) acid. PGA = poly(glycolic) acid. Biomaterials i Artificial Natural Metals Ceramics* Polymers* Composites Collagen/Elastin ' Degradable and nondegradable variants bulk properties (together with the design) determine the strength (mechanical) of an implant - intrinsic properties: elastic modulus, yield stress, and ultimate stress - these parameters determine the stiffness, deformability, and strength - fatigue - cyclic stresses (much smaller than ultimate tensile stress) surface properties are important for interactions of implant with biological systems - should not be toxic, carcinogenic, pyrogenic, cytotoxic, or antigenic to living cells - surface energy - important factor in the establishment of cell adhesion Biomaterial in the living environment ■ interaction of biosystems with biomaterial surfaces can be desirable - enhanced integration in the body ■ noninteracting surfaces - when deposition of biological material (biofouling or bioadhesion) is undesirable ■ control over bioadhesion - encourage adhesion of host cells but discourage adhesion of infectious bacteria - a common cause of implant failure ■ biodegradation - production of polymeric and ceramic biomaterials whose degradation rates can be controlled ■ devices that in time can be replaced by native tissue Biological processes implant = intrusion of a foreign object - initiates a response of the body - wound healing - soft x hard tissues (scars) formation of thrombus - incoming fluids and blood, activation of platelets and coagulation cascade, polymerization of fibrin = clot matrix for future incoming cells inflammation initiated as release of vasodilators, chemoattractants, and other mediators - platelet-derived growth factor (PDGF) - tumor growth factor-beta (TGF-|3) by platelets activation of the complement cascade within the coagulating fluid surrounding the implant recruitment of inflammatory and other cells (chemotaxis), - development of new blood vessels (angiogenesis), and overall cell regulation nonspecific defense mechanisms by cells and factors - granulocytes, monocytes, and the complement system - resident inflammatory cells (macrophages and mast cells) try to eliminate intruders - if necessary, specific immune responses - production of antibodies by B lymphocytes and/or activation of cytotoxic T lymphocytes can be initiated Reparative phase formation of new tissue requires the activation and/or proliferation of distinct cell types, resulting in the replacement of lost or damaged tissue soft tissue healing - fibroblasts and endothelial cells - formation of new extracellular matrix, angiogenesis - nutrients and oxygen for proliferating cells hard tissue - ossification (bone formation), two ways: - intramembranous ossification is carried out by osteoprogenitor cells present in the cambium layer of the periosteum - endochondral ossification occurs at and overlies the defect site and undifferentiated mesenchymal cells attracted from tissues surrounding the defect become committed cartilage-producing cells, mineralization of the cartilage tissue leads to bone formation Biointerface ■ initial contact of implant and host relies on non-cellular interactions ■ newly introduced implant is surrounded by liquid - water mono / multi layer - biomaterial surface - hydrophobic or hydrophilic (cell adhesion) ■ ions (Na+, CI-) appear ■ followed by adsorption of proteins - denaturation might occur - these adsorbed modified proteins can be recognized as a foreign material stimulating reactions ■ living cells are coming ■ final result of implantation: (1) integration, (2) extrusion, (3) resorption, (4) encapsulation 14 Contribution of "nano" ■ making more bio-friendly (biocompatible) surfaces through nanostructuring - contacts for (oriented) adhesion of both extracellular matrix proteins (ECM) and cells ■ Nanofabrication methods Type of System Materials Resolution Lithography Silica, silicon, silicon nitride, x, y, and z to 10 nm silicon carbide Colloidal resist Silica, silicon, silicon nitride, silicon carbide x, y, and z to 5 nm Self-organizing or Polymer demixing, self-assembling particles In 10-nm range self-assembling and monolayers, other self-assembling systems Soft lithography Any fairly large molecule xand y to 200 nm, z to one monolayer Biomirriicry Many Actual native dimensions Photolithography Material substrate Resist Material substrate Light source Mask Resist Material substrate 1. Coating II II II II Resist Material substrate I 3. Development Negative tone etching - pits, grooves, and other topographies of controlled shape and size deposition of a thin film - coating the exposed area with a desired solution, from which the solvent evaporates or in which the particles (molecules) organize themselves in a specific