Smart bio-nanoparticle elements Number:7,393,924 from the United States Patent and Trademark Office (PTO) owispatent

Title: Smart bio-nanoparticle elements

Abstract: The invention in suitable embodiments is directed to isolated bio-nanoparticle elements and isolated bio-nanoparticle platforms employing such isolated bio-nanoparticle elements. In one aspect, the isolated bio-nanoparticle elements are formed with purified Clathrin and or with purified coatomer I/II self-assembling protein molecules.

Patent Number: 7,393,924 Issued on 07/01/2008 to Vitaliano,   et al.

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Inventors:	Vitaliano; Franco (Boston, MA), Vitaliano; Gordana (Boston, MA)
Appl. No.:	11/024,424
Filed:	December 30, 2004

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Related U.S. Patent Documents

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	Application Number	Filing Date	Patent Number	Issue Date
	60534354	Jan., 2004

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Current U.S. Class:	530/350
Current International Class:	C07K 14/00 (20060101)

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References Cited [Referenced By]

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U.S. Patent Documents

5427767	June 1995	Kresse et al.
6756039	June 2004	Yeates et al.
7033834	April 2006	Valerio et al.
7037520	May 2006	Smyth Templeton
7048949	May 2006	Sligar et al.
7060291	June 2006	Meers et al.
7063860	June 2006	Chancellor et al.
7101532	September 2006	Aikawa et al.
7101570	September 2006	Hope et al.
7108863	September 2006	Zalipsky et al.
7112330	September 2006	Buonamassa et al.
7112337	September 2006	Huang et al.
7268116	September 2007	Liang

Foreign Patent Documents

	WO 2004/001019		Dec., 2003		WO

Other References

Antonny et al., Self-assembly of minimal COPII cages, EMBO Rep. Apr. 2003; vol. 4, pp. 419-424. cited by examiner . Cheng, et al, Cryo-electron Tomography of Clathrin-coated Vesicles: Structural Implications for Coat Assembly, J. Mol. Biol. (2007) 365, 892-899. cited by other . DeNardo, et al, Effect of Molecular Size of Pegylated Peptide on the Pharmacokinetics and Tumor Targeting in Lymphoma-Bearing Mice, Clinical Cancer Research, vol. 9, 2003. cited by other . Fath, et al, Structure and Organization of Coat Proteins in the COPII Cage, Cell 129, 1325-1336, Jun. 29, 2007. cited by other . Fotin, et al, Molecular model for a complete clathrin lattice from electron cryomicroscopy, Nature , vol. 432, Dec. 2, 2004. cited by other . Fotin, et al, Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy, Journal of Structural Biology, 156 (2006) 453-4. cited by other . Guo and Linstedt, COPII-Golgi protein Interactions regulate COPII coat assembly and Golgi size, The Journal of Cell Biology, vol. 174, No. 1, Jul. 3, 2006 53-63. cited by other . Matsuoka, et al. Surface structure of the COPII-coated vesicle, PNAS Nov. 20, 2001 vol. 96 No. 24 13706-13709. cited by other . Sanford and Kumar, New proteins in a materials world, Current Opinion in Biotechnology 2005, 16:416-421. cited by other . Smith, et al. Clathrin coats at 21 .ANG. resolution: a cellular assembly designed to recycle multiple membrane receptors, EMBO Journal vol. 17 No. 17 pp. 4943-4953, 1998. cited by other . Yokoyama and Ryan, Highly cooperative control of endocytosis by clathrin, unpublished, Department of Biochemistry, Weill Medical College of Cornell University. cited by other . Crowther, et al, 1976, On the structure of coated vesicles. J. Mol. Biol., 103, 785-798. cited by other . R.A. Crowther and B. M. F. Pearse, Assembly and Packing of Clathrin into Coats, The Journal Of Cell Biology, vol. 91 Dec. 1981 790-797. cited by other.

Primary Examiner: Mondesi; Robert B.

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Claims

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In view of the foregoing, what is claimed is:

1. An isolated bio-nanoparticle element comprising a cage element defining a cavity, up to 100 nanometers in diameter, formed from a plurality of self-assembling, purified Clathrin protein molecules and or purified coatomerI/II protein molecules and one or more types of cargo elements located within the cavity, wherein at least one of the elements, under the guidance of one or more types of externally and or self-directed methods, executes one or more types of functions and or effects one or more ends, in vivo and or in vitro.

2. An isolated bio-nanoparticle element according to claim 1, comprising one or more active and or passive methods for capturing and or positioning one or more types of cargo elements within the cavity.

3. An isolated bio-nanoparticle element according to claim 1, comprising cage and one or more methods for internally and or externally to functionalizing cage and or one or all or a subset of cargo elements.

4. An isolated bio-nanoparticle element according to claim 1, wherein cage and or one or all or a subset of cargo elements respond to one or more types of internal and or external stimuli.

5. An isolated bio-nanoparticle element according to claim 1, wherein the one or more types of cargo elements, we a plurality of cargo elements.

6. An isolated bio-nanoparticle element according to claim 1, comprising one or more types of coatings on part or the entirety of cage and or one or all or a subset of cargo elements.

7. An isolated bio-nanoparticle element according to claim 1, wherein cage, and or one or all or a subset of cargo elements, are physically and or functionally incorporated in whole or in part in one or more types of elements.

8. An isolated bio-nanoparticle element according to claim 1, wherein cage, and or one or all or a subset of cargo elements are stable and or viable for an approximate and or specified period of time.

9. An isolated bio-nanoparticle element according to claim 1, wherein the self-assembling plurality of protein molecules is comprised of Clathrin molecule, in whole or in part.

10. An isolated bio-nanoparticle element according to claim 1, wherein the self-assembling plurality of protein molecules is comprised of a coatomer I/II molecules, in whole or in part.

11. An isolated bio-nanoparticle element according to claim 1, wherein the cage is substantially greater than one nanometer in diameter.

12. An isolated bio-nanoparticle element according to claim 1, wherein the cage is at least about 50 nanometers in diameter.

