SEMICONDUCTOR QUANTUM DOTS FOR EFFICIENT DELIVERY AND INTRACELLULAR IMAGING OF siRNA

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Serial

Number 60/918,979, entitled "Semiconductor Quantum Dots for Efficient Delivery and Intracellular Imaging of Silencing RNA" filed on March 20, 2007, the entirety of which is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NIH Grants Nos. U54CA119338 awarded by the U.S. National Institutes of Health of the United States government. The government has certain rights in the invention

FIELD OF THE DISCLOSURE

The present disclosure relates generally to nanostructures, and relates more particularly, to bioconjugated nanostructures.

BACKGROUND RNA interference (RNAi) is one of the most powerful technologies for sequence-specific suppression of genes and has potential applications ranging from functional gene analysis to therapeutics. Low immunogenicity and oncologic properties of non-viral vehicles are preferred for efficient and specific in vitro or in vivo drug or siRNA, delivery. Various strategies, therefore, have been developed based on nanoparticles (nanoparticles), and polymers such as liposome, gold, and silica nanoparticles, peptides, and cationic and biodegradable polymers. However, the delivery efficiency of such vehicles has proven to be less than desired.

In addition to the failings of currently used vehicles, a frequently encountered limitation of all existing delivery vehicles is the lack of an intrinsic signal for long term and realtime imaging of siRNA transport, which could provide vitally important information on rational design and engineering of DNA and RNA carriers. To overcome this limitation in information, organic fluorophores are usually used to label liposome, silica nanoparticles, cationic polymers and the like. However, the photobleaching associated with essentially all organic dyes prevents long-term tracking or imaging of oligonucleotides. Electron-dense gold nanoparticles are visible under transmitted electron microscope (TEM) and can provide the highest imaging resolution. This method is suitable only for fixed cells. Imaging live cells in realtime is nearly impossible. The semiconductor quantum dots (QDs) of the present

disclosure were advantageously used to study siRNA delivery because of their intrinsic fluorescence and the unique optical properties (e.g., tunable emission, photostability and brightness).

Fluorescence and electron microscopy of QD labeled proteins in cells and tissue sections has been correlated due to the visibility in TEM. Surprisingly, however, besides the fluorescence and electron density that allow intracellular siRNA imaging, QDs of the present disclosure can also serve as a highly efficient scaffold for siRNA delivery, which can be attributed to their size, shape and surface. Despite the current use QDs in bioimaging applications, the application and usefulness of these advantageous structural properties had not been realized, but which will enable broad applications in drug delivery and discovery, pharmacokinetic and pharmacodynamic studies.

SUMMARY Briefly described, embodiments of this disclosure, among others, encompass nanostructures, methods of preparing nanostructures, methods of modifying the expression from a gene by delivering an siRNA species to the interior of a targeted cell. The disclosure encompasses nanostructures that comprise a quantum dot and a hydrophobic protection structure. The hydrophobic protection structure includes a capping ligand and an amphiphilic copolymer, where the hydrophobic protection structure encapsulates the quantum dot. An siRNA species may be attached to the exterior surface of a hydrophilic protection surface of the nanostructure.

The nanostructure may passively attach to the cell membrane of a target cell and be internalized or, by means of a target-specific probe, be actively directed to a specific target cell or tissue before internalization. The siRNA may then contact the target gene of its transcription product, thereby modifying the expression of the gene, either increasing the expression or decreasing the level of expression. By including in the nanostructure a semiconductor quantum dot, it is also possible to monitor the progression of the nanostructure and the molecules attached thereto, within an intact host animal, tissue or targeted cell. One aspect of the disclosure encompasses nanostructures comprising a quantum dot, a hydrophobic protection structure comprising a capping ligand and an amphiphilic copolymer, wherein the hydrophobic protection structure encapsulates the quantum dot, and an siRNA molecule. In one embodiment of the nanostructures according to the disclosure, the siRNA may be disposed on the hydrophobic protection surface.

In various embodiments of this aspect of the disclosure, the amphiphilic copolymer may be selected from the group consisting of an amphiphilic block copolymer, an amphiphilic random copolymer, an amphiphilic alternating copolymer, an amphiphilic periodic copolymer, or any combination thereof. In one embodiment of the disclosure, the amphiphilic copolymer may be a block copolymer is selected from the group consisting of a diblock copolymer, a triblock copolymer, and a combination thereof. In another embodiment, amphiphilic block copolymer may comprise an ABC triblock structure having grafted 8-carbon alkyl side chains. In one particularly advantageous embodiment of the disclosure, the ABC triblock structure may comprise a poly-butylacrylate segment, a poly-ethylacrylate segment, and a poly-methacrylic acid segment.

In the various embodiments of the present disclosure, the quantum dot may comprise a core and a cap, wherein the core of the quantum dot may be selected from the group consisting of a NA-VIA semiconductor, a IMA-VA semiconductor, a IVA-IVA semiconductor, and a IVA-VIA semiconductor.

In one embodiment, the core of the quantum dot is a IIA-VIA semiconductor. In other embodiments of the disclosure, the core of the quantum dot may be selected from the group consisting of CdS, CdSe, CdTe, ZnSe, ZnS, PbS, PbSe, or a combination thereof. In one embodiment of the nanostructure of the disclosure, the quantum dot may comprise CdTe/CdSe. In another embodiment, the core of the quantum dot is CdSe.

In the embodiments of the nanostructures of the disclosure, the cap may be a IIA-VIA semiconductor of high band gap. In one embodiment, the cap is ZnS. In the embodiments of the nanostructures of the present disclosure, the capping Iigand may comprise tri-octylphosphine oxide.

In this aspect of the nanostructures of the disclosure, the nanostructures may further comprise a targeting probe, wherein the probe may be selected from the group consisting of an antibody, a polypeptide, a polynucleotide, a drug molecule, an inhibitor compound, or a combination thereof, and wherein the targeting probe may have an affinity for a marker on the surface of a target cell.

In the embodiments of the present disclosure, the targeting probe may be disposed on the hydrophobic protection structure. In one embodiment, the probe is a tumor-targeting Iigand. Another aspect of the present disclosure is methods of preparing a nanostructure, comprising providing a nanoparticle, forming a hydrophobic protection

structure around the nanoparticle that includes at least one compound selected from a capping ligand, an amphophilic copolymer, and combinations thereof, and contacting the nanoparticle with an siRNA, whereby the siRNA attaches to the outer surface of the nanoparticle. One embodiment of this aspect of the disclosure further comprises attaching a cell targeting probe to the hydrophobic protection structure, wherein the probe is selected from an antibody, a polypeptide, a polynucleotide, a drug molecule, an inhibitor compound, or a combination thereof.

In the embodiments of this aspect of the disclosure, the nanoparticle is a quantum dot wherein the core of the quantum dot may be selected from the group consisting of a MA-VIA semiconductor, a MIA-VA semiconductor, a IVA-IVA semiconductor, and a IVA-VIA semiconductor, and wherein the hydrophobic protection structure may comprise a capping ligand and an amphiphilic copolymer, and wherein the amphiphilic copolymer may be a block copolymer selected from a diblock copolymer, a triblock copolymer, and combinations thereof.

In embodiments of the method of the disclosure, the quantum dot may comprise a core and a cap, wherein the core of the quantum dot may be selected from the group consisting of a MA-VIA semiconductor, a IMA-VA semiconductor, a IVA-IVA semiconductor, and a IVA-VIA semiconductor. In one embodiment, the core of the quantum dot is a MA-VIA semiconductor. In embodiments of the disclosure, the core of the quantum dot may be selected from the group consisting of CdS, CdSe, CdTe, ZnSe, ZnS, PbS, PbSe, or a combination thereof.

In one embodiment, the quantum dot comprises CdTe/CdSe. In another embodiment, the core of the quantum dot is CdSe. In embodiments of the disclosure, the cap may be a MA-VIA semiconductor of high band gap. In one embodiment of the disclosure, the cap is ZnS.

In embodiments of the method according to the disclosure, the capping ligand may comprise tri-octylphosphine oxide, and the amphiphilic block copolymer may be an ABC triblock structure comprising a poly-butylacrylate segment, a poly- ethylacrylate segment, and a poly-methacrylic acid segment.

Another aspect of the present disclosure is a method of modifying the expression of a gene in a target cell, comprising contacting a target cell with at least one nanostructure comprising a semiconductor quantum dot, a hydrophobic protection structure including at least one compound selected from a capping ligand, an amphiphilic copolymer, or a combination thereof, wherein the hydrophobic protection structure encapsulates the quantum dot, and an siRNA species, wherein

the molecules of the siRNA are disposed on the exterior surface of the nanostructure, and wherein the nanostructure enters the cell, and wherein the siRNA species modifys the expression of a gene to which the siRNA is targeted.

In embodiments of this aspect of the disclosure, the nanostructure may further comprise a cell targeting probe, wherein the cell targeting probe directs the nanoparticle to the targeted cell. In embodiments of the methods according to this aspect of the disclosure, the target cell may be a cultured cell linear a cell in a tissue of a host.

Embodiments of this aspect of the disclosure may further comprise detecting the nanostructure in a cell or in a host. In one embodiment, the nanostructure is detected by fluorescence.

In the various embodiments of the method of this aspect of the disclosure, it is contemplated that the presence of the target in the subject corresponding to the probe by detecting the nanoparticle. BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

FIG. 1 illustrates an exemplar embodiment of a nanostructure with siRNA molecules attached thereto.

FIG. 1B illustrates the effects of the partial derivatization of carboxylate groups into tertiary amines.

FIG. 1C illustrates the spread of hydrodynamic diameters of the nanostructures of the disclosure. FIG. 1 D illustrates the TEM image of nanostructures of the disclosure.

FIG. 2 illustrates a comparison between siRNA-QDs of the disclosure and other transfection agents with siRNA on the expression of cyclophilin B in MB 231 cells.

FIG. 3A illustrates the cytotoxic effect of various carriers on cultured cells. FIG. 3B illustrates the cytotoxic effect of various carriers on cultured cells versus time.

FIG. 3C illustrates the cytotoxic effect of various carriers on cultured cells versus carrier concentration.

FIG. 4A illustrates the kinetics of QD-siRNA uptake as shown by fluorescence of the QDs.

FIG. 4B illustrates migration and assembly of siRNA-QDs to the nuclei of cells.

FIGS. 5A-5B illustrate the progress of formation of endocytotic vesicle formation during siRNA-QD uptake by cells. FIGS 6A-6F illustrate decreases in cyclophilin B expression following contact of cancer cells with siRNA using a variety of carrier delivery vehicles. FIG. 6A: MCF- 7 cells; FIG. 6B, NIH3T3 cells; FIG. 6C, PC-3 cells; FIG. 6D, NCI-H cells; FIG. 6E, HeLa cells; FIG. 6F, OVCAR-3 cells.

FIG. 7 illustrates toxicity of delivery vehicles to various cell lines. FIG. 8 schematically illustrates a nanoparticle according to the disclosure having siRNA and targeting probes attached to the hydrophilic protection layer.

The drawings are described in greater detail in the description and examples below.

The details of some exemplary embodiments of the methods and systems of the present disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent to one of skill in the art upon examination of the following description, drawings, examples and claims. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like

those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. "Consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated. Definitions

"DNA" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form, or as a double- stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

The term "expressed" or "expression" as used herein refers to the transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene. The term "expressed" or "expression" as used herein also refers to the translation from said RNA nucleic acid molecule to give a protein, an amino acid sequence or a portion thereof.

The term "modify the level of gene expression" as used herein refers to generating a change, either a decrease or an increase in the amount of a transcriptional or translational product of a gene. The transcriptional product of a gene is herein intended to refer to a messenger RNA (mRNA) transcribed product of a gene and may be either a pre- or post-spliced mRNA. Alternatively, the term

"modify the level of gene expression" may refer to a change in the amount of a protein, polypeptide or peptide generated by a cell as a consequence of interaction of an siRNA with the contents of a cell. For example, but not limiting, the amount of a polypeptide derived from a gene may be reduced if the corresponding mRNA species is subject to degradation as a result of association with an siRNA introduced into the cell.

As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be single-stranded or double- stranded, but advantageously is double-stranded DNA. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. A "nucleoside" refers to a base linked to a sugar. The base may be adenine (A), guanine (G) (or its substitute, inosine (I)), cytosine (C), or thymine (T) (or its substitute, uracil (U)). The sugar may be ribose (the sugar of a natural nucleotide in RNA) or 2-deoxyribose (the sugar of a natural nucleotide in DNA). A "nucleotide" refers to a nucleoside linked to a single phosphate group.

As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides may be chemically synthesized and may be used as primers or probes. Oligonucleotide means any nucleotide of more than 3 bases in length used to facilitate detection or identification of a target nucleic acid, including probes and primers.

The term "quantum dot" (QDs) as used herein refers to semiconductor nanocrystals or artificial atoms, which are semiconductor crystals that contain anywhere between 100 to 1 ,000 electrons and range from about 2-10 nm. Some QDs can be between about 10-20 nm in diameter. QDs have high quantum yields, which makes them particularly useful for optical applications. QDs are fluorophores that fluoresce by forming excitons, which can be thought of the excited state of traditional fluorophores, but have much longer lifetimes of up to 200 nanoseconds. This property provides QDs with low photobleaching. The energy level of QDs can be controlled by changing the size and shape of the QD, and the depth of the QDs'

potential. One of the optical features of small excitonic QDs is coloration, which is determined by the size of the dot. The larger the dot, the redder, or more towards the red end of the spectrum the fluorescence. The smaller the dot, the bluer or more towards the blue end it is. The bandgap energy that determines the energy and hence the color of the fluoresced light is inversely proportional to the square of the size of the QD. Larger QDs have more energy levels which are more closely spaced, thus allowing the QD to absorb photons containing less energy, i.e. those closer to the red end of the spectrum. Because the emission frequency of a dots dependent on the bandgap, it is therefore possible to control the output wavelength of a dot with extreme precision. Colloidally prepared QDs are free floating and can be attached to a variety of molecules via metal coordinating functional groups. These groups include but are not limited to thiol, amine, nitrile, phosphine, phosphine oxide, phosphonic acid, carboxylic acids or other ligands. By bonding appropriate molecules to the surface, the quantum dots can be dispersed or dissolved in nearly any solvent or incorporated into a variety of inorganic and organic films.

