Therapeutic Nanodevices: Introduction, Applications and Barriers

Let us make an in-depth study of the therapeutic nanodevices. The below given article will help you to learn about the following things:- 1. Introduction to Therapeutic Nanodevices 2. Synthetic Approaches: ‘Top-Down’ Versus ‘Bottom-Up’ Approaches for Nanotherapeutic Device Components 3. Technological and Biological Opportunities 4. Applications for Nanotherapeutic Devices and 5. Barriers to Practice and Prospects.

Introduction to Therapeutic Nanodevices:

Therapeutic nanotechnology offers minimal­ly invasive therapies with high densities of function concentrated in small volumes, fea­tures that may reduce patient morbidity and mortality. Unlike other areas of nanotechnol­ogy, novel physical properties associated with Nano scale dimensionality are not the raison d’etre of therapeutic nanotechnology, where­as the aggregation of multiple biochemical (or comparably precise) functions into controlled Nano architectures.

Multi functionality is a hallmark of emerging Nano therapeutic de­vices, and Multi functionality can allow Nan­o therapeutic devices to perform multi-step work processes, with each functional compo­nent contributing to one or more Nano-device subroutine such that, in aggregate, subrou­tines sum to a cogent work process.

Canoni­cal Nano therapeutic subroutines include targeting to sites of disease, dispensing mea­sured doses of drug (or bioactive compound), detection of residual disease after therapy and communication with an external clinician/ operator. Emerging Nano therapeutics thus blur the boundaries between medical devices and traditional pharmaceuticals. Assembly of therapeutic Nano devices generally exploits ei­ther (bio) material self-assembly properties or chemo selective bio conjugation techniques, or both.

Given the complexity, composition, and the necessity for their tight chemical and structural definition inherent in the nature of Nano therapeutics, their cost of goods might .exceed that of (already expensive) biologist. Early therapeutic Nano devices will likely be applied to disease states which exhibit signifi­cant unmet patient need (cancer and cardio­vascular disease), while application to other disease states well-served by conventional therapy may await perfection of nontherapeutic design and assembly protocols.

Design of Therapeutic Nanodevices:

The biotechnology industry historically has focused on production of individual soluble protein and nucleic acid molecules for phar­maceutical use, with only limited attention paid to functional supra-molecular structures. This bias toward free molecules lies in the face of the obvious importance of integrated supra-molecular structures in biology and, to the casual observer, may seem an odd gap in attention and emphasis on the part of practic­ing biotechnologists.

The bias toward single molecule, protein therapeutics, however, fol­lows from the fact that biotechnology is an industrial activity, governed by market con­siderations. Of the myriad potential thera­peutics that might be realized from biotech­nology, single protein therapeutics are among the easiest to realize from both technical and regulatory perspectives and so warrant ex­tensive industrial attention. This is changing, however, and more complex entities (actual supra-molecular therapeutic devices) have and will appear with increasing frequency in the twenty-first century.

New top-down and bottom-up materials derived from micro/nanotechnology provide the opportunity to complement the traditional limits of bio­technology by providing scaffolds that can support higher level organization of multiple biomolecules to perform work activities they could not perform as free, soluble molecules. Such supra-molecular structures have been called Nano biotechnological devices or semi­synthetic Nano devices and figure prominent­ly in therapeutic nanotechnology.

Incorporation of Biomolecules:

In gen­eral, design of Nano devices is similar to de­sign of other engineer structures, providing that the special properties of the materials (relating to their Nano scale aspects such as quantum, electrical, mechanical, biological properties, etc.), as well as their impact in therapy, are considered.

Therapeutics can in­teract with patients on multiple levels, rang­ing from organism to molecular, but it is rea­sonable to expect that most Nano therapeutics will interface with patients at the Nano scale at least to some extent. Typically, this means in­teraction between therapeutics and biological macromolecules, supra-molecular structures and organelles, which, in turn, often dictates the incorporation of biological macromol­ecules (and other bio-structures) into Nano devices.

Incorporating biological structures into (Nano biological) devices presents special challenges that do not occur in other aspects of engineering practice. Unlike fully synthetic devices, semi-biolog­ical Nano devices must incorporate pre-fabricated biological components (or derivatives thereof), and therefore the intact Nano devices are seldom made entirely de novo.

As a corollary, knowledge of properties of biologi­cal device components is often incomplete (as they were not made by human design), and, therefore, the range of activities inherent in any Nano biological device design may be much less obvious and less well-defined than it is for fully synthetic devices. Further com­plicating the issue, the activities of biological molecules are often multifaceted (many genes and proteins exhibit pleiotropic activities), and the full range of functionality of individ­ual biological molecules in interactions with other biological systems (as in Nano therapeutics) is often not known.

This makes design and prototyping of biological Nano devices an empirically intensive, iterative process. Bio­logical macromolecules have properties, par­ticularly those relating to their stability that can limit their use in device contexts. In gen­eral, proteins, nucleic acids, lipids, and other biomolecules are more labile to physical in­sult than are synthetic materials.

With the possible exceptions of topical agents or oral delivery and endosomal uptake of Nano therapeutics (both involving exposure to low pH), patients can tolerate conditions encountered by Nano biological therapeutics in vivo, and device liability in the face of physical insult is generally a major consideration only in ex vivo settings (relating to storage, sterilization, ex vivo cell culture, etc.). Living organism re­model themselves constantly in response to stress, development, pathology, and external stimuli.

For instance, epithelial tissues and blood components are constantly eliminated and re­generated, and bone and vasculature are con­tinuously remodelled. The metabolic facilities responsible (circulating and tissue-bound proteases and other enzymes, various clear­ance organs, the immune system, etc.) can potentially process biological components of Nano biological therapeutic devices as well as endogenous materials, leading to partial or complete degradation of Nano therapeutic structure, function, or both.

Furthermore, the host immune and wound responses protect the host against pathogenic organism incur­sions by mechanisms that involve sequester­ing and degrading the pathogens. Nano biological therapeutics is subject to the actions of these host defense systems and to normal remodeling processes. Conversely, instability of active bio components can offer a valuable and simple way to delimit the activity of Nano therapeutics containing biomolecules.

Nanotherapeutic Design Paradigms:

Sev­eral early attempts to codify the canonical properties of ideal Nano biological devices, and therapeutic Nano devices in particular, have been made. In general, Nano biological devices contain biological components that retain their function in new (device) contexts.

In other words, one must abstract enough of a functional biological unit from its native context to allow it to perform the function for which it was selected. If one wishes, for example, to appropriate the specific antigen recognition property of an antibody for a device function (say, in targeting), it is not necessary to incorporate the entire 150,000 atomic mass unit (AMU) antibody, the bulk of which is devoted to functions other than antigen recognition, but it is critical to incor­porate the approximately 20,000 AMU of the antibody essential for specific antibody-anti- gen binding.

Device function is the result of the summed and various activities of biologi­cal and synthetic device components, though functional biological components generally exist in the context of higher order systems that support the organisms of which they are a part. The control the Nano biological device designer can exert on the relative organization of biological device components allows biomolecules abstracted from their native context and incorporated in Nano biotechnological devices to contribute to functions entirely different from those they performed in their organismal contexts.

All of these fea­tures are illustrated in the bacmid, or Bac-to- Bac, system, a commercially available molec­ular cloning device. This system configures prokaryotic genetic elements from multiple sources into a device for producing recom­binant eukaryotic viruses, a function that is unprecedented in nature.

The system is fea­sible because of the modularity of the genetic elements involved and because of the strict control of the relative arrangement of genetic elements allowed by recombinant DNA tech­nology. Analogous devices based on bacterial and eukaryotic regulatory elements to pre-program the micro and Nano scale architec­tural properties and physiological behavior of living things are now being realized.

Bacmid provides an example of a nontherapeutic Nano biological device and illustrates some specific design approaches for build­ing functional devices with bio-components. Hypothetical properties of Nano scale devices specifically for therapeutic purposes have been codified.

In Nano therapeutic applica­tions, devices should be non-invasive and target therapeutic payloads to sites of disease to maximize therapeutic benefit while mini­mizing undesired side effects. This, of course, implies the existence of therapeutic effector functions in these Nano devices, to give de­vices the ability to remediate a physiologically undesirable condition.

Beyond that, several attributes relate to sensing of biomolecules, cells, or physical conditions (sensing disease itself, identification of residual disease, and, potentially, targeting capacity, responding to intrinsic or externally supplied triggers for payload release).

Other properties relate to communication between device subunits (for instance, between sensor and effector domains of the device) or between the device and an external operator (external triggering and data documentation capability). With appropriate design, device functions can be modular.

As discussed below, this approach allows construction of Nano therapeutic plat­forms as opposed to single one-off devices that are capable of only one therapeutic task. The vision of Nano scale therapeutic platforms arose from collaboration between the National Cancer Institute (NCI) and the Na­tional Aeronautics and Space Agency (NASA), USA. NASA is concerned with minimal mass therapeutics: therapeutics, along with almost everything else used by astronauts, must be launched from Earth.

NCI is interested in early detection of disease to improve prog­nosis. Since this requires screening a popu­lation predominantly of healthy patients, the screening mode must be minimally invasive. The considerations of both agencies might be met with ultra-small (micro or Nano scale) multi-potent therapeutic devices.

Furthermore, the proposed therapeutic platforms should not only remediate unde­sired physiological conditions but also have the capacity to recognize them and report them. Extensive capability for molecular recognition and communication with exter­nal clinicians/operators is integral to the re­quirements of NASA and NCI.

These capaci­ties would allow drugs or other therapeutic interventions to be provided in a controlled fashion, to maximize benefit and minimize side effects, and is the essence of ‘smart’ therapeutics.

Though substan­tial progress has been made in device design and realization, no fully realized multi-potent Nano scale therapeutic platform has yet been commercialized, but the therapeutic platform paradigm, in which devices are modular, with functional tasks segregated into individual modules, has potential to be extremely pow­erful.

Classes of broadly similar devices could be tailored to specific disease states by inter­changing modules (targeting modules, drugs dispensers, etc.) as appropriate to the disease state or therapeutic course. Thus, the hypo­thetical device represents a possible therapeu­tic platform composed of functional modules that could be for use in particular indications or in specific individuals.

Utility and Scope of Therapeutic Nano Devices:

Therapeutic nanotechnology will be useful, of course, when the underlying biol­ogy of the disease states involved is amenable to intervention at the Nano scale. As we will discuss, while several disease states and phys­iological conditions (cancer, vaccination, cardiovascular disease, etc.) are particularly accessible to Nano scale interventions, some Nano technological approaches may be ap­plicable more broadly.

Much as was the case with the introduction of recombinant protein therapeutics over the last 20 years, Nano therapeutics may present regulatory and pharma-co-economic challenges related to their nov­elty and their cost of goods (COGs).

Synthetic Approaches: ‘Top-Down’ Versus ‘Bottom-Up’ Approaches for Nanotherapeutic Device Components:

Synthesis of nanomaterial’s is commonly thought of in terms of top-down’ or ‘bottom- up’ processes. Top-down approaches begin with larger starting materials and, in a more or less controlled fashion (depending on the technique), remove material until the desired structure is achieved. Most micro fabrication techniques for inorganic materials (lithography and milling techniques, etc.) fit this de­scription.

In contrast, bottom-up approaches begin with smaller subunits that are assem­bled, again with varying levels of control, de­pending on technique, into the final product. Key examples of materials made by bottom-up approaches include some inorganic struc­tures. This includes ‘handmade’ structures created by using direct atomic or molecular placement by force microscopy; structures built using various eposi­tion or growth methods, as well as all polym­erization methods of synthesis. Thus, almost all biologic macromolecules and most bio­genic structures, including mineralized bio- materials, are made by bottom up methods. To make the distinction between top-down and bottom-up approaches more concrete, we will consider an example of each.

