HK1158930A - Particle compositions with a pre-selected cell internalization mode - Google Patents

Description

Particulate compositions having preselected cellular internalization patterns

Statement regarding federally sponsored research

Some studies underlying the present invention were supported by Federal funds, department of defense fund No. W81XWH-04-2-0035 and NASA fund No. SA 23-06-017. The united states government may have certain rights in the invention.

Technical Field

The present invention relates generally to micro-and nanoparticle compositions and their uses, and more particularly to micro-and nanoparticle compositions having a pre-selected specific cellular internalization pattern.

Background

Endocytosis is a general term that defines the process by which a cell imports and/or exports selected extracellular substances, such as molecules, viruses, particles and microorganisms, and targets them to specific organelles within the cytoplasm. Endocytosis can occur through a variety of pathways, including clathrin-mediated and caveolar-mediated endocytosis, phagocytosis, clathrin-and caveolar-independent endocytosis. The specific endocytosis pathway may depend on the size and nature of the extracellular substance, see, e.g., Conner s.d. and s.l.schmid.2003.nature 422: 37-44. For example, activation of a cytoplasmic crypt with a characteristic size of 50-60nm, i.e., plasma membrane invagination, can aid in cytoplasmic crypt-mediated endocytosis; clathrin-mediated endocytosis requires concentrations of transmembrane receptors and their binding ligands on the plasma membrane to result in the formation of vesicular cages (vesicular cage) with characteristic sizes up to several hundred microns (100-500 nm); phagocytosis involves specific cell surface receptors and signal transduction cascades that form cell membrane bulges that eventually encapsulate micron-sized foreign material (> 1 μm). Endocytosis, independent of clathrin and caveolae, may be accompanied by the formation of invaginated vesicles smaller than 100 nm.

Particle endocytosis can be very important in several fields, such as virology, drug and gene delivery, and nanotopography, see for example Marsh m, and a.helenius.2006.cell124: 729 to 40; vasir j.k., and v.labhasetwar.2006.expert opin.drug deliv.3: 325- > 344; oberdorster g., e.oberdorster, j.oberdorster.2005.113: 823-39.

For nano-sized particles, e.g. natural particles such as enveloped viruses, or artificial particles such as biomimetic particles, the most efficient internalization mechanism is probably receptor-mediated endocytosis, in which molecules (ligands) distributed on the particle surface bind to antagonistic molecules (receptors) expressed on the cell membrane, which eventually bends invaginates to encapsulate foreign substances, see e.g. Marsh m. and a. helenius.2006.cell 124: 729 to 40; smith a.e., a.helenius.2004.science 304: 237-42. These receptors can accumulate at the invagination site by surface diffusion, and without this, endocytosis does not occur, or takes a much longer time to complete.

Summary of The Invention

One embodiment provides a method of formulating a particulate composition having a preselected cellular internalization mode, the method comprising a) selecting a cell having a surface receptor; and b) obtaining particles having i) a surface moiety having an affinity for or capable of binding to said receptor, and ii) a shape, wherein the surface distribution of said surface moiety and said shape are effective to achieve a preselected cellular internalization pattern for the selected cell.

Another embodiment is a method of treating or monitoring a physiological condition comprising administering to a subject in need thereof an effective amount of a particulate composition having a preselected cellular internalization pattern.

Another embodiment is a particle composition having a preselected cellular internalization mode.

Drawings

Fig. 1 is a schematic illustration of a particle having an elliptical cross-section. Ligand molecules on the surface of the particle can interact with receptor molecules on the surface of the cell membrane.

FIG. 2 shows the evolution of the elliptical-cylindrical particles, showing a half-wrap time of 0.5 τ_wAnd the package length ratio x_{Maximum of}/R₁With respect to the aspect ratio Γ (in the range 0.9 to 4), R₂Immobilization was at 50 nm.

FIG. 3 is a gamma_{Minimum size}And R₂Wherein Γ is_{Minimum size}Is a fixed R corresponding to a minimum of half-wrap time₂The value of Γ. FIG. 3 is also Γ_{Minimum size}Half wrap time and R₂A graph of the relationship (c).

