Related U.S. Application Data 10 страница

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In some embodiments, a therapeutically effective amount of an inventive vaccine nanocarrier composition is delivered to a patient and/or animal prior to, simultaneously with, and/or after diagnosis with a disease, disorder, and/or condition. In some embodiments, a therapeutic amount of an inventive vaccine nanocarrier composition is delivered to a patient and/or animal prior to, simultaneously with, and/or after onset of symptoms of a disease, disorder, and/or condition. In certain embodiments, a therapeutic amount of an inventive vaccine nanocarrier composition is administered to a patient and/or animal prior to exposure to an infectious agent. In certain embodiments, a therapeutic amount of an inventive vaccine nanocarrier composition is administered to a patient and/or animal after exposure to an infectious agent. In certain embodiments, a therapeutic amount of an inventive vaccine nanocarrier composition is administered to a patient and/or animal prior to exposure to an addictive substance or a toxin. In certain embodiments, a therapeutic amount of an inventive vaccine nanocarrier composition is administered to a patient and/or animal after exposure to an addictive substance or a toxin.

In some embodiments, the pharmaceutical compositions of the present invention are administered by a variety of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), transdermal, mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/ or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. In certain embodiments, the composition is administered orally. In certain embodiments, the composition is administered parenterally. In certain embodiments, the composition is administered via intramuscular injection.

In certain embodiments, vaccine nanocarriers which delay the onset and/or progression of a disease, disorder, and/or condition (e. g., a particular microbial infection) may be administered in combination with one or more additional therapeutic agents which treat the symptoms of the disease, disorder, and/or condition. For example, the vaccine nanocarriers may be combined with the use of an anti-cancer agent, anti-inflammatory agent, antibiotic, or anti-viral agent.

The invention provides a variety of kits comprising one or more of the nanocarriers of the invention. For example, the invention provides a kit comprising an inventive nanocarrier and instructions for use. A kit may comprise multiple different nanocarriers. A kit may comprise any of a number of additional components or reagents in any combination. According to certain embodiments of the invention, a kit may include, for example, (i) a nanocarrier comprising at least one immunomodulatory agent, wherein the at least one immunomodulatory agent is capable of stimulating both a T cell and/or В cell response, at least one targeting moiety, and/or at least one immuno stimulatory agent; (ii) instructions for administering the nanocarrier to a subject in need thereof. In certain embodiments, a kit may include, for example, (i) at least one immunomodulatory agent, wherein the at least one immunomodulatory agent is capable of stimulating both a T cell and В cell response; (ii) at least one targeting moiety; (iii) at least one immunostimulatory agent; (iv) a polymeric matrix precursor; (v) lipids and amphiphilic entities; (vi) instructions for preparing inventive vaccine nanocarriers from individual components (i)-(v).

In some embodiments, the kit comprises an inventive nanocarrier and instructions for mixing. Such kits, in some embodiments, also include an immuno stimulatory agent

and/or an antigen. The nanocarrier of such kits may comprise an immunomodulatory agent (e. g., a T cell antigen, such as a universal T cell antigen) and/or a targeting moiety. The T cell antigen and/or the targeting moiety may be on the surface of the nanocarrier. In some embodiments, the immunomodulatory agent and the antigen are the same. In some embodiments, they are different.

In any of the foregoing embodiments described above, the word conjugated means covalently or noncovalently conjugated, unless the context clearly indicates otherwise. In any of the foregoing embodiments described above, the word encapsulated means physically trapped within, whether by admixture, by a shell surrounding a core, by covalent bonding internal of the surface of the nanocarrier, and the like.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: Combined vaccine targeting strategy for optimal humoral and cellular immune response. The composite vaccine carries internal T cell antigens, adjuvants (not shown) and targeting moieties for DCs, FDC and SCS-Mph together with surface antigen for В cell recognition. Upon s. c. or i. m. injection, the material reaches lymph nodes via draining lymph vessels and accumulates on each APC (for clarity, only APC-specific targeting moieties are shown, but each APC acquires the entire complex). DCs internalize and digest the complex and present antigenic peptides in MHC class I and class II to CD8 and CD4 T cells, respectively. The activated T cells differentiate into effector/memory (JEffiMem)^cell^s that mediate cellular immune responses. T^ cells provide help to В cells that were initially stimulated by antigen on SCS-Mph and in the process have acquired and processed T cell antigens for restimulation оГГ_//лThe help provided by T_FH cells allows the development of a GC reaction during which В cells proliferate and generate high- aflinity antibodies.