The term "siRNA" refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can vary in length (generally 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5' or 3 ¹ end of the sense strand and/or the antisense strand. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region. siRNA may be divided into five (5) groups (non-functional, semi-functional, functional, highly functional, and hyper-functional) based on the level or degree of silencing that they induce in cultured cell lines. In this context, "non-functional siRNA" are defined as those siRNA that induce less than 50% (<50%) target silencing. "Semi-functional siRNA" induce 50-79% target silencing. "Functional siRNA" are molecules that induce 80- 95% gene silencing. "Highly-functional siRNA" are molecules that induce greater than 95% gene silencing. "Hyperfunctional siRNA" are a special class of molecules. For purposes of this document, hyperfunctional siRNA are defined as those molecules that: (1) induce greater than 95% silencing of a specific target when they are transfected at subnanomolar concentrations (i.e., less than one nanomolar); and/or (2) induce functional (or better) levels of silencing for greater than 96 hours. These relative functionalities (though not intended to be absolutes) may be used to compare siRNAs to a particular target for applications such as functional genomics, target identification and therapeutics.

siRNAs trigger host cell RNA degradation mechanisms in a sequence- specific manner. They may therefore be used to inactivate endogenous RNAs or pathogen RNA such as, but not only, HIV-1 RNA. It is preferred that there be not more than one mismatch (mismatches are defined as not including G: U pairs) in each double-stranded region, more preferably no mismatches, and most preferred that the double stranded region(s) be perfectly matched. Where the targeted molecule is variable (e.g., HIV-1 RNA), highly conserved regions should be targeted. A family of variants can be targeted provided they do not have more than one mismatch with one or other of the strands of the double-stranded region of the siRNA molecule. For longer siRNA molecules, several short duplexes may be joined, allowing targeting of multiple genes, which is preferred for targets with higher variablity. The siRNA duplexes may also be produced from longer RNA transcripts by splicing or self-cleaving means, for example by incorporating self-cleaving ribozymes between or flanking the duplex regions. siRNA molecules are easily formed from DNA molecules having an inverted repeat structure. Alternatively, siRNA duplexes may be formed from two RNA molecules with complementary regions. siRNA molecules with double-stranded regions of greater than 30 base pairs can be used if they are nuclear localized, e.g., if they are made without signals for cytoplasmic export such as polyadenylated sequences. Until recently, siRNA had not been shown to work in human cells. Recently, however, siRNA (also called iRNA, or RNAi, or hairpin RNA) has been shown to inhibit HIV-1 replication in T lymphocytes. siRNA molecules targeted to the viral LTR ₁ or the accessory vif and nef genes inhibited early and late steps of HIV replication in cell lines and primary lymphocytes. siRNA has also been successfully targeted to other viruses and can be targeted against endogenous genes.

The term "target" is used in a variety of different forms throughout this document and is defined by the context in which it is used. "Target mRNA" refers to a messenger RNA to which a given siRNA can be directed against. "Target sequence" and "target site" refer to a sequence within the mRNA to which the sense strand of an siRNA shows varying degrees of homology and the antisense strand exhibits varying degrees of complementarity. The phrase "siRNA target" can refer to the gene, mRNA, or protein against which an siRNA is directed. Similarly, "target silencing" can refer to the state of a gene, or the corresponding mRNA or protein. The term "transfection" refers to a process by which agents are introduced into a cell. The list of agents that can be transfected is large and includes, but is not limited to, siRNA, sense and/or anti-sense sequences, DNA encoding one or more

genes and organized into an expression plasmid, proteins, protein fragments, and more. There are multiple methods for transfecting agents into a cell including, but not limited to, electroporation, calcium phosphate-based transfections, DEAE- dextran-based transfections, lipid-based transfections, molecular conjugate-based transfections (e.g., polylysine-DNA conjugates), microinjection and others.

The term "cancer", as used herein shall be given its ordinary meaning and is a general term for diseases in which abnormal cells divide without control. Cancer cells can invade nearby tissues and can spread through the bloodstream and lymphatic system to other parts of the body. There are several main types of cancer, for example, carcinoma is cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream. Lymphoma is cancer that begins in the cells of the immune system. When normal cells lose their ability to behave as a specified, controlled and coordinated unit, a tumor is formed. Generally, a solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas (some brain tumors do have cysts and central necrotic areas filled with liquid). A single tumor may even have different populations of cells within it with differing processes that have gone awry. Solid tumors may be benign (not cancerous), or malignant (cancerous). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors. Representative cancers include, but are not limited to, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head & neck cancer, leukemia, lung cancer, lymphoma, melanoma, non-small-cell lung cancer, ovarian cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, thyroid cancer, gastric cancer, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma, glioblastoma, ependymoma, Ewing's sarcoma family of tumors, germ cell tumor, extracranial cancer, Hodgkin's disease, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, liver cancer, medulloblastoma, neuroblastoma, brain tumors generally, non-Hodgkin's lymphoma, osteosarcoma, malignant fibrous histiocytoma of bone, retinoblastoma, rhabdomyosarcoma, soft tissue sarcomas generally, supratentorial primitive neuroectodermal and pineal tumors, visual pathway and hypothalamic glioma, Wilms' tumor, acute lymphocytic leukemia, adult acute myeloid leukemia, adult non-Hodgkin's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, esophageal cancer,

hairy cell leukemia, kidney cancer, multiple myeloma, oral cancer, pancreatic cancer, primary central nervous system lymphoma, skin cancer, small-cell lung cancer, among others.

A tumor can be classified as malignant or benign. In both cases, there is an abnormal aggregation and proliferation of cells. In the case of a malignant tumor, these cells behave more aggressively, acquiring properties of increased invasiveness. Ultimately, the tumor cells may even gain the ability to break away from the microscopic environment in which they originated, spread to another area of the body (with a very different environment, not normally conducive to their growth) and continue their rapid growth and division in this new location. This is called metastasis. Once malignant cells have metastasized, achieving cure is more difficult.

Benign tumors have less of a tendency to invade and are less likely to metastasize. Brain tumors spread extensively within the brain but do not usually metastasize outside the brain. Gliomas are very invasive inside the brain, even crossing hemispheres. They do divide in an uncontrolled manner, though. Depending on their location, they can be just as life threatening as malignant lesions. An example of this would be a benign tumor in the brain, which can grow and occupy space within the skull, leading to increased pressure on the brain.

Cardiovascular disease, as used herein, shall be given its ordinary meaning, and includes, but is not limited to, high blood pressure, diabetes, coronary artery disease, valvular heart disease, congenital heart disease, arrthymia, cardiomyopathy, CHF, atherosclerosis, inflamed or unstable plaque associated conditions, restinosis, infarction, thromboses, post-operative coagulative disorders, and stroke. Inflammatory disease, as used herein, shall be given its ordinary meaning, and can include, but is not limited to, autoimmune diseases such as arthritis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, other diseases such as asthma, psoriasis, inflammatory bowel syndrome, neurological degenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, vascular dementia, and other pathological conditions such as epilepsy, migraines, stroke and trauma.

Autoimmune disease, as used herein, shall be given its ordinary meaning, and includes, but is not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, biliary cirrhosis, bullous pemphigoid, canavan disease, cardiomyopathy, celiac sprue-

dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, diffuse cerebral sclerosis of Schilder, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Fuch's heterochromic iridocyclitis,

Graves' disease, Guillain-Barr, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP) ₁ IgA nephropathy, insulin dependent diabetes, intermediate uveitis, juvenile arthritis, lichen planus, lupus, Mnire's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, nephrotic syndrome, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary Agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, vasculitis, vitiligo, VKH (Vogt-Koyanagi-Harada) disease, Wegener's granulomatosis, anti-phospholipid antibody syndrome (lupus anticoagulant), Churg-Strauss (allergic granulomatosis), dermatomyositis/polymyositis, Goodpasture's syndrome, interstitial granulomatous dermatitis with arthritis, lupus erythematosus (SLE, DLE, SCLE), mixed connective tissue disease, relapsing polychondritis, HLA-B27 asssociated conditions including ankylosing spondylitis, psoriasis, ulcerative colitis, Reiter's syndrome, and Uveal diseases.

Viral disease, as used herein, shall be given its ordinary meaning, and includes target viruses such as, but not limited to, paramyxo-, picoma-, rhino-, coxsackie-, influenza-, herpes-, adeno-, parainfluenza-, respiratory syncytial-, echo-, corona-, Epstein-Barr-, cytomegalo-, varicella zoster, and hepatitis (e.g., variants including hepatitis C Virus (HCV), Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), Hepatitis D Virus (HDV), Hepatitis E Virus (HEV), Hepatitis F Virus (HFV), Hepatitis G Virus (HGV), Human immunodeficiency). Neurological conditions, as used herein, shall be given its ordinary meaning, can be generally classified into three classes: those disease with ischemic or hypoxic mechanisms; neurodegenerative diseases (see Adams et a/, Principles of Neurology, 1997, 6 ^th Ed., New York, pp 1048); and neurological and psychiatric diseases associated with neural cell death. Diseases with ischemic or hypoxic mechanisms can be further subclassified into general diseases and cerebral ischemia. Examples of such general diseases

involving ischemic or hypoxic mechanisms include myocardial infarction, cardiac insufficiency, cardiac failure, congestive heart failure, myocarditis, pericarditis, perimyocarditis, coronary heart disease (stenosis of coronary arteries), angina pectoris, congenital heart disease, shock, ischemia of extremities, stenosis of renal arteries, diabetic retinopathy, thrombosis associated with malaria, artificial heart valves, anemias, hypersplenic syndrome, emphysema, lung fibrosis, and pulmonary edema. Examples of cerebral ischemia disease include stroke (as well as hemorrhagic stroke), cerebral microangiopathy (small vessel disease), intrapartal cerebral ischemia, cerebral ischemia during/after cardiac arrest or resuscitation, cerebral ischemia due to intraoperative problems, cerebral ischemia during carotid surgery, chronic cerebral ischemia due to stenosis of blood-supplying arteries to the brain, sinus thrombosis or thrombosis of cerebral veins, cerebral vessel malformations, and diabetic retinopathy.

Neurodegenerative disease can include, but is not limited to, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, Wilson's disease, multi-system atrophy, Alzheimer's disease, Pick's disease, Lewy-body disease, Hallervorden-Spatz disease, torsion dystonia, hereditary sensorimotor neuropathies (HMSN), Gerstmann-Straussler-Schanke- r disease, Creutzfeld-Jakob-disease, Machado-Joseph disease, Friedreich ataxia, Non-Friedreich ataxias, Gilles de Ia Tourette syndrome, familial tremors, olivopontocerebellar degenerations, paraneoplastic cerebral syndromes, hereditary spastic paraplegias, hereditary optic neuropathy (Leber), retinitis pigmentosa, Stargardt disease, and Kearns-Sayre syndrome.

Examples of neurological and psychiatric diseases associated with neural cell death include septic shock, intracerebral bleeding, subarachnoidal hemorrhage, multiinfarct dementia, inflammatory diseases (e.g., vasculitis, multiple sclerosis, and Guillain-Barre-syndrome), neurotrauma (e.g., spinal cord trauma, and brain trauma), peripheral neuropathies, polyneuropathies, epilepsies, schizophrenia, metabolic encephalopathies, and infections of the central nervous system (e.g., viral, bacterial, fungal).

The nanostructures of the present disclosure are advantageous for the delivery of small siRNA oligonucleotides to a cell for use in such as, but not limited to, gene expression studies, protein studies, and the therapeutic gene expression for the treatment of a disease such as cancer, autoimmune disease, cardiovascular disease and the like. In particular, the nanostructures can be used in in-vivo diagnostic and/or therapeutic applications such as, but not limited to, targeting and/or

imaging of diseases and/or conditions (e.g., identify the type of disease, locate the proximal locations of the disease, and deliver drugs to modify gene expression in targeted or diseased cells (e.g., cancer cells) in living animals. The nanostructures in combination with spectral imaging can be used for multiplexed imaging and detection of genes, proteins, and the like, in single living cells.

Most advantageously, the nanostructures of the present disclosure may be used for the delivery of oligonucleotides, and in particular siRNAs to a cell to modify the expression of a gene, either by modifying transcription from the gene to an rmRNA, or translation therefrom. Embodiments of the nanostructure include, but are not limited to, a nanoparticle (e.g., quantum dots, metal particles and metal oxide particles) and a hydrophobic protection structure that encapsulates the nanoparticle. In addition, the nanostructure can include, but is not limited to, a bio-compatibility compound (e.g., polyethylene glycol (MW about 500 to 50,000 and 1000 to 10,000), dextran, and derivatives such as amino-dextran and carboxy-dextran, and polysaccharides) and a probe (e.g., polynucleotide, polypeptide, a therapeutic agent, and/or a drug). The bio-compatibility compound, the probe and an siRNA are substantially disposed (e.g., attached to the surface of the hydrophobic protection structure and/or attached within the hydrophobic protection structure) on the hydrophobic protection structure. The hydrophobic protection structure includes a capping ligand and/or a amphiphilic copolymer (e.g., amphiphilic block copolymers, amphiphilic random copolymers, amphiphilic alternating copolymers, amphiphilic periodic copolymers, and combinations thereof).

In one embodiment, the nanostructure can include two or more nanoparticle or two of more types of nanoparticle. In addition, the nanostructure can include a hydrophobic protection structure having two or more copolymers (e.g., two or more block copolymers). Further, the nanostructure can include multiple nanoparticle and multiple copolymers (e.g., block copolymers). In addition, the nanostructure can include two or more different types of probes having different functions. Furthermore, the nanoparticle and the copolymers (e.g., block copolymers) can be assembled into micro and macro structures.

FIGS. 1 A and 8 illustrate schematic exemplar embodiments of nanostructures bearing an siRNA according to the present disclosure. The nanostructure includes, but is not limited to, a nanoparticle having a hydrophobic protection structure that encapsulates the QD nanoparticle. In addition, the nanostructure can include, but is not limited to, a bio-compatibility compound and an siRNA molecule(s).

The hydrophobic protection structure may include a capping ligand layer and/or a copolymer layer (e.g., amphiphilic block copolymer). The following illustrative examples will use amphiphilic block copolymers, but other copolymers such as, but not limited to, amphiphilic random copolymers, amphiphilic alternating copolymers, amphiphilic periodic copolymers, and combinations thereof, can be used in combination with block copolymers, as well as individually or in any combination. In addition, the term "amphiphilic block copolymer" will be termed "block copolymer" hereinafter.

The nanostructure can include a number of types of nanoparticle such as, but not limited to, a semiconductor nanoparticle. In particular, semiconductor quantum dots suitable for use in the nanostructures of the present disclosure are described in more detail below and in U.S. Patent 6,468,808 and International Patent Application WO 03/003015, which are incorporated herein by reference.