13. An isolated bio-nanoparticle element according to claim 1, wherein the cage is at least about 100 nanometers in diameter.

14. An isolated bio-nanoparticle element according to claim 1, wherein the elements comprise a platform comprised of a plurality of isolated bio-nanoparticle elements each having a cage defining a cavity formed from a plurality of self-assembling purified protein molecules, and one or more cargo elements of one or more types located within the cavity, in vitro and or in vivo.

15. An isolated bio-nanoparticle element according to claim 1, wherein cage is empty and includes no cargo elements.

16. An isolated bio-nanoparticle element according to claim 1, wherein cage produces ordered scaffolding, creating self-assembling multi-layer structures having one or more dimensions.

17. An isolated bio-nanoparticle element according to claim 1, wherein cage, and or one or all or a subset of cargo elements, form non-permeable, permeable, and or semi-permeable cavities.

18. An isolated bio-nanoparticle element according to claim 1, wherein cage has icosahedral geometry.

19. An isolated bio-nanoparticle element according to claim 1, wherein cage and or one or all or a subset of the cargo elements are comprised of recombinant and or synthetic biological elements, in whole or in part.

20. An isolated bio-nanoparticle element according to claim 1, wherein cage and or one or all or a subset of the cargo elements effect one or more self-modifying behaviors and actions of one or more types.

21. An isolated bio-nanoparticle element according to claim 1, wherein cage inhibits charge transfer between cage and its enclosed cargo and or prevents cage distortion.

22. An isolated bio-nanoparticle element according to claim 1, wherein more than one bio-nanoparticle element is physically and or functionally linked together.

23. An isolated bio-nanoparticle element according to claim 1, wherein the one or more types of externally and or self directed methods, executing one or more types of functions comprise a plurality of methods and or functions.

24. An isolated bio-nanoparticle element according to claim 7, wherein, the incorporated elements comprise a plurality of elements, in vitro and or in vivo.

25. An isolated bio-nanoparticle element according to claim 14, wherein the plurality of cargo elements of a subset of the bin-nanoparticle elements are a plurality of cargo elements.

26. An isolated bio-nanoparticle-element according to claim 14, wherein, a plurality of bio-nanoparticle elements are physically and or functionally incorporated in whole or in part in one or more types of elements, in vitro and or in vivo.

27. An isolated bio-nanoparticle element according to claim 26, wherein, the incorporated elements comprise a plurality of element.

28. An method for forming an isolated bin-nanoparticle element comprising forming a cage defining a cavity, up to 100 nanometers in diameter, formed from a plurality of self-assembling purified Clathrin protein molecules and or purified coatomerI/II protein molecules and one or more types of cargo elements located within the cavity, and, wherein at least one of the elements, under the guidance of one or more externally and or self-directed methods, executes one or more functions and or effects one or more ends in vivo and or in vitro.

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Description

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FIELD OF THE INVENTION

This application claims priority to Jan. 6, 2004, USPTO Application No. 60/534,354, with the provisional title, "Intelligent Bio-Structures". The invention relates generally to the field of nanoparticles, and more specifically, in one embodiment, to smart bio-nanoparticle elements formed from materials comprised of self-assembling protein molecules. In another embodiment, the invention relates to a multipurpose, multifunction nanoscale bio-nanoparticle platform, such as a drug discovery platform, electronics platform, information processing platform, telecommunication platform and the like, using such smart bio-nanoparticle (SBN) elements.

BACKGROUND OF THE INVENTION

Structures at the nanometer scale are sometimes referred to as nanoparticles. One example of nanoparticles is Fullerenes, which are the third allotropic form of carbon and form nanoparticles that may be an empty cage or the cage may contain cargo. The latter cage form is usually termed an endohedral Fullerene. Nanoscale endohedral Fullerenes can be used to stabilize reactive species inside the Fullerene cage, as in N@C60 or Sc2C2@C84. In addition, doped endohedral Fullerenes offer electronic and magnetic properties and might also be applied to electronics and information processing. Endohedral Fullerenes also have potential for biomedical applications such as targeted drug delivery, as well as other application areas.

In one application area, for example, the ability of endohedral Fullerenes to sequester one or more metal atoms, which may be toxic, inside the Fullerene cage has led to a research effort aimed at exploring their potential as contrast-enhancing agents for magnetic resonance imaging. Contrast agents enhance the quality of MRI images, aiding in the detection and diagnosis of injuries or abnormalities in the human body. The leading commercial MRI contrast agents are gadolinium (III) chelates such as gadolinium-diethylenetriaminepentaacetic acid, also known by its brand name Magnevist. Gadolinium III (Gd3+) works so well because of its unique electronic structure--it is the only ion with seven unpaired electrons. Once injected into the body, Gd3+ can magnetically "tickle" water protons present in tissues, accelerating their relaxation between radio-frequency pulses. Faster relaxation leads to higher signal intensity and therefore greater contrast in the MRI images. Encapsulating the gadolinium inside a Fullerene cage might prove safer, and such endohedrals potentially offer additional advantages. For example, the trimetallic-nitride-containing endohedral Fullerenes can accommodate three metal atoms inside each cage, potentially offering a more potent agent.

But before such endohedral Fullerenes can be tested and used in vivo, they must be made water-soluble. All endohedral Fullerenes exhibit extreme hydrophobicity, which must be overcome for many applications, especially for in vivo medical applications. One way to overcome this problem is to attach hydroxyl groups to the outer surface of the Fullerene cage. Compared with Magnevist, a commercially available contrast agent, prepared polyhydroxylated Gd@C82 can provide as much as 20 times better signal enhancement for water protons at much lower gadolinium concentration.

Although promising, there are problems with C82 endohedral Fullerenes. Most studies of metallofullerenes have centered on C82 isomers primarily because their solubility allows them to be more easily separated from empty Fullerenes and purified using high performance liquid chromatography (HPLC). But studies of polyhydroxylated Gd@C82 in rats revealed a significant, and potentially harmful uptake of the material by the reticular endothelial system, such as the lung, liver, and spleen. A similar uptake pattern of a polyhydroxylated derivative of Ho@C82 has been observed in these tissues, as well as in bone.