FIG. 4 shows the evolution of the elliptical-cylindrical particles, showing a half-wrap time of 0.5 τ_wAnd the package length ratio x_{Maximum of}/R₁Fixed volume (R) in relation to the aspect ratio Γ (in the range 0.9 to 4)_s＝50nm)。

Detailed Description

Related document

The following research papers and patent documents, the entire contents of which are incorporated herein by reference, may be helpful in understanding the present invention:

1) PCT publication No. WO 2007/120248, published 10/25/2007;

2) PCT publication No. WO 2008/041970, published 4 months and 10 days 2008;

3) PCT publication No. WO 2008/021908, published 2.21.2008;

4) U.S. patent application publication No. 2008/0102030, published on 5/1/2008;

5) U.S. patent application publication numbers 2003/0114366;

6) U.S. patent application No. 12/034,259, filed on month 2, 20, 2008;

7) U.S. patent application No. 12/110,515, filed on 28/4/2008;

8) tasciotti et al, Nature Nanotechnology, vol 3, 151-;

9) decuzzi and Ferrari, Biomaterials 28, 2007, 2915-;

10) decuzzi and Ferrari, Biophysic Journal, 94, 2008, 3790-.

Definition of

"A" or "an" means one or more unless stated otherwise.

Unless otherwise indicated, the terms "endocytosis" and "endocytosis" refer to receptor-mediated endocytosis and receptor-mediated endocytosis, respectively.

"microparticle" refers to a particle having a maximum dimension of 1 micron to 1000 microns, or 1 micron to 100 microns as described in some embodiments.

"nanoparticle" refers to particles having a largest dimension of less than 1 micron.

"phagocytosis" refers to the ingestion of large (characteristic size over 2 microns) particles by specialized phagocytes, including macrophages, monocytes, and neutrophils. Phagocytosis involves the formation of bulges in the cell membrane, eventually encapsulating foreign particles.

"receptor-mediated endocytosis" or RME refers to a cell internalizing particle having distributed on its surface moieties, such as ligands, that bind to an antagonist moiety (receptor) expressed on the cell membrane. RME involves cell membrane bending, resulting in complete encapsulation of the particle by the cell membrane, and eventual internalization of the particle by the cell. Particles internalized by RME are smaller in characteristic size than particles internalized by phagocytosis. RME is not limited to phagocytes.

"biodegradable" refers to a material that dissolves or degrades in a physiological medium or a biocompatible polymeric material that degrades under physiological conditions by physiological enzymes and/or under chemical conditions.

Disclosure of Invention

The present inventors recognized the importance of particle shape in receptor-mediated endocytosis. Accordingly, the present invention provides a method of formulating a particle composition having a preselected mode of cell internalization, which can include selecting a target cell and obtaining a particle having a surface moiety on its surface, which has affinity for or can bind to a surface receptor on the surface of the target cell. The distribution of the surface moieties on the surface of the particle and the shape of the particle are effective to achieve a preselected cellular internalization pattern of the selected cell.

The preselected cellular internalization mode can be selected from an "endocytosis" or "non-endocytosis" mode. "endocytosis" refers to the mode in which a subject-mediated endocytosis particle can be completely enveloped by a cell membrane and eventually internalized, and "non-endocytosis" refers to the mode in which a particle can be at most partially enveloped by a cell membrane

In some embodiments, the preselected cellular internalization mode can be frustrated endocytosis or partial endocytosis. By "frustrated endocytosis" is meant a mode in which the particle is only partially encapsulated by the cell membrane and cannot be internalized by the cell.

Particles having a preselected cellular internalization pattern can be used to treat and/or monitor physiological conditions. In this case, a target site affected by a physiological condition and having a surface receptor on its cell surface in a subject, e.g. a mammal, preferably a human, may be selected and an effective amount of a composition comprising particles having a pre-selected mode of internalization for the cells of the target site is administered to the subject. The physiological condition may be, for example, a disease, such as cancer or inflammation.

In general, the selected cells can be any type of cell having surface receptors on its surface. In many embodiments, the selected cell can be a mammalian cell, such as a human cell.

The particular internalization mode selected can depend on the desired particle application. For example, when the particles contain a substance that is desired to be delivered into the cell, such as an imaging agent or a therapeutic agent, an endocytic mode may be preferred. On the other hand, when the particles contain substances that are not desired to be delivered into the cell, non-endocytosis or impaired endocytosis may be preferred. An example of this would be a multi-stage delivery vector as described in PCT publication No. WO 2008/021908, in which the primary particles (containing the secondary particles inside) of the vector serve to recognize and adhere to a target site in the endothelium without being internalized by endothelial cells. After adhering, the primary particles may release the secondary particles.