The nanostructure can include quantum dots such as, but not limited to, luminescent semiconductor quantum dots. In general, quantum dots include a core and a cap, however, uncapped quantum dots can be used as well. The "core" is a nanometer-sized semiconductor. While any core of the IIA-VIA, IIIA-VA or IVA-IVA, IVA-VIA semiconductors can be used in the context of the present disclosure, the core must be such that, upon combination with a cap, a luminescent quantum dot results. A IIA-VIA semiconductor is a compound that contains at least one element from Group NB and at least one element from Group VIA of the periodic table, and so on. The core can include two or more elements. In one embodiment, the core is a IIA-VIA, IIIA-VA or IVA-IVA semiconductor that ranges in size from about 1 nm to 20 nm. In another embodiment, the core is more preferably a IIA-VIA semiconductor and ranges in size from about 2 nm to 10 nm. For example, the core can be CdS, CdSe, CdTe, ZnSe, ZnS, PbS, PbSe or an alloy.

The "cap" is a semiconductor that differs from the semiconductor of the core and binds to the core, thereby forming a surface layer on the core. The cap can be such that, upon combination with a given semiconductor core a luminescent quantum dot results. The cap should passivate the core by having a higher band gap than the core. In one embodiment, the cap is a IIA-VIA semiconductor of high band gap. For example, the cap can be ZnS or CdS. Combinations of the core and cap can include, but are not limited to, the cap is ZnS when the core is CdSe or CdS, and the cap is CdS when the core is CdSe. Other exemplary quantum does include, but are not limited to, CdS, ZnSe, CdSe, CdTe, CdSe _x Te _1-x , InAs, InP, PbTe, PbSe, PbS, HgS, HgSe, HgTe, CdHgTe, and GaAs.

The wavelength emitted {i.e., color) by the quantum dots can be selected according to the physical properties of the quantum dots, such as the size and the material of the nanocrystal. Quantum dots are known to emit light from about 300 nanometers (nm) to 1700 nm (e.g., UV, near IR, and IR). The colors of the quantum dots include, but are not limited to, red, blue, green, and combinations thereof. The color or the fluorescence emission wavelength can be tuned continuously. The wavelength band of light emitted by the quantum dot is determined by either the size of the core or the size of the core and cap, depending on the materials which make up the core and cap. The emission wavelength band can be tuned by varying the composition and the size of the QD and/or adding one or more caps around the core in the form of concentric shells.

The intensity of the color of the quantum dots can be controlled. For each color, the use of 10 intensity levels (0,1 , 2, ...9) gives 9 unique codes (10 ¹ - 1), because level "0" cannot be differentiated from the background. The number of codes increase exponentially for each intensity and each color used. For example, a three color and 10 intensity scheme yields 999 (10 ³ -1) codes, while a six color and 10 intensity scheme has a theoretical coding capacity of about 1 million (10 ⁶ - 1). In general, n intensity levels with m colors generate (n ^m - 1) unique codes. Use of the intensity of the quantum dots has applications in nanostructures including a plurality of different types of quantum dots having different intensity levels and also in nanostructures including a plurality of different types of quantum dots having different intensity levels that are included in a porous material. The quantum dots are capable of absorbing energy from, for example, an electromagnetic radiation source (of either broad or narrow bandwidth), and are capable of emitting detectable electromagnetic radiation at a narrow wavelength band when excited. The quantum dots can emit radiation within a narrow wavelength band (FWHM, full width at half maximum) of about 40 nm or less, thus permitting the simultaneous use of a plurality of differently colored quantum dots with little or no spectral overlap.

The synthesis of quantum dots is well known and is described in U.S. Patent Nos. 5,906,670; 5,888,885; 5,229,320; 5,482,890; 6,468,808; 6,306,736; 6,225,198, efc, International Patent Application WO 03/003015, (all of which are incorporated herein by reference) and in many research articles. The wavelengths emitted by quantum dots and other physical and chemical characteristics have been described in US Patent 6,468,808 and International Patent Application WO 03/003015 and will not be described in any further detail. In addition, methods of preparation of

quantum dots are described in US Patent 6,468,808 and International Patent Application WO 03/003015 and will not be described any further detail. As mentioned above, the hydrophobic protection structure of the nanostructures according to the present disclosure includes the capping ligand and/or the block copolymer. In particular, when the nanoparticle is a quantum dot, the hydrophobic protection layer includes the capping ligand and the block copolymer, where the capping ligand and the block copolymer interact with one another to form the hydrophobic protection structure. As such, the capping ligand and the block copolymer are selected to form an appropriate hydrophobic protection structure. For example, the block copolymer and the nanoparticle can interact through interactions such as, but not limited to, hydrophobic interactions, hydrophilic interactions, pi-stacking, etc., depending on the surface coating of the nanoparticle and the molecular structure of polymers.

The capping ligand caps the nanoparticle (e.g., quantum dot) and forms a layer on the nanoparticle, which subsequently bonds with the block copolymer to form the hydrophobic protection structure. The capping ligand can include compounds such as, but not limited to, an O=PR ₃ compound, an O=PHR ₂ compound, an O=PHR ₁ compound, a H ₂ NR compound, a HNR ₂ compound, a NR ₃ compound, a HSR compound, a SR ₂ compound, and combinations thereof. "R" can be a C ₁ to C ₁₈ hydrocarbon, such as but not limited to, linear hydrocarbons, branched hydrocarbons, cyclic hydrocarbons, substituted hydrocarbons (e.g., halogenated), saturated hydrocarbons, unsaturated hydrocarbons, and combinations thereof. Preferably, the hydrocarbon is a saturated linear C ₄ to C ₁₈ hydrocarbon, a saturated linear C ₆ to C ₁₈ hydrocarbon, and a saturated linear C ₁₈ hydrocarbon. A combination of R groups can be attached to P, N, or S. In particular, the chemical can be selected from tri-octylphosphine oxide, stearic acid, and octyldecyl amine.

As mentioned above, the copolymer includes, but is not limited to, amphiphilic block copolymers, amphiphilic random copolymers, amphiphilic alternating copolymers, amphiphilic periodic copolymers, and combinations thereof. The amphiphilic random copolymer can include, but is not limited to random copolymer poly(methyl acrylate-co-acrylic acid); random copolymer poly(methyl methacrylate- co-n-butyl acrylate); random copolymer poly(methyl methacrylate-co-hydroxypropyl acrylate); random copolymer poly(styrene-co-p-carboxyl chloro styrene); random copolymer poly(styrene-co-4-hydroxystyrene); random copolymer poly(styrene-co-4- vinyl benzoic acid); random copolymer poly(styrene-co-4-vinyl pyridine); (and combinations thereof. The amphiphilic alternating copolymer can include, but is not

limited to, poly(maleic anhydride-alt-1-octadecene), poly(maleic anhydride-alt-1- tetradecene), alternating copolymer poly(carbo tert.butoxy α-methyl styrene-alt- maleic anhydride) and alternating copolymer poly(carbo tert.butoxy norbornene-alt- maleic anhydride), and combinations thereof. The block copolymer includes amphiphilic di- and or triblock copolymers. In addition, the copolymer can include hydrocarbon side chains such as, but not limited to, 1-18-carbon aliphatic side chains, 1-18-carbon alkyl side chains, and combinations thereof. Furthermore, the di or tri block copolymers have at least one hydrophobic block and at least one hydrophilic block. The following is in an exemplary list of amphiphilic random and alternating copolymers: random copolymer poly(dimethyl siloxane-co-diphenyl siloxane); random copolymer poly(methyl acrylate-co-acrylic acid); random copolymer poly(methyl methacrylate-co-n-butyl acrylate); random copolymer poly(methyl methacrylate-co-t- butyl acrylate); random copolymer poly(methyl methacrylate-co-hydroxypropyl acrylate); random copolymer poly(tetrahydrofuranyl methacrylate-co-ethyl methacrylate); random copolymer poly(styrene-co-4-bromostyrene); random copolymer poly(styrene-co-butadiene); random copolymer poly(styrene-co-diphenyl ethylene); random copolymer poly(styrene-co-t-butyl methacrylate); random copolymer poly(styrene-co-t-butyl-4-vinyl benzoate); random copolymer poly(styrene-co-p- carboxyl chloro styrene); random copolymer poly(styrene-co-p-chloromethyl styrene); random copolymer poly(styrene-co-methyl methacrylate); random copolymer poly(styrene-co-4-hydroxystyrene); random copolymer poly(styrene-co-4-vinyl benzoic acid); random copolymer poly(styrene-co-4-vinyl pyridine); alternating copolymer poly(carbo tert.butoxy α-methyl styrene-alt-maleic anhydride); alternating copolymer poly(carbo tert.butoxy norbornene-alt-maleic anhydride); alternating copolymer poly(α- methyl styrene-alt-methyl methacrylate); and alternating copolymer poly(styrene-alt- methyl methacrylate).

The following in an exemplary list of amphiphilic copolymers: poly((meth)acrylic acid) based copolymers (e.g., poly(acrylic acid-b-methyl methacrylate); poly(methyl methacrylate-b-acrylic acid); poly(methyl methacrylate-b- sodium acrylate); poly(sodium acrylate-b-methyl methacrylate); poly(methacrylic acid-b-neopentyl methacrylate); poly(neopentyl methacrylate-b-methacrylic acid); poly(t-butyl methacrylate-b-ethylene oxide); poly(methyl methacrylate-b-sodium methacrylate); and poly(methyl methacrylate-b-N,N-dimethyl acrylamide)), polydiene and hydrogenated polydiene based copolymers (e.g., poly(butadiene(1 ,2 addition)-b- methylacrylic acid; poly(butadiene(1 ,4 addition)-b-acrylic acid); poly(butadiene(1 ,4

addition)-b-sodium acrylate); poly(butadiene(1 ,4 addition)-b-ethylene oxide; poly(butadiene(1 ,2 addition)-b-ethylene oxide); poly(butadiene(1 ,2 addition)-b- ethylene oxide)-hydroxy benzoic ester terminal group; 4-methoxy benzyolester terminated poly(butadiene-b-ethylene oxide) diblock copolymer; poly(butadiene-b-N- methyl 4-vinyl pyridinium iodide); poly(isoprene-b-N-methyl 2-vinyl pyridinium iodide); poly(isoprene-b-ethylene oxide) (1 ,4 addition); poly(isoprene-b-ethylene oxide) (1 ,2 and 3,4 addition); poly(propylene-ethylene-b-ethylene oxide); and hydrogonated poly(isoprene-b-ethylene oxide) (1 ,2 addition)), hydrogentated diene based copolymers {e.g., poly(ethylene-b-ethylene oxide) and poly(isoprene-b-ethylene oxide)), poly(ethylene oxide) based copolymers (e.g., poly(ethylene oxide-b-acrylic acid); poly(ethylene oxide-b-ε-caprolactone); poly(ethylene oxide-b-6-(4'- cyanobiphenyl-4-yloxy)hexyl methacrylate); poly(ethylene oxide-b-lactide); poly(ethylene oxide-b-2-hydroxyethyl methacrylate); poly(ethylene oxide-b-methyl methacrylate); poly(-methyl methacrylate-b- ethylene oxide); poly(ethylene oxide-b- methacrylic acid); poly(ethylene oxide-b-2-methyl oxazoline); poly(ethylene oxide-b- propylene oxide); poly(ethylene oxide-b-t-butyl acrylate); poly(ethylene oxide-b- tetrahydrofurfuryl methacrylate); and poly(ethylene oxide-b-N,N- dimethylethylmethacrylate)), polyisobutylene based copolymers (e.g., poly(isobutylene-b-ethylene oxide)), polystyrene based copolymers (e.g., poly(styrene-b-acrylic acid); poly(styrene-b-sodium acrylate); poly(styrene-b- acrylamide); poly(p-chloromethyl styrene-b-acrylamide); poly(styrene-co-p- chloromethyl styrene-b-acrylamide); poly(styrene-co-p-chloromethyl styrene-b-acrylic acid); poly(styrene-b-cesium acrylate); poly(styrene-b-ethylene oxide); poly(4- styrenesulfonic acid-b-ethylene oxide); poly(styrene-b-methacrylic acid); poly(styrene-b-sodium methacrylate); poly(styrene-b-N-methyl 2-vinyl pyridinium iodide); and poly(styrene-b-N-methyl-4-vinyl pyridinium iodide)), polysiloxane based copolymers (e.g., poly(dimethylsiloxane-b-acrylic acid)), poly(2-vinyl naphthalene) based copolymers (e.g., poly(2-vinyl naphthalene-b-acrylic acid)), poly (vinyl pyridine and N-methyl vinyl pyridinium iodide) based copolymers (e.g., poly(2-vinyl pyridine-b- ethylene oxide); poly(N-methyl 2-vinyl pyridinium iodide-b-ethylene oxide); and poly(N-methyl 4-vinyl pyridinium iodide-b-methyl methacrylate)).

The following in an exemplary list of amphiphilic diblock copolymers: poly(meth)acrylate based copolymers (e.g., poly(n-butyl acrylate-b-methyl methacrylate); poly(n-butyl acrylate-b-dimethylsiloxane-co-diphenyl siloxane); poly(t- butyl acrylate-b-methyl methacrylate); poly(t-butyl acrylate-b-4-vinylpyridine); poly(2- ethyl hexyl acrylate-b-4-vinyl pyridine); poly(t-butyl methacrylate-b-2-vinyl pyridine);