Production of Nanoporous Membranes by Micro Fabrication Methods: A Top-Down Approach:

Lithography can be summarized by three basic steps:

1. Pattern Design (generation of masks),

2. Pattern definition (exposure) and

3. Pattern transfer (etching/lift-off).

Optical lithography uses masks to form patterns on resist/substrate surfaces to produce features. The technique’s power lies in its reproduc­ibility and its capacity to manufacture via highly parallel processes. The key limitation of photolithography lies in the fact that reso­lution of features is diffraction-limited by the wavelength of light used. To address this limi­tation, short-wave radiation (i.e. X-rays with wavelengths of about 1nm wavelength) can be generated by synchrotron or other sources (from X-ray tubes, discharge plasma, or laser plasma), controlled, and focused for use in X-ray lithographic techniques.

The process is identical conceptually to optical lithography but requires special masks and resists ame­nable to the high-frequency radiation used. Combinations of filters and mirrors can pro­duce resolutions in feature size of less than 100 nm, with fabrication throughputs con­gruent with those of other optical lithogra­phy processes. Limited access to synchrotron sources in turn limits the wide application of the method, however.

The relatively long wavelengths used in conventional photoli­thography are generally unsuitable for forma­tion of Nano scale features unless some clever technical expedient, like the use of a sacrifi­cial layer is employed. As we will discuss in consideration of ap­plications of Nano therapeutic devices, there are numerous potential applications for tun­able Nano pore membranes. For certain applications, such as immune isolation, the distribution of pore sizes must be tight and nearly perfect. Until the late 1990s, this was an unattainable objective.

The key technical innovation facilitating the micro fabrication of highly defined Nano porous membranes was an approach featuring a sacrificial oxide layer sandwiched between two structural lay­ers that ultimately is etched away to define the pore pathway and diameter. The sacrificial layer (SiO₂) is sandwiched between a silicon wafer and a poly silicon layer: pore channel diameter is determined by the SiO₂ thickness.

In the process (Fig. 10.1), the top of a silicon wafer is doped with boron to increase its mechanical robustness, and p⁺ silicon is overlaid. Pore exit holes are plasma etched through the p⁺ and doped material. In the pore dimension-determining step, SiO₂ is grown on the wafer by dry thermal oxidation, a process allowing control of oxide layer thickness to within 1 nm. This oxide constitutes the critical sac­rificial layer. A thick poly-silicon layer (again, boron-doped for mechanical strength) is then deposited over the oxide.

Pore entries holes are etched by plasma etch, offset from the exit pores. Ultimately, the off­set will require the diffusion pathway of the finished structure to pass through a ‘bottle­neck’ whose diameter is determined by the thickness of the sacrificial layer.

The backside of the wafer is anisotropically etched to ex­pose the doped layer of the wafer (now the bottom of the membrane structure). Pores are opened by removal of exposed sacrificial layer using concentrated hydrofluoric acid.

Synthesis of Poly (Amido) Amine (PAMAM) Dendrimers: a Bottom-Up Approach:

PAMAM dendrimers are remarkably defined synthetic molecules made using polymer chemistry (Fig 10.2). Their unique structural attribute is their fractal geometry, and their unique physical property is their high mono-disparity.

Clever use of orthogonal conjuga­tion strategies in their synthesis drives their mono-disparity. Orthogonal conjugation in­volves the mutually exclusive reactivity of the chemical specificities present in the reaction: when the orthogonal reactants are present in a vessel and the reaction is carried at ap­propriate stoichiometry, only a mono-disperse single product is formed.

PAMAM dendrim­ers are among the most mono-disperse syn­thetic materials available and, within limits, their sizes, surface chemistries, and shapes can be controlled at the synthetic level. To a greater or lesser extent, these desirable mate­rial properties of dendrimers are caused by an orthogonal synthetic strategy.

PAMAM dendrimers are usually built from an initiator core (an amine) in sequential shells, called generations (i.e., G₀, G₁, G₂, etc.). Each generation comprises two synthetic half- steps, each of which is self-limiting in that the reagents performing each addition step are reactive only with the distinct chemical func­tionalities added to the growing dendrimer in the previous addition reaction.

Reagent is added until the chemical functionalities from the previous step are fully consumed (Fig. 10.2). In the first synthetic half-step, methyl acrylate is added to the amine precursor, add­ing a carboxy-terminated functionality to all amine branch points. Polymer intermediates terminating in such carboxy functionalities are called ‘half-generation’.

In the second half-step, the generation is completed. Ethyl­ene diamine is added, resulting in a branched amine-terminated adduct for each carboxy-late group of the half-generation precursor. In general, before proceeding to downstream synthetic operations, products of individual reactions are purified so that only function­alities incorporated into growing dendrimer are available for subsequent reactions.

Un­der ideal conditions, only a single chemical structure can result from each generation of growth, and with adequate purification be­tween addition reactions; in the absence of limitations on the completeness of each reac­tion (such as steric limitations which become manifest in dendrimers of generation 6 and above), dendrimers of each generation are perfectly identical to each other.

The Limits of Top-Down and Bottom-Up Distinctions with Respect to Nano-Materials and Nanodevices:

Some materials can be produced by alternate means, some of which are bottom-up, some of which are top down approaches. For instance, carbon nanotubes can be synthesized in an arguably top-down approach from graphite sheets in an arc oven or can be grown bot­tom-up, by a metal-catalysed polymerization method.

Additionally, not all finished mate­rials can be classified as either top-down or bottom-up: synthetic protocols can contain both steps. For instance, while proteins are synthesized from lower molecular weight amino acid precursors by chemically or bio­logically mediated polymerization (bottom- up), they are often made as precursor mol­ecules that are processed to a final product by chemical or enzymatic cleavage (top-down).

While the question of whether any given ma­terial is made top-down or bottom-up can be ambiguous, the synthetic provenance of multicomponent Nano devices can be even more so. In analogy to biogenic materials, synthetic polymers are bottom-up materials per se, but fabrication of raw polymeric materials into final device architectures often involves top- down steps. As we will see, therapeutic Nano devices frequently contain both synthetic and biologic components, and strict top-down and bottom-up categorization of these de­vices is often not applicable.

Technological and Biological Opportunities:

This section considers selected enablers for therapeutic Nano devices. Some are purely technological: nanomaterial self-assembly properties, bio conjugation methods, engi­neered polymers for conditional release of therapeutics, external triggering strategies, and so forth. Others relate to disease-state tissue or cell-specific biology that can be exploited by Nano therapeutics, such as the emerging vascular address system and intrin­sic triggering approaches. In association with those applications, we will consider addition­al biological opportunities for Nano scale ap­proaches specific to particular disease states.

Assembly Approaches:

Assembly of components into devices is ame­nable to multiple approaches. In the case of devices comprising a single molecule or pro­cessed from a single crystal (some micro fabricated structures, single polymers, or grafted polymeric structures) assembly may not be an issue. Integration of multiple, separately micro fabricated components may sometimes be necessary (as in the immune isolation cap­sule discussed below) and may sometimes drive the need for assembly, even for silicon devices. Furthermore, many therapeutic Nano devices contain multiple, chemically diverse components that must be assembled precisely to support their harmonious contribution to device function.

‘One-off’ Nanostructures and Low Throughput Construction Methods:

Direct-write technologies can obtain high (nanome­ter scale) resolution. For instance, electron-beam (e-beam) lithography is a technique requiring no mask, and that can yield reso­lutions on the order of tens of nanometers, depending on the resist materials used.

Resolution in e-beam lithography ultimately is limited by electron scattering in the resist and electron optics, and like most direct-write ap­proaches, e-beam lithography is limited in its throughput. Parallel approaches involving si­multaneous writing with up to 1,000 shaped e-beams are under development and may mit­igate limitations in manufacturing rate.

Force microscopy approaches utilize an ultrafine cantilever tip (typically with point diameters of 50 nm or less, Fig. 10.3) in contact with, or tapping, a surface or a stage. The technique can be used to image molecules, to analyse molecular biochemical properties (like ligand receptor affinity), or to manipulate materials at Nano scale.

In the latter mode, force mi­croscopy has been used to manipulate atoms to build individual nanostructures since the mid-1980s. This has led to construction of structures that are precise to atomic levels of resolution (Fig. 10.3), though the manufac­turing throughput of ‘manual’ placement of atoms by force microscopy is limited.

Dip-pen nanolithography (DPN) is a force microscopy methodology that can achieve high resolution features (features of 100 nm or less) in a single step. In DPN, the AFM tip is coated with molecules to be deployed on a surface, and the molecules are transferred from the AFM tip to the surface as the coat­ed tip contacts it.

DPN also can be used to functionalize surfaces with two or more con­stituents and is well suited for deployment of functional biomolecules on synthetic surfac­es with Nano scale precision. DPN suffers the limitations of synthetic throughput typical of AFM construction strategies.

Much as multi-beam strategies might im­prove throughput in e-beam lithography, multiple tandem probes may increase assem­bly throughput for construction methods that depend on force microscopy significantly, but probably not sufficiently to allow manufac­ture of bulk quantities of nanostructures, as will likely be needed for consumer Nano thera­peutic devices.

As standard of care evolves in­creasingly toward tailored courses of therapy and individual therapeutics become increas­ingly multi-capable and powerful, however, relatively low throughput synthesis/assembly methods may become more desirable. For the moment, though, ideal manufacturing ap­proaches for Nano therapeutic devices resem­ble either industrial polymer chemistry, occurring in bulk, in convenient buffer systems, or in massively parallel industrial micro fabrication approaches.

In any case, therapy for a single patient may involve billions of billions of individual Nano therapeutic units, so each individual Nano therapeutic structure must require only minimal input from a human synthesis/manufacturing technician.

Self-Assembly of Nanostructures:

Self-assembly has been long recognized as a po­tentially critical labor-saving approach to construction of nanostructures, and many organic and inorganic materials have self-assembly properties that can be exploited to build structures with controlled configurations.

Self-assembly processes are driven by thermodynamic forces and generally result in structures that are not covalently linked. Intra/intermolecular forces driving assembly can be electrostatic or hydrophobic interac­tions, hydrogen bonds, and van der Waals interactions between and within subunits of the self-assembling structures and the assembly environment. Thus, final configurations are limited by the ability to ‘tune’ the properties of the subunits and control the assembly en­vironment to generate particular structures.

Self-assembly of carbon nanostructures:

Carbon nanotubes (Fig. 10.4) spontane­ously assemble into higher order structures (Nano ropes) as the result of hydropho­bic interactions between individual tubes. Multi-wall carbon nanotubes (MWCNT) are well-known structures that can be viewed as self-assembled, nested structures of nano­tubes with tube diameters decreasing serially from the outermost to innermost tubes.

The striking resemblance that MWCNT have to macro scale bearings has been noted and ex­ploited. MWCNT linear bearings can be actu­ated by application of mechanical force to the inner nanotubes of the MWCNT assembly Actuation causes the assembly to undergo a reversible telescoping motion. Interestingly, these linear MWCNT bearings exhibit essentially no wear as the result of friction between bearing components.

C₆₀ fullerenes and single wall carbon nano­tubes (SWCNT) also spontaneously assemble into higher order nanostructures called ‘pea-pods’ in which fullerene molecules are encapsulated in nanotubes. The fullerenes of pea- pods modulate the local electronic properties of the SWCNT in which they are encapsulat­ed and may allow tuning of carbon nanotube electrical properties.