FIG. 2: SCS-Mph bind lymph-bome viral particles and present them to follicular В cells. (A) Immunohistochemical staining of the cortex of a mouse popliteal lymph node stained with anti-CD169 and counter-stained with wheat germ agglutinin. The lymph node was harvested 30 minutes after footpad injection of red fluorescent vesicular stomatitis virus (VSV). In the subcapsular sinus of the draining lymph node, the red virus colocalized exclusively with CD169⁺macrophages. (B) Electron micrograph of a lymph node macrophage (Mph) and a follicular В cell (Bl) below the floor of the subcapsular sinus (SCSI) 30 minutes after VSV injection shows VSV at the surface and within a phagolysosome of the Mph and at the interface between Mph and В cells (arrowheads). (C) Injection of VSV into the footpad of untreated mice (B6) results in rapid downregulation of surface-expressed IgM on virus-specific В cells, a sign of В cell activation. Depletion of SCS-Mph after footpad injection of clodronate liposomes (CLL) abolished В cell activation, indicating that SCS-Mph are essential to present particulate antigen to В cells.

FIG. 3: An exemplary liposome nanocarrier with a lipophilic immunomodulatory agent incorporated in the membrane, and a hydrophilic immunomodulatory agent encapsulated within the liposome.

FIG. 4: An exemplary nanoparticle-stabilized liposome nanocarrier with a lipophilic immunomodulatory agent

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incorporated into the membrane, and a hydrophilic immunomodulating agent encapsulated within the liposome.

FIG. 5: An exemplary liposome-polymer nanocarrier with a lipophilic immunomodulatory agent incorporated into the membrane, and a hydrophobic immunomodulating agent encapsulated within the polymeric nanoparticle.

FIG. 6: An exemplary nanoparticle-stabilized liposome- polymer nanocarrier with a lipophilic immunomodulatory agent incorporated into the membrane, and a hydrophobic immunomodulating agent encapsulated within the polymeric nanoparticle.

FIG. 7: An exemplary liposome-polymer nanocarrier containing reverse micelles with a lipophilic immunomodulatory agent incorporated into the membrane, and a hydrophilic immunomodulatory agent encapsulated within the reverse micelles.

FIG. 8: An exemplary nanoparticle-stabilized liposome- polymer nanocarrier containing reverse micelles with a lipophilic immunomodulatory agent incorporated into the membrane, and a hydrophilic immunomodulatory agent encapsulated inside the liposome.

FIG. 9: An exemplary lipid-stabilized polymeric nanocarrier with a hydrophilic immunomodulatory agent conjugated to the lipid monolayer, and a hydrophobic immunomodulatory agent encapsulated inside the polymer core.

FIG. 10: An exemplary lipid-stabilized polymeric nanocarrier containing reverse micelles with a hydrophilic immunomodulatory agent conjugated to the lipid monolayer, and a hydrophilic immunomodulatory agent encapsulated inside the polymer core.

FIG. 11: Capture of lymph-bome VSV by SCS macrophages. (A) MP-IVM micrographs of VSV in a popliteal LN (numbers: minutes after footpad injection; scale bar: 100 pm). (B) VSV accumulation in a C57BL/6—»Act(EGFP) recipient 3 hours after injection (scale bar: 50 pm). (C) Electron micrographs of VSV in LN 5 minutes after injection. Center micrograph is shown schematically (left) and at higher magnification (right). Arrowheads identify VSV particles (scale bars: 2 pm). (D) Confocal micrographs of VSV-draining LN (30 minutes). Scale bars: 100 pm (left), 15 pm (right). (E) VSV titers in popliteal LNs 2 hours after injection into wildtype, C3-deficient or CLL-depleted mice. ***: p< 0. 001 (two-way ANOVA, Bonferroni’s post-test). (F) VSV capture in DH-LMP2a mice. *: p< 0. 05 (unpaired t-test). (G) VSV titers after footpad injection in untreated and CLL-treated mice (one of two similar experiments; n=3). ProxLN: inguinal, paraaortic LNs; BrachLN: brachial LN. (H) Viral titers in lymph, spleen and blood after TD cannulation; *: p< 0. 05 (unpaired t-test). Horizontal bars in (E-H) indicate means.