poiy(2-hydroxyl ethyl acrylate-b-neopentyl acrylate); poly(2-hydroxyl ethyl methacrylate-b-neopentyl methacrylate); poly(2-hydroxyl ethyl methacrylate-b-n-butyl methacrylate); poly(2-hydroxyl ethyl methacrylate-b-t-butyl methacrylate); poly(methyl methacrylate-b-acrylonitrile); poly(methyl methacrylate-b-t-butyl methacrylate); poly(isotactic methyl methacrylate-b-syndiotactic methyl methacrylate); poly(methyl methacrylate-b-t-butyl acrylate); poly(methyl methacrylate-b-trifluroethyl methacrylate); poly(methyl methacrylate-b-2-hydroxyethyl methacrylate with cholesteryl chloroformate); poly(methyl methacrylate-b-disperse red 1 acrylate); poly (methyl methacrylate-b-2-hydroxyethyl methacrylate); poly(methyl methacrylate-b-neopentyl acrylate); and poly(methacrylate-b-2-pyranoxy ethyl methacrylate)), polydiene based copolymers (e.g., poly(butadiene(1 ,2 addition)- b-i-butyl methacrylate); poly(butadiene(1 ,2 addition)-b-s-butyl methacrylate); poly(butadiene(1 ,4 addition)-b-t-butyl acrylate; poly(butadiene(1 ,2 addition)-b-t-butyl acrylate; poly(butadiene(1 ,2 addition)-b-t-butyl methacrylate); poly(butadiene(1 ,4 addition)-b-ε-caprolactone); poly(butadiene((1 ,4 addition)-b-dimethylsiloxane); poly(butadiene(1 ,4 addition)-b-methyl methacrylate) (syndiotactic); poly(butadiene(1 ,2 addition)-b-methyl methacrylate); poly(butadiene(1 ,4 addition)-b- 4-vinyl pyridine; poly(isoprene(1 ,4 addition)-b-methyl methacrylate(syndiotactic)); poly(isoprene(1 ,4 addition)-b-2-vinyl pyridine; poly(isoprene(1 ,2 addition)-b-4-vinyl pyridine); and poly(isoprene(1 ,4 addition)-b-4-vinyl pyridine)), polyisobutylene based copolymers (e.g., poly(isobutylene-b-t-butyl methacrylate); poly(isobutylene-b-ε- caprolactone); poly(isobutylene-b-dimethylsiloxane); poly(isobutylene-b-methyl methacrylate); poly(isobutylene-b-4-vinyl pyridine), polystyrene based copolymers (e.g., poly(styrene-b-n-butyl acrylate); poly(styrene-b-t-butyl acrylate); poly(styrene-b- t-butyl acrylate), broad distribution; poly(styrene-b-disperse red 1 acrylate); poly(p- chloromethyl styrene-b-t-butyl acrylate); poly(styrene-b-N-isopropyl acrylamide); poly(styrene-b-n-butyl methacrylate); poly(styrene-b-t-butyl methacrylate); poly(styrene-b-cyclohexyl methacrylate); poly(styrene-b-2-cholesteryloxycarbonyloxy ethyl methacrylate); poly(styrene-b-N,N-dimethyl amino ethyl methacrylate); poly(styrene-b-ethyl methacrylate); poly(styrene-b-2-hydroxyethyl methacrylate); poly(styrene-b-2-hydroxypropyl methacrylate); poly(styrene-b-methyl methacrylate); poly(styrene-b-methylmethacrylate); poly(styrene-b-n-propyl methacrylate); poly(styrene-b-butadiene(1 ,4 addition)); poly(styrene-b-butadiene(1 ,2 addition)); poly(styrene-b-isoprene(1 ,4 addition)); poly(styrene-b-isoprene(1 ,2 addition or 3,4 addition)); poly(styrene-b-isoprene(1 ,4 addition)), hydrogenated; tapered block copolymer poly(styrene-b-butadiene); tapered block copolymer poly(styrene-b-

ethylene); poly(styrene-b-ε-caprolactone); poly(styrene-b-l-lactide); poly(styrene-b- dimethylsiloxane), trimethylsilane endgroup; poly(styrene-b-dimethylsiloxane), silanol endgroup; poly(styrene-b-methyl phenyl siloxane); poly(styrene-b- ferrocenyldimethylsilane); poly(styrene-b-t-butyl styrene); poly(styrene-b-t- butoxystyrene); poly(styrene-b-4-hydroxyl styrene); poly(4- amino benzyl-b-styrene); poly(styrene-b-2-vinyl pyridine); poly(styrene-b-4-vinyl pyridine); and poly(α- methylstyrene-b-4-vinyl pyridine), polyvinyl naphthalene) based copolymers (e.g., poly(2-vinyl naphthalene-b- n-butyl acrylate), poly(2-vinyl naphthalene-b- t-butyl acrylate); poly(2-vinyl naphthalene-b- methyl methacrylate); and poly(2-vinyl naphthalene-b- dimethylsiloxane)), polyvinyl pyridine) based copolymers (e.g., poly(2-vinyl pyridine-b-ε-caprolactone); poly(2-vinyl pyridine-b-methyl methacrylate); and poly(4-vinyl pyridine-b-methyl methacrylate)), poly (propylene oxide-b-ε- caprolactone) (e.g., poly (propylene oxide-b-ε-caprolactone)), polysiloxane based copolymers (e.g., poiy(dimethylsiloxane-b-n-butyl acrylate); poly(dimethylsiloxane-b- t-butyl acrylate); poly(dimethylsiloxane-b-t-butyl methacrylate); poly(dimethylsiloxane- b-ε-caprolactone); poly(dimethylsiloxane-b-6-(4'-cyanobiphenyl-4-yloxy)hexyl methacrylate); poly(dimethylsiloxane-b-1-ethoxy ethyl methacrylate); poly(dimethylsiloxane-b-hydroxy ethyl acrylate); and poly(dimethylsiloxane-b-methyl methacrylate)), adipic anhydride based copolymers (e.g., poly(ethylene oxide-b- adipic anhydride); polypropylene oxide-b-adipic anhydride); poly(dimethyl siloxane- b-adipic anhydride); poly(methyl methacrylate-b-adipic anhydride); and poly(2-vinyl pyridine-b-adipic anhydride)).

The following in an exemplary list of amphiphilic a-b-a triblock copolymers: poly((meth)acrylate) based triblock copolymers (e.g., poly(n-butyl acrylate-b-9,9-di-n- hexyl-2,7-fluorene -b-n-butyl acrylate); poly(t-butyl acrylate-b-9,9-di-n-hexyl-2,7- fluorene -b-t-butyl acrylate); poly(acrylic acid-b-9,9-di-n-hexyl-2,7-fluorene -b- acrylic acid); poly(t-butyl acrylate-b-methyl methacrylate-b-t-butyl acrylate); poly(t-butyl acrylate-b-styrene-b-t-butyl acrylate); poly(methyl methacrylate-b-butadiene(1 ,4 addition)-b-methyl methacrylate); poly(methyl methacrylate-b-n-butyl acrylate-b- methyl methacrylate); poly(methyl methacrylate-b-t-butyl acrylate-b-methyl methacrylate); poly(methyl methacrylate-b- t-butyl methacrylate acid-b-methyl methacrylate); poly(methyl methacrylate-b-methacrylic acid-b-methyl methacrylate); poly(methyl methacrylate-b-dimethylsiloxane-b-methyl methacrylate); poly(methyl methacrylate-b-9,9-di-n-hexyl-2,7-fluorene -b-methyl methacrylate); poly(methyl methacrylate-b-styrene-b-methyl methacrylate); poly(trimethylamonium iodide ethyl methacrylate-b-9,9-di-n-hexyl-2,7-fluorene-b- trimethylamonium iodide ethyl

methacrylate); poly(N,N-dimethyl amino ethyl methacrylate-b-9,9-di-n-hexyl-2,7- fluorene-b-N,N-dimethyl amino ethyl methacrylate); and poly(N,N-dimethyl amino ethyl methacrylate-b-propylene oxide-b-N,N-dimethyl amino ethyl methacrylate)), polybutadiene based triblock copolymers (e.g., poly(butadiene(1 ,4 addition)-b- styrene-b-butadiene(1 ,4 addition))), poly(oxirane) based triblock copolymers (e.g., poly(ethylene oxide-b-9,9-di-n-hexyl-2,7-fluorene -b-ethylene oxide); poly(ethylene oxide-b-propylene oxide-b-ethylene oxide); poly(ethylene oxide-b-styrene-b-ethylene oxide); and poly(propylene oxide-b-dimethyl siloxane-b-propylene oxide)), polylactone and polylactide diblock copolymers (e.g., poly(lactide-b-ethylene oxide-b- lactide); poly(caprolactone-b-ethylene oxide-b-caprolactone); and alpha,-ω diacrylonyl terminated poly(lactide-b-ethylene oxide-b-lactide)), polyoxazoline based triblock copolymers (e.g., poly(2-methyl oxazoline-b-dimethyl siloxane-b-2-methyl oxazoline))), polystyrene based triblock copolymers (e.g., poly(styrene-b-acrylic acid- b-styrene); poly(styrene-b-butadiene (1 ,4 addition) -b-styrene); poly(styrene-b- butadiene (1 ,2 addition) -b-styrene); poly(styrene-b-butylene-b-styrene); poly(styrene-b-n-butyl acrylate-b-styrene); poly(styrene-b-t-butyl acrylate-b-styrene); poly(styrene-b-9,9-di-n-hexyl-2,7-fluorene-b-styrene); poly(styrene-b-ethyl acrylate-b- styrene); poly(styrene-b-isoprene-b-styrene); poly(styrene-b-ethylene oxide-b- styrene); poly(styrene-b-4-vinyl pyridine-b-styrene); and poly(styrene-b-dimethyl siloxane-b-styrene)), polyvinyl pyridine) based triblock copolymers (e.g., poly(2-vinyl pyridine-b-butadiene(1 ,2 addition)-b-2-vinyl pyridine); poly(2-vinyl pyridine-b-styrene- b-2-vinyl pyridine); and poly(4-vinyl pyhdine-b-styrene-b-4-vinyl pyridine).

The following in an exemplary list of amphiphilic a-b-c triblock copolymers: poly(styrene-b-butadiene-b-methyl methacrylate) (e.g., poly(styrene-b-butadiene-b- methyl methacrylate)), poly(styrene-b-butadiene-b-2-vinyl pyridine) (e.g., poly(styrene-b-butadiene-b-2-vinyl pyridine)), poly(styrene-b-t-butyl acrylate-b-methyl methacrylate) (e.g., poly(styrene-b-t-butyl acrylate-b-methyl methacrylate)), poly(styrene-b-isoprene-b-glycidyl methacrylate) (e.g., poly(styrene-b-isoprene-b- glycidyl methacrylate)), poly(styrene-b-2-vinyl pyridine-b-ethylene oxide) (e.g., poly(styrene-b-2-vinyl pyridine-b-ethylene oxide)), poly(styrene-b-anthracene methyl methacrylate-b-methymethacrylate) (e.g., poly(styrene-b-anthracene methyl methacrylate-b-methymethacrylate)), poly(styrene-b-t-butyl acrylate-b-2-vinyl pyridine) (e.g., poly(styrene-b-t-butyl acrylate-b-2-vinyl pyridine)), and poly(styrene-b- t-butyl methacrylate-b-2-vinyl pyridine) (e.g., poly(styrene-b-t-butyl methacrylate-b-2- vinyl pyridine)).

The following in an exemplary list of amphiphilic funtionalized diblock and triblock copolymers: amino terminated poly(dimethylsiloxane-b-diphenylsiloxane); amino terminated poly(styrene-b-isoprene); amino terminated poly(ethylene oxide-b- lactone); hydroxy terminated poly(styrene-b-2-vinyl pyridine); hydroxy terminated polystyrene-b-poly(methyl methacrylate); α-hydroxy terminated poly(styrene-b- butadiene(1 ,2-addition)); 4-methoxy benzyolester terminated poly(butadiene-b- ethylene oxide) diblock copolymer; succinic acid terminated poly(butadiene-b- ethylene oxide) diblock copolymer; α,ω-disuccinimidyl succinate terminated poly(ethylene oxide-propylene oxide-ethylene oxide); cabinol at the junction of poly(styrene-b-isoprene(1 ,4 addition)); and silane at the junction of poly(styrene-b-2- vinyl pyridine).

In addition, the following is an exemplary list of amphiphilic block copolymers: poly(1-vinylpyrrolidone-co-vinyl acetate); poly(ethylene-co-propylene-co-5- methylene-2-norbornene); poly(styrene-co-acrylonitrile); poly(2-vinylpyridine-co- styrene); poly(ethylene-co-methacrylic acid) sodium salt; poly(acrylonitrile-co- butadiene-co-styrene); polyvinyl chloride-co-vinyl acetate-co-maleic acid); poly(ethylene-co-vinyl acetate); poly(ethylene-co-ethyl acrylate); poly(4-vinylpyridine- co-styrene); polyvinyl butyral-co-vinyl alcohol-co-vinyl acetate); poly(methyl methacrylate co-methacrylic acid); poly-(vinyl chloride-co-vinyl acetate-co- hydroxypropyl acrylate); Luviquat ^® HM 552; polyvinyl chloride-co-vinyl acetate-co- vinyl alcohol); poly(styrene-co-divinylbenzene); poly(DL-lactide-co-glycolide); poly(acrylonitrile-co-methyl acrylate); poly[(vinyl chloride-co-(1-methyl-4- vinylpiperazine)]; poly(2-isopropenyl-2-oxazoline-co-methyl methacrylate); poly(tetrafluoroethylene oxide-co-difluoromethylene oxide) α,ω-diol, ethoxylated; polytdimethylsiloxane-co-methyKS-hydroxypropyOsiloxanej-graf f-polyCethylene glycol) methyl ether; poly(acrylonitrile-co-methacrylonitrile); poly(ethylene-co-i-butene); poly(vinylidene fluoride co-hexafluoropropylene); poly(ethylene-co-i-octene); poly(ethylene-co-methyl acrylate); poly(acrylonitrile-co-butadiene), amine terminated; poly(perfluoropropylene oxide-co-perfluoroformaldehyde); poly(butyl methacrylate- co-isobutyl methacrylate); poly(styrene-co-maleic anhydride), partial isooctyl ester, cumene terminated; poly(acrylonitrile-co-butadiene-co-acrylic acid), dicarboxy terminated; polyvinyl alcohol-co-ethylene); poly(dimethylsiloxane-co- methylphenylsiloxane); poly(styrene-co-maleic anhydride); poly(Bisphenol A-co- epichlorohydrin); poly(styrene-co-butadiene); poly[(R)-3-hydroxybutyric acid-co-(ft)- 3-hydroxyvaleric acid]; polyvinyl alcohol-co-vinyl acetate-co-itaconic acid); poly(methylstyrene-co-indene), hydrogenated; poly(4-vinylphenol-co-2-hydroxyethyl