According to the Unites States Environmental Protection Agency (2003), which is funding research on nanoparticle toxicity, there is a serious lack of information about the human health and environmental implications of manufactured nanomaterials, e.g., nanoparticles, nanotubes, nanowires, Fullerene derivatives, and other nanoscale materials. Little is known about the fate, transport, and transformation of nanosized materials after they enter the environment. As the production of manufactured nanomaterials increases and as products containing manufactured nanomaterials are disposed of, these materials could have harmful effects as they move through the environment.

Forthcoming study results, including those funded by the EPA, may not be encouraging for pharmaceutical applications of Fullerenes like imaging contrast agents, because the metal-containing agent must be excreted without long-term retention in tissues. Unwanted organ and tissue retention also applies to targeted drug delivery systems using endohedral Fullerenes, which are about one nanometer in diameter. Organ and tissue retention issues also raise environmental concerns in general when in vitro endohedral Fullerenes or carbon nanotubes containing a metal or a toxic substance are free-floating in the air and are either inhaled into the lungs where they are absorbed into the body, and/or are absorbed into the body through contact with the skin.

Another drawback of C82 endohedral Fullerenes is that they are difficult to make in large quantities and with high purity, which is necessary for pharmaceutical applications and non-medical applications, like nanoscale electrical circuits. Generally, the success rate of creating endohedral Fullerenes is only about 1:10,000. This very poor success rates also leads to high costs. Endohedral Fullerenes cost as much as $1,000 per gram, in comparison to empty C60 cages, which cost only about $30 per gram.

Metallofullerenes in the C60 family, such as Gd@C60, have not been seriously considered for pharmaceutical applications because its members are generally insoluble and air-sensitive. On the plus side, M@C60 compounds can be produced in a carbon arc in yields up to 10 times higher than soluble M@C82 species. Water-soluble and fully air stable Fullerene fractions that are largely Gd@C60 have been experimentally produced. However, this material may contain several different isomers, rendering it unfit for many applications.

Besides using metal atoms, molecular clusters, and reactive species for medical imaging, chemists doing NMR spectroscopy have also encapsulated noble-gas atoms inside Fullerene cages and studied the interactions between the host and guest. In addition to Fullerene-caged helium, neon, argon, krypton, and xenon have also been put into Fullerenes, making unusual and highly stable noble-gas compounds in which no formal bond exists between the noble gas and the surrounding carbons. These compounds typically are made by heating the Fullerene in the presence of a suitable gas at 650.degree. C. and 3,000 atmospheres. Under these conditions, though, no more than one in 1,000 Fullerene cages ends up with a noble-gas atom inside, making large scale production infeasible, as well as very costly.

Aside from helium for NMR spectroscopy, xenon (129Xe) is the only other noble-gas isotope having a spin of one-half, which makes the nucleus easily observable using NMR spectroscopy. But all endohedral Fullerenes suffer from a severe cargo carrying limitation, as the hollow core of the endohedral Fullerene is only seven to eight angstroms in diameter. Therefore, when trying to force xenon into C60, you get three to five times less xenon inside than helium, because xenon is so much larger. Such a tight fit brings into play another negative aspect common to all endohedral Fullerenes: the cage is highly conductive. When charge transfer to the Fullerene cage occurs, it distorts.

For example, xenon's 5p electrons are much closer to and interact much more strongly with the Fullerene's p electrons. When you alter the cage environment in any way, such as by making a Fullerene adduct, the cage may pucker slightly and the dimensions may change. The modified cage or the new group on the outside will interact very strongly with the enclosed cargo in ways that aren't easy to describe or predict. This cage distortion is not specific to xenon, and may occur with any enclosed particles that interact with the Fullerene cage. Endohedral Fullerene charge transfer and subsequent cage distortion is unacceptable in commercial and medical applications because results will not be consistent and predictable, and may also be harmful and injurious in some circumstances, like in vivo applications. This cage distortion drawback also potentially entails significant legal and medical liability issues.

The ability of endohedral Fullerenes to encapsulate various types of cargo is also limited. Apart from noble gases, the encapsulated metal atom can only be an alkali metal, alkaline earth metal, Sc, Y, U, or a lanthanide metal, with the most unusual of these species being Sc3N@C80, which has a nitride nitride-bridged Sc3N cluster inside a Fullerene. Most of the other metals in the periodic table do not form endohedral metallofullerenes, but rather form insoluble metal carbides and other unextractable materials.

Along with their limited cargo carrying capacity; charge transfer to the cage; organ and tissue retention; extreme hydrophobicity; and their difficulty of manufacture and very high cost, their cargo type limitations further limit the commercial and scientific potential of endohedral Fullerene-based endohedrals, for example, in the fabrication of nanoscale electronic integrated circuits.

Nanoscale integrated circuits from endohedral Fullerenes ("NICE"), apparently resolves at least one of these problems, namely, fabrication. NICE is the only known methodology that has been shown to produce macroscopic amounts of metal-containing endohedral Fullerenes. NICE uses a unique resistless proximal probe-based nanolithography technique to produce thin films of Fullerenes containing metal atoms. The films are characterized by laser desorption mass spectrometry and optical spectroscopy (IR and UV-vis absorption) among other methods. It is possible to dissolve the material and separate the endohedral compound from the empty Fullerenes and other material in the films. In this way macroscopic amounts of purified endohedral Fullerenes can be prepared, which up to now have been Li@C60. Material production and yield optimization for C60 endohedrally doped with other alkalis (Na, K) and the lanthanide La will also be developed at some point with NICE. A further innovative aspect of NICE is the additive approach taken to nanofabrication that uses a shadow mask technique whereby complex patterns such as rings and intersecting lines are readily produced. With the NICE method, the material composition of the as-deposited line can be varied, allowing for the formation of junctions within a single layer.