In many embodiments, the selected cell may be a non-phagocytic cell, i.e., a cell that is incapable of phagocytosis. Examples of phagocytic cells, i.e., cells that undergo phagocytosis, include neutrophils, monocytes, and macrophages.

In some embodiments, the selected cell may be an endothelial cell, e.g., a vascular endothelial cell, and the target site may be a vascular site, e.g., a plugged (coapted) vessel, a vascular tone vessel, or a renormalized vessel. For cells that fill the vasculature, the surface receptor may be the angiopoietin 2 receptor; in the case of cells of the angiogenic vessel, the surface receptor may be Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor or an endothelial marker such as alpha_vβ₃An integrin; in the case of cells that re-normalize the vasculature, the surfaceThe receptor can be carcinoembryonic-related cell adhesion molecule 1(CEACAM1), endothelin-B receptor (ET-B), vascular endothelial growth factor inhibitor glatiramer (gravin)/AKAP12, protein kinase A and protein kinase C scaffold protein.

The surface portion of the particle surface may be complementary to a receptor on the surface of the selected cell, and the surface portion may be, for example, an antibody, aptamer or ligand having affinity for or capable of binding to the receptor on the surface of the selected cell membrane. In some embodiments, the surface moiety may comprise a moiety specific for a selected cell membrane surface receptor. In some embodiments, the surface moiety may comprise a moiety that is not specific for a selected cell membrane surface receptor. In other embodiments, the surface moiety may comprise both specific and non-specific moieties.

The particles having a preselected internalization mode can be part of a composition that can further comprise particles that do not have a preselected internalization mode. The number of particles having a preselected internalization mode can be at least 10% or at least 25% or at least 50% or at least 75% or at least 90% of the total number of particles in the composition.

In many embodiments, the particles may be non-spherical particles. In some embodiments, the particles may be particles having a circular cross-section. In some embodiments, the particles may be elliptical particles. In other embodiments, the particles may be cylindrical particles having an elliptical cross-section in a direction perpendicular to the axis of the cylinder.

In some embodiments, the largest characteristic dimension of the particle, for example the major axis length of a particle having an elliptical cross-section, may be less than 2 microns, less than 1 micron, or less than 800nm, or from 5nm to 500nm, 5nm to 800nm, 5nm to 1 micron, 10nm to 800nm, 10nm to 500nm, 20nm to 1 micron, 20nm to 800nm, 20nm to 500nm, 50nm to 1 micron, 50nm to 800nm, or 50nm to 500 nm.

Preferably the largest characteristic dimension of the particle is significantly smaller than the characteristic dimension of the selected cell. The largest characteristic dimension of the particle may be at least 3 times smaller, at least 5 times smaller, at least 10 times smaller, at least 20 times smaller, at least 30 times smaller, at least 50 times smaller, at least 100 times smaller, at least 200 times smaller, at least 300 times smaller, at least 500 times smaller, at least 100 times smaller than the characteristic dimension of the selected cell. The characteristic dimension of the selected cells may range from about 5 microns to about 40 microns, or from about 10 microns to about 30 microns.

In some embodiments, the resulting particle may be a particle having a convex lower surface, the local curvature and the local surface density of the moiety, e.g. ligand, being such that the particle is efficiently internalized by subject-mediated endocytosis, meaning that the local curvature κ is less than the curvature of maximal endocytosis κ for the local surface density of the moiety_max. The maximum endocytosis curve can depend on the binding energy between the moiety on the surface of the particle and the receptor on the surface of the selected cell membrane, the bending energy factor of the selected cell membrane, the surface density of the moiety on the particle, and the density of the receptor on the selected cell membrane. Methods for assessing maximum endocytosis curvature are disclosed below.

In some embodiments, the local surface density of the portion on the convex surface may be greater than the surface density of the portion on other portions of the particle surface, thus making the distribution of the surface portion on the particle surface non-uniform.

For particles having an elliptical cross-section, obtaining particles having a preselected mode of cellular internalization can include obtaining particles having an aspect ratio corresponding to the selected mode.

For example, figure 1 shows an elliptical cross-section of an elliptical-cylindrical particle interacting with a membrane via specific ligand receptor binding. The surface density of the ligands on the particle surface is m_lAnd the surface density of receptors on the cell membrane surface is m_r。

The elliptical cross-section can be represented by R₁And R₂Characterisation that they are ellipsesHalf the length of the axis of the circle. The aspect ratio of an ellipse may be defined as R ═ R₁/R₂. The cross-sectional area of the particles is A ═ pi R₁R₂＝πΓR₂ ²＝πR_s ²Wherein R is_sIs the radius of a particle with a circular cross-section having the same cross-sectional area as an elliptical particle.