methacrylate); poly(styrene-co-maleic anhydride), cumene terminated; poly(methyl methacrylate-co-ethylene glycol dimethacrylate); poly(ethylene-co-propylene); poly(styrene-co-maleic acid), partial isobutyl/methyl mixed ester; poly(Bisphenol A- co-epichlorohydrin), glycidyl end-capped; poly(methyl methacrylate-co-methacrylic acid); poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-acrylonitrile); poly(propylene-co-tetrafluoroethylene); poly(butyl methacrylate-co-methyl methacrylate); poly(dimethylsiloxane-co-alkylmethylsiloxane); poly(acrylic acid-co- acrylamide) potassium salt; poly(oxymethylene-co-1 ,3-dioxepane); poly(chlorotrifluoroethylene-co-vinylidene fluoride); poly(melamine-co-formaldehyde), acrylated solution; poly(pentafluorostyrene-co-glycidyl methacrylate); poly(1 ,1 ,1 ,3,3,3-hexafluoroisopropyl methacrylate-co-glycidyl methacrylate); poly(2,2,3,4,4,4,-hexafluorobutyl methacrylate-co-glycidyl methacrylate); poly(2,2,3,3,3-pentafluoropropyl methacrylate-co-glycidyl methacrylate); poly[(propylmethacryl-heptaisobutyl-PSS)-co-(A7-butylmethacr ylate)]; poly(pyromellitic dianhydride-co-4,4'-oxydianiline), amic acid solution; poly(terf-butyl methacrylate-co- glycidyl methacrylate); poly[(propylmethacryl-heptaisobutyl-PSS)-co- hydroxyethylmethacrylate]; poly[(m-phenylenevinylene)-co-(2,5-dioctoxy-p- phenylenevinylene)]; poly[(methylmethacrylate)-co-(9-anthracenylmethyl methacrylate)]; poly[(methylmethacrylate)-co-(2-naphthylacrylate)]; poly[methylmethacrylate-co-(7-(4-trifluoromethyl)coumarin methacrylamide)]; poly[(methylmethacrylate)-co-(9-anthracenylmethyl acrylate)]; poly[(methylmethacrylate)-co-(9/-/-carbazole-9-ethylmethacry late)]; poly[(propylmethacryl-heptaisobutyl-PSS)-co-(methylmethacryl ate)]; poly[(isobutylene-atf-maleic acid), ammonium salt)-co-(isobutylene-atf-maleic anhydride)]; poly(ethylenecarbonyl-co-propylenecarbonyl); poly[4,5-difluoro-2,2- bis(trifluoromethyl)-1 ,3-dioxole-co-tetrafluoroethylene]; poly(dimethylsiloxane-co- diphenylsiloxane), trimethylsilyl terminated; poly(dimethylsiloxane-co- methylhydrosiloxane), trimethylsilyl terminated; poly(dimethylsiloxane-co- diphenylsiloxane), divinyl terminated; poly(styrene-co-methy! methacrylate); poly(styrene-co-α-methylstyrene); poly(1 ,4-cyclohexanedimethylene terephthalate- co-ethylene terephthalate); Amberjet™ 4200; poly[dimethylsiloxane-co-methyl(3- hydroxypropyl)siloxane]-graff-poly(ethylene glycol) [3-(trimethylammonio)propyl chloride] ether solution; poly[dimethylsiloxane-co-methyl(3-hydroxypropyl)siloxane]- graft-poly(ethylene/propylene glycol); poly(ethylene-co-butyl acrylate); poly(ethylene- co-ethyl acrylate-co-maleic anhydride); poly(ethyl methacrylate-co-methyl acrylate); poly(ethylene-co-1-butene-co-1-hexene); poly(melamine-co-formaldehyde),

isobutylated solution; poly[Bisphenol A carbonate-co-4,4'-(3,3,5- trimethylcyclohexylidene) diphenol carbonate]; poly(acrylamide-co-acry!ic acid); poly(styrene-co-maleic acid), partial sec-butyl/methyl mixed ester; poly(4- hydroxybenzoic acid-co-6-hydroxy-2-naphthoic acid); poly[butylene terephthalate-co- poly(alkylene glycol) terephthalate]; poly(ethylene-co-vinyl acetate-co-methacrylic acid); poly(melamine-co-formaldehyde), methylated; poly(acrylonitrile-co-butadiene), dicarboxy terminated; polyvinyl chloride-co-vinyl acetate-co-2-hydroxypropyl acrylate); poly(tetrafluoroethylene oxide-co-difluoromethylene oxide) α,ω-diol; poly(melamine-co-formaldehyde), butylated solution; poly[(phenyl glycidyl ether)-co- formaldehyde]; poly(acrylamide-co-diallyldimethylammonium chloride) solution; poly(tetrafluoroethylene-co-perfluoro(propylvinyl ether)); poly(4-vinylpyridine-co-butyl methacrylate); poly(dimer acid-co-alkyl polyamine); poly(1-vinylpyrrolidone-co-2- dimethylaminoethyl methacrylate), quaternized solution; poly(methyl methacrylate- co-ethyl acrylate); Luviquat ^® FC 550; poly(vinyltoluene-co-α-methylstyrene); poly(epichlorohydrin-co-ethylene oxide-co-allyl glycidyl ether); poly(dimethylsiloxane- co-methylhydrosiloxane); polybutadiene-graft-poly(methyl acrylate-co-acrylonitrile); poly(styrene-co-maleic anhydride), partial 2-butoxyethyl ester, cumene terminated; poly(dimethylamine-co-epichlorohydrin) solution; poly(ethylene-co-acrylic acid); poly(acrylamide-co-acrylic acid) partial sodium salt; poly(hexafluoropropylene oxide- co-difluoromethylene oxide) monoalkylamide; poly(1-vinylpyrrolidone-co-2- dimethylaminoethyl methacrylate) solution; poly(acrylic acid-co-maleic acid) sodium salt; poly(ethylene-co-acrylic acid, zinc salt); poly(ethylene-co-tetrafluoroethylene); poly(2,2,2-trifluoroethyl methacrylate-co-glycidyl methacrylate); poly(pentabromophenyl acrylate-co-glycidyl methacrylate); poly(2,2,3,3,4,4,4- heptafluorobutyl methacrylate-co-glycidyl methacrylate; poly[methylmethacrylate-co- (disperse yellow 7 methacrylate)]; poly(2,2,3,3-tetrafluoropropyl methacrylate-co- glycidyl methacrylate); poly(pentabromophenyl methacrylate-co- glycidylmethacrylate); poly[methylmethacrylate-co-(Disperse Orange 3 methacrylamide)]; poly[((S)-~()-1-(4-nitrophenyl)-2-pyrrolidinemethyl)acrylate -co- methylmethacrylate]; poly[(methylmethacrylate)-co-(Disperse Red 13 methacrylate)]; poly[(methylmethacrylate)-co-(Disperse Red 13 acrylate)]; poly[methylmethacrylate- co-(λ/-( 1-naphthyl)-λ/-phenylacrylamide)]; poly[(propylmethacryl-heptaisobutyl-PSS)- co-styrene]; poly(pyromellitic dianhydride-co-thionin); poly(ethylene glycol)-co-4- benzyloxybenzyl alcohol, polymer-bound; poly[(isobutylene-a/f-maleimide)-co- (isobutylene-atf-maleic anhydride)]; poly[dimethylsiloxane-co-(3- aminopropyl)methylsiloxane]; poly[dimethylsiloxane-co-[3-(2-(2-

hydroxyethoxy)ethoxy)propyl)methylsiloxane]; poly(vinylidene chloride-co- acrylonitrile-co-methyl methacrylate); poly(ethylene-co-1 ,2-butylene)diol; poly(DL- lactide-co-caprolactone) (40:60); poly(methyl methacrylate-co-butyl methacrylate); poly(tetrafluoroethylene oxide-co-difluoromethylene oxide)α,ω-diol bis(2,3- dihydroxypropyl ether); poly[dimethylsiloxane-co-(2-(3,4- epoxycyclohexyl)ethyl)methylsiloxane]; polyvinyl chloride-co-isobutyl vinyl ether); poly(indene-co-coumarone); poly(styrene-co-4-bromostyrene-co-divinylbenzene); poly(ethylene-co-butyl acrylate-co-carbon monoxide); polyvinyl acetate-co-butyl maleate-co-isobornyl acrylate) solution; poly(3,3',4,4'-benzophenonetetracarboxylic dianhydride-co-4,4'-oxydianiline/1 ,3-phenylenediamine), amic acid (solution); poly(tetrafluoroethylene-co-vinylidene fluoride-co-propylene); poly(ethylene-co- methacrylic acid) lithium salt; poly(styrene-co-butadiene-co-methyl methacrylate); poly(vinylidene chloride-co-vinyl chloride); poly(styrene-co-maleic acid), partial isobutyl ester; poly[4,4'-methylenebis(phenyl isocyanate)-alt-1 ,4- butanediol/poly(ethylene glycol-co-propylene glycol/polycaprolactone]; poly(ethylene- co-methacrylic acid); poly(isobutylene-co-maleic acid) sodium salt; poly(ethylene-co- methacrylic acid) zinc salt; poly(4-styrenesulfonic acid-co-maleic acid) sodium salt; poly(acrylonitrile-co-butadiene-co-acrylic acid), glycidyl methacrylate diester; poly(urea-co-formaldehyde), butylated solution; poly(ethylene-co-methyl acrylate-co- glycidyl methacrylate); poly[(phenyl glycidyl ether)-co-dicyclopentadiene]; poly[(o- cresyl glycidyl ether)-co-formaldehyde]; poly(urea-co-formaldehyde), methylated; poly(acrylic acid-co-maleic acid) solution; poly(3-hydroxybutyric acid-co-3- hydroxyvaleric acid); poly(p-toluenesulfonamide-co-formaldehyde); poly(styrene-co- allyl alcohol); poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-styrene); poly(acrylonitrile-co-butadiene); poly(4-vinylphenol-co-methyl methacrylate); polyldimethylsiloxane-co-methyKS-hydroxypropylJsiloxanel-gra ff-po^ethylene-ran- propylene glycol) methyl ether; poly(hexafluoropropylene oxide-co-difluoromethylene oxide) monoamidosilane; poly(dimethylamine-co-epichlorohydrin-co- ethylenediamine) solution; poly(ethylene-co-butyl acrylate-co-maleic anhydride); poly(trimellitic anhydride chloride-co-4,4'-methylenedianiline); poly[methylmethacrylate-co-(Disperse Orange 3 acrylamide)]; poly[((S)-~()-1-(4- Nitrophenyl)-2-pyrrolidinemethyl)methacιγlate-cc>-methy lmethacrylate]; poly[(propylmethacryl-heptaisobutyl-PSS)-co-(f-butylmethacry late)]; poly[(methylmethacrylate)-co-(2-naphthylmethacrylate)]; poly[methylmethacrylate-co- (fluoresceinO-acrylate)]; poly[methylmethacrylate-co-(fluoresceinθ-methacrylate)]; poly{[2-[2',5'-bis(2"-ethylhexyloxy)phenyl]-1 ,4-phenylenevinylene]-co-[2-methoxy-5-

(2'-ethylhexyloxy)-1 ,4-phenylenevinylene]}; poly[(methylmethacrylate)-co-(Disperse Red 1 acrylate)]; poly(4-hydroxy benzoic acid-co-ethylene terephthalate); poly^inylidene chloride-co-acrylonitrile); poly(dimethylsiloxane-co-diphenylsiloxane), dihydroxy terminated; poly(1 ,4-butylene adipate-co-1 ,4-butylene succinate), extended with 1 ,6-diisocyanatohexane; poly(dicyclopentadiene-co-p-cresol); poly[ethyl acrylate-co-methacrylic acid-co-3-(1 -isocyanato-1 -methylethyl)-α- methylstyrene], adduct with ethoxylated nonylphenol solution; poly(epichlorohydrin- co-ethylene oxide); poly(Bisphenol A-co-4-nitrophthalic anhydride-co-1 ,3- phenylenediamine); poly(ethylene-co-methyl acrylate-co-acrylic acid); poly(propylene-co-i-butene); Nylon 6/66; poly(ethylene-co-acrylic acid) sodium salt; poly(ethylene-co-vinyl acetate-co-carbon monoxide); poly(melamine-co- formaldehyde), methylated/butylated (55/45); poly(maleic acid-co-olefin) sodium salt solution; poly(tetrafluoroethylene oxide-co-difluoromethylene oxide) α,ω- diisocyanate; poly(lauryl methacrylate-co-ethylene glycol dimethacrylate); poly[(phenyl isocyanate)-co-formaldehyde]; poly[2,6-bis(hydroxymethyl)-4- methylphenol-co-4-hydroxybenzoic acid]; poly(tetrafluoroethylene oxide-co- difluoromethylene oxide) α,ω-dicarboxylic acid; poly[methylmethacrylate-co- (Disperse yellow 7 acrylate)]; poly[(methylmethacrylate)-co-(9H-carbazole-9- ethylacrylate)]; poly[methylmethacrylate-co-(λ/-(1 -naphthyl)-λ/- phenylmethacrylamide)]; poly[(methylmethacrylate)-co-(Disperse Red 1 methacrylate)]; poly(L-lactide-co-caprolactone-co-glycolide); poly[methylmethacrylate-co-(7-(4-thfluoromethyl)coumarin acrylamide)]; poly[dimethylsiloxane-co-methyl(3,3,3-thfluoropropyl)siloxan e]; poly[dimethylsiloxane-co-methyl(stearoyloxyalkyl)siloxane]; poly(hexafluoropropylene oxide-co-difluoromethylene oxide) alcohol, ethoxylated phosphate; poly(ethylene-co- 1 ,2-butylene) mono-ol; poly[dimethylsiloxane-co-methyl(3-hydroxypropyl)siloxane]- graff-tetrakis(1 ,2-butylene glycol); poly(1 ,4-butylene adipate-co-polycaprolactam); poly(vinyl acetate-co-crotonic acid); poly(tert-butyl acrylate-co-ethyl acrylate-co- methacrylic acid); poly(i-vinylpyrrolidone-co-styrene); poly(tetrafluoroethylene oxide- co-difluoromethylene oxide)-α,ω-bis(methyl carboxylate); poly(vinylidene chloride-co- methyl acrylate); poly(acrylonitrile-co-vinylidene chloride-co-methyl methacrylate); poly(styrene-co-maleic anhydride), partial cyclohexyl/isopropyl ester, cumene terminated; poly(4-ethylstyrene-co-divinylbenzene); poly(dimethylsiloxane-co-dimer acid), bis(perfluorododecyl) terminated; poly(styrene-co-maleic anhydride), partial propyl ester, cumene terminated; poly(dimer acid-co-ethylene glycol), hydrogenated; poly(ethylene-co-glycidyl methacrylate); poly[dimethylsiloxane-co-methyl(3-

hydroxypropyl)siloxane]-graff-poly(ethylene glycol) 3-aminopropyl ether; poly(dimer acid-co-1 ,6-hexanediol-co-adipic acid), hydrogenated; poly(3,3',4,4'- biphenyltetracarboxylic dianhydride-co-1 ,4-phenylenediamine), and amic acid solution; and poly[λ/,λ/'-bis(2,2,6,6-tetrannethyl-4-piperidinyl)-1 ,6-hexanediamine-co- 2,4-dichloro-6-morpholino-1 ,3,5-triazine].

In particular, the block copolymer may comprise an ABC triblock structure having a poly-butylacrylate segment, a poly-ethylacrylate segment, and a poly- methacrylic acid segment, for example. The block copolymer can include a diblock and/or triblock copolymer having two or more different poly-aliphatic-acrylate segments. In addition, the block copolymer can include a diblock and/or triblock copolymer having two or more poly-alkyl-acrylate segments.

The nanostructure can be attached to a probe molecule. The probe molecule can be any molecule capable of being linked to the nanostructure either directly or indirectly via a linker. The probe molecule can be attached by any stable physical or chemical association to the nanostructure directly or indirectly by any suitable means.