But NICE does not address the issues of limited cargo carrying capacity of endohedral Fullerenes, which is limited to just one to three atoms. Nor does NICE overcome the complex issues of charge transfer and endohedral cage distortion, which depend on certain quantum parameters of molecules, such as: point set groups, energies of electron levels, dipole (multipole) moments, electron affinity, ionization potential, molecular orbitals, electron density, electrostatic-potential derived charges, bond orders, net atomic charges, free valences, total energy, energy of formation, singlet and triplet UV/Visible spectra, IR and Raman spectra, polarizabilities, hyperpolarizabilities, magnetic moments, NMR properties, geometry optimization, atoms in molecules properties, etc. Nor does NICE overcome the issues of tissue and organ retention of potentially toxic endohedral Fullerenes when used in vivo, or the potentially harmful results of environmental exposure to endohedral Fullerenes. Nor does NICE overcome the extreme hydrophobicity of endohedral Fullerenes. Finally, NICE, which fabricates endohedral Fullerenes carrying metal cargo, does not fabricate Fullerenes that carry noble gases, and also does not overcome the fundamental cargo material type limitations of endohedral Fullerenes.

Methodologies such as NICE typically involve a "top down" assembly approach, and employ some form of lithography and replication. Top down approaches can be time consuming, expensive and exacting, and wasteful of materials if not performed correctly.

Another type of nanostructure, also sometimes referred to as a nanoparticle, consists of liposomes (spherical vesicles) that have been used as an alternative to in vivo endohedral Fullerenes because of the unique advantages of liposomes, which include their ability to protect their in vivo cargo from degradation, their ability to target their cargo, which can be a drug to the site of action, and to reduce the toxicity of side effects.

Another type of in vivo nanoparticle is comprised of lipids that has a surfactant agent and a cosurfactant agent and may also contain therapeutic agents, and possibly a steric acid. These lipid-based cages are 40 to 150 nanometers in size. These nanoparticles may be used to deliver entrapped agents across various biological barriers, such as the transmucosal passage, and also to overcome the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (CSG). As a general aspect, certain classes of surfactants have been shown to be effective at crossing these biological barriers and for allowing passage into the brain and CSF of various kinds of coated vesicles and nanoparticles having entrapped agents.

Another example of an in vivo nanoparticle is a therapeutic agent delivery system comprising a capsid formed from a coat protein of a bacteriophage selected from the group consisting of MS-2, R17, fr, GA, Q.beta, and SP, and with a foreign moiety enclosed in the capsid. The foreign moiety cargo is of a size sufficiently small to be enclosed in the capsid and the foreign moiety is linked to a RNA sequence comprising a translational operator of the bacteriophage. The translational operator binds to the coat protein during formation of the capsid.

The foregoing in vivo nanoparticle delivery approaches, including others in the prior art show promise as biological encapsulation methods and have the potential for becoming effective therapeutic agent delivery systems, especially those systems using surfactants. However, all of the foregoing biological cages, just like endohedral Fullerenes and others in the prior art, also suffer from various limitations that are unique to their various material compositions. For example, developmental work on liposomes and lipid nanoparticles has been limited, due to their inherent problems such as low encapsulation efficiency, rapid leakage of water-soluble drugs in the presence of blood components, and poor storage stability.

Furthermore, studies have concluded that the use of liposomes is an inefficient method of gene transfer for gene therapy, which incorporates functional genes into the cell to replace the action of dysfunctional genes. Inside of the body, liposomes fuse with cell membranes and deliver DNA to the cell via diffusion. However, studies have concluded that the use of liposomes is an inefficient method of gene transfer. Not only is plasmid size limited but gene loading is also poor: only one in every 100-10,000 liposomes contain intact DNA. In addition, many liposomes are taken into the cell by endocytosis, instead of releasing their DNA by diffusion. This leads to excessive breakdown of the DNA and results in poor transfection efficiency.

For many of these reasons, drug delivery systems using nanoparticles comprised of biodegradable polymers are emerging as one of the most widely used systems because of their numerous strengths, such as ease of fabrication, well-understood materials, and the ability to attach targeting moieties and barrier-passing surfactants.

Polymeric delivery systems were first used to provide the controlled release of many common drugs. Early polymers used were non-biodegradable and had to be surgically removed once its drug had been released. To avoid this inconvenience, researchers began searching for biodegradable alternatives. Successful controlled release systems have increased patient compliance in the administration of malaria drugs and several types of contraceptives in developing countries where patients have limited access to their physicians. In addition, such systems have also made improvements in the veterinary field, simplifying drug administration to animals. Controlled drug release has many advantages, including the ability to supply more constant drug levels, enable more efficient utilization of the drug, and the ability to locally deliver the agent and confine it to that area. In addition, decreased costs and frequency of administration add to the attractive features of biodegradable drug delivery systems.

Biodegradable polymers are generally divided into two categories: surface eroding polymers and bulk-eroding polymers. In surface-eroding polymers, erosion is confined to the polymer surface. In bulk-eroding polymers, erosion occurs throughout the entire cross-section of the polymer. However, the wide majority of polymers erode by a combination of both mechanisms. Degradation leads to erosion and is achieved by polymer chain scission, usually by hydrolysis.

For example, one biodegradable polymer nanoparticle approach uses sub-150 nm nanoparticles capable of transporting and releasing therapeutic agents, such as nucleic acids. DNA release in gene therapy applications is initially controlled by surface erosion followed by bulk erosion: as the backbone bonds hydrolize, channels form in the polymer allowing water to reach the interior of the nanoparticle. As water penetrates, bulk erosion occurs and the DNA is released. In one example, a biodegradable polymer nanosphere surface has attached to it a targeting moiety. In another nanoparticle embodiment, a biodegradable polymer nanosphere surface has attached to it a masking moiety. In yet another embodiment both targeting and masking moieties are attached to a nanosphere surface. In another biodegradable polymer example, surfactants have also been applied. For example, one nanoparticle mechanism uses biodegradable polybutylcyanoacrylate nanoparticles overcoated with polysorbate 80 for the purposes of crossing the BBB and CSF and delivering therapeutic agents.