The geometry of the particles may be characterized by a plurality of pairs of parameters, e.g. R₁And R₂Γ and R₁Γ and R₂Γ and R_sOr Γ and V, where V is the particle volume.

In one case, by Γ and R₂The shape parameter that determines the preselected internalization mode may be a specific R when defining the geometric characteristics of the particle₂The aspect ratio Γ.

If the aspect ratio of the particles of a particular minor axis half-length is less than a first critical value Γ ═ R (R ═ R)₂κ_max)^-1/2Then a particle with an elliptical cross-section may be in the "non-endocytic" mode, where κ_maxIs the maximum endocytosis curvature, which is defined below.

If the aspect ratio of the particles of a given minor axis half length is not greater than a second critical value Γ ″ -, R₂κ_maxThe particle may be in a frustrated endocytosis mode.

A particle of a particular minor axis half-length may be in endocytic mode if its aspect ratio is greater than a first critical value Γ' and less than a second critical value Γ ″.

Maximum endocytosis curvature κ_maxCan be defined as the inverse minimum endocytic radius calculated for selected cells for spherical particles with the same ligands on the surface as non-spherical particles or particles with a circular cross-section.

The minimum endocytic radius and maximum endocytic curvature can be estimated using the following formula (1):

in the above equation, C is relative to k_BLigand-receptor binding energy of T, wherein k_BIs the Boltzmann constant, and T is the temperature of the cell and target site (expressed in Kelvin, absolute temperature). C depends on the particular ligand-receptor pair. In particular, the first and second (c) substrates,is the equilibrium dissociation constant of the ligand-receptor interaction at the cell/particle interface.Can be estimated from the following relationshipWherein K_dAre the equilibrium dissociation constants of the same ligand-receptor pair (e.g., can be determined experimentally in solution); h is the thickness of the localized region to which the ligand-receptor site is limited. In many cases, h may be equal to about 10 nm.

B is the bending energy factor of the cell membrane and can be measured, for example, by Hochmuth, r.m., j.biomech., 33: 15-22, 2000, as determined by the method described in detail.

m_rIs the average surface density on the receptor, and m can be determined using methods known to those skilled in the art when the particles do not interact with the cells_r. For example, methods similar to those used in Panes J. et al, am.J.Physiol.1995; 269(6Pt 2): radiolabelled monoclonal antibodies complementary to the intercellular adhesion molecule 1 receptor as detailed in H1955-64, in vivo m is determined_r. Alternatively, m can be measured using a fluorescently labeled monoclonal antibody complementary to the receptor_r. Such a fluorescently labeled monoclonal antibody may be a phycoerythrin-labeled antibody as described in U.S. Pat. No. 4,520,110.

m_lIs a ligand m_lCan be varied by controlling the surface functionalization conditions of the particles and/or by varying the size of the ligand molecules. The actual surface density of the ligands on the particles can be verified by radioactive assays using flow cytometry (cytofluorimetry) or radiolabeled challenge molecules.

For particles with a uniform distribution of surface ligands, the local surface density and the average surface density may be the same.

In certain embodiments, the minimum radius of endocytosis and the maximum curvature of endocytosis can be assessed by a method such as described in U.S. patent application No. 12/034,259 "endocytic particles" (filed 2/20/2008), which is incorporated herein by reference in its entirety.

FIG. 2 shows the evolution of the elliptical-cylindrical particles, showing a half-wrap time of 0.5 τ_wAnd the package length ratio x_{Maximum of}/R₁In relation to the aspect ratio Γ (ranging from 0.9 to 4), as evaluated by a theoretical model (as described by Decuzzi and Ferrari, Biophysical Journal, 94, 2008, 3790-₂＝50nm。x_{Maximum of}Is the projection of the particle envelope length on the x-axis in fig. 1. At x_{Maximum of}/R₁When 1, at τ_wFull wrapping occurs. In FIG. 2, Γ < Γ'_crAt time (about 0.9 in this case), the wrapping cannot begin, resulting in τ_wInfinite, x_{Maximum of}/R₁The ratio is zero. For Γ'_cr＜Γ＜Γ”_crMiddle value Γ in (1), ratio x_{Maximum of}/R₁Always 1, meaning that the particle is fully internalized, 0.5. tau_wIncreasing almost linearly with increasing Γ. Gamma < gamma'_crWhen x_{Maximum of}/R₁Decreases, reaches a minimum, and then increases steadily as Γ increases.