The potentially fine-level control of nanotube properties may prove useful in nanotube-containing electrical de­vices, particularly in cases wherein nanotubes are serving as molecular wires. In this capac­ity, carbon nanotubes have been incorporated into FETs (field effect transistors, discussed below), and other molecular electronic struc­tures.

Ultimately, these architectures may result in powerful, ultra-small computers to provide the intelligence of ‘smart’, indwelling Nano therapeutic devices. In general, though, fullerenes, nanotubes carbon Nano ropes, nanotube bearings, and peapods have yet to find extensive biological application, in part because of their extreme hydrophobicity, presumably poor biocompatibility, and high chemical stability. But controlled derivatization of nanotubes may be possible through a number of approaches including controlled of bond strain to render individual carbons of the tubes selectively chemically reactive (so-called Mechano synthesis).

Carbon nano­tubes and fullerenes, however, owe many of their remarkable properties (chemical stabil­ity, mechanical robustness, and some electrical properties) to the fact that all the valences of the constituent carbon atoms (except those at the ends of nanotubes that are ‘open’) are sat­isfied. Thus, to derivatize carbon nanostruc­tures is to degrade them, a fact one must con­sider when the design purposes that drove
incorporation of carbon structures into a therapeutic depends on the chemical perfec­tion of the material.

Self-Assembly of Materials Made by Tra­ditional Polymer Chemistry:

In the realms of drug delivery and biomedical micro and Nano devices, the most familiar self-assem­bled structures are micelles. These structures are formed from the association of block co­polymer subunits (Fig. 10.5), each individual subunit containing hydrophobic and hydrophilic domains.

Micelles spontaneously form when the concentration of their subunits ex­ceeds the critical micelle concentration (cmc) in a solvent in which one of the polymeric do­mains is immiscible. The cmc is determined by the immiscible polymeric domain and can be adjusted by control of the chemistry and length of the immiscible domain, as well as by control of solvent conditions.

Micelles formed at low concentrations from low-cmc polymers are stable at high dilution. Micelles formed from polymer monomers with high cmcs can dissociate upon dilution, a phe­nomenon that might be exploited to control release of therapeutic cargos. If desired, mi­celles can be stabilized by covalent cross-linking to generate shell-stabilized structures.

The size dispersity and other properties of micelles can be manipulated by control of solvent conditions, incorporation of excipients (to modulate polymer packing proper­ties), temperature, and agitation. From the standpoint of size, reasonably mono-disperse preparations (polydispersity of 1-5%) of Nano scale micellar structures can be pre­pared.

The immense versatility of industrial polymer chemistry allows micellar struc­tures to be tuned chemically to suit the task at hand. They can be modified for targeting or to support higher order assembly proper­ties. They can be made to imbibe therapeutic or other molecules for delivery and caused to dissociate or disgorge themselves of payloads at desired times or bodily sites under the in­fluence of local physical/chemical conditions.

The tenability of these and other properties at the level of monomeric polymer subunits as well as the level of assembled higher order structures make micelles potentially power­ful Nano scale drug delivery and imaging ve­hicles. Fractal materials, such as dendritic poly­mers exhibit packing properties that can be exploited to assemble higher order aggregate structures called ‘tecto (dendrimers)’. In fact, these self-assembly properties are being ex­ploited in oncological Nano therapeutics.

In principle, these self-assembling therapeutic complexes need not be pre-formed prior to administration. Individual functional mod­ules of the therapeutic assembly might be administered sequentially, potentially to tailor therapies more precisely to individual patient responses.

Stoichiometric Control and Self-Assembly:

As the preceding examples demonstrate, self- assembly approaches sometimes do not feature precise control of subunit identity and stoichiometry in the assembled complexes. This can be a limitation when the stoichi­ometry and relative arrangement of differen­tiate individual subunits is critical to device function.

Stoichiometry is less an issue when the self-assembling components are identical and functionally fungible, as in the synthetic, peptidyl anti-infective. In the anti-infective architecture, individual peptide components are flat, circular molecules. The planar character of the toroidal subunits is a consequence of the alternating chirality of alternating D-L amino acids (aas) in the primary sequence of the peptide rings.

Alternating D and L aas is not possible in proteins made by ribosomal synthesis. Ribosomes recognize and incorpo­rate into nascent polypeptides only L amino acids, and so, as the result of aa chirality and bond strain, peptides made by ribosomes cannot be made flat, closed toroid’s like those of the peptidyl anti-infective.

Much as in α-helical domains of ribosomal synthesized proteins, however, the aa R-groups (which are of varying hydrophobic or hydrophilic chemical specificities) are ar­ranged in the plane of the closed D, L rings extending out from the center of the rings. Hydrogen bonds between individual rings govern self-assembly of the toroid’s into rod like stacks, while the R-groups dominate in­teractions between multiple stacks of toroid’s and other macromolecules and structures.

The planar toroidal subunits can be admin­istered as monomers and self-assembly into multi-toroid at the desired site of action (in biological membranes). But the peptide toroid’s R-groups are chemically tuned so that the rod structures into which they spontaneously assemble intercalate preferentially in specific lipid bilayers (i.e. in pathogen vs. host membranes).

Moreover, the assembled rods may undergo an additional level of self-assembly into nultirod structures, spanning pathogen membranes. Whether as single rod or mul­tiple rod assemblies, membrane intercalation by stacked toroid’s reduces the integrity of pathogen membranes selectively, and there­fore particular toroid species exhibit selective toxicity to specific pathogens.

These toroidal, synthetic antibiotics and other Nano scale an­timicrobials represent critically needed, novel antibacterial agents. Resistance to traditional, microbially derived antibiotics often is tied to detoxifying functions associated with sec­ondary metabolite synthesis; these detoxify­ing functions are essential for the viability of many antibiotic producing organisms.

The genes encoding such detoxifying functions are rapidly disseminated to other microor­ganisms, accounting for the rapid evolution of drug resistant organisms that has bedev­iled antimicrobial chemotherapy for the last 25 years. Synthetic Nano scale antibiotics, like the peptide toroid’s and the N8N antimicrobi­al Nano emulsion, act by mechanisms entirely distinct from those of traditional secondary metabolite antibiotics, and no native detoxifying gene exists.

Therefore, novel Nano scale antimicrobials may not be subject to the un­fortunately rapid rise in resistant organisms associated with most secondary metabolite antibiotics, though this remains to be seen. As bacterial infection continues to re-emerge as a major cause of morbidity and mortality in the developed world, consequence of in­creasing antibiotic resistant pathogens, novel Nano scale antibiotics will become more im­portant.

Biomolecules in Therapeutic Nanodevices—Self-Assembly and Orthogonal Conju­gation:

Biological macromolecules undergo self-assembly at multiple levels, and like all instances of such construction, biological self-assembly processes are driven by ther­modynamic forces. Some biomolecules un­dergo intermolecular self-assembly (as in protein folding from linear peptide sequenc­es). Higher order structures are, in turn, built by self-assembly of smaller self-assembled subunits (for instance, structures assembled by hybridization of multiple oligonucleotides, enzyme complexes, fluid mosaic membranes, ribosomes, organelles, cells, tissues, etc.).

Proteins are Nano random copolymers of 20 chemically distinct amino acid (aa) subunits. The precise order of aas (i.e., via interac­tions between aa side chains) drives the lin­ear polypeptide chains to form specific sec­ondary structures (the α helices and β sheet structures).

The secondary structures have their own preferences for association, which, in turn, leads to the formation of the tertiary and quaternary structures that constitute the folded protein structures. In its entirety, this process produces consistent structures that derive their biological functions from strict control of the deployment of chemical speci­ficities (the aa side chains) in three-dimen­sional space.

Biomolecules can be used to drive assembly of nanostructures, either as free molecules or conjugated to heterologous Nano-materials. For instance, three dimensional nanostruc­tures can be made by DNA hybridization. Such DNA-nanostructures can exhibit tightly controlled topographies but limited integrity in terms of geometry, due to the flexibility of DNA strands. Oligonucleotides, antibodies and other specific biological affinity reagents can also use to assemble nanostructures.

Of­ten the domains of biomolecules responsible for assembly and recognition are small, continuous, and discrete enough that they can be abstracted from their native context as mod­ules and appended to other Nano-materials of interest to direct formation of controlled Nano scale architectures.

Several orthogonal bio conjugate approaches have arisen from the field of protein semi-synthesis. These pro­tein synthetic chemistries allow site-specific conjugation of polypeptides to heterologous materials in bulk, as the result of conjuga­tion between exclusively, mutually reactive electrophile-nucleophile pairs (analogous to dendrimer synthesis discussed above). They have been applied to the synthesis of mul­tiple therapeutic Nano devices.

As described above, proteins are profoundly dependent on their three-dimensional shapes: chemical de-privatization at critical aa sites can profoundly impact protein bioactivity. Because conjuga­tion can be directed to pre-selected sites via orthogonal approaches, and since the sites of conjugation in the protein can be chosen because the proteins involved tolerate ad-ducts at those positions, proteins coupled to Nano-materials by such orthogonal method­ologies often retain their biological activity.

In contrast, protein bioactivity in conjugates generally is lost or profoundly impaired when proteins are coupled to Nano-materials using promiscuous chemistries. For instance, pro­teins such as cytokines (and other protein hormones) elicit their effects by interacting with a receptor, and a large fraction of their surface (20% or more) is involved in receptor binding, directly or indirectly.

Promiscuous chemistries [1-ethyl-3-(3-diamethylamino-propyl) carbodiimide or EDC, conjugation] used to conjugate cytokines to nanoparticles tend to inactive hIL-3 and other cytokines whereas the same protein/particle bio-conjugates retain bioactivity if judiciously chosen orthogonal conjugation strategies are used.

Proteins for which only a small portion of their surfaces contribute to the interesting portions of their bioactivities, such as some enzymes or intact antibodies may be some­what less sensitive to promiscuity of the bio-conjugate strategy used, but the benefits of orthogonal conjugation strategies can also apply to these protein bio-conjugates. The po­tential utility of orthogonal conjugation for incorporation of active biological structures into semi-synthetic Nano devices is becoming more fully recognized and cannot be overes­timated.

Targeting: Delimiting Nanotherapeutic Action in Three-Dimensional Space

Delivery of therapeutics to sites of action is a key strategy to enhance clinical benefit, particularly for drugs useful within only narrow windows of concentration because of their toxicity. Diverse targeting approaches are available, ranging from methods exploiting differential extravasations limits of vascu­lature of different tissues, sizes, and surface chemistry preferences for cellular uptake; preferential partition of molecules and par­ticles into specific tissues-by virtue of their charges, sizes, surface chemistry, or extent of opsonisation; or the affinity of biologi­cal molecules decorating the Nano device for counter-receptors on the cells or tissues of interest.

The Reticuloendothelial System and Clearance of Foreign Materials:

Physical properties such as surface chemistry and particle size can drive targeting of Nano-materials (and presumably Nano devices contain­ing them) to some tissues. For instance, the pharmacokinetic (Pk) and bio distribution (Bd) properties of many drugs and Nano-materials are driven by their clearance in urine, which is in turn governed by the filtration preferences of the kidney. Most molecules making transit into urine have masses of less than 25 to 50 kilo-daltons (kDa; 25-50 kDa particles corresponding loosely to effective diameters of about 5 nm or less) and are pref­erably positively charged; these parameters are routinely modulated to control clearance rates of administered drugs.

Clearance of low molecular weight (Nano) materials in urine can be suppressed by tuning their molecular weights and effective diameters, typically ac­complished by chemical conjugation, to poly­mers such as poly (ethylene glycol). Polymer conjugation (pegylation) has been applied to many different materials and may provide some degree of charge shielding. Pegylation also increases effective molecular weights of small materials above the kidney exclusion limit, diverting them from rapid clearance in urine.