The advantages and benefits of using biodegradable polymer nanospheres, including those that use surfactants and targeting moieties, are significant for in vivo targeted drug delivery. But they also create new classes of problems, and also do not overcome some existing ones, some of which issues are enumerated herein:

First, therapeutic agent models using in vivo biodegradable polymer nanospheres, liposomes, lipids, and caspid delivery nanoparticles, as well as endohedral Fullerenes, and others in the prior art still consist of an in vitro model being applied to an in vivo system, and clinical drug trials may show that promising in vitro results do not positively transfer to in vivo environment, especially in humans. This results in significant lost opportunity costs, as well as wastes large amounts of time, resources, and capital.

Second, side effect profiles are not satisfactorily addressed by the foregoing in vivo targeted delivery systems and in the prior art, and side effects may in fact be exacerbated because a highly potent concentration of a therapeutic agent will be delivered to highly targeted areas of interest. One possible consequence of in vivo targeted delivery systems is that dosing regimens, especially off-label use, may have to be significantly recalibrated by health care givers, necessitating new training and learning.

Third, the ability to cross various biological barriers into the brain and CSF, for example, using surfactants, and to deliver in vivo targeted concentrations of both small and large molecule payloads past these barriers will raise a host of new issues concerning agent efficacy, dosing and side effect profiles. As a consequence, individual patient factors such as genotype, phenotype, age, gender, ethnicity etc., may come into play more than ever, and these factors are not addressed by delivery systems in the prior art. Furthermore, once biological barriers to the brain and the CSF are commonly breached--especially by large molecule payloads that heretofore were not possible to typically deliver--new short and or long-term biological effects may also come into play and create important biological changes at the inter-cellular and intra-cellular level. Therefore, new, highly targeted drug regimens will need to be closely monitored and controlled after agent delivery for maximum efficacy and patient safety, and such monitoring and critical adjustments will need to be done on the fly and in vivo if they are to be maximally effective. However, all the foregoing in vivo delivery systems and others in the prior art lack such an in vivo ability to intelligently monitor, control, react, and dynamically adjust cellular processes after delivery of their agent payload to a target, as well as fail to take into account unique, individual patient factors.

Fourth, the materials comprising the foregoing delivery systems and others in the prior art are "dumb" materials. Although they may necessarily follow the control laws that regulate in vivo biochemical reactions and physiological processes, current in vivo delivery systems and others in the prior art do not feature or are not comprised of materials having the innate ability or characteristics to utilize and or leverage these control laws to intelligently respond to changing in vivo conditions. For example, the materials of the foregoing delivery systems and others in the prior art do not manifest an in vivo capability or the intelligence to dynamically alter a prescribed course of agent delivery in the face of an unexpected biological and or drug interaction, and in that sense, the regulatory control laws actually work against these dumb delivery systems. Once these dumb materials are set in motion, they cannot alter their course of behavior and are therefore highly static, fixed function systems.

Fifth, all the foregoing in vivo delivery systems and others in the prior art, with the possible exception of Fullerenes, lack structural persistence. Once the nanoparticles find their target and deliver their cargo, their job is finished and their various types of coatings rapidly disband, which means that the functionality of these various agent delivery systems and others in the prior art is severely time constrained. This temporal constraint represents a significant nanoparticle design limitation. Structural persistence for a period of time is a highly desirable quality for any nanoparticle or nanostructure as it permits the addition of temporal-based functionalities to the nanoparticles. For example, it may be highly advantageous for a nanoparticle to loiter for some period in an area after its initial agent delivery in order to monitor the situation and to potentially make a decision to deliver more agent cargo for improved agent efficacy.

Sixth, the ability to have multiple targeting moieties and one or more types of agents, while possible, is not currently practical with all of the foregoing systems, including others in the prior art. Multiple targets presume either on the fly smart target prioritization for a single cargo type or multiple cargo types that can be intelligently orchestrated and delivered in a dynamic environment--qualities that all of the foregoing delivery systems and others in the prior art currently lack.

Seventh, precise, highly ordered placement of cargo elements with minimal inter-cargo spacings is not possible with any of the foregoing in vivo agent delivery systems and others in the prior art, which are basically hollow nanospheres with no internal structural elements except for the cargo they may be carrying. Internal precision ordering of such agent cargo within the nanoparticle can, for example, enable the precise, intelligently controlled spatial and or temporal release of agents. Minimal inter-cargo spacings within the nanoparticle also afford the ability to tightly pack agents, especially mixed agents types--e.g., a diagnostic agent and a therapeutic agent--into the same nanoparticle with minimal interference between agents. Precision ordering and spacing within a nanoparticle is therefore in of itself an integral component of a targeted system, amplifying and extending the capabilities of agents carried within the nanoparticle.

Eighth, all current delivery systems and others in the prior art are limited to carrying cargo just within their cavities. Currently, they have no capabilities for building aggregated complexes of self-assembled structures that dynamically bind together one or more elements, some of which may be heterogeneous and external to the nanoparticle, into complex systems having one or more external elements, cavities and payload types. Current in vivo delivery systems and others in the prior art therefore do not make possible the assembly of sophisticated, complex nanostructures that fully exploit all the manifold possibilities of targeted agent delivery.

Ninth, there is no provision or capability for programming algorithmically-driven behaviors into the current targeted agent delivery systems and others in the prior art, with the possible exception of endohedral Fullerenes, and their capability for becoming smart, programmable and or self-directed systems to perform complex and sophisticated tasks in vivo is therefore severely limited, if not impossible.

Tenth, there is currently no provision or capability for integrating current agent delivery systems and others in the prior art, with the possible exception of endohedral Fullerenes, into other smart devices and mechanisms either in vivo or in vitro, either functionally or logically, including with other devices and operators at a distance (e.g., telemedicine), thereby limiting their overall therapeutic, diagnostic, therapeutic and system expansion capabilities.