FIG. 12: Characterization of CD169⁺ macrophages in peripheral LNs. (A-C) Lineage marker expression analysis of pooled mononuclear cells from LNs of naive C57BL/6 mice. (A) After gating on the CD169⁺ population (middle panel), cells were analyzed for expression of the two macrophage-associated surface markers, I-Ab (MHC class II) and CDllb (bottom panel). Staining with an isotype control for anti-CD169 is shown in the top panel. (B) CD169⁺I- Ab⁺CDllb⁺ cells were further analyzed for expression of CD68, F4/80, CDllc, and Gr-1. Gates were drawn to identify marker+ cells, except for CDllc staining where the marker was positioned to identify conventional CD1 Ic⁷'" ¹⁷' dendritic cells (overlay). Numbers indicate percentage of CD169⁺I-Ab⁺CDllb⁺ cells under the histogram gate. Data are representative of 3-5 experiments with similar results. (C) Quantitative analyses of data in panel (B), error bars represent SEM. (D-G) Confocal micrographs of popliteal

LNs from naive C57BL/6 mice showing co-expression of selected markers on CD169⁺ cells (arrowheads). Scale bars: 125 pm in the left column and 20 pm in all other columns.

FIG. 13: Morphological changes in popliteal LNs following CLL treatment. (A) Confocal micrographs of popliteal LNs (top three rows) and spleens (bottom row) of untreated control mice (-CLL, left column) and animals that had received CLL footpad injections 6-10 days earlier. CLL treatment depleted CD169⁺ macrophages in the LN (top row), but not in spleens; Lyve-1⁺ medullary lymphatic endothelial cells (second row) and cortical CD1 Ic⁷'" ¹⁷' dendritic cells (third row) were not affected. (B) Cellular subset frequency in popliteal LNs with and without CLL treatment, data are from n=3 mice and shown as mean±SEM; *: p< 0. 05, **: p< 0. 01; unpaired student’s t-test. (C) Frequency of different I-Ab⁺CD1 lb⁺ leukocyte subsets in popliteal LNs at 6-10 days after footpad injection of 50 pl CLL. Each symbol represents pooled popliteal LNs from one mouse. Subset frequencies among total mononuclear cells in popliteal LNs were assessed by flow cytometry after gating on I-Ab+CDllb+ cells as shown in FIG. 12A. (D) Immunohistochemical analysis of popliteal LNs without treatment (-CLL) or 7 days after footpad injection of CLL (+CLL). Scale bars: 300 mm. (E) Ultrastructure of the SCS in a representative popliteal LN 7 days after CLL treatment and 5 minutes after footpad injection of 20 pg VSV-IND. Note the complete absence of SCS macrophages and viral particles. Scale bar: 2 pm.

FIG. 14: Retention of fluorescent viruses and latex nanoparticies in popliteal LNs. (A) Confocal micrographs of popliteal LNs 30 minutes after footpad injection of Alexa- 568-labeled adenovirus (AdV). Frozen sections were stained with FITC-a-CD169 and Alexa-647-a-B220 to identify В cells. Scale bars: 100 pm (left panel) and 15 pm (right panel). (B) Transmission electron micrographs of AdV particles captured by a SCS macrophage. The top panel shows an annotated schematic drawing of the low magnification overview (middle panel). The boxed area in the middle panel is enlarged in the lower panel, arrowheads denote electron- dense, spherical AdV particles. Scale bars: 2 pm (top and middle panel) and 1 pm (lower panel). (C-D) Confocal micrographs of popliteal LNs from C57BL/6 mice 30 minutes after footpad injection of 20 pg Alexa-568 labeled UV-inactivated AdV (C) or W (D). Fluorescent viruses accumulated in the cortical SCS above В follicles identified by FITC-a-B220 staining and also in the medulla where viruses were not only bound by CD169⁺ macrophages, but also by LYVE-1⁺ lymphatic endothelial cells. Scale bars indicate 125 pm (left panel) and 25 pm (right panel). (E) Confocal micrograph of a popliteal LN 30 minutes after hind footpad injection of Alexa-568 labeled VSV and approximately 10¹¹ Crimson Fluospheres (200 nm diameter). Frozen LN sections were counter-stained with FITC-a-CD169. Note that the Latex beads, unlike VSV, were poorly retained in draining LNs. Scale bar: 125 pm.