Coating foreign particles with serum pro­teins (opsonization) is the first step in the clearance of foreign materials. Opsonized particles are recognized and taken up by tis­sue dendritic cells (DCs) and specific clear­ance organs. These tissues (thymus, liver, and spleen, constituting the organs of the reticu­loendothelial system or RES) extract materi­als from circulation by both passive diffusion and active processes (receptor-mediated endocytosis).

Charge-driven, receptor-mediat­ed uptake of synthetic Nano-materials occurs in the RES and can result in partition of posi­tively charged nanoparticles into the RES. For instance, PAMAM dendritic polymers ex­hibit high positive charge densities related to the large number of primary amines on their surfaces.

In experimental animals, bio distribution of unmodified PAMAM dendrimers is limited nearly exclusively to RES organs. This unfavorable bio distribution can be modu­lated by capping’ the dendrimers (i.e., de-rivatizing the dendrimer to another chemical specificity, such as carboxy or hydroxyl func­tionalities). Despite legitimate applications of targeting to the kidney and the RES (for instance in glomerular disease), intrinsic tar­geting to clearance sites is of interest primar­ily as a technical problem that impedes thera­peutic delivery to other sites.

In such cases, numerous targeting strategies are available, some of which depend on synthetic Nano-materials properties to minimize uptake of Nano therapeutic devices by clearance systems and maximize delivery to desired sites. Targeting via biological affinity reagents decorating the surfaces of therapeutic Nano devices may be the most direct approach.

Nanotherapeutic Targeting Exploiting Bi­ological Affinity Properties:

Tissue-specific delivery by biological affinity requires the presence of tissue-specific surface features, most commonly proteins or glycoproteins (tissue-specific antigens). Historically, the search for tissue-specific antigens for drug targeting, whether associated with tumors or other cells, organs or tissues, has been ardu­ous and not entirely gratifying.

The primary problems are specificity (few antigens are uniquely present in any single tissue), avail­ability (some tissues may not have their own unique antigenic signature or marker), and therapeutic extravasation or directed migra­tion in tissue spaces (markers in tissue may not be accessible from vasculature).

An excit­ing recent development in biochemical tar­geting is the discovery of a vascular address system. The vascular address system has been characterized by administering a peptide phage display to library animals, resecting in­dividual organs, and extracting phage from the vasculature of the isolated organs.

Amaz­ingly, phage isolated from different organs ex­hibited distinct consensus presented peptide sequences, indicating that the vasculature of individual organs presented unique cognate receptors, each bound by a different short (ten amino acids or fewer) consensus peptide sequence that had been affinity selected from the phage display library.

Furthermore, the affinity-selected peptides have the capacity to tether Nano to micro scale particles to the site of their cognate receptors. Site-specific drug delivery using the vascular address system has already been demonstrated: it has further been used to target an apoptotic (cytocidal) agent to the prostate and to direct destruction of the organ in an animal model.

Mapping of the vascular address system is currently underway and holds the promise of specific delivery of therapeutic agents to vasculature of specific organs. It remains to be seen whether each organ has a single mo­lecular marker constituting its address that is amenable to binding a single peptide se­quence; organs may instead have unique con­stellations of antigenic markers.

If so, specific targeting may be possible using multiple pep­tides, each peptide binding its cognate recep­tor on target organ vasculature very weakly. Peptides used in such a multivalent target­ing strategy would be chosen to reflect the unique constellation of address markers pres­ent in the target tissue. Affinities of cognates for linear peptides are often very low, though their aggregate affinity may be substantially higher than that of any peptide-vascular ad­dress cognate alone.

Under ideal conditions, the affinity of such a multi-peptide, multi-cognate complex should be the equivalent of the products of the affinities of each constituent peptide for its individual constituent cognate. Such multivalent interaction avidities can be extremely high (and the corresponding effec­tive affinity constants are also high) but sel­dom fully realize their theoretical maximums. It should be noted that most of the vascular addresses identified to date deliver materials to the organ vasculature: extravasation and access of organ tissue spaces by Nano-thera­peutics remains a separate issue.

Triggering: Delimiting Nanotherapeutic Action in Space and Time:

Controlled triggering of therapeutic action is the other side of the targeting coin. If the site and time of Nano therapeutic delivery cannot be adequately controlled, the site of therapeutic action can be delimited by spa­tially or temporally-specific triggering. The triggering event might drive release of active therapeutic from a reservoir, or chemical or physical processing of drug materials from an inert to an active form (inert administrations that are converted to active form at a specific time or place drugs which are activated by a chemical reaction occurring their sites of ac­tion are called pro-drugs—Fig. 10.6).

Three major triggering strategies are widely used: external stimuli, intrinsic triggering, and sec­ondary signaling (multicomponent systems). Triggering strategies require Nano therapeutic delivery devices to be sensitive to a controlled triggering event, or a spatially/temporally in­trinsic triggering event mediated by the host. Obviously, the triggering event itself must be tolerable to the patient.

Nanotherapeutic Triggering Using Exter­nal Stimuli:

External stimuli are provided by an external Nano device operator/clinician usually in the form of a site-specific energy input, typically light, ultrasound, or magnetic or electrical fields. Organic polymeric struc­tures are very amenable to interaction with these energy sources. For instance, micellar structures can be reversibly dissociated with ultrasound, in which case they disgorge their contents or expose their internal spaces to the environment during ultrasound pulses (20 to 90 kilohertz range).

The process has been used to control release of cytotoxin (doxoru­bicin) from micelles, and short ultrasound transients might be used for pulsatile or in­termittent exposure of patients to therapeu­tics. Light is another popular external trigger­ing modality. Bioactive materials can be covalently associated to a Nano scale-delivery vehicle by photo-labile linkages, or micelles can be constructed so that their permeability is altered as the result of exposure to light.

In the latter case, light input can cause photo-polymerization resulting in micelle compaction that drives release of therapeutic cargo or photo-oxidation, which causes loss of mi- cellar integrity to release encapsulated mate­rials. The ability of light to penetrate dense tissues is a clear limitation to this approach. This concern can be accommodated by poly­mer systems responsive to wavelengths that penetrate tissue efficiently (usually in the red- infrared region of the spectrum), or by use of systems such as fiber optics to deliver light to deep tissues.

Externally applied magnetic fields can also be used to control Nano therapeutic activity. For instance, eddy currents induced by alter­nating magnetic fields can heat Nano-metallic particles and their immediate vicinity, an ap­proach that has been successfully applied to control the bioactivity of individual biologi­cal macromolecules.

Colloidal gold particles are covalently conjugated to biomolecules (nucleic acid, or NA, duplexes or proteins), and alternating magnetic fields are used to induce heating sufficient to cause dissocia­tion of hybridized NA structures or denaturation of protein three-dimensional struc­tures.

Within certain parameters, the process is reversible and so allows the construction of semi-biological assembly or release switches. The modality has clear application to tempo­rally specific triggered release, but the cur­rent inability to direct magnetic fields to pre­selected tissue locations may limit its use for spatially specific triggering.

Nanotherapeutic Triggering by Intrinsic Physiological Conditions:

Intrinsic trigger­ing is a pro-drug strategy that depends on the conditions at the desired site of action to— triggers the activity of the Nano therapeutic. Much as the three-dimensional conforma­tion of—and, therefore the activity of—pro­teins can be controlled in external trigger­ing strategies, proteins can be engineered to make their conformations sensitive to intrin­sic conditions at their desired sites of activ­ity. For instance, non-covalent complexes of a diphtheria toxin (DT) protein variant and a bio-specific antibody have been engineered for specific toxicity to cells that can take up the complexes.

The bi-specific antibody recognizes both a cell-specific surface receptor as well as the DT in an inactive (nontoxic) configuration. Antibody binding to the receptor results in receptor-mediated endocytosis of the com­plex in target cells. As a result of the lower pH of the endosomal compartment (reach­ing pH 4-5 in endosomes, as opposed to the constant pH 7-8 in circulation) into which the bound complex is taken up, the DT vari­ant protein undergoes a three-dimensional conformational shift to its toxic form. This conformation of DT is not recognized by the bi-specific antibody, so the toxin dissociates from the complex and kills the cell.

Hypoxia triggered pro-drugs have also been developed that are activated either by chemical reduc­tion or by enzymatic activities induced in hypoxic tissues. Extreme hypoxia is a unique feature of neoplastic tissue, so these strategies have clear application in oncology. Clinical manipulation of the extent of tumor oxy­genation is possible and may also be possible in other tissues. If so, redox state dependent triggering approaches may have applications beyond cancer.

Enzyme-activated delivery (EAD) is anoth­er pro-drug-like triggering strategy in which the properties of a Nano scale drug-delivery vehicle are altered at the site of action by an enzymatic activity endogenous to that site. Most typically, this involves a liposomal or micellar nanostructure from which designed pendent groups can be cleaved by metabolic enzymes (alkaline phosphatase, phospholipases, proteases, glycosidic enzymes, etc.) that are highly expressed at the site of thera­py.

Nanostructures are designed so that enzy­matic cleavage of the pendent group causes a conformational or electrostatic change in the polymeric components of the delivery device, rendering the micellar structure fusogenic, leaky, or causing partial or complete dissocia­tion of the structure. This has the result of de­livering or releasing therapeutic payloads at pre-selected sites.

Nanotherapeutic Triggering Using Second­ary Signaling:

Secondary signaling or multi-component delivery systems are more com­plex delivery strategies. These systems can feature site-specific and systemic delivery of one or more components. One class of such systems is the so-called ADEPTS devices (an­tibody directed enzyme-pro-drug therapy). An ADEPTS system features affinity-based tar­geting of an enzymatic pro-drug activator to a desired site, followed by systemic administra­tion of a pro-drug. ADEPTS are used primarily for oncology, and so the pro-drug involved is generally one that can be activated to cytotox­icity.

Since pro-drug dosing is systemic, pro-d­rug toxicity must be minimal in the inactive form, and the activating catalyst must not be present at locations where cytotoxicity would be deleterious. To address this issue, the Nano-therapeutic designer can chose to engineer an activation mechanism that has no physiologi­cal analogue in the host. In addition, tuned, high liability of the active drug can potentially delimit the site and extent of cytotoxic effect mediated by ADEPTS systems.

Other secondary signaling systems rely on DNA hybridization to assemble therapeutic components into catalytically active com­plexes at their sites of action; in this system, the individual components lack therapeutic activity until they are brought within a few nanometers of each other by hybridization to a target single-stranded nucleic acid molecule.

At this point, the catalytic moiety and the pro-drug moieties are sufficiently close in space to allow catalytic cleavage of the active component from the pro-drug. This strategy makes no provision for delivery of drug and catalyst bio-conjugates to the site neither of interest, nor for their transit across biologi­cal membranes. The system is similar to the DNA hybridization-driven fluorescence transfer system.

Alternatively, secondary signaling systems can exploit competitive displacement of therapeutics from a carrier structure. In this case, a non-covalent complex of engineered antibody and plasminogen activator (PA) is tethered to blood and fibrin clots by antibody affinity for fibrin.

PA is released from the bound complex (to dissociate the clot) using bolus systemic administration of a nontoxic binding competitor for PA to the antibody complex. The strategy establishes a high lo­cal concentration of PA at clot sites, efficiently dissolving clots and potentially minimizing systemic side effects.

Layering Strategies for Fine Control of Nanotherapeutic Action:

It should be clear that many of these approaches may be broad­ly applicable to trigger events other than drug release or to drive assembly or disassembly of therapeutic nanostructure in situ. It should also be clear that these approaches are often complementary, and that multiple approaches can be used in single Nano therapeutic devices. Layering targeting and triggering approaches tends to make devices more complex, but it also allows clinician/operators to intervene at multiple points in therapy, potentially leading to finer control of the therapeutic process and better clinical outcomes.