Thus, there exists a need for an improved nano-structure element that overcomes the limitations of in vivo and in vitro endohedral Fullerenes, as well as overcomes the limitations of biodegradable polymer nanospheres, liposomes, lipid-formed systems, caspids and other agent delivery systems in the prior art for in vivo applications.

SUMMARY OF THE INVENTION

The invention, in one aspect, remedies the deficiencies of the prior art by providing a nanoscale smart bio-nanoparticle (SBN) element, which may be employed in a scalable, smart bio-nanoparticle platform. An SBN platform according to the invention may be used, for example, in biomedical, electronics, telecommunications, and information processing applications.

In one embodiment, the SBN element is formed from one or more nanoscale cargo elements contained within a self-assembling protein cage. In some configurations, cargo elements include one or more metals. In other embodiments, the cargo elements are exclusively non-metal cargo elements that may include gases, as well as other cargo elements like drugs, optics, polymers, etc. One advantage of the invention is that it inhibits charge transfer between the cage and its enclosed cargo and prevents cage distortion. Another advantage of inhibiting charge transfer is that it reduces limitations on the make up of enclosed cargo elements. According to one feature, the SBN element is formed using a "bottom-up" fabrication approach. According to such an approach, various self-assembling and self-directed approaches are employed. Using such an approach, the SBN platform can be formed from the ground up, one element at a time, for highly specific nano-scale tasks. Another advantage of the "bottom-up" fabrication approach is that it reduces the amount of superfluous material that surrounds each cargo element within the cavity, reducing the cargo element's exposure to contaminant background radiation and thereby improving the functional effectiveness of the cargo. A further advantage of the bottom-up self-assembly of the SBN element is that it enables the precise, highly ordered placement of cargo elements with minimal inter-cargo spacings, thus avoiding a significant drawback to the use of endohedral Fullerenes, and also of other prior art endohedral Fullerene approaches, such as precise ion implantation through masks, and manipulation of single atoms using probes. In addition, the invention can maintain its structural integrity at room temperature in vitro and vivo, which eliminates the need for elaborate structure stabilizing mechanisms, like cooling systems. In one embodiment, the cavity defined by the cage is larger than those described in the Fullerene art, so the invention can incorporate a larger variety and number of cargo elements. According to another feature, the proteins that form the cage can be bio-engineered using commercially available biotechnology tools and other tools and techniques known in the art to contain different cargo elements, which makes the invention more versatile and cost-effective than the existing Fullerene art. Unlike existing Fullerene systems where the cargo elements must be inserted into an existing structure, the invention, in one embodiment, provides individual protein molecules that self-assemble around the cargo elements to form the cage, which makes the addition of cargo elements easier.

Another advantage of the invention is that the SBN protein material does not exhibit extreme hydrophobicity. A further advantage of the invention is that it provides a protein structure that can be bio-engineered to prevent in vivo SBN and or cargo uptake by organs, tissue, and bone and the SBN and or its cargo are secreted quickly and easily. In the converse, another advantage is that the SBN protein material and or its cargo can be bio-engineered for highly selective uptake by targeted cells, tissue, organs, bone, as well as other organic and inorganic matter.

Unlike current nanoparticle in vivo agent delivery systems, and also of other prior art, the invention's biological model is consistent from in vitro to in vivo, making drug discovery safer, more efficacious, more time and cost effective, and overall, a much more rapid process. Further unlike existing nanoparticle in vivo agent delivery systems, and also of other prior art, the invention, in one embodiment, provides for individual patient factors such as genotype, phenotype, age, gender, ethnicity etc., to be taken into account and factored into dosing and administration consideration. Also unlike existing nanoparticle in vivo agent delivery systems, and of other prior art, the invention, in one embodiment, provides for dynamic, in vivo dosing regimens that significantly reduce drug side effect profiles. And further unlike existing nanoparticle in vivo agent delivery systems, and of other prior art, the invention, in one embodiment, provides for the ability to intelligently monitor, control, react, and further adjust biological processes after delivery of agent cargo payload, and to do so on the fly and in vivo. Further unlike existing nanoparticle in vivo agent delivery systems, and of other prior art, the invention, in one embodiment, provides a structure that maintains its structural integrity long enough to do useful work for a time certain period. Also unlike existing nanoparticle in vivo agent delivery systems, and of other prior art, the invention, in one embodiment, provides bottom-up self-assembly of the SBN element and the precise, highly ordered placement of cargo elements with minimal inter-cargo spacings. Further unlike existing nanoparticle in vivo agent delivery systems, and of other prior art, the invention, in one embodiment, provides for the ability to attack multiple in vivo targets on the fly using smart target prioritization for a single cargo type and or multiple cargo types that can be intelligently orchestrated and delivered in a dynamic in vivo environment. Also unlike existing nanoparticle in vivo agent delivery systems, and of other prior art, the invention, in one embodiment, provides for smart materials that may deliberately exploit and leverage regulatory control laws, and these smart materials may dynamically and interactively respond to changing in vivo environments using various self-directed behaviors. Also unlike existing nanoparticle in vivo agent delivery systems, and of other prior art, the invention, in one embodiment, provides for the ability for building aggregated, complex self-assembled structures that dynamically bind together one or more exogenous, heterogeneous elements into complex systems having one or more cavities and payload types. The invention therefore makes possible the assembly of smart, complex delivery vehicles that fully exploit all the possibilities of targeted agent delivery systems.

Further unlike existing nanoparticle in vivo agent delivery systems, and of other prior art, the invention, in one embodiment, provides for a capability for targeted agent delivery systems that deliberately leverage and utilize biological control laws and may act as smart, self-directed systems. Moreover, unlike existing nanoparticle in vivo agent delivery systems, and of other prior art, the invention, in one embodiment, provides a capability for its integration into other smart devices and mechanisms, some of which may be heterogeneous, either in vivo or in vitro, and either functionally or logically, including with other devices and operators at a distance, thereby significantly enhancing the overall capabilities of the invention.