FIG. 15: Effect of CLL footpad injection on VSV distribution in draining LNs. Confocal micrographs show the localization of fluorescent VSV particles in popliteal LNs without (A) or 7 days after (B) CLL treatment. В follicles were identified by FITC-a-B220 staining. In the medulla (boxed area), VSV was bound by LYVE-1⁺ cells that were not affected by CLL treatment. Scale bars: 125 pm (left column) and 25 pm (right column).

FIG. 16: SCS macrophages present lymph-derived AdV to follicular В lymphocytes. (A) Confocal micrograph of CD169⁺ macrophages in the SCS above a В follicle in a popliteal LN. Frozen sections were counterstained with

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29 wheatgerm agglutinin (WGA) to identify extracellular matrix and with a-B220 to detect В cells. Note that some В cells reside in the SCS, and one В cell appears to migrate between the follicle and the SCS (arrowhead). Scale bar: 25 pm. (B) Electron micrograph and (C) schematic drawing of a SCS macrophage and surrounding cells in a popliteal LN 30 minutes after footpad injection of AdV. Scale bar: 2 pm. The boxes drawn in (C) indicate areas of higher magnification shown in panels (D) and (E). These panels show two examples of AdV particles at the interface between the SCS macrophage and В cells (arrowheads). Asterisks denote other macrophage-associated AdV particles. Scale bars: 500 nm.

FIG. 17: Macrophage-mediated transfer of lymph-bome VSV across the SCS floor alters virus-specific В cell behavior. (A) Electron micrographs and schematic drawing (middle) showing a macrophage penetrating the SCS floor of a popliteal LN 30 minutes after VSV injection. Scale bars: 10 pm (left) and 2 pm (right). Arrow: vacuole with digested VSV. Arrowheads: virions in contact zone between macrophage and В cells. (B) MP-IVM of polyclonal and VI10YEN В cells in popliteal LNs. Scale bars: 50 pm. (C) Regional ratios of VI10YEN В cells/control В cells following VSV injection. Results are from 3 movies/group. (D, E) Localization of VI10YEN В cells in popliteal LNs relative to the SCS. **: p< 0. 01 (one-way ANOVA with Bonferroni’s post-test).

FIG. 18: Characteristics of VSV serotypes and VSV-IND- specific VI10YEN В cells. (A) SDS-PAGE gels (12%) of purified VSV lysates. Top: VSV-IND and VSV-NJ. The N and P proteins со-migrate in VSV-NJ, approximate molecular weights are shown in parentheses. (B) Binding of Alexa- 488 labeled VSV-IND (middle row) or VSV-NJ (bottom row) to В cells from C57BL/6 mice (left column) or VI10YEN mice (right column). The upper row shows control staining with the anti-idiotypic antibody 35. 61 to the VI10YEN BCR (Dang and Rock, 1991, J- Immunol., 146: 3273). (C) Intracellular calcium flux in Cl)43'" '^{! 1} purified, Fluo-LOJO loaded В cells from VI10YEN mice (upper row) or C57BL/6 mice (lower row). Events were collected continuously over time, asterisks indicate the timepoint when antibodies or virus were added. Virus particles were used at 1000/B cell, anti-IgM-(Fab)₂ at 10 pg/10⁶В cells. (D) Neutralization assay for total Ig and IgG in serum of C57BL/6 mice 4 and 10 days after immunization by footpad injection of 10 pg UV-VSV or UV-VSV-AlexaFluor-488- IND. (E) Calcium flux in VI10YEN В cells exposed to supernatant from VSV stocks. Supernatant was generated by ultracentrifugation through a sucrose cushion resulting in approximately 10, 000-fold reduction in viral titers and was used on В cells either undiluted (top right) or at 1: 100 dilution (bottom right). As a control, VSV stock solution was diluted to equivalent viral titers (MOI; left panels). The results demonstrate the presence of antigenic VSV-G that is not associated with virus particles in our virus preparation.