Sensing Modalities:

The need for ‘smart’ therapy is a key theme of therapeutic nanotechnology and pharmacology as a whole. Drugs with narrow therapeu­tic windows should be delivered only to their desired site of action and be pharmacologi­cally active only when that activity is needed. These strategies can limit undesired second­ary effects of therapy, some of which can be debilitating or life threatening.

One possible approach to this issue is the incorporation of sensing capability (specifically, the capacity to recognize appropriate contexts for thera­peutic activity) into Nano therapeutic devices. Sensing capability may allow self-regulation of a therapeutic device, reporting to an exter­nal clinician/device operator, or both.

In the context of our discussion of therapeutic Nano and micro scale devices, we will consider pri­marily electrical and electrochemical sensor systems, particularly micro fabricated (Field Effect Transistor or FET, and cantilever) and conducting polymer sensors.

Sensor Systems:

Sensing is predominantly higher-order device functionality, depending on multiple device components though one could argue that some targeting/triggering strategies, particularly targeting by bio-affinity and intrinsic triggering strategies, must, a priori, incorporate at least limited sensing ca­pability. But biosensors, as they are typically considered, are multifunctional, multicomponent devices.

Usually a biosensor system is composed of signal transducer, sensor inter­face, biological detection (bio-affinity) agent, and an associated assay methodology, with each system component governed by its in­herent operational considerations. The trans­ducer component determines the physical size and portability of the biosensor system.

Signal transducers are moieties that are sen­sitive to a physical-chemical change in their environment and that undergo some detect­able change in chemistry, structure, or state as the result of analytic (the thing to be sensed) recognition.

Analytes for Nano therapeutic application could be biomolecules, like pro­teins, small molecules (organic or inorganic), ions (salts or hydrogen ions), or physical con­ditions (such as redox state, temperature). Interfaces are the sensor components that interact directly with the analyte. For sensor use in Nano therapeutic devices, immobilized or otherwise captured biological molecules (proteins, nucleic acids) often constitute the sensor interface.

Whatever the chemical na­ture of the interface, it determines the selec­tivity, sensitivity, and stability of the sensing system and also is a dominant determinant of sensor operational limits. Assay method­ology determines the need (or lack thereof) for analyte tracers, the number of analytical reagents, and the complexity and rapidity of the sensing process. Nano therapeutics are of interest at least in part because they can be minimally invasive, low complexity, yet ro­bust and accurate; convenient assay methods are therefore highly desirable.

Cantilever Biosensors:

Micromechanical cantilevers transducer sensed events by me­chanical means. Both changes in the resonant frequency and deflection of cantilevers result­ing from analyte binding or dissociation can be conveniently and sensitively detected.

These changes in cantilever state can be conveniently detected by optical, capacitive, interferometry, or piezoresistive/piezoelectric methods, among others. Micro fabricated cantilever dimensions range from micron to sub-micron range, with potential for further dimensional optimization (by carbon Nano-tubes appended to them, for instance).

They are operationally versatile and can be used in air, vacuum, or liquid, although they suffer some degradation in performance in liquid media. Like most micro machined structures, they can be batch fabricated and conveniently multiplexed. Cantilevers used in atomic force microscopy approaches can be used to study individual bio-molecular interactions.

In this approach, cantilever tips are derivatized with biomolecules (effectively, one member of a receptor/counter-receptor pair), and the tip-bound biomolecule is allowed to bind its counter-receptor (itself bound on a surface). Under non-equilibrium conditions (i.e., con­ditions that result in thermodynamically ir­reversible change in analyte molecular struc­tures), the force required to disrupt single molecular interactions can be measured and related to classical biochemical parameters of receptor binding.

The method has been ap­plied to interactions between hormones and their receptors, sugars and lectins, as well as hybridizing DNA strands. Cantilever systems sensitively detect changes in mass at their surfaces: changes as small as mass densities of 0.67 ng/cm^-3 are theoretically detectable. This can allow detection of binding of extremely small objects to the cantilever and has been applied to detection and enumeration of prokaryotic and eukaryotic cells, as well as small numbers of macromolecules.

The in­corporation of biological receptors or affinity reagents on the cantilever surface can drive specific binding events for particular sensing tasks. Micro cantilevers are also highly sensi­tive to temperature, detecting changes as low as 10-5 K; they can also detect small changes in pH.

Field Effect Transitor Biosensors:

Field ef­fect transitor (FET) architectures are another sensing architecture that can be conveniently produced by micro-nanofabrication. FETs consist of a current source, a current drain, a conductive path (sensing channel) between them, and a sensing gate to which a bias can be applied.

Analyte binding to the sensing channel induces a charge transfer resulting in a dipole between the surface and the under­lying depletion region of the semiconductor: current that passes between the source and drain of a semiconductor FET is quite sensi­tive to the charge state and potential of the surface in the connecting channel region.

Moving a standard silicon FET from deple­tion to strong inversion (i.e., shifting the sur­face potential by >~ 0.5 eV) requires less than ~ 10-7 C/cm² or ~ 6×1012 charges/cm², cor­responding to transfer of 6.25×1011 e/cm². With FETs of 2,000 square micro-meters, detection of biological analytes in sub-Nano molar concentrations is easily feasible.

Specificity for binding of macromolecular analytes of interest can be provided by deployment of biological affinity reagents in the FET sensing channel. Submicron FETs are routinely manufactured; use of carbon Nano-tubes in FETs will offer still greater miniaturization.

Carbon nanotubes also have ex­cellent mechanical properties and chemical stability in addition to potentially tunable electrical properties, making them highly de­sirable electrode/Nano-electrical materials for any number of Nano electrical applications. Biomolecules can be bound to carbon Nano- tubes, particularly in FET and Nano electrode applications.

Most biomolecules bound to carbon nanotubes are not covalently bound and do not exhibit direct electrical commu­nication with the nanotube, though redox enzymes bound to nanotubes and other con­ductive nanomaterial’s may.

Flavin adenine dinucleotide (FAD) and flavo-enzyme glucose oxidase (Gox) both display quasi-reversible one electron transfer when absorbed onto un-annealed carbon nanotubes in glassy car­bon electrodes. Gox, so immobilized, retains its substrate-specific (glucose) oxidative ac­tivity, leading to applications in sensing cir­culating glucose for diabetes and, perhaps, to a strategy of harvesting electrical power from metabolic energy.

Conducting Polymers and Sensor Biocompatibility:

Biocompatibility of most metallic structures is limited at best; metal structures rapidly foul with serum proteins (i.e., become opsonized), undergo electrochemical degra­dation, or have other problematic properties. Polymer chemistry, however, has the capacity to tune composition to enhance biocompat­ibility properties and is commonly used to make synthetic surfaces more biologically tolerable. Electrically conductive polymers are potentially attractive in this context.

Poly­meric materials are available with intrinsic conductivities comparable to that of metals [up to 1.5 x 107 (Ωm)-1 which by weight, is about twice that of copper]. Significant conductivity has been documented for a dozen or so polymers, including polyacetylene, poly- paraphenylene, polypyrrole, and polyaniline, doped with various impurities.

Careful con­trol of doping can tune electrical conductivity properties over several orders of magnitude. Conductive polymeric materials may be ex­tremely well-suited to bio sensing applica­tions. The polymeric materials themselves are often compatible with proteins and other biomolecules in solution.

Furthermore, poly­mers are amenable to very simple assembly of sensor transducer-interface components by deposition of the polymer and trapped pro­tein (sensor interface) directly on a metallic micro of Nano electrode surface. Combined with a facile Nano electrode array micro fabrication method, simultaneous conducting polymer-interface protein deposition may of­fer an extremely simple way to fabricate mul­tiplexed sensor arrays.

These electrochemical and electro physical sensing modalities are of little use in and of themselves: they must communicate with ei­ther other device components (such as drug dispensing effector components) or with ex­ternal observers or operators.

Sensor coupling in autonomously operating devices produced by micro fabrication can be done directly, and the coupling linkages incorporated into the fabrication protocol. Coupling with biologi­cal device components can also be direct, as when conductive materials are conjugated to biomolecules, and can directly modulate their activity. Sensor-device coupling can also be indirect, through electrochemically pro­duced mediator molecules.

Sensors might be independent of Nano therapeutics and report conditions at the site of therapeutic action to an external operator, who would use any one of the external triggering strategies discussed above to engage therapy when and where ap­propriate.

Communication/sensor interroga­tion might be accomplished most crudely by direct electrical wiring of sensors to an exter­nal observation station. Alternatively, if the event to be detected is transduced optically, as in colorimetric smart polymers that change optical properties in the presence of analyte, sensors might be interrogated by fiber optics.

These modalities are most applicable to sen­sor arrays delivered to the site of interest by a catheter. The use of a catheter may be justified in some therapeutic applications, but it is an invasive procedure and is not optimal.

We have already seen multiple examples of pro-drugs that are activated by cleavage of an inhibitory domain from the complex. One non-invasive approach to communication with external operators could exploit this phenomenon by detecting the cleaved frag­ment in bodily fluids. If the cleaved moiety cleared through urine, the extent of drug acti­vation could be monitored noninvasively via urinalysis. There is no a priori need to con­nect the cleavage event to drug activation. For instance, an operator might administer a cat­alyst that cleaves a detectable material from a Nano therapeutic.

If this secondary signaling moiety was in­dependently targeted to the desired site of therapy, presence of the detectable cleaved product would provide information regard­ing the bodily location of the therapeutic Nano device. The cleaved product would not be detect­able unless the therapeutic and secondary signaling moiety co-localized at a single site. Other sophisticated communication involves ultrasound or electro-magnetic radiation to carry information. Application of these sorts of modalities currently occurs in in vivo im­aging approaches.

Imaging Using Nanotherapeutic Contrast Agents:

Imaging is a minimally invasive procedure that allows visualization of organs and tissues following the administration of a detectable moiety (contrast agent). The contrast agent is then exposed to some condition that in­teracts with the contrast agent so as to pro­duce an emission or response detectable to an external monitoring device.

Nano-sized particles (5-100 nm in diameter) have found application as contrast-enhancing agents for medical imaging modalities such as magnetic resonance imaging. MRI currently provides cross-sectional and volumetric images with high spatial resolution (< 1 mm) and is po­tentially applicable to many clinical purpos­es, though enhanced imaging capabilities are desirable. For instance, blood flow measure­ments of healthy and diseased arteries can be quantified better by the aid of improved con­trast agents that highlight blood flow at the vessel wall.

Similarly, some tissues (certain tumors) do not exhibit strong contrast within the MRI field and cannot be readily identified or characterized at present. It is of great inter­est, therefore, to develop Nano scale particles that provide enhanced contrast for many applications.

Magnetic Resonance Imaging (MRI):

The basics: Objects to be imaged are exposed to a strong magnetic field and a well-defined ra­dio frequency pulse. The external magnetic field (BO) serves to loosely align protons either with (lower energy level) or against (high energy level) the field, the difference between the two energy levels being proportional to BO.

Once the protons are separated into these two populations, a short multi-wavelength burst (or pulse) of radio frequency energy is applied. Any particular proton will absorb only the frequency that matches its particu­lar energy (the Larmor frequency).

This reso­nance absorption is followed by the excitation of protons from the low to high energy level and of equivalent protons moving from high to low energy levels. After the radio frequen­cy pulse, protons rapidly return to their origi­nal equilibrium energy levels. This process is called relaxation and involves the release of absorbed energy. Once equilibrium is again established, another pulse can be applied.