conformation (selfassembly) Colloidal resist Colloidal dispersion i Etching Thin film deposition I I Q Q Q Q o g no ooo o| Removal of colloidal particles ■ colloidal particles of different materials and sizes down to 5 nm can be produced and subsequently dispersed over a biomaterial surface ■ the adsorbed particles can be used as a template for patterning the underlying surface ■ both etching and deposition are feasible ■ removal of particles provides the desired pattern Soft lithography ■ hard template based on Si ■ soft template based on elastomer ■ patter transfer to the target ■ often combined with SAM Resist 'master' PDMS casting PDMS inverse replica Positive replica Microcontact printing Microfluidic patterning Biomimetic approaches create an implant surface which is not (or to a lesser extent) recognized as foreign by the host constituents of the natural cellular environment (ECM proteins) can help - domains influence cell behavior - interactions of the receptor family of integrins - including proliferation, migration, morphological change, gene expression, and cell survival by intracellular signaling how to achieve: - physical adsorption (van der Waals or electrostatic interactions) - physical entrapment (use of a barrier) - covalent attachment (also more sophisticated such as covalent linking to polymeric networks) not necessarily complete proteins, significant peptides Arg-Gly-Asp (RGD) amino acid sequence - cell-binding domain of fibronectin, serves as a ligand for integrin receptor (a5pi) Calcium phosphates natural hard tissues comprise precipitated minerals - also used for biomimetic biomaterial surfaces hydroxyapatite surface coatings for bone implants Ca phosphates are bioactive - allow dynamic interactions favoring bone formation with implant surroundings deposition techniques: magnetron sputtering plasma spraying electrostatic spray deposition generation of nanostructured coatings Topographical nanostructures ■ increase in surface area - greater potential for tissue integration (mechanical interlocking, contact guidance) ■ tested shapes - grooves, pits, ridges, cliffs, tunnels, steps, waves, wells, tubes, nodes, pillars, pores, spheres, cylinders,... ■ cell responses - orientation, extension, movement, and activation - phosphorylation, actin polymerization, mRNA expression, phagocytic activity ■ control over cellular alignment (including cellular extenstions) ■ precise effects still rather unclear Contact guidance ■ rat dermal fibroblasts on microgrooved substrates ■ cells become oriented and elongate along the surface grooves ■ specific role for biomaterial surface nanotopography has been demonstrated for growing nerve tissue ■ achieved control over the outgrowth of neurites from the cell bodies of neurons - both the sites at which they emerge from cell bodies and directions - surface roughness is usually welcome, too 18 Protein / peptide surfaces ■ signalling to cells to modulate spatial behavior ■ examples of immobilized peptides Peptide ECM Molecule Source Application RGD Multiple ECM molecules, Enhance bone and cartilage tissue e.g., fibronectin, formation in vitro and in vivo; vitronectin, laminin, regulate neurite outgrowth in vitro collagen, and and in vivo; promote myoblast thrombospondin adhesion, proliferation, and differentiation; enhance endothelial cell adhesion and proliferation IKVAVYIGSR Laminin Regulate neurite outgrowth in vitro RNIAEIIKDI and in vivo Recombinant fibronectin Fibronectin Promote formation of focal contacts fragment (FNIII7_10) in preosteoblasts Ac-GCRDGPQ- Common MMP Encourage cell-mediated GIWGQDRCG substrates, e.g., proteolytic degradation, collagen, fibronectin, remodeling, and bone and laminin regeneration in vivo Topography vs. chemistry ■ nanotechnological structures: ■ isotropic that do not differ chemically from the intrinsic substratum ■ anisotropic nanotechnological structures using patterns of molecules chemically different from those of the intrinsic substratum ■ reactions of cells to similar topographies on chemically different biomaterial surfaces are comparable ■ major role of topography, modulated by the chemical surface coating Health issues of nanomaterials linked to multiple factors including chemical composition, size, shape, and surface chemistry entrance paths - inhalation through the respiratory tract, ingestion, injection into the blood stream, transportation via the skin historically - industrial manufacturing, gas exhaust from vehicles, coal, asbestos, man-made mineral fibers such as fiberglass, ambient particles in the atmosphere data available, but not yet conclusive who should be involved - scientists, industry, regulatory agencies, citizens interests groups, the public - ... not to repeat the fear of GMOs