FIG. 3 is a gamma_{Minimum size}And R₂Wherein Γ is_{Minimum size}Is corresponding to half wrapping timeFixed R of small value₂The value of Γ. FIG. 3 is also Γ_{Minimum size}Half wrap time and R₂A graph of the relationship (c). Same date and Γ'_crAnd Γ "_crAre shown in Table 1.

TABLE 1

R₂，nm Γ_{Minimum size} (0.5τ_w)_{Minimum size}Of seconds Γ'_cr Γ″_cr

38.35(＝R_{Maximum water}) 1 ∞ 1 1

50 0.96 41.60 0.87 1.30

75 0.80 78.33 0.71 1.95

100 0.68 123.4 0.62 2.61

150 0.56 243.7 0.50 3.91

300 0.40 823.6 0.36 7.82

500 0.32 2105.2 0.28 13.0

With particle size R₂Of high aspect ratio Γ_{Minimum size}(minimum internalization time) from 1 (R)₂＝R_{Minimum size}) Reduced to 0.3 (R)₂＝500nm)。

In some cases, the two parameters that describe the geometric characteristics of the elliptical-cylindrical particle may be Γ and R_s。

In this case, the shape parameter that determines the pre-selected cellular internalization mode may be a specific R_sThe aspect ratio of the values Γ.

If the aspect ratio of the particles of a particular minor axis half-length is less than the first critical value Γ₁’＝(R_sκ_max)^-2/3Particles with an elliptical cross-section may then be in a "non-endocytic" mode, where κ_maxIs the maximum endocytosis curvature, which is defined below.

If the aspect ratio of the particles of the particular minor axis half-length is not greater than the second critical value Γ₁”＝(R_sκ_max)^2/3The particle may be in a frustrated endocytosis mode.

If the aspect ratio of the particles of a particular minor axis half-length is greater than a first critical value Γ₁And is less than a second critical value₁", the particle may be in endocytic mode.

FIG. 4 shows the evolution of the elliptical-cylindrical particles, showing a half-wrap time of 0.5 τ_wAnd the package length ratio x_{Maximum of}/R₁In relation to the aspect ratio Γ (ranging from 0.9 to 4), as evaluated by a theoretical model (as described by Decuzzi and Ferrari, Biophysical Journal, 94, 2008, 3790-_s＝50nm。

Type of particle

The type of particle having the preselected internalization mode is not specifically limited. For example, the particles may be liposomes, polymer-based particles, silicon-based and silica-based particles, quantum dots, nanogold shells, dendrimers (dendrimers), or viral particles.

In some embodiments, particles having a shape effective to achieve a preselected internalization mode can be manufactured. In other embodiments, particles having a shape effective to achieve a preselected internalization mode can be selected from a library of particles having a wide distribution of shapes and/or sizes. Zetasizer from Malvern Instruments, Worcestershire, United Kingdom, for example, may be utilized^TMThe nano-series instrument is fed from the particle libraryIn row selection, the instrument is capable of determining the geometric dimensions of the particles.

The particles can be made using a variety of methods. In general, manufacturing methods that can control the size and shape of the particles may be preferred.

In some embodiments, the particles can be fabricated by a top-down microfabrication process or a nanofabrication process, such as photolithography, electron beam lithography, X-ray lithography, deep UV lithography, or nanolithography. An advantage of using a top-down manufacturing process may be that such a process may be used for large-scale production of uniformly sized particles.

These particles may have a targeting moiety, such as a ligand, aptamer or antibody, on the surface. For example, the ligand may be chemically attached to a suitable reactive group on the surface of the particle. Protein ligands can be attached to amino-and thiol-reactive groups under conditions effective to form thioether and amide bonds. Methods for attaching antibodies or other polymeric binders to inorganic or polymeric supports can be found, for example, in Taylor, R. eds., "principles and Applications of Protein Immobilization proteins and Applications", pp 109110 (1991).

For example, when particles are prepared by a top-down micro-or nano-fabrication method, a non-uniform surface distribution of surface portions can be achieved. For example, a substrate from which the particles are made may be patterned with a coating that resists deposition of the ligand so that the particles have at least two different surface areas: one resistant to ligand deposition and the other not. When the substrate is subsequently contacted with a solution containing the ligand, the substrate may release the ligand to produce particles with a heterogeneous distribution of ligand.