Data is collected by positioning a receiver perpendicular to the transmitter: relaxation energy release induces a detectable, quan­tifiable signal (i.e., in amplitude, phase, and frequency) at the receiver coil. Since multiple protons in multiple chemical environments are involved, the signal at the receiver in­cludes many frequencies.

The received signal consists of multiple, superimposed signals (called the free induction decay or FID) sig­nal, resulting from the relaxation of multiple, chemically distinct protons. FID is converted from the time domain to frequency domain (by Fourier transformation), within which individual proton types can be identified. It­erations of this procedure in two or three di­mensions can create a high-resolution image of anatomical cross section or volume.

Intrinsic factors affecting image quality include the proton density of the tissues, lo­cal blood flow, and two relaxation time con­stants: longitudinal relaxation time (T₁) and transverse relaxation time (T₂). Control of T₁ and T₂ relaxation effects are most critical for high-resolution MR images. T₁ relaxation measures energy transfer from an excited proton to its environment.

In tissues, protons of fats and cholesterol molecules (relatively movement-constrained macromolecules) re­lax efficiently after a pulse and exhibit a short T₁ time. Water in solutions (a small molecule tumbling relatively freely in solution) has a much longer T₁ time. T₂ relaxation measures the duration of coherency between resonat­ing protons after a pulse, prior to their return to equilibrium.

Tightly packed, solid tissues with closely interacting hydrogen nuclei relax more quickly than loosely structured liquids, so tissues such as skeletal muscle have a short T2s, while cerebrospinal fluid has a very long T2. Intrinsic factors are manipulated through extrinsic factors, such as the external magnet­ic field strength, the specific pulse sequence, etc., allowing the collection of meaningful MR images.

Nanoparticle contrast agents:

Signal in­tensity in tissue is influenced linearly by pro-Ron density, while changes in T1 or T2 result in exponential changes in signal intensity. T1 and T2, therefore, are manipulated to enhance imaging by administration of exogenous con­trast-enhancing agents.

MRI contrast agents are divided into paramagnetic, ferromag­netic, or super paramagnetic materials. Metal ion toxicity is an unfortunate consequence of physiologic administration of contrast agents but can be mitigated somewhat by complexation of the metals with organic molecules.

Paramagnetic metals used for enhanced MRI contrast (gadolinium, Gd, iron, Fe, chromium, Cr, and manganese, Mn) have permanent magnetic fields, though the mag­netic moments of individual domains are unaligned. Upon exposure to an external magnetic field, individual domain moments become aligned, generating a strong local field (up to 104 gauss).

Paramagnetic metal ions interact with water molecules, causing an enhanced relaxation of the water mol­ecules via tumbling of the water-metal com­plex, dramatically decreasing the T1 value for the water molecules and enhancing the pro­ton signal. Contrast enhancement by Para magnetics is thus due to the indirect effect the contrast agent has on water and its magnetic resonance properties.

Ferromagnetic and super paramagnetic ma­terials both contain iron (Fe) clusters, which generate magnetic moments 10 to 1,000 times greater than do individual iron ions. Clusters greater than 30 nm in diameter are ferromagnetic, whereas smaller particles are super paramagnetic. Ferromagnetic materi­als maintain their magnetic moment after the external field is removed, but super paramagnetic materials lose their magnetic field after the field is removed, as do paramagnetic. Both ferromagnetic and super paramagnetic substances minimize the proton signal by shortening T2, resulting in negative contrast (i.e., darkening of the image).

Nanobiotechnological Contrast Agent Design:

First generation contrast agents of­ten contained a signal metal ion/complex, whereas emerging agents incorporate Nano scale metal clusters, crystals, or aggregates, sometimes encapsulated within a synthetic or biopolymer matrix or shell. These metal cluster agents improve contrast effects and, hence, output MR images profoundly. Fur­thermore, surface chemical groups (from the matrix or shell) can be derivative to improve biocompatibility or allow targeting to a tissue or site of interest.

Typically particles are prepared from col­loidal suspension where metallic cores are thoroughly mixed with the matrix material before being aggregated out of solution with a non-solvent. Dextran (a polymer of 1, 6-β-D- glucose) is a typical matrix used in commer­cial imaging reagents: Combidex is coated with 10,000 molecular weight dextran, Feridex has an incomplete, variable dextran coat­ing, and Resovist is coated with carboxydextran.

Other polymeric materials are also used (oxidized starch), and matrix-less particles are also produced. Nanoparticle contrast agents must be purified under tightly controlled conditions (generally by centrifugation or high pressure liquid chromatography) and accurately characterized (for size dispersity by light scattering, chromatography, photon correlation, or electron microscopy) to assure the reproducibility of imaging agent produc­tion lots.

Elemental analysis, X-ray powder diffraction, and Mossbauer spectroscopy have also been used to characterize metallic cores. Tight definition of the finished particles en­sures accurate correlation between structural properties of imaged materials and prior in vitro and in vivo studies, allowing collection and interpretation of meaningful images.

Me­tallic cores generally range from 4 to 20 nm in diameter, while coated particles can be up to 100 nm or more in diameter. Compared to larger cores, however, nanoparticles less than 20 nm in diameter exhibit considerably lon­ger blood half-life and improved T1 and T2 relaxivity effects.

Clearance of nano­particles via the RES is a critical problem usu­ally approached by surface modifications to mitigate nonspecific adsorption of proteins to the biomaterial surface (i.e., the opsonization of synthetic materials). Neutral, hydrophilic surfaces tend to adsorb less serum protein than hydrophobic or charged surfaces.

Bisphosphonate and phosphorylcholine derived thin film coatings have been applied to nanoparticles to stabilize iron oxide particles against pH, opsonization, and aggregation. Such thin films do not fully eliminate protein adsorption, and dense layers or thick brushes of polysaccharides or hydrophilic polymers may more effectively avoid opsonization. Other, as yet incompletely understood, bio­logical factors influence the use of nanoparti­cles in vivo. For instance, a direct correlation between the circulating half-life of nanoparti­cles (i.e., their T1/2s) and age of animals used has been reported.

The observed increase in T1/2 may be correlated to age-related chang­es in phagocytic activity. Local environments also influence particle stability: iron oxide particles are degraded at a pH of 4.5 or less, a condition sometimes attained in some in­tracellular vesicles.

First generation contrast agents were primarily blood-pool agents that moved freely through the entire vasculature. Targeting contrast-enhancing nanoparticles to sites of interest can reduce heavy metal toxicity associated with the commonly used agents by diminishing the dose required to obtain an acceptable image.

Contrast agent targeting can also provide enhanced diagnos­tic information. For instance, nanoparticles that bind to molecular fibrin at a clot site on a vessel wall have been developed, potentially allowing differentiation between vulnerable and stable atherosclerotic plaques. Similarly prognostically valuable data regarding dis­ruptions of the blood-brain barrier (BBB) have been visualized with MRI as the result of delivery of contrast-enhancing nanoparticles to the affected site.

Applications for Nanotherapeutic Devices:

Nano therapeutic de­vices are novel, emerging therapeutics with properties not fully understood or predict­able. Nano therapeutics, therefore, must be justifiable on at least two levels. As we have seen, the nature of the therapeutic task and the state of current Nano scale-materials tech­nology make the incorporation of biological macromolecules unavoidable for much Nano therapeutics.

Proteins, for example, typically are substantially more expensive than small molecule therapeutics, and precise nanostruc­tures containing proteins will be more costly still. Nano therapeutics must justify their high COGs.

Secondly, as new therapeutic modali­ties, Nano therapeutics may carry significantly larger risks than those associated with more conventional therapies. Expensive, novel moi­eties—such as Nano biotechnological thera­peutic devices— are, therefore, most likely to be accepted for treatment of conditions that not only are accessible to intervention at the Nano scale but also for which existing thera­peutic modalities have acknowledged short­comings in patient morbidity or mortality.

We have selected two disease states sufficient­ly grave and sufficiently un-served to warrant Nano therapy: cancer and cardiovascular dis­ease. Modulation of immune responses and vaccination is our third application area.

Nanotherapeutic Devices in Oncology:

The economic burden imposed by cancer is immense, measuring in the billions of dollars annually. Existing therapies such as surgical resection, radiotherapy, and chemotherapy have profoundly limited efficacy and fre­quently provide unfavorable outcomes as the result of catastrophic therapeutic side effects. Additionally, the biology, chemistry, and physics of cancer, in general, and solid tumors in particular, provide therapeutic avenues ac­cessible only by Nano scale therapeutics. Oncology is thus an ideal arena for emerging Nano technological therapies.

Tumor Architecture and Properties:

Tu­mors as tissues are relatively chaotic structures exhibiting vast structural heterogeneity as a function of both time and space. In healthy tissues, vasculature resembles a regular mesh in which the mean distance of tissue spaces to the nearest vessel is tightly controlled and highly uniform.

On the other hand, the vas­culature of tumors resembles a percolation network containing regions experiencing vastly different levels of perfusion. Tumors of 1 mm³ or larger typically contain measurably hypoxic domains, with pO₂ values as much as two- to three- fold lower than in normal tis­sue.

High levels of hypoxia are characteristic of enhanced metastatic potential and tumor progression. Also due to insufficiency of per­fusion, tumors frequently contain necrotic domains. The average tortuosity of vascu­lar flow paths in tumors is also much greater than in healthy tissue, and transient throm­botic events lead to enhanced resistance to flow and on-going vascular remodeling.

Aside from its plasticity, tumor vasculature itself is highly irregular, may be incompletely lined with endothelial cells, and often exhibits significantly higher extravasation limits (the highest molecular weight of materials that can leave the vasculature and diffuse into the interstitial spaces) than normal vascula­ture. Tumor tissue is also poorly drained by lymphatic’s, so extravagated materials tend to remain in situ in tumors and are not cleared efficiently.

These biological phenomena all differenti­ate tumor tissue from normal tissue and can be exploited in therapy. For instance, the spe­cial vascular integrity and lymphatic drainage properties of tumors constitute the Enhanced Permeability and Retention effect (EPR). EPR presents an obvious opportunity for interven­tion with Nano scale therapeutics.

Extravasa­tion limits for normal tissues are variable, but structures larger than a few nanometers in diameter do not leave circulation efficiently in most tissues, whereas tumor vasculature frequently allows agrees of materials in the tens to hundreds of nanometer range.

Fur­ther, Nano-materials, once extravagated, are not cleared by lymphatic drainage. EPR pro­vides tumor targeting that does not depend on biological affinity reagents: tumor tis­sue provides preferential depot sites for extravagated drug delivery devices. Targeting to a desired site of action is highly desirable for cytotoxic therapeutics, and EPR provides the basis for a growing class of polymer thera­peutics. That said, EPR and targeting by bio­logical affinity are not necessarily mutually exclusive.

A number of antigens more or less specifically related to tumors are known (tu­mor associated antigens or TAAs), and as dis­cussed above tumor/organ-specific vascular addresses might be exploited for delivery of therapeutics. For instance, various biological reagents that recognize TAAs (i.e., antibody to carcinoembryonic antigen, transferrin) have been conjugated to Nano scale contrast agents to enhance contrast agent localization to tumoral sites.

Cardiovascular Applications of Nanotherapeutics:

The cardiovascular system is one of the most dynamic and vital systems of the body. In principle, every cell in the body is accessible to the bloodstream via the vasculature. If controlled navigation of, and extravasation from, the vasculature can be accomplished, there­fore, the potential of Nano therapeutic devic­es is virtually unlimited.

But as mentioned throughout this article, opsonization and immune clearance, as well as targeting and triggering of therapeutics, remain key issues to address in order to facilitate wide application of Nano therapeutic devices to cardio­vascular disease. In this section we provide a brief overview of categories and examples of Nano therapeutic devices used in blood-contacting applications.