FIG. 19: VSV-induced adhesion of VI10YEN В cells to ICAM-1 and VCAM-1. (A, B) Adhesion of purified naive and VSV-IND activated (30 minute exposure) VI10YEN В cells to plastic plates coated with the indicated concentrations of recombinant ICAM-1-Fc (A) or VCAM-1-Fc (B). Pooled data of two triplicate experiments are shown. Horizontal bars represent means. (C, D) Confocal micrographs of ICAM-1 and VCAM-1 expression in popliteal LNs of C57BL/6mice. Scale bars: 50 (E) Adhesion of purified naive wildtype and VI10YEN В cells to plastic dishes coated with the indicated pfu-equivalent concentrations of UV-inacti- vated VSV-IND. Data represent means±SEM of triplicates.

FIG. 20: SCS macrophages are required for early activation of VSV-specific В cells in LNs. (A) Confocal micrograph shows MHC-II colocalization with VSV-IND (30 minutes after injection) in VIlOYENxMHCII(EGFP) В cells at the SCS (arrowhead), not the deep follicle (asterisk). Scale bar: 25 pm. (B) Distance of VSV-associated and VSV-free VIlOYENxMHCII(EGFP) В cells to the SCS. Horizontal lines: medians. (C) BCR expression kinetics on VI10YEN and (D) polyclonal В cells after VSV-IND footpad injection. (E) BCR expression on VI10YEN cells in CLL-treated or untreated popliteal LNs after VSV-IND injection (20 pg). Mean fluorescence intensities were normalized to virus-free values (dashed line). Means±SEM (3-5 mice). (F) Confocal micrograph of VI10YEN В cells in control and (G) CLL-treated popliteal LNs 6 hours after VSV-IND injection (0. 4 pg). Scale bar: 125 pm. (H) VI10YEN В cell frequency at T/B borders and in follicles 6 hours after VSV-IND injection at indicated doses. Means±SEM; n=3-4 follicles/2 mice; *: p< 0. 05; **: p< 0. 01; ***: p< 0. 001 (t-test).

FIG. 21: VI10YEN В cell motility in draining LNs following virus injection. Median 3D instantaneous velocities of wildtype (triangles) and VI10YEN В cells (circles) in deep follicles and the SCS/superficial follicle about 5-35 min after VSV footpad injection. Horizontal bars represent means; *: p< 0. 05; **: p< 0. 01 (one-way ANOVA with Bonferroni’s post test). Note that specific В cells slow down throughout the entire follicle, likely as a consequence of free VSV-G in our preparation (see FIG. 18). Control experiments showed similar В cell motility parameters in CLL- treated and nontreated popLNs.

FIG. 22: Timecourse of activation marker induction on VI10YEN В cells in virus-draining and non-draining LNs following injection of VSV-IND. VI10YEN В cells were fluorescently tagged with CMTMR and transferred to naive mice that were injected 18 hours later with 20 pg UV- inactivated VSV-IND (time 0 hours). The draining popliteal LN (popLN) and a distal brachial LN (brachLN) were harvested after the indicated time intervals to generate single-cell suspensions. CD69 and CD86 expression on В cells was assessed by flow cytometry after gating on (A) B220⁺CMTMR⁺VI10YEN cells or (B) B220⁺CMTMR“ endogenous control В cells.

FIG. 23: Confocal (left and middle columns) and MP- IVM micrographs (right column) of popliteal LNs of mice that had received adoptive transfers of a mixture of CMTMR-labeled VI10YEN В cells and CMAC-labeled polyclonal В cells (in the right column). On the following day, 20 pg UV-inactivated VSV-IND was injected in a footpad and the draining popliteal LNs were either surgically prepared for MP-IVM or harvested for confocal analysis of frozen sections at the indicated time points. MP-IVM images show that VSV-specific, but not polyclonal В cells made contact with VSV in the SCS as early as 30 minutes after virus injection. VI10YEN В cells relocated to the T/B border at 6 hours following injection. Scale bars: 150 pm in the left column and 25 pm in the other columns.

FIG. 24: In vivo targeting of SCS-Mph using Fc fragments from human IgG. (A) The FACS histograms on the left document the binding of fluorescent PEG-PLGA nanoparticles (-100 nm diameter) to lymph node macrophages. (B) Fc-nanoparticle (NP) targets SCS-Mph and follicular dendritic cells.

FIG. 25: Identification of the chemokine receptor CX3CR1 (fractalkine receptor) on macrophages in lymph node subcapsular sinus (SCS), but not in macrophages in the medulla. The micrograph on the right is a 3D projection of

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a lymph node from a double-knockin mouse where green fluorescent protein (GFP) is expressed in the CX3CR1 locus, while red fluorescent protein (RFP) reports the expression of another chemokine receptor, CCR2. SCS-Mph are readily identifiable by their prominent green fluorescence, while medullary macrophages express primarily RFP.