In some embodiments, the particles can have one or more channels connecting the reservoir to the surface. In some embodiments, the reservoirs and channels can be pores in the body of the particle. In this case, the particles may comprise a porous or nanoporous material. The pores of the porous or nanoporous material can be controlled to achieve a desired loadingActive agent and/or desired release rate. The nanoporous material having a controlled pore size can be an oxide material, such as SiO₂、Al₂O₃Or TiO₂. Nanoporous oxide particles, also known as sol-gel particles, can be made, for example, in Paik j.a. et al, j.mater.res, volume 17, 8 months 2002. The nanoporous material having a controlled pore size may also be nanoporous silicon. Nanoporous silicon particles can be produced, for example, in Cohen m.h. et al, Biomedical Microdevices 5: 3,253-259, 2003.

In some other embodiments, the particles may be completely devoid of any channels. Such particles may comprise, for example, biodegradable materials. For example, the particles may be composed of metals (e.g., iron, peptides, gold, silver, platinum, copper), alloys, and oxides thereof. The biodegradable material may also be a biodegradable polymer such as polyorthoesters, polyanhydrides, polyamides, polyalkylcyanoacrylates, polyphosphazenes and polyesters. Exemplary biodegradable polymers are found, for example, in U.S. patents 4,933,185, 4,888,176, and 5,010,167. Specific examples of such biodegradable polymeric materials include poly (lactic acid), polyglycolic acid, polycaprolactone, polyhydroxybutyrate, poly (N-palmitoyl-trans-4-hydroxy-L-prolinate), and poly (DTH carbonate).

In certain embodiments, the particle itself may be the active agent.

Active agent

The active agent may be a therapeutic compound and/or an imaging agent. The choice of the polymeric active agent will depend on the desired application.

The therapeutic agent may be a physiologically or pharmacologically active substance that produces a desired biological effect in the foraminous vasculature of a subject, such as a mammal or human. Therapeutic agents may be inorganic or organic compounds, including peptides, proteins, nucleic acids, and small molecules. The therapeutic agent may take various forms, for example, uncharged molecules, molecular complexes, pharmacologically acceptable salts such as hydrochloride, hydrobromide, sulfate, laurate, palmitate, phosphate, nitrite, nitrate, borate, acetate, maleate, tartrate, oleate, salicylate, and the like. For acidic therapeutic agents, metal salts, amine salts, or organic cation salts, such as quaternary ammonium salts, may be employed. Drug derivatives such as bases, esters and amides may also be used as therapeutic agents. The water-insoluble therapeutic agents may be used in the form of their water-soluble or base derivatives, which in either case or by delivery, are capable of being converted by the action of enzymes, hydrolyzed by body pH, or converted by other metabolic processes to the original therapeutically active form.

The therapeutic agent may be a chemotherapeutic agent, an immunosuppressive agent, a cytokine, a cytotoxic agent, a nucleolytic compound, a radioisotope, a prodrug of a receptor and an activating enzyme, or any combination thereof, which may be naturally occurring or produced by synthetic or recombinant means.

Drugs that suffer from classical multidrug resistance, such as vinca alkaloids (e.g., vinblastine and vincristine), anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin-D), and microtubule stabilizing drugs (e.g., paclitaxel) may be particularly suitable for use as the therapeutic agent.

Cancer chemotherapeutic agents may also be preferred therapeutic agents. Useful cancer chemotherapeutics include nitrogen mustards, nitrosoureas, ethyleneimines, alkyl sulfonates, tetrazines, platinum compounds, pyrimidine analogs, purine analogs, antimetabolites, folic acid analogs, anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors, and hormonal agents. Exemplary chemotherapeutic agents are actinomycin-D, Ikrainian (Alkeran), Ara-C, anastrozole, asparaginase, BiCNU, bicalutamide, bleomycin, busulfan, capecitabine, carboplatin, carmustine, CCNU, chlorambucil, cisplatin, cladribine, CPT-11, cyclophosphamide, cytarabine, cytosine arabinoside, cyclophosphamide, dacarbazine, D-actinomycin, daunomycin, dexrazoxane, docetaxel, doxorubicin, DTIC, epirubicin, ethylenimine, Poposide, floxuridine, fludarabine, fluorouracil, flutamide, fotemustine, gemcitabine, herceptin, hexamethonium, hydroxyurea, flavobilin, ifosfamide, irinotecan, lomustine, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, carboplatin, carmustine, carm, Oxaliplatin, paclitaxel, pamidronic acid, pentostatin, plicamycin, procarbazine, rituximab, steroids, streptozocin, STI-571, streptozocin, tamoxifen, temozolomide, teniposide, tetrazine, thioguanine, thiotepa, toludex, topotecan, trioxane (Treosulphan), tritrexate, vinblastine, vincristine, vindesine, vinorelbine, VP-16, and aroada.