In one embodiment, the probe molecule has an affinity for one or more target molecules (e.g., cancer cell) for which detection (e.g., determining the presence of and/or proximal position within the vessel (body)) is desired. If, for example, the target molecule is a nucleic acid sequence, the probe molecule should be chosen so as to be substantially complementary to the target molecule sequence, such that the hybridization of the target and the probe occurs. The term "substantially complementary," means that the probe molecules are sufficiently complementary to the target sequences to hybridize under the selected reaction conditions. The probe molecule and the target molecule can include, but are not limited to, polypeptides (e.g., protein such as, but not limited to, an antibody (monoclonal or polyclonal)), nucleic acids (both monomeric and oligomeric), polysaccharides, sugars, fatty acids, steroids, purines, pyrimidines, drugs (e.g., small compound drugs), ligands, or combinations thereof. Advantageously, the probe is an antibody or a ligand compatible with, and capable of binding to, a target molecule on the surface of a cell such as, but not limited to, a cancer cell.

Use of the phrase "polypeptide" or "protein" is intended to encompass a protein, a glycoprotein, a polypeptide, a peptide, and the like, whether isolated from nature, of viral, bacterial, plant, or animal (e.g., mammalian, such as human) origin, or synthetic, and fragments thereof. A preferred protein or fragment thereof

includes, but is not limited to, an antigen, an epitope of an antigen, an antibody, or an antigenically reactive fragment of an antibody.

In one embodiment, the nanostructure can include at least two different types of probes, one being a targeting probe that targets certain cells or compounds associated with a condition and/or disease, while the second probe is an siRNA intended to modify the expression of a gene. In this manner, the nanostructure acts as a detection component, a delivery component to the cells of interest, and a delivery component for the siRNA. The detection of the nanoparticle can be used to ensure the delivery of the nanostructure to its intended destination, to provide a research tool for the optimization of siRNA modification of a gene expression in a cell, as well as the quantity of nanostructures delivered to the destination.

The present disclosure provides methods of targeting one or more target cells in a sample or a subject (e.g., mammal, human, cat, dog, horse, etc.), and in particular, to deliver an siRNA to a target cell in vivo. For example, the nanostructure can be used to detect and modify the proliferation of tumor cells in an animal using the nanostructures, according to the present disclosure.

It should also be noted that nanostructures could be used for the detection of, as part of treatment (e.g., drug delivery), as an indication of delivery to one or more targets (e.g., cancers), and combinations thereof, conditions and/or diseases such as, but not limited to, cancers, tumors, neoplastic diseases, autoimmune diseases, inflammatory diseases, metabolic conditions, neurological and neurodegenerative diseases, viral diseases, dermatological diseases, cardiovascular diseases, an infectious disease, and combinations thereof.

In one embodiment, a single nanoparticle coated with block copolymers, or nanoparticle-polymer composites containing one or more nanoparticles and at least one species of siRNA, can be injected into subjects (e.g., humans, domesticated animals, and cattle) as a probe or deliver the siRNA to a primary tumor. These nanostructures can be linked to a bio-compatible compounds (e.g., PEG and dextran) for long-circulating "passive targeting" reagents, and/or linked to bio-affinity probes (e.g., antibody, antigen, peptide, oligonucleotide, small molecule ligand, and drugs) for "active" targeting of primary tumor.

It should be noted that a cell can be pre-labeled (e.g., in vitro and in vivo) with nanostructures and/or microstructures. For example, cells can be labeled with nanoparticle-block copolymer microstructures in vitro through immuno staining, adsorption, microinjection, cell uptake, and the like. The cells then can be monitored in vitro, or traced in vivo with the nanoparticles as a tracer, fluorescence, magnetic,

combinations thereof, and the like, while the expression of a gene may be modified by the siRNA.

Block copolymers can be used to control the degradation of nanoparticle. For example, block copolymers can be used to either protect (make bio-compatible) the nanoparticle against degradation in biological conditions, especially for in vivo applications, or control the degradation rate/degree of the nanostructure, by varying the molecular structure of the block copolymer.

Now having described the embodiments of the nanostructure in general, the following is a non-limiting illustrative example of an embodiment of the present disclosure. This example is not intended to limit the scope of any embodiments of the present disclosure, but rather is intended to provide specific experimental conditions and results. Therefore, one skilled in the art would understand that many experimental conditions can be modified, but it is intended that these modifications be within the scope of the embodiments of the present disclosure. Multifunctional nanoparticle probes based on semiconductor quantum dots

(QDs) have been developed for cancer targeting and imaging in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer, and linking this amphiphilic polymer to tumor-targeting ligands and drug- delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mice indicate that the QD probes can be delivered to tumor sites by both enhanced permeation and retention and by antibody binding to cancer-specific cell surface biomarkers. The use of both subcutaneous injection of QD-tagged cancer cells and systemic injection of multifunctional QD probes resulted in the sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. This example also reports the integration of a whole-body macro-illumination system with wavelength-resolved spectral imaging for efficient background removal and precise delineation of weak spectral signatures. These results raise new possibilities for ultrasensitive and multiplexed imaging of molecular targets in vivo.

As schematically illustrated in FIGS. 1A and 8, core-shell CdSe-ZnS quantum dots may be protected by both a coordinating ligand (TOPO) and an amphiphilic polymer coating. Due to strong hydrophobic interactions between TOPO and the polymer hydrocarbon, these two layers "bond" to each other and form a hydrophobic protection structure that is resistant against hydrolysis and enzymatic degradation even under complex in vivo conditions. In contrast to simple polymers and amphiphilic lipids used in previous studies, the methods described herein use a high- molecular-weight (MW = about 100 kD) copolymer with an elaborate ABC triblock

structure and a grafted 8-carbon (C-8) alkyl side chain. This triblock polymer includes a polybutylacrylate segment (hydrophobic), a polyethylacrylate segment (hydrophobic), a polymethacrylic acid segment (hydrophilic), and a hydrophobic hydrocarbon side chain. This polymer can disperse and encapsulate single TOPO- capped QDs via a spontaneous self-assembly process. As a result, the QDs are protected to such a degree that their optical properties (e.g., absorption spectra, emission spectra, and fluorescence quantum yields) did not change in a broad range of pH (1 to 14) and salt conditions (0.01 to 1 M) or after harsh treatment with 1.0 M hydrochloric acid (PEG-linked QDs). Under in vivo conditions, QD probes can be delivered to tumors by both a passive targeting mechanism and an active targeting mechanism. In the passive mode, macromolecules and nanometer-sized particles are accumulated preferentially at tumor sites through an enhanced permeability and retention (EPR) effect. This effect is believed to arise from two factors: (a) angiogenic tumors that produce vascular endothelial growth factors (VEGF) that hyperpermeabilize the tumor-associated neovasculatures and cause the leakage of circulating macromolecules and small particles; and (b) tumors lack an effective lymphatic drainage system, which leads to subsequent macromolecule or nanoparticle accumulation. For active tumor targeting, antibody-conjugated quantum dots have been used to target a prostate-specific cell surface antigen, PSMA. Previous research has identified PSMA as a cell surface marker for both prostate epithelial cells and neovascular endothelial cells. PSMA has been selected as an attractive target for both imaging and therapeutic intervention of prostate cancer. Accumulation and retention of PSMA antibody at the site of tumor growth is the basis of radioimmunoscintigraphic scanning (e.g., ProstaScint scan) and targeted therapy for human prostate cancer metastasis.

For example, the QD -siRNA probes according to the present disclosure may be conjugated to a PSMA monoclonal antibody, J591 , which recognizes the extracellular domain of PSMA, were first evaluated for binding to PSMA in prostate cancer cell lines, lmmunocytochemical data confirmed strong and specific binding of the PSMA Ab J591-conjugated QD probes to a human prostate cancer cell line, C4- 2, which is known to express PSMA on the cell surface. Control studies using QD- PEG (without antibody) showed only a low level of nonspecific cell binding to C4-2 cells. Additional control studies using PC-3 cells, a PSMA negative human prostate cancer cell line, also showed the absence of QD binding. These results establish

that the PSMA antibody-QD conjugates retain their PSMA binding activity and specificity.

To investigate the behavior of QD-PSMA Ab conjugated probes in living animals, the following were examined: their specific uptake and retention, background or nonspecific uptake, blood clearance, and organ distribution as well as their relationship to QD surface modifications. Nonspecific QD uptake and retention took place primarily in the liver and the spleen, with little or no QD accumulation in the brain, the heart, the kidney, or the lung. This pattern of in vivo organ uptake and distribution was similar to that of dextran-coated magnetic iron oxide nanoparticles. For polymer-encapsulated QDs with excess COOH groups, no tumor targeting was observed, indicating nonspecific organ uptake and rapid blood clearance. For polymer-encapsulated QDs with surface PEG groups, the rate of organ uptake was reduced and the length of blood circulation was improved, leading to slow accumulation of the nanoparticles in the tumors. For QDs encapsulated by PEG and bioconjugated with PSMA antibody, the nanoparticles were delivered and retained by the tumor xenografts, but nonspecific liver and spleen uptake was still apparent.

The QD nanostructures according to the present disclosure are particularly advantageous because they are highly stable against in vivo degradation, are efficient in the delivery of siRNAs to the interior of a cell and the progress in the uptake of the particles both by a host animal or human and the tissues and cells therein may be tracked . An important feature is a high-molecular-weight triblock copolymer, which completely encapsulates TOPO-QDs and forms a stable hydrophobic protection layer around single QDs.

On the hydrophilic surface of this polymer layer, there is a large number of functional groups (e.g., about 400 to 500 carboxylic acids groups) that may be derivatized and which allow the attachment of both diagnostic and therapeutic agents. With small-molecule ligands such as synthetic organic molecules, short oligonucleotides and peptides, many copies of the same ligand can be linked to single dots, leading to multivalent QD-target binding. Previous research has shown that properly designed multivalent ligands can increase the binding affinity by 10 orders of magnitude.

The polymer-encapsulated QD probes are in an excellent size range for in vivo tumor targeting. With small peptide-dye conjugates, rapid extravasation often leads to blood clearance of the probe in less than one minute. The circulation or retention time can be improved by attaching small probes to macromolecules or nanoparticles, a strategy widely used in drug delivery research. Indeed, the

described work indicates that PEG-shielded QDs are able to circulate in blood for as long as about 48-72 hours, with a half decay time of about 5-8 hours. At the same time, these probes are small enough for efficient binding to cell surface receptors, for internalization through endocytosis or peptide translocation, and for passing through the nuclear pores to enter the cell nucleus (using nuclear-localization peptides) (FIG. 8A, top right). However, the penetration depth of QDs into solid tumors will be limited, at least in part, by their nanometer sizes.

The unique optical properties of QDs also provide new opportunities for multicolor imaging and multiplexing. For example, multicolor imaging will allow intensity ratioing, spatial colocalization, and quantitative target measurements at metastatic tumor sites. Optical encoding strategies are also possible based on the use of multiple colors and multiple intensity levels. This combinatorial approach has been demonstrated for tagging a large number of genes, proteins, and small- molecule libraries. In addition to wavelength and intensity, lifetime fluorescence imaging represents a new dimension. Because the excited state lifetimes (about 20- 50 ns) of QDs are nearly one order magnitude longer than that of organic dyes (about 2-5 ns), QD probes should be suitable for fluorescence lifetime imaging (FLIM) of cells, tissue specimens, and living animals.

The current use of orange/red-emitting quantum dots is not optimized for tissue penetration or imaging sensitivity. Extensive work in tissue optics has shown deep tissue imaging (millimeters to centimeters) requires the use of far-red and near- infrared light in the spectral range of about 650-900 nm. This wavelength range provides a "clear" window for in vivo optical imaging because it is separated from the major absorption peaks of blood and water. Based on tissue optical calculations, it is estimated that the use of near-infrared-emitting quantum dots should improve the tumor imaging sensitivity by at least 10-fold, allowing sensitive detection of about 10- 100 cancer cells. Toward this goal, a new class of alloyed semiconductor quantum dots having cadmium selenium telluride, with tunable fluorescence emission up to about 850 nm and quantum yields up to 60%. Together with core-shell CdTeCdSe type-ll materials, the use of near-infrared-emitting QDs should bring major improvements in tissue penetration depth and cell detection sensitivity.

A remaining issue is the QD's toxicity and metabolism in vivo. Recent work indicates that CdSe QDs are highly toxic to cells under UV illumination for extended periods of time. This is understandable because UV-irradiation often dissolves the semiconductor particles, releasing toxic cadmium ions into the medium. In the absence of UV irradiation, QDs with a stable polymer coating are essentially nontoxic

to cells (no effect on cell division or ATP production). In vivo studies also confirmed the nontoxic nature of stably protected QDs. This is perhaps not surprising because the polymer protection layer is so stable that the QD core would not be exposed to the outside environment. Consistent with this conclusion, the uptake of dextran- protected iron oxide nanoparticles (up to 10 million particles per cell) does not significantly reduce cell viability, and that the injection of micelle-protected QDs (up to 2 billion per embryo cell) does not affect frog embryo development. Up to 3 million QDs in a single cancer cell did not appreciably reduce its viability or growth.

At the present, however, little is known about the mechanism of metabolism or clearance of QD probes injected into living animals. For the polymer- encapsulated QDs, chemical or enzymatic degradations of the semiconductor cores are unlikely to occur. But the polymer-protected QDs might be cleared from the body by slow filtration and excretion through the kidney.

The present disclosure involves the development of a new class of polymer- encapsulated QD probes having siRNA molecules attached thereto, and optionally a target probe, for the efficient delivery of the siRNA to a cell for modification of a gene expression in the cell. The compositions of the present disclosure are advantageous for cancer targeting and imaging in vivo. These probes are bright, stable, and have a versatile triblock copolymer structure that is well suited for conjugation to additional diagnostic and therapeutic agents. In vivo imaging results indicate the QD probes can be targeted to tumor sites through both passive and active mechanisms, but passive targeting is much slower and less efficient than active targeting. When combined with wavelength-resolved imaging, the QD probes allow sensitive and multicolor imaging of cancer cells in living animals. The use of near-infrared-emitting quantum dots should improve both the tissue penetration depth and imaging sensitivity. In accordance with the described study, quantum dots could be integrated with targeting, imaging, and therapeutic agents to develop "smart" nanostructures for noninvasive imaging, diagnosis, and treatment of cancer, cardiovascular plaques, and neurodegenerative disease. The structural design of the novel class of siRNA carriers of the present disclosure are composed of a QD core, a hydrophobic protection layer and a periodic amphiphilic polymer layer with precisely controlled ratios of carboxylic anions and tertiary amine cations as schematically shown in FIG. 1A. In these carriers, siRNA molecules are advantageously absorbed onto a QD surface via electrostatic attractions.