Cardiovascular Tissue Engineering:

A us­able, immunologically compatible artery for coronary bypass is not always available, so synthetic or semi-synthetic artery substitutes are highly valuable. Tissue engineering ap­proaches might mitigate the lack of acceptable homologous artery, as long as an acceptable artery substitute can be produced.

Current tissue engineering approaches involve syn­thesis of three dimensional, porous scaffolds that allow adhesion, growth, and prolifera­tion of seeded cells to generate a functional vessel. Cellular organization and growth in synthetic scaffolds are often chaotic and ran­dom, and sufficient blood-supply is seldom achieved within the scaffold. These condi­tions often result in death of the seeded cells and compromise the integrity of the vascular prosthesis.

Micro fabrication techniques have allowed miniaturization of the tissue engineering scaf­folds. These techniques allow the molecular- level control of cellular adhesion and propagation by control of surface topography, surface chemistry, and arrangement of morph genic and proliferative signaling ligands and cy­tokines.

Micro fabrication schemes are being developed for the elucidation of parameters necessary for cellular attachment and orien­tation on well-defined silicon and polymer-based surfaces. One particularly powerful application of controlled cellular growth is for the development of artificial capillaries to remediate vascular disease.

Micro-machined silicon or polymer channels of the dimen­sions of capillaries have shown promise as effective conduits to direct endothelial cells to form tubes through which a fluid (blood) could eventually flow. MEMS technology and Nano scale control of molecular events and interactions has also been applied to the de­velopment of cardiovascular sensors. For instance, blood cell counters have successfully detected red and white blood cells and show promise for quick, reliable blood cell analy­sis.

Additionally, force transducers have been developed to measure the contractile force of a single cardiac myocyte. Basic research into cellular function and response to stimuli such as these will eventually support more efficacious and robust cardiovascular tissue engineering approaches.

Nanoparticulate Carriers for Therapy and Imaging:

Two major cardiovascular applica­tions of nanoparticles are targeted contrast agents for imaging (MRI, CT, X-ray, etc.) and targeted drug delivery vehicles to address vascular lesions (atherosclerotic plaques) and other vascular disease states.

In either appli­cation, nanoparticle surfaces must be appro­priately modified to avoid RES clearance and to drive interaction with the desired effector cells. Decades of research in the area of molec­ular and cell biology have provided research­ers with many tools to make the appropriate protein component selection for Nano therapeutic contrast agents. For instance, knowing that the pro-coagulant protein tissue factor is expressed only at the surface of injured en­dothelial cells provides a means to differen­tiate healthy from diseased vasculature.

It also provides a method to target disease sites. Similar sorts of information regarding disease and normal vasculature at specific tissue sites can be critically important for targeting specialized vascular beds, such as that which occurs at the BBB. Several groups have begun efforts to develop nanoparticles that selec­tively pass across the BBB to deliver chemo-therapeutic or other drugs to the brain, and this may improve outcomes from stroke.

With regard to MRI approaches, particles used for targeted contrast enhancement in specific tissues or vascular beds may be quite powerful for detection of unstable athero­sclerotic plaques and quantitative analysis of blood flow. For instance, nonspecific accumulation of nanoparticles within human atherosclerotic plaques has been reported and may ultimately provide a way to distin­guish vulnerable and stable plaques.

Such data might be used to direct therapy, with the intent, as always, to improve therapeutic out­come. Furthermore, Nano therapeutic devices have been designed to include both contrast and drug delivery functions. Single-platform, multifunctional particles for cardiovascular therapy represent a cutting edge in the car­diovascular field. These delivery vehicles are being designed and improved continual for longer circulation time, more efficient target­ing, degradability, and biocompatibility.

Nanotherapeutics and Specific Host Immune Response:

Immune responses directed to immuno-gens (molecules or structures capable of eliciting specific immune responses, also called anti­gens) are a pervasive aspect of the responses of vertebrates to exposure to foreign mac­romolecules, proteins, particles, and organ­isms.

Undesired antibody response; in par­ticular, is the single most important hurdle for clinical use of recombinant proteins. On the other hand, undesired cellular immune responses are key mediators of frequently devastating disorders for patients, whether the responses are directed to self (as in au­toimmune disease) or to foreign antigens (as in transplant rejection).

As we shall see, host immune responses to Nano therapeutic devic­es are a critical determinant of their efficacy. Any strategy (including the biotechnological strategies) that allows controlled manipula­tion of immune responses either to augment desirable responses, as in vaccines, or to miti­gate deleterious responses holds substantial potential benefit.

Basic Immunology:

Specific immune re­sponses are directed to individual macro­molecules or assemblages of them and can be categorized into cellular responses, mediated by cytotoxic T-lymphocytes (CTLs), and hu­meral responses, mediated by soluble proteins (found in blood serum and other biological fluids) secreted by B-cells (antibodies). Re­gardless of the effector (CTLs or antibodies), specific responses are directed to individual molecular features of antigens called epitopes.

The Three Types of Epitopes are:

Cytotoxic T- cell (CTL) epitopes, Helper T-cell epitopes, and B-cell epitopes.The first two types are peptides that must be presented on cell sur­faces in the context of the class I (in the case of CTL epitopes for CTL responses) or class II (in the case of T-helper epitopes, involved in both antibody and CTL responses) major his­tocompatibility complex (MHC) molecules.

B-cell epitopes are a chemically and structur­ally heterogeneous group of epitopes and include synthetic molecules as well as peptides. In addition to CTL and B-cell epitopes, which are recognized by immune effectors, T-helper epitope are recognized by regulatory compo­nents of the immune system (helper T-cells). Vibrant CTL and antibody responses require immuno-gens containing T-helper epitopes along with either B-cell or CTL epitopes.

While epitope-presenting MHC class I and class II complexes must be present on cell surfaces to trigger immune responses, the ma­chinery for antigen, processing and loading of MHC class I or class II proteins for antigen presentation reside in distinct membrane- bound compartments in the cell. For instance, processing for class I presentation (to trigger CTL responses) occurs through the cytosol, but charging of MHC class 1 complexes is accomplished in the endoplasmic reticulum.

On the other hand, processing for class II presentation (to augment antibody and CTL responses), as well as the loading of class II, occur in endosomal compartments. Although these are membrane bounded compartments in the cytosol, topographically the interiors of endosomal compartments are part of the extracellular environment and are not inside the cell at all.

In the case of both class I and class II, the antigen presenting complexes are moved to the cell surface after charging. The moving of molecules between different cel­lular locations (called trafficking) is tightly regulated by and driven by specific cellular proteins, systems, and structures.

Proper traf­ficking is essential to assure that all cellular components assume their correct position in the cellular organization, which in this case, is to assure antigens are directed to the com­partments that will (most often) produce most protective immune responses. These complex antigen processing and presentation systems can be perturbed and exploited to offer novel opportunities in vaccine design.

Nanotherapeutic Vaccines: Eliciting De­sired Host Immune Responses:

Both thera­peutic and prophylactic vaccines are naturals for Nano biological approaches. Therapeutics that modulate immune responses (antibody, CTL responses, or both); vaccines typical­ly produce desired immunity to disease or pathogens. Ideally they are chemically well defined, safe, and induce long term (prefera­bly, multiyear) protective immune responses. Recent developments in immune biology have opened opportunities for Nano biological vac­cine design.

Particulate Vaccines:

The immune system, through dendritic cells (DCs), other antigen presenting cells (APCs), and immune effec­tors, performs on-going surveillance for non- self-antigens using the MHC antigen presen­tation system described above.

It has long been known that APCs sample serum for antigens that they process and present using the MHC class II pathway for antibody pro­duction. Research of this country has shown that DCs, macrophages, and other APCs also take up particulate materials and process and present the constituent peptide epitopes via the MHC class I pathway to trigger specific CTL responses.

Previous dogma held that only endogenous antigens (such as those produced in virally infected cells) could be presented through the class I MHC pathway. Both soluble and particulate antigens can be presented via the class I pathway, but particu­late antigens are orders of magnitude more potent than the same immuno-gens formulat­ed in soluble form.

APCs are not particularly fastidious regarding chemical composition of the particles they take up: latex beads as well as iron beads have been used. Self-assembled nanoparticles made of lapidated epitope pep­tides also deliver orders of magnitude of bet­ter immunization than do soluble formula­tions of the same peptides.

The specific size for optimal CTL responses is not fully defined but clearly is in the nanometer to micrometer size range. One could imagine a systematic exploration of optimum size and composi­tional preferences for uptake by APCs using such chemically and morphologically tunable polymeric nanomaterial’s as dendrimers and tectodendrimers.

Nanobiological Design of Nano Therapeutic Vaccines:

Antigen traf­ficking determines which specific MHC com­plex (class I or class II) epitopes are presented on and, therefore, which immune effectors are produced in the subsequent response. The process, while tightly regulated, is amenable to intervention using Nano therapeutic Vac­cines.

Nano biological design strategies can take advantage of the numerous proteins and peptides now known to mediate the traffick­ing of carried materials to specific sites. In the device context, these trafficking moieties might assure delivery of antigens to the ap­propriate cellular compartments to trigger desired class I or class II mediated responses.

Vaccine polypeptides, for instance, might be fused or formulated with pathogen poly­peptides that will deliver them across epithe­lium from outside the host for use in mucosal vaccination. Still other peptides are known to carry injected materials across the cell envelope into the cytosol.

Alternatively, vaccine entities taken up by receptor-mediated endocytosis (into endosomal vasicles, the tra­ditional site of uptake for antigens destined for MHC class II presentation) could be shut­tled into the cytosol (and hence to the class I MHC pathway) by the incorporation of pep­tide domains of bacterial toxins that mediate endosomal escape to cytoplasm.

Potentially, Nano biological therapeutic design strategies can combine knowledge of polymer chemis­try, Nano-materials, chemistry, cell, molecular, and immuno-biology to generate highly effec­tive Nano therapeutic vaccines.

Nanotechnology and Modulation of Im­mune Responses:

On the other hand, protein immunogenicity represents a critical limita­tion to the therapeutic use of recombinant proteins, particularly when specific antibody responses to the therapeutics are engendered. The consequences of antibody responses to proteins are variable, ranging from no effect to decidedly negative consequences to the host.

Antibodies can neutralize therapeutic bioactivity, rendering drug entities inactive in re­peated rounds of therapy, distort drug Pk and Bd properties, and, in the worst case, trigger autoimmune responses with cross-reactive host antigens (host antigens that share some identity with the immunizing epitopes).

Antibody Responses to Nanotherapeutic Devices:

Antibody responses to protein components are a major challenge for Nano biological therapeutic devices in vivo. Pro­tein components of bio conjugates to diverse Nano-materials can not only retain their in­trinsic immunogenicity but, worse, can ren­der the otherwise non-immunogenic syn­thetic Nano-materials to which they are linked capable of inducing antibody responses.

The mechanism of immunogenicity seems to be haptenization, a well-known phenomenon in immunology. Haptenization occurs when antigens containing only 3-cell epitopes are covalently linked to proteins containing T- helper epitopes, thereby creating hybrid an­tigens that are fully immunogenic with re­spect to antibody responses. As a result of the chemically repetitive structure of many syn­thetic Nano-materials, the antibodies directed to them have interesting properties.

Most notably, antibodies generated to nanostruc­tures are often cross-reactive to other chemi­cally similar nanostructures. For example, antibodies raised using haptenized fullerenes also recognize higher generation PAMAM dendrimers (G1, G2, G3, etc.).