In general, in one aspect, the invention features an SBN element that includes a cage defining a cavity in which one or more cargo elements are located. The cage is formed from a plurality of self-assembling protein molecules. In a further embodiment, at least one of the cages includes a cargo element.

In one embodiment, one or more SBN elements and or SBN-related elements and in any combination include synthetic materials that are organic and or inorganic in composition.

In various embodiments of the invention, the cage is substantially larger than one nanometer in diameter, including sizes that can exceed about 50 or even about 100 nanometers in diameter. According to one embodiment, the self-assembling cage is a functional substitute for C.sub.60, C.sub.80, C.sub.82, and other types of empty Fullerene cages, as well as a substitute for cargo carrying endohedral Fullerenes. Furthermore, empty cage Fullerenes, carbon nanotubes, cargo carrying endohedral Fullerenes and other nanoparticles such as liposomes, caspids, lipid-formed vesicles, polymer, polybutylcyanoacrylate, and cetyl alcohol nanoparticles, but not limited to such, may be carried as ordered cargo within the self-assembling protein cage.

The relatively large size of the cage and its structural components allows for a large and wide variety of possible cargo elements, especially in contrast to the limited cargo capacity of Fullerenes. The SBN and or cargo elements may also modify, process, manipulate, encode and decode, input, output, transmit, communicate, store and or read information using techniques known in the art, in vivo and in vitro.

Preferably, the cage has faceted geometry. In some embodiments the cage is symmetric with respect to a plane. In one embodiment, ordered cargo elements are linearly positioned at vertices along a single plane using circulant ordering. In one particular embodiment, the self-assembling protein molecules that make up the cage are clathrin molecules, which may be biologically engineered. In another embodiment, the self-assembling protein molecules that make up the cage are coatamer protein molecules, which also may be biologically engineered.

According to another feature, once inside the SBN, cargo is protected from the external environment, and the SBN is stable with respect to dissociation and any cargo toxicity is sequestered from the surrounding environment.

According to one feature, an SBN element and or cargo elements may use a reservoir of unassembled SBN materials and or cargo materials to re-supply, reassemble and regenerate defective and or destroyed and or inoperable SBN and or cargo elements.

In some configurations, the cage contains a single cargo element, while in other configurations it contains multiple cargo elements. In some cases, each of the cargo elements is or includes a metal. Alternatively, some of the cargo elements are or include non-metal elements.

According to one feature, the cargo elements may include one or more research, therapeutic, diagnostic, assay, or prosthetic agents. Such agents may be, for example, nano-structured and/or may include chemical, biological and/or metallic materials. The agents may be or include organic or inorganic materials or a combination thereof.

According to one SBN embodiment, a cargo element contains an ion with one or more unpaired electrons. According to one in vivo application for enhanced medical imaging, paramagnetic lanthanide or transition metal ion complexes are cargo elements that decrease the NMR relaxation times of nearby proton nuclei of H2O molecules, leading to brighter images and enhanced contrast between areas containing the contrast agent and the surrounding tissues.

According to another feature, one or more cargo elements may be or include nanoscale assay systems, diagnostic devices and agents, sensors, therapeutic devices and agents, and/or prostheses, in any combination. Some or all of the cargo elements may operate under the control and influence of other SBN elements, and altogether may comprise a scalable, nanoscale information-processing platform for SBN-based biomedicine.

According to another feature, the SBN uses its material properties and directed self-assembly to address all issues associated with nanotechnology information processing devices: Atomic and molecular scale device design, their interconnection, nanofabrication and circuit architectures.

In another configuration, the invention features the ability to intelligently respond to in vivo and or in vitro environmental conditions and manifest special functions. Protein molecules used by the invention can perform a physical form of bio-computation via their dynamic (energy-consuming) self-assembly actions. They compute, not by moving electrons around in transistor circuits, but by moving molecules around and adding or removing them from constantly modified nanostructures. Mere molecules can act in complex, coordinated fashion--they are executing a kind of physical "computer program". The invention may use this bio-mechanism to develop self-directed, self-assembly processes. The invention in one embodiment yields a new and novel way to build incredibly small structures that manifest "bio-intelligence." The seat of this intelligence within the invention materials lies in their deliberate utilization and taking advantage of biological control laws.

The various control laws that regulate biochemical reactions and physiological processes often display features that allow biomolecules or biological structures to perform more tasks than are reasonably expected from a simple mechanical device. A distinctive hallmark of the intramolecular dynamics of biomolecules is the concerted and interlocking steps of conformational changes that lead to a purposeful action: each part fits spatially and each step fits temporally (kinetically) with an element of anticipation of the purposeful outcome. These concerted and interlocking steps are sometimes referred to as closed entailment loops (Rosen). The overall process exhibits intentionality that is conducive to the suggestion of a master hand behind the design. Of course, the evolutionary mechanism, and now bio- and genetic engineering replace the need of a master hand. In one embodiment, the invention takes deliberate advantage of these biological control laws, and via the use bio- and genetic engineering methods known in the art makes use of these control laws to regulate complex in vivo and in vitro biochemical reactions and physiological processes. An example of biological control laws at work is the automatic self-directed, self-assembly in vitro and in vivo of Clathrin and coatamer proteins, which are both highly complex structures.

In the invention, the artificially-induced and or natural binding of an SBN element to a cell membrane, other tissue, and or an in vitro element such as, for example, but not limited to, a pathogen and the resulting biological processes and interactions may lead to a series of deliberately controlled, extended, modulated, purposefully, and or self-directed behaviors of bioengineered SBN materials that provide real time, self-adaptive, self-regulatory in vivo and or in vitro behaviors on the part of the SBN. The deliberately extended and or modified behaviors, functionalities, and characteristics of a bioengineered SBN constitute a smart bio-computer that is an analog to electronic processing systems directed by software-based algorithms.