Amphiphilic copolymers have been reported as useful for QD solublization and bioconjugation for cellular and molecular imaging applications. These structures utilize a dense layer of carboxylic groups on the surface to minimize non-specific interaction between nanoparticles and cells (which are also negatively charged). In the carriers of the present disclosure, the carboxylate groups are partially derivatized into tertiary amines to provide an overall positive charge on a QD surface, thereby enabling the capture of negatively charged siRNA molecules, as illustrated in FIG. 1 B.

While it would appear advantageous to increase the amount of positive charge carried by the QDs so that they may carry more siRNA molecules, it has been found that the zeta potential or the ratio of the amine / carboxylate is critical in the nanoparticle stability, siRNA transfection efficiency and cytotoxicity effect. At a zeta potential value of +19.4 mV before siRNA binding and a zeta potential of about +8.5 mV after binding, the QDs offer the highest siRNA delivery efficiency, yet still maintain good stability in biologic buffers and cell culture media. In contrast, less tertiary amine modification results in low or no transfection effect, whereas a higher modification degree leads to particle aggregation in cell culture media and increased toxicity. A key finding is that even with the 'zwitterionic' surface, QDs do not form aggregates in solution. While not wishing to be bound by any one theory, this colloidal stability could be attributed to the even distribution of positive and negative charges on QD surface rather than formation of positive and negative patches.

Dynamic light scattering measurements show that the QD-polymer hybrid structure has a hydrodynamic radius of 6.5 nm, as shown in FIG. 1C, which agrees with previously reported values [NatBiotech 22, 969, which is incorporated hereby by reference]. Considering a QD core may be 5 nm in diameter, as determined from TEM images shown in FIG. 1 D, 1 nm hydrophobic ligands (TOPO) on both sides, and 1.5 nm amphiphilic polymer coating on top of the TOPO molecules, the overall particle size is about 10 nm in diameter. The larger hydrodynamic radius suggests a strong interaction between the highly-charged surface coating polymer and solvent. Previous experiments focused on DNA plasmids, which are much bigger (approximately 1 ,000 KDa) than the nanoparticles of the present disclosure.

As a result of this size mismatch, it requires many nanoparticles to work together, and forming clusters with the DNA plasmid for successful transfection. This aggregation process often leads to nanoparticle aggregates of heterogeneous size distribution and substantially limits their uses as an in vivo delivery vehicle. In contrast, for small siRNA molecules, QDs may serve as a structure scaffold for

siRNA assembly, and they do not form aggregates, as shown by dynamic light scattering measurements. On average, each QD nanocrystal carries 1-2 siRNA molecules for the optimum transfection efficiency. With more siRNA molecules attached, the overall positive charge on QD surface would be reduced by the negatively charged oligonucleotides. This would prevent the final structure from adsorbing onto a cell surface; on the other hand, less siRNAs would result in competition between free QDs and QD-siRNA complexes.

Based on the geometric/size constraints and the ligand coupling efficiencies (about 40-50%, experimentally determined by using fluorescently labeled ligands), it has been estimated that each dot contains about 200 TOPO molecules, about 4 to 5 triblock copolymer molecules, about 5 to 6 PEG molecules, and about 5 to 6 antibody molecules. High-sensitivity fluorescence imaging showed "blinking" signals when a dilute solution (10 ^'12 M) of the QD bioconjugate was spread on a glass surface. This blinking behavior is characteristic of single quantum systems such as single dye molecules and single QDs, indicating that the triblock copolymer has efficiently dispersed the dots into single particles. Preliminary TEM results also revealed that the QD probes consisted of single particles, with little or no aggregation. It is worth noting, however, that QD blinking has no adverse implications for in vivo tumor imaging because the tumor cells are labeled with a large population (up to millions) of QDs, far from the single-dot regime.

Geometric calculation also indicates that the total surface area of a QD of about 6 nm in radius is about 100 nm ² , and a typical siRNA double strand is about 7 nm in length and can cover about 14 nm ² on a single QD. With 1-2 siRNA molecules only, QDs would still have enough surface area to efficiently interact with cells, while more adsorbed siRNAs could block the angles QDs approaching cells.

It is contemplated that the nanostructures of the present disclosure are advantageously effective when contacted with a variety of cell types. Specific targeting to a particular cells or in vivo can also be achieved by simultaneously linking a QD with siRNAs and a targeting probe. As schematically shown in FIG. 8, for example, the siRNA molecules can be attached to the QD surface and targeting probes may be linked to QDs via a PEG spacer.

One aspect of the disclosure encompasses nanostructures comprising a quantum dot, a hydrophobic protection structure comprising a capping ligand and an amphiphilic copolymer, wherein the hydrophobic protection structure encapsulates the quantum dot, and an siRNA molecule. In one embodiment of the nanostructures

according to the disclosure, the siRNA may be disposed on the hydrophobic protection surface.

In various embodiments of this aspect of the disclosure, the amphiphilic copolymer may be selected from the group consisting of an amphiphilic block copolymer, an amphiphilic random copolymer, an amphiphilic alternating copolymer, an amphiphilic periodic copolymer, or any combination thereof. In one embodiment of the disclosure, the amphiphilic copolymer may be a block copolymer is selected from the group consisting of a diblock copolymer, a triblock copolymer, and a combination thereof. In another embodiment, amphiphilic block copolymer may comprise an ABC triblock structure having grafted 8-carbon alkyl side chains. In one particularly advantageous embodiment of the disclosure, the ABC triblock structure may comprise a poly-butylacrylate segment, a poly-ethylacrylate segment, and a poly-methacrylic acid segment.

In the various embodiments of the present disclosure, the quantum dot may comprise a core and a cap, wherein the core of the quantum dot may be selected from the group consisting of a HA-VIA semiconductor, a IIIA-VA semiconductor, a IVA-IVA semiconductor, and a IVA-VIA semiconductor.

In one embodiment, the core of the quantum dot is a IIA-VIA semiconductor. In other embodiments of the disclosure, the core of the quantum dot may be selected from the group consisting of CdS, CdSe, CdTe, ZnSe, ZnS, PbS, PbSe, or a combination thereof.

In one embodiment of the nanostructure of the disclosure, the quantum dot may comprise CdTe/CdSe. In another embodiment, the core of the quantum dot is CdSe. In the embodiments of the nanostructures of the disclosure, the cap may be a

IIA-VIA semiconductor of high band gap. In one embodiment, the cap is ZnS.

In the embodiments of the nanostructures of the present disclosure, the capping ligand may comprise tri-octylphosphine oxide.

In this aspect of the nanostructures of the disclosure, the nanostructures may further comprise a targeting probe, wherein the probe may be selected from the group consisting of an antibody, a polypeptide, a polynucleotide, a drug molecule, an inhibitor compound, or a combination thereof, and wherein the targeting probe may have an affinity for a marker on the surface of a target cell.

In the embodiments of the present disclosure, the targeting probe may be disposed on the hydrophobic protection structure. In one embodiment, the probe is a tumor-targeting ligand.

Another aspect of the present disclosure is methods of preparing a nanostructure, comprising providing a nanoparticle, forming a hydrophobic protection structure around the nanoparticle that includes at least one compound selected from a capping ligand, an amphiphilic copolymer, and combinations thereof, and contacting the nanoparticle with an siRNA, whereby the siRNA attaches to the outer surface of the nanoparticle.

One embodiment of this aspect of the disclosure further comprises attaching a cell targeting probe to the hydrophobic protection structure, wherein the probe is selected from an antibody, a polypeptide, a polynucleotide, a drug molecule, an inhibitor compound, or a combination thereof.

In the embodiments of this aspect of the disclosure, the nanoparticle is a quantum dot wherein the core of the quantum dot may be selected from the group consisting of a MA-VIA semiconductor, a IMA-VA semiconductor, a IVA-IVA semiconductor, and a IVA-VIA semiconductor, and wherein the hydrophobic protection structure may comprise a capping ligand and an amphiphilic copolymer, and wherein the amphiphilic copolymer may be a block copolymer selected from a diblock copolymer, a triblock copolymer, and combinations thereof.

In embodiments of the method of the disclosure, the quantum dot may comprise a core and a cap, wherein the core of the quantum dot may be selected from the group consisting of a MA-VIA semiconductor, a IMA-VA semiconductor, a IVA-IVA semiconductor, and a IVA-VIA semiconductor. In one embodiment, the core of the quantum dot is a MA-VIA semiconductor. In embodiments of the disclosure, the core of the quantum dot may be selected from the group consisting of CdS, CdSe, CdTe, ZnSe, ZnS, PbS, PbSe, or a combination thereof. In one embodiment, the quantum dot comprises CdTe/CdSe. In another embodiment, the core of the quantum dot is CdSe.

In embodiments of the disclosure, the cap may be a MA-VIA semiconductor of high band gap. In one embodiment of the disclosure, the cap is ZnS.

In embodiments of the method according to the disclosure, the capping ligand may comprise tri-octylphosphine oxide, and the amphiphilic block copolymer may be an ABC triblock structure comprising a poly-butylacrylate segment, a poly- ethylacrylate segment, and a poly-methacrylic acid segment.

Another aspect of the present disclosure is a method of modifying the expression of a gene in a target cell, comprising contacting a target cell with at least one nanostructure comprising a semiconductor quantum dot, a hydrophobic protection structure including at least one compound selected from a capping ligand,

an amphiphilic copolymer, or a combination thereof, wherein the hydrophobic protection structure encapsulates the quantum dot, and an siRNA species, wherein the molecules of the siRNA are disposed on the exterior surface of the nanostructure, and wherein the nanostructure enters the cell, and wherein the siRNA species modifies the expression of a gene to which the siRNA is targeted. In embodiments of this aspect of the disclosure, the nanostructure may further comprise a cell targeting probe, wherein the cell targeting probe directs the nanoparticle to the targeted cell. In embodiments of the methods according to this aspect of the disclosure, the target cell may be a cultured cell line or a cell in a tissue of a host.

Embodiments of this aspect of the disclosure may further comprise detecting the nanostructure in a cell or in a host. In one embodiment, the nanostructure is detected by fluorescence.

The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.

It should be emphasized that the embodiments of the present disclosure, particularly, any "preferred" embodiments, are merely possible examples of the implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure, and the present disclosure and protected by the following claims.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 ⁰ C and 1 atmosphere.

Example 1

Core-shell quantum dots (ZnS-capped CdSe) were synthesized according to procedures known in the art. A high-temperature coordinating solvent, tri-n- octylphosphine oxide (TOPO), was used for the synthesis, leading to high-quality QDs that were capped by a monolayer of TOPO molecules. These dots were highly fluorescent (about 60% quantum yields) and monodispersed (about 5% size variations). QD-encoded microbeads were prepared by using 0.5 μm mesoporous microbeads in butanol, and were isolated and purified as reported previously. A thblock copolymer consisting of a poly-butylacrylate segment, a poly- ethylacrylate segment, and a poly-methacrylic acid segment was purchased from Sigma (St. Louis, MO). At a molecular weight of about 100,000 daltons, this polymer contains more than 1000 total monomer units, with a weight distribution of 23% methacrylic acid and 77% combined butyl and ethyl acrylates. For encapsulating QDs, about 25% of the free carboxylic acid groups were derivatized with octylamine (a hydrophobic side chain). Thus, the original polymer dissolved in dimethylformamide (DMF) was reacted with n-octylamine at a polymer/octylamine molar ratio of 1 :40, using ethyl-3-dimethyl amino propyl carbodiimide (EDAC, 3-fold excess of n-octylamine) as a cross-linking reagent. The product yields were generally greater than 90% due to the high EDAC coupling efficiency in DMF (determined by a change of the free octylamine band in thin layer chromatography). The reaction mixture was dried with a ratovap (Rotavapor R-3000, Buchi Analytical Inc, Delaware). The resulting oily liquid was precipitated with water, and was rinsed with water 5 times to remove excess EDAC and other by-products. After vacuum drying, the octylamine-g rafted polymer was re-suspended in an ethanol/chloroform mixture, and was stored for use. Example 2

Surface modification and bioconjugation: Using a 3:1 (v/v) chloroform/ethanol solvent mixture, TOPO-capped quantum dots were encapsulated by the amphiphilic tri-block polymer. A polymer-to-QD ratios of 5 to 10 was used because molecular geometry calculations indicated that at least 4 polymer molecules would be required to completely encapsulate one quantum dot. Indeed, stable encapsulation (e.g., no aggregation) was not achieved at polymer/dot ratios less than 4:1. After vacuum drying, the encapsulated dots were suspended in a polar solvent (aqueous buffer or ethanol), and were purified by gel filtration. Standard procedures were then used to crosslink free carboxylic acid groups (about 100 on each polymer molecule) with amine-containing ligands such as amino-PEGs (Sunbio, Korea), peptides, and

antibodies. Briefly, the polymer-coated dots were activated with 1 mM EDAC at pH 6 for 30 min. After purification, the activated dots were reacted with amino-PEG at a QD/PEG molar ratio of 1 :50 at pH 8 for 2 hours, generating PEG-linked probes. Alternatively, the activated dots were reacted with PEG at a reduced QD/PEG ratio of 1 :6 at pH 8 for 20 min, and then with a tumor-targeting antibody at a QD/antibody molar ratio of 1 :15 for 2 hours. The final QD bioconjugates were purified by column filtration or ultracentrifugation at 100,000 g for 30 min. After resuspension in PBS buffer (pH 7), aggregated particles were removed by centrifugation at 6000 g for 10 mins. Example 3

Fluorescence imaging: In vivo fluorescence imaging was accomplished by using a macro-illumination system (Lightools Research, Encinitas, CA), designed specifically for small animal studies. True-color fluorescence images were obtained using dielectric long-pass filters (Chroma Tech, Brottleboro, VT) and a digital color camera (Optronics, Magnafire SP, Olympus America, Melville, NY). Wavelength- resolved spectral imaging was carried out by using a spectral imaging system (CRI, Inc., Woburn, MA) comprising a optical head that includes a liquid crystal tunable filter (LCTF, with a bandwidth of 20 nm and a scanning wavelength range of 400 to 720 nm), an optical coupler and a cooled, scientific-grade monochrome CCD camera, along with image acquisition and analysis software. The tunable filter was automatically stepped in 10 nm increments from 580 to 700 nm while the camera captured images at each wavelength with constant exposure. Overall acquisition time was about 10 seconds. The 13 resulting TIFF images were loaded into a single data structure in memory, forming a spectral stack with a spectrum at every pixel. With spectral imaging software, small but meaningful spectral differences could be rapidly detected and analyzed.

Autofluorescence spectra and quantum dot spectra were manually selected from the spectral image using the computer mouse to select appropriate regions. Spectral unmixing algorithms (available from CRI, Inc., Woburn, MA) were applied to create the unmixed images of "pure" autofluorescence and "pure" quantum dot signal, a procedure that takes about one second on a typical personal computer. When appropriately generated, the autofluorescence image should be uniform in intensity regardless of the presence or absence of quantum-dot signals (as is the case in FIG. 6A through 6D). The identification of valid spectra for unmixing purposes need only be performed initially, as the spectra can be saved in spectral libraries and re-used on additional spectral stacks.