FIG. 26: SCS-Mph express the chemokine receptor CX3CR1. The graph shows a FACS plot of a single cell suspension from a lymph node of a knockin mouse that was genetically engineered to express GFP from the CX3CR1 locus. SCS-Mphs are identified by staining with a soluble receptor, CRFc, which binds macrophages in the SCS, but not the medulla. The CRFc negative CX3CR1-expressing (i. e., GFP-high) cells are conventional dendritic cells that express this chemokine receptor.

FIG. 27: Fluorescent micrographs of frozen sections from mouse popliteal lymph nodes 24 h after footpad injection of 0. 2 pm diameter Latex beads surface modified with either amine (left and middle panel) or carboxy moieties (right panel). Both sets of beads were purchased from Invitrogen (Cat. no. F8763 and F8805). Sections on left and right were counterstained with anti-CDl 69. Images are oriented so that the medulla (weak, diffuse staining with anti-CD169) faces to the right and the subcapsular sinus (SCS) region (bright anti-CD169) faces to the left. Note that the red amine modified particles prominently localize to the SCS, while blue carboxy modified beads are primarily retained in the medulla.

FIG. 28: (A) Antigen-bearing targeted nanoparticles are highly immunogenic and induce high antibody titers. (B) The induced immune response elicited by nanoparticle vaccines confers potent protection from a lethal dose of VSV.

FIG. 29: In vivo T cell activation by immunomodulatory nanoparticles. (A) Effect of NPs on CD4 T cell activation. (B) Effect of NPs on CD8 T cell response mixed with CpG adjuvant (TLR9 agonist). (C) Effect of co-encapsulated adjuvant on CD8 T cell activation.

FIG. 30: Shows an exemplary nicotine conjugation strategy-

FIG. 31: Shows an exemplary R848 conjugation strategy.

DEFINITIONS

Abused substance: As used herein, the term “abused substance” is any substance taken by a subject (e. g., a human) for purposes other than those for which it is indicated or in a manner or in quantities other than directed by a physician. In some embodiments, the abused substance is a drug, such as an illegal drug. In certain embodiments, the abused substance is an over-the-counter drug. In some embodiments, the abused substance is a prescription drug. The abused substance, in some embodiments, is an addictive substance. In some embodiments, the abused substance has mood-altering effects, and, therefore, includes inhalants and solvents. In other embodiments, the abused substance is one that has no mood-altering effects or intoxication properties, and, therefore, includes anabolic steroids. Abused substances include, but are not limited to, cannabinoids (e. g., hashish, marijuana), depressants (e. g., barbituates, benodi- azepines, flunitrazepam (Rohypnol), GHB, methaqualone (quaaludes)), dissociative anesthetics (e. g., ketamine, PCP), hallucinogens (e. g, LSD, mescaline, psilocybin), opioids and morphine derivatives (e. g., codeine, fentanyl, heroin, morphine, opium), stimulants (amphetamine, cocaine, Ecstacy (MDMA), methamphetamine, methylphenidate (Ritalin), nicotine), anabolic steriods, and inhalants. In some

embodiments, the abused substance for inclusion in a nanocarrier is the complete molecule or a portion thereof.

Addictive substance: As used herein, the term “addictive substance” is a substance that causes obsession, compulsion, or physical dependence or psychological dependence. In some embodiments, the addictive substance is an illegal drug. In other embodiment, the addictive substance is an over-the-counter drug. In still other embodiments, the addictive substance is a prescription drug. Addictive substances include, but are not limited to, cocaine, heroin, marijuana, methamphetamines, and nicotine. In some embodiments, the addictive substance for inclusion in a nanocarrier is the complete molecule or a portion thereof.

Amino acid: As used herein, term “amino acid, ” in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” or “natural amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, “non-natural amino acid” encompasses chemically produced or modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, and/or substitution with other chemical groups that can change the peptide’s circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. The term “amino acid” is used interchangeably with “amino acid residue, ” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e. g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

Antibody: As used herein, the term “antibody” refers to any immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. Such proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. As used herein, the terms “antibody fragment” or “characteristic portion of an antibody” are used inter-

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