In one SBN embodiment, intramolecular dynamics of biomolecules and the concerted and interlocking steps of conformational changes lead to deliberately purposeful actions. For example, each SBN element fits spatially and each step in a process fits temporally (kinetically) with an element of anticipation of the purposeful outcome. In one example embodiment, conformational changes and or non-covalent bonding of an SBN structural element to a cell membrane may lead to the precise dispatch and sequenced delivery of selected cargo agents from the SBN structure to the target cell. Alternatively, a new series of interlocking steps between a part of a cell membrane and all or a subset of the materials comprising an SBN structure may cause the cessation of agent delivery to the target cell. In another example case, the spatially and temporally defined control events between the cell and the bioengineered SBN materials may cause the SBN structure to release diagnostic and monitoring agents to determine the most appropriate course of therapeutic action. The calculated utilization of biological control laws by SBN materials may, for example, provide for a sophisticated drug delivery system that provides optimal dosing by altering its drug delivery behavior and also producing minimal side effect profiles.

In one SBN embodiment, graphs are used. By utilizing graphs and Lie algebras, including Clifford algebras, geometrically-derived algorithms produce one or more elements whose geometric structures possesses certain desired properties and capabilities for an SBN and constituent SBN materials. Graphs, geometrical pictures, simple or complex, are used in mathematical models for the study of the production of SBN proteins and their spatial folding, with applications to drug design and genomics. Graphs provide a simple beginning to many complicated objects. In such a situation one hopes to show how combinatorial properties of the graph are reflected in the resulting object--does the simple object "control" the complicated one? The complicated objects in this instance are highly infinite operator algebras, like Infinite-dimensional Lie algebras. Even elementary examples illustrate the passage from graph to algebra, like the construction of a tree, and while the boundary of a tree is space, the boundary of a graph is an algebra.

According to another feature, the invention is an improvement over in vivo biodegradable nanoparticles in the prior art because in some invention embodiments it may use molecular-imprint technology. Molecular imprinting, or biomimetic chemistry that deals with imitations of natural binding entities, such as enzymes and antibodies, involves preorganization of polymerizable monomers around an imprinting molecule. Following polymerization and removal of the imprint molecule, the solid polymer contains binding sites that are complementary in size and shape to the template. This cavity, in one embodiment, can facilitate catalysis of certain reactions and may also be used for shape selective separations. In other embodiments, imprinted polymers embodiments may facilitate the fabrication of SBN membrane materials to achieve selective diffusion; as chromatographic supports for the separation of enantiomers and oligonucleotides by SBN elements; to provide the recognition element for SBN chemical sensors, and for the synthesis of polymeric materials that mimic biological receptors that are targeted by SBN elements and or play a role in the design of new drugs. Other SBN embodiments may utilize imprinted membranes and thin films that also function as an artificial cell wall for the selective transport of targeted drugs, peptides and biologically important molecules.

According to another feature, biodegradable films may also be used as a pliable template for biological elements, which elements are pressed into a biodegradable film and then removed, leaving a physical mold of the biological element's shape. The film can then be hardened and used by an SBN to detect that particular biological element, which may be, but is not limited to, a particular receptor, protein, or cell, since its complex imprint shape on the film will bind only to that particular biological element. Molecular imprinting polymerization renders a polymer matrix with a series of cavities, or imprints, that are complementary in size, shape and position of chemical functionalities to a template molecule. These imprints enable the polymer matrix to rebind the template molecule selectively from mixtures of closely related compounds. One illustrative invention embodiment provides for a molecular-level process for biodegradable capsule production and produces nanocapsules with surface feature sizes at the molecular level.

In one embodiment, the molecular-level imprint process provides for a smart targeting system using biodegradable nanocapsules for delivery of one or more SBN cargo elements and or one or more non-SBN elements in vivo or in vitro. In another embodiment, molecular imprinting is used for the production of molecule-specific cavities that mimic the behavior of natural receptor binding sites, without the temperature sensitivity of the natural systems. Artificial polymers may be built for any target molecule. The polymers are prepared in the presence of a template molecule that interacts with the polymer network via ionic, covalent or hydrogen bonding interactions. Target specificity of the correct docking position for an imprinted nanocapsule embodiment is provided by the geometric pattern on the nanocapsule that locks onto its complementary shape on the docking site, forming a transient complex. The shape of the docking pattern matters more than non-covalent bonds, because, with the exception of electrostatic interactions, non-covalent bond interactions are extremely short ranged (no more than a few angstroms). Various biodegradable materials may be used in various embodiments for imprinting, including, but not limited to, bio-base materials, polymers, biological elements, and other materials such as biodegradable plastics and films.

In one application, SBN elements perform targeted agent delivery in vivo or in vitro, wherein the agents are one or more research, diagnostic, therapeutic, prosthetic, and or assay agents.

In one application, SBN elements perform targeted agent delivery in vivo or in vitro using as appropriate ligands, and or targeting moieties, and or other vectors.

In another application, an intelligent, nanoscale, cell-sized platform comprised of SBN elements performs smart molecular-level and or cellular-level target site loitering, monitoring, repair, construction and or dynamic, interactive control of systems, in vitro and in vivo. SBN biomedical platforms include, but are not limited to, imaging, sensor, genetic and protein assay, diagnostic, drug delivery, prosthetic, and intra-cellular, tissue, organ, and or circulatory engineering, modification and repair platforms, including implantable defibrillators, pacemakers, coronary stents, and angioplasty devices and or systems.

According to one illustrative configuration, one or more cargo elements that interfere with a SBN's operation if carried in the same protein cage as another cargo element type is instead carried in a separate SBN element protein cage that exclusively carries non-interfering cargo elements, thereby inhibiting disruptive interference with SBN operations. Such non-interfering-only cargo cages may be functionally and or physically linked with other SBN element cages carrying other cargo element types.

In another aspect, the protein cage features no cargo elements at all. According to one embodiment, empty self-assembling cages include highly ordered scaffolding for self-assembling multi-layer, multi-dimensional, multi-SBN element and multi-non SBN element systems.

In another aspect, the face