Cells and tissue sections were examined by using an inverted Olympus microscope (IX-70) equipped with a digital color camera (Nikon D1), a broad-band ultraviolet (330-385 nm) light source (100-W mercury lamp), and a long-pass interference filter (DM 400, Chroma Tech, Brattleboro, VT). Wavelength-resolved spectra were obtained by using a single-stage spectrometer (SpectraPro 150, Roper Scientific, Trenton, NJ). Example 4

To evaluate the siRNA efficiency of a QD delivery vehicle, a model gene silencing experiment was designed using the human breast cancer cell line MDA-MB 231 and targeting the cyclophilin B gene. Cyclophilin B is recommended as a positive silencing control in human cell lines, and is associated with the secretory pathway. This gene is abundantly expressed in most cells and because it is nonessential, knockdown of the corresponding mRNA does not affect cell viability. In comparison with three most commonly used transfection reagents, lipofectamine, TRANSIT-TKO™ and JETPEI™ (using the optimal amount of each transfection reagent as determined by the manufacturer's protocols, and siRNA at a constant concentration), FIG. 2 illustrates that for MB 231 cells, QDs nearly suppressed cyclophilin B expression completely and are 16 times more efficient than the average of the other three reagents. Similar results have also been observed using other types of cell lines including breast cancer MCF7, prostate cancer PC3, cervical carcinoma HeIa, fibroblast NIH3T3, lung cancer NCI-H, and ovarian cancer OVCAR-3, as shown in FIG. 7.

This surprisingly high delivery efficiency may be explained by the unique structural properties of QDs'. First, QD-siRNA complexes remained single, and the small sizes facilitated their diffusion and entrance into cells. Secondly, after the nanostructures were endocytosed intracellularly, both the tertiary amines and carboxylic groups on the QD surface play important roles in breaking small cellular compartments, such as endosomes and lysomomes, and thus releasing the siRNA into the cytoplasm. At low pH values carboxylic and amine groups are protonated, and at high pH values they will be de-protonated. Therefore, this zwitterionic surface would behave like a buffer system that can quickly neutralize excess protons in lysosomes and lead to a net influx of chloride ions. The osmotic pressure building along this proton buffering process would eventually rupture the endosomes and release its contents. Similarly, conventional cationic polymer carriers, such as PEI, are believed to escape endosomes through the same mechanism, a process known as "proton sponge

effect" [PNAS 92, 7297, which is incorporated by reference ]. Nanoparticles may also cause steric hindrance in enzymatic functions and thus protect the attached oligonucleotides from enzymatic degradation [JACS 123, 7626; PNAS 99, 5018; JACS 125, 7168, each of which is incorporated by reference]. Thirdly, due to QDs' hydrophobic surface coating after synthesis, they are solublized with amphiphilic polymers, whose hydrophobic domains bond to QD surface tightly via hydrophobic interactions. The hydrophilic groups can render QDs water soluble. The periodic block copolymer as used in the QDs of the current model experiment had a molecular weight of approximately 7,500 Da and multiple polymer molecules are required to make a QD water soluble. Through this self assembly process, it's was unlikely that the original QD surface would be completely encased by polymers. Possible hydrophobic patches could then interact with cell membrane lipid bilayers and facilitate its entrance into cytoplasm. It has been reported that tuning the hydrophobicity of PEI molecules by alkylation can enhance the transfection efficiency by up to 400 times. The results of the model experiment demonstrate that QDs of the present disclosure have advantageous properties when compared to other non-viral DNA and RNA delivery technologies. Example 5

The possible cellular toxicity of the novel transfection reagents of the present disclosure was examined. This is a particularly important issue for semiconductor QDs, because of a concern that toxic Cd ²⁺ ions could be released over time.

Both time- and dose- dependent studies, shown in FIG. 3, have shown that QDs are much less toxic than the traditional transfection reagents. This effect is especially significant over extended incubation times or at higher concentrations. It is likely that the cytotoxicity effect is unlikely to arise from Cd ²⁺ ions because the stable polymer coating layer protects QDs from exposure. Resistance of the block polymer coatings to extreme chemical conditions was shown previously [NatBiotech 22, 969, which is incorporated by reference]. Furthermore, other studies have suggested that for chemically stable nanoparticles, the colloid stability against aggregation plays an important role in their toxicity level. QDs may maintain single structure in biological buffers and cell culture media as is shown by in vivo cell imaging, and thus exhibit low toxicity effect. It is also possible that the surface ligands could also make a difference, even for chemically inert nanoparticles such as gold. Anionic groups are substantially less toxic than cationic groups. Although the net charge of QDs was positive, many of the surface ligands were carboxylic acids,

which would act as a proton buffer while maintaining a low toxicity effect. Similar results have been observed in the other six types of cells, as shown in FIG. 7. However, at an elevated concentration of QDs (5 - 1Ox of the optimal), cell proliferation was slowed down, possibly due to interference with microtubule dynamics. Example 6

To investigate the intracellular behavior of QD-siRNA complexes, its uptake, movement and localization in live cells using fluorescence microscopy was examined. The intrinsic fluorescence and photostability of QDs allowed continuous imaging of the delivery vehicle in realtime. FIG. 4A, for example, shows the kinetics of QD-siRNA uptake. The complexes attached to the cell membrane immediately after mixing (T < 1 min), which is manifested as a bright ring structure. Subsequent incubation over a period of 4 hours allowed the complexes to enter and accumulate inside the cells (bright interior), suggesting an efficient transportation across the plasma membrane. The complexes quickly migrated toward, and accumulated around, the cell nucleus, a behavior observed as soon as 30 min after mixing with cells and completed around 4 hours, as shown in FIG. 4B.

The QD-siRNA complexes, once having entered the cells, undergoes active and directional transport and move several orders of magnitude faster than would be the case by random diffusion. Their trajectory and velocity were very similar to active transportation driven by molecular motors. To better characterize this ballistic behavior, QDs' unique optical properties were combined with a confocal microscope equipped with a Nipkow spinning disk and a cellline stably expressing GFP- microtubule. Although QDs are photostable, fluorescent proteins such as GFP are subjected to quick photobleaching. The Nipkow technology not only offered the capability of fast confocal image acquisition for studies involving dynamic biological process, but also reduced photobleaching and phototoxicity problems. Co- localization of QD travel paths with GFP fluorescence indicated that the complexes walk along microtubules. The movement is likely mediated by both dynein and kinesin, because of the bilateral pattern, although the overall direction is inward and toward the cell nucleus.

To confirm the observation of cytoskeleton-based movement, cells were treated with the actin and microtubule disrupting drugs, cytochalasin and nocodazole. Cytochalasin has no apparent effect on the complex transport when added independently; whereas 10 μM nocodazole almost completely stopped the

fast and directional movement within 1 hour post-treatment. When the two drugs were added together, nocodazole took effect much faster and the complexes stopped moving as fast as 30 mins. The complexes only underwent local and random thermomotion, which confirmed that they indeed move along microtubules. Example 7

The characteristic intermittent fluorescence of QDs did not interfere with the fast imaging technique, because QDs are typically imaged in groups. After the QD- siRNAs entered cells through endocytosis, as shown in FIG. 5A, many copies of the complexes were confined in a small endosomal compartment. Assuming the fluorescence 'on' and 'off rates for QDs were roughly 50:50, the chance that 'n' copies of QDs stay in the 'off state simultaneously is (1/2) ⁿ . This number becomes statistically negligible if 'n' is larger than 10. To further understand the transport mechanism, it was necessary to determine if the complexes stayed inside endosome shuttle and traveled along microtubule, or first escaped endosome and then walked by themselves. For example, PEI mediated delivery has been suggested to travel inside endosomes [Biochimica et Biophysica Acta 1514, 21 , which is incorporated herein by reference]; whereas adenoviruses are believed to escape the endosome before moving along microtubules toward the nucleus [Hum Gene Ther 11 , 151 , whicih is incorporated herein by reference]. Both mechanisms are possible for QD- siRNA complexes, because 1) endosomes are known to walk along microtubule, and 2) single QDs have been observed diffuse inside cells and aided by a toxin that doesn't involve endosome formation walking on microtubules (data not shown).

High resolution TEM and fluorescence imaging revealed that the complexes attached to the cell surface and quickly formed endocytotic vesicles (FIGS. 5A and 5B). Over extended time, the vesicles entered cells and some of them could even form multivesicular structures, as shown in FIGS. 5C and 5D. When the nanoparticles localized around the perinuclear region, they formed clusters inside big vesicles similar to late-stage endosomes that typically merged together (shown in FIGS. 5E and 5F). Using spinning disk confocal microscope, it was observed that the complex transport may be particularly active around the microtubule organization center (MTOC), as shown in the insert panel of FIG. 5E.

All these behaviors are characteristics of endosome dynamics and suggest that the QD complexes may be transported inside endosomes toward the nucleus before released into cytoplasm, similar to PEIs mediated transfection. The exact timing when the endosomes merge and rupture, and QD-siRNA separation are not clear.

Example 8

QD Nanoparticle Engineering: Highly luminescent quantum dots were synthesized as described above. The purified QDs (100 nM) were mixed with poly(maleic anhydride-alt-1-tetradecene) (Sigma Aldrich 10 mM) in chloroform and slowly dried under vacuum. The transparent thin film formed was then sonicated in aqueous buffer (pH 8.5), and the water-soluble QDs were purified from excess polymer molecules with a filtration column (first concentrated with ultracentrifugation). QDs were then mixed with N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (Sigma Aldrich EDAC) and N ₁ N- Dimethylethylenediamine (Sigma Aldrich, 1000 X of QDs) in desired amounts. Due to the variations in chemical purity and quality, (EDAC degrades over time), the actual amount of EDAC is determined empirically until the zeta potential of the final QD nanoparticle reaches about +8.5 mV. The modified QDs were purified with dialysis (MWCO = 3000), and stored at 4 ⁰ C for future uses. Example 9

Cells Transfection

(a) Cell lines: MDA-231 (MDA- breast carcinoma), MCF-7 (breast carcinoma), HeLa (cervical carcinoma), NIH3T3 (mouse fibroblasts), NCI-H 460 (NCI-H-lung carcinoma), OVCAR-3 (ovarian carcinoma), PC-3 (prostate carcinoma). (b) Transfection: siRNA transfection was performed using Lipofectamine

2000 (Invitrogen Corp., Carlsbad, CA), TRANSIT-TKO™ (TranslT) (Mirus Bio Corp., Madison, Ml), JETPEI™ (Qbiogene, Morgan Irvine, CA) transfection reagents, and modified QDs. Briefly, 3 x 10 ⁴ cells/well were plated into 24-well plates overnight to achieve 60-80% confluent monolayers. On the day of transfection, cultured cells were washed and pre-incubated for 40 mins with OPTIMEM™ (Invitrogen Corp., Carlsbad, CA) without serum or antibiotics. Lipofectamine (2 μl/well) and TRANSIT™ (2 μl/well) were diluted in 500 μl of OPTIMEM™ at manufacturer's recommended concentrations, and incubated for 15 mins at room temperature. Then 10OnM of siRNA directed against human cyclophilin B (Dharmacon Inc., Chicago, IL) was added to the mixture of transfection reagent and medium, mixed by pipeting and incubated for additional 20 mins at room temperature.

For transfection with JETPEI™, transfection reagent and siRNA were initially diluted in 50 μl of 15OnM NaCI, vortex for 10 sec, mixed together and vortex for 10 sec followed by 20 mins incubation at room temperature. 50-10OnM QDs were mixed with siRNA vortex for 10 sec and incubated for 20 mins at room temperature. Before the transfection, 500μl of OPTIMEM™ was added to the QDs-siRNA

complexes and mixed by pipeting. The siRNA-transfection reagents complexes were then added to each well and the cells were incubated at 37 ⁰ C for 18 hrs. Complete DMEM (10% FBS) was then added to the cells to and incubated for 36 hrs at 37 ⁰ C in the CO ₂ incubator. Note that QDs could also be used with complete media and 200 nM. QDs typically offered the best transfection results. Example 10

Immuno-blotting: Transfected cells were lysed using RIPA lysis buffer containing 1 % lgepal-630, 0.5 % deoxycholate, 0.1 % SDS, 1 mM PMSF and 1 μg/ml of each leupeptin, aprotinin and pepstatin in phosphate buffered saline (PBS). Lysates were centrifuged, supernatant collected and protein measured by a standard Bradford assay. Equal amounts of protein were loaded and separated on 11 % SDS-PAGE then transferred to nitrocellulose membranes and blocked with 5% milk blocking buffer for 2 hrs. The blocked membrane was incubated with rabbit polyclonal anti-human cyclophilin B antibodies (Abeam, Cambridge, MA). The membranes were washed in TTBS and probed with HRP-linked labeled goat anti- rabbit secondary antibodies (Cell Signaling Inc., Beverly, MA). The blot was developed using an ECL kit (Amersham Corp., Arlington Heights, IL). The membrane was then exposed to Kodak X-OMAT film for 10 -30 sec. Example 11 QDs Gel Motility Assay: Equal amounts of QD+, QD- (625 and 545nm) were loaded into 0.5% acrylamid gel and electrophoresed in PBS running buffer (ph 7.4) at 60V for 2 hrs. Multicolor gel images were documented with a macro-imaging system (Lightools Research, Encinitas, CA). Example 12 Cytotoxicity assay: A Sulforhodamine B (SRB) assay was used according to the method by Skehan et a/., (J. Natl. Cancer Inst. (1990), 82:1107-1112). Briefly, cells were collected by trypsinization, counted and plated at a density of 10 ⁴ cells/well in 96-well flat-bottomed microtiter plates (100 μl of cell suspension/well). In the chemosensitivity assay, Lipo, TranslT, JetPEI and QD+ were tested at scalar concentrations ranging from 0 to 10x of optimal transfection dose. Optimal transfection dose was the concentration of transfected reagents recommended by manufacturer for optimal transfection results (described in cell transfection section) and were referred as 1 x. 1 * concentration of QD+ was 10OnM. Transfection reagents in the different concentrations were diluted in OPTIMEM™ (serum free) and incubated for 6-48 hrs. Each experiment was repeated three times. The optical density of treated cells was determined at a wavelength of 540 nm by means of a

fluorescence plate reader. Cytocidal effect of drugs was calculated according to the formula OD zero - OD treated / OD zero * 100%. The 'OD zero' depicts the cell number at the moment of drug addition, and the 'OD treated' reflected the cell number in treated wells on the day of the assay. It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations, and are merely set forth for a clear understanding of the principles of this disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.