Presumably, cross-reactivity results from the presence of common B-cell epitopes in the immuno-gen used and in structurally similar cross-reactive antigens. Thus, patients immunized with any therapeutic containing one haptenized Nano- structure could potentially raise antibodies that would recognize carbon nanotubes, and antibodies raised to haptenized generation 0 (G0) PAMAM dendrimers also recognize any Nano therapeutic containing a material struc­turally similar to the haptenized nanomaterial. This is a potentially serious problem, in light of the documented ability of antibody responses to interfere with therapy by neu­tralizing drug or distorting Pk and Bd proper­ties.

Molecular weights of serum antibodies are around 150,000 AMU with an effective diameter in excess of 10 nm. Many of the desirable properties of Nano therapeutics derive from their precisely controlled sizes, and size-dependent proper­ties like EPR targeting could be jeopardized if the therapeutic to be delivered were Nano-covalently complexed with antibodies directed to its synthetic components.

Due to long-term persistence of memory immune responses, exposure to haptenized nano therapeutics could endanger patient responsiveness to structurally similar materials for the duration of their lives. Obviously, immunogenicity is a critical challenge to the Nano biological thera­peutic paradigm that has yet to be satisfacto­rily addressed.

Polymer Conjugation to Mitigate Immune Responses:

There are multiple approaches to mitigating immunogenicity, some of which are applicable to Nano biotechnological therapeutic devices. Polymer conjugation, particularly conjugation of proteins to poly (ethylene glycol) (PEG, a flexible, biologically well tolerated hydrophilic polymer) has been used to control immunogenicity of a broad class of proteins and synthetic molecules since the mid-1970s.

Typically, pegylation involves covalent linkage (often by promiscu­ous conjugation chemistry) of variably num­bers (usually two to six polymer molecules) of size poly-disperse (usually 3,000 to 10,000 AMU) linear or branched PEG chains to the potential immunogenic. This approach results in poly-disperse bio conjugates, which flies in the face of the molecular level-structural precision often considered essential for nano­technology. Still, pegylation may be useful in some limited Nano biological applications.

The mechanism of molecule-specific im­mune-suppression remains obscure, despite research since early 1980s. The flexibility of the PEG strands allows them to wrap around the molecules they are conjugated to, perhaps delimiting access of immune effectors and immune processing machinery to the poten­tial immuno-gens.

Pegylated molecules exhib­it dramatically enhanced apparent diameters and vastly enhanced solubility in aqueous buffer. Both of these phenomena result in substantially diminished clearance of pegy­lated molecules by the RES and may account for the retention of in vivo bioactivity despite the apparent steric hindrance that may result from wrapping PEG moieties around their protein bio conjugate partners.

Pegylation-related, bio-conjugate specific immunosuppres­sive phenomena cannot be fully accounted for by sterics, though Immunological experi­ments in syngeneic mice show that the protein-specific immunosuppression associated with pegylated proteins is transmissible from one animal to another by surgically harvested splenocytes. This finding is more consistent with a mechanism of suppression involving immunologically induced tolerance of for­eign antigens as opposed to one caused by simple steric considerations.

Synthetic Nanoporous Membranes for Immunoisolation:

A second approach to avoiding immune responses to Nano devices or bio components is immunoisolation by encapsulation. In this strategy, potentially immunogenic structures (Nano devices, cells, macromolecules) are sequestered from high molecular weight, large host immune ef­fectors, and immuno surveillance, while small molecules, such as glucose and small proteins, diffuse freely.

While conceptually simple, immuno isolation is technically chal­lenging in that the number of pores must be high to maximize flux, and the pores must be highly uniform in dimension. Because of the exquisite sensitivity of the immune system, immuno isolation must be nearly perfect if it is to work at all.

Particularly in the case of im­muno isolation of cells for xenografts, if only a tiny fraction (as few as 1 %) of the pores of an immune isolation capsule are large enough to admit immune effectors (for instance, an­tibodies and complement components, ex­clusion of which requires pores of 50 nm or less), or the viability of the graft will be com­promised. Thus, the strategy requires fabri­cation of membranes with pores that have very tightly controlled dispersities in terms of their maximum diameters. Until recently, immunoisolation for xenografts has failed for lack of such membranes.

The technological innovations that made the synthesis of these high-quality Nano-po­rous membranes were described above. The Nano-porous membranes can be used to form immunoisolation capsules when either two such membranes are joined so that they bind an enclosed space or that a Nano-porous mem­brane is used to enclose a compartment of some sort in a therapeutic structure.

The en­closed spaces can be charges with functional Nano devices or, for that matter, living cells. Such immunoisolation capsules charged with heterologous pancreatic islet cells have dem­onstrated immense promise for regulating normoglycemia in diabetic animal models.

Biogenic, Nanoporous, Immune Isolation Membranes:

The novel micro fabrication strategy has made it possible to make these highly defined membranes in an industrial process, and they are applicable to numerous biomedical applications beyond immuno iso­lation. Still the fabrication process is rigorous, and an alternative biogenic method to derive selectively porous silicon capsules has been pro­posed. Diatoms are encapsulated in biogenic amorphous silicon shells (called frustules), which the living cells secrete.

Frustules must necessarily be porous to allow the cells to take on nutrients and excrete wastes, and the po­rosity, pore dimensions, and other micro and Nano scale morphological frustule features are tightly controlled at genetic and physi­ological levels. Using techniques well known to microbiologists (manipulation of culture conditions), geneticists (mutagenesis), and cell biologists (optically driven cell sorting in flow systems), it is theoretically possible to select and propagate diatoms whose frustule morphology, size, and pore characteristics fall within pre-selected limits.

Frustules can be isolated intact from diatom cultures, so bio­genic production of silicon membranes with porosity characteristics similar to the micro fabricated membranes discussed above may be feasible. Though they have yet to be real­ized, immuno isolation capsules made from genetically/physiologically tuned frustules have been proposed: one wonders what other bio mineralized structures eventually might also be put to Nano biotechnological use.

Barriers to Practice and Prospects:

The ef­fort to produce Nano scale therapeutic devices is clearly highly interdisciplinary. As we have seen, it touches on numerous established dis­ciplines, encompassing elements of physiol­ogy, biotechnology, bio conjugate chemistry, electrical engineering, and materials science, to name just a few of the fields involved. Ob­viously, this broad sweep of knowledge is dif­ficult for any one investigator to masterfully.

The breadth of the effort constitutes just one of the major barriers to entry in the field. Other challenges include the raw complex­ity of biology, the fashion in which biologists hold and distribute information, and cultural differences between engineers and biological scientists.

Complexity in Biology:

Biology is characterized by particularity: nu­ances of biological systems are often unique to the system at hand and highly idiosyncratic. This follows from the fact that biological systems are not purpose-built, as are designed devices, but rather arose as the consequence of evolutionary processes. Evolution is a high­ly chaotic business, with the outcome of any given evolutionary process highly sensitive to initial conditions (populations of organ­isms subjected to selection, other organisms in the environment, biology of all organisms involved, resource availability, other environ­mental factors, etc.)

Moreover, many of the variables affecting natural selection processes are non-static and change, as a function of time or space or both, while selection is exert­ed against a population of organisms. Condi­tions leading to one evolutionary adaptation or another are therefore seldom duplicated exactly, so that individual adaptive features are idiosyncratic, with elements relating not only to their biological functions but also to their evolutionary history.

Individual systems and adaptive features thereof can be almost baroquely complex because the structures themselves arose under unique conditions, under unique selective pressure, and from unique initial biological systems. The extent of the complexity of biological systems is ap­parent even in individual macromolecular constituents of biological systems and can be made clear by comparison of synthetic nano­structures to biological nanostructures with similar dimensional aspects.

SWCNT (single wall carbon nanotubes) have a minimal diameter of about 1.3 nm, and many proteins likewise have diameters of few nms. SWCNTs are regular and homogeneous polymeric structures composed entirely of carbon atoms.

Proteins, on the other hand, are not polymers of one repeating subunit, but rather are non-random copolymers of 20 chemically distinct amino acids (aas). The order of the aas drives specific folding events that produce three-dimensional structures that, in turn, present specific aas and their side chains at specific positions in space. This control of aa position in space drives protein activity: small changes in aa sequence can perturb structure and function profoundly.

Therefore, though they fall within the same broad size regime, proteins are structurally and functionally much more complex than SWCNTs. The extent of biological complexity can be glimpsed when one consider that indi­vidual living things are ordered aggregates of multiple macromolecules and supra-molecu­lar structures (potentially, billions and bil­lions of them), belonging to distinct chemical classes (lipids, proteins, nucleic acids, etc.), with each individual macromolecule being at least as complex in structure and function.

The nearly irreducible complexity of biologi­cal systems is a central fact of practice in the biological sciences and drives the way biolo­gists gather and disseminate information. It is the key informant of the culture of biologists.

Dissemination of Biological Information:

The product biologists generate, then, is information, rather than devices or structures. Additionally, as we have just seen, biology is a ferociously complex discipline. As a practi­cal matter, biological data itself is often much more rich (and often more ambiguous) than data from harder scientific endeavors.

The data can be so very rich that biologists must make choices as to what data is relevant to a given phenomenon and, therefore, what data they will publish. With absolutely no intent to conceal or mislead, biologists are often driven to publish only a small fraction of the data they gather.

Generally biologists to publish data they believe to be of broad scientific sig­nificance. Typically, the chosen information does not include data that might be critical to technology development, frequently mak­ing the biological literature an inadequate resource for engineers.

The omitted informa­tion thus becomes lore. That is to say, the in­formation is critical for the practice or devel­opment of a given technology but usually is not accessible to persons outside the field (as many Nano therapeutic device producers may be). This can be illustrated by recent experi­ences around phage display-mediated affinity maturation of four helical bundle proteins.

Four helical bundle proteins are loosely relat­ed small proteins consisting of four α-helices arrange in a specific configuration. They in­clude insulin, growth hormone, most cytok­ines, and various other molecules involved in cell-to-cell signaling. They have a wide range of therapeutically valuable properties that might be made even more desirable if their potencies could be enhanced. There is, thus, significant interest in variant four-helical bundle proteins with improved bioactivities.

Phage display is a method that can be used to sort protein variant libraries for variants with enhanced affinity for a target recep­tor. This is usually accomplished by iterative rounds of affinity selection on the receptor followed by propagation of selected phage. As rounds of selection and propagation precede, the mean affinity of the selected variants for the receptor increases (so-called affinity mat­uration).

Affinity maturation by phage display can be used to identify variant proteins from librar­ies that have enhanced biological activities, providing that affinity for receptor is limiting in the overall activity of the parent protein.

Phage display affinity maturation has been successfully used to select some enhanced activity variants of four helical bundle proteins but is not applicable to engineering increased activity for all four helical bundle proteins. The reason for the inapplicability of display methods to engineer high-activity human growth hormone (hGH) variants became ap­parent in a recent publication from Genentech.

In the case of hGH, the affinity of the parent molecule for receptor is in excess of that required to drive maximal biological responses. This fact was inferred after a multiyear effort (beginning in the late 1980s and culminating in 1999) to derive more active hGH proteins, involving literally thousands of hGH variants.

Many of the variants did indeed exhibit en­hanced affinity for the hGH binding protein (receptor), but none exhibited significantly enhanced biological activity. Each data point (i.e., the biological activity of each individual variant) essentially constituted a failure of a technology to provide a desired result and was therefore seen, not unreasonably, as negative (and therefore uninteresting) data by the investigators.

Consequently, the information was not broadly disseminated until a signifi­cant conclusion could be presented (i.e., that biological responses to hGH are not limited by ligand-receptor affinity). Thus, between the late 1980s and the 1999 publication, this information was known primarily and unam­biguously only to the investigators involved, even though it would have been critical to any technologist planning to affinity mature hGH by phage display for enhanced activity.