Useful cancer chemotherapeutic agents also include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzotepa, carboquone, meturedepa, and uredepa; aziridine and melamines including hexamethylmelamine, tritylamine, triethylphosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards, such as chlorambucil, chlorophosphamide, estramustine, ifosfamide, mechlorethamine hydrochloride, melphalan, novibahin, phenylacetylcholesterol mustine, prednimustine, trofosfamide, uracil mustard; nitroureas, such as carmustine (Cannustine), pyritinose, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as doxorubicin, actinomycin, antromycin, azaserine, bleomycin, C-actinomycin, calicheamicin, Carabicin (Carabicin), carminomycin, carvacmycin, chromomycin, D-actinomycin, daunomycin, ditobicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin (idambicin), mosaic, mitomycin, mycophenolic acid, norubicin, olivomycin, pelomycin, bofilomycin (potfiromycin), puromycin, doxorubicin, roxydicin, streptodorcin, streptonigrin, streptozotocin, ubenizomib, netstastin, and zorubicin; anti-generationThank-sening drugs such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine and 5-FU; androgens such as carpoterone, drostarone propionate, epitioandrostanol, meperitane (rnephiostatne), and testolactone; anti-adrenergic agents, such as aminoglutethimide, mitotane, and trostane; folic acid supplements such as folic acid; acetic acid glucurolactone; (ii) an aldehydic phosphoramide; (ii) aminolevulinic acid; amsacrine; berrabucil (besrabucil); a bisantrene group; idatrosa; desphosphamide (defofamine); colchicine; diazaquinone; alfuzinin (elfornitine); eletamine; etoglut; gallium nitrate; a hydroxyurea; (ii) mushroom polysaccharides; lonidamine; mitoguazone; mitoxantrone; mopidanol; diamine nitracridine; pentostatin; methionine; pirarubicin; podophyllinic acid; 2-ethyl hydrazide; procarbazine;lezoxan; cilostase (Sizofran); a germanium spiroamine; alternarionic acid; a tri-imine quinone; 2, 2' -trichlorotriethylamine; uratan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; adding cytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxols, e.g. taxolBristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (Taxel @)RPR company, Andony, France (Rhone-Poulenc Rorer, Antony, France)); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; (ii) a platinum analog, wherein the platinum analog,such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; novier; dihydroxy anthraquinone; (ii) teniposide; daunomycin; aminopterin; (ii) an aptamer; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoic acid; an esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of the above drugs. The invention also includes anti-hormonal agents, such as anti-estrogen agents, for modulating or inhibiting the effects of hormones on tumors, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5) -imidazole, 4-hydroxy tamoxifen, trovaxifen, Keoxifene (Keoxifene), onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of the above drugs.

Cytokines may also be used as therapeutic agents. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; (ii) prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); a liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; a muller tube inhibiting substance; a mouse gonadotropin-related peptide; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factors, such as NGF-beta; platelet growth factor; transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); an osteoinductive factor; interferons such as interferon- α, - β, and- γ; colony Stimulating Factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-csf (gcsf); interleukins (IL), such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factors, such as TNF- α or TNF- β; and other polypeptide factors, including LIF and Kit Ligand (KL). The term "cytokine" as used herein includes proteins of natural or recombinant cell culture origin, as well as biologically active equivalents of the native sequence cytokines.

The imaging agent may be a substance that provides imaging information about a target site within the body of an animal, such as a mammal or human. The imaging agent may comprise a magnetic material, such as iron oxide, for magnetic resonance imaging. In optical imaging, the active agent may be, for example, a semiconductor nanocrystal or quantum dot. In optical coherence tomography, the imaging agent may be metal (e.g., gold or silver) nanocage particles. The imaging agent may also be an ultrasound contrast agent, such as micro or nanobubbles, or iron oxide micro or nanoparticles.

Composition comprising a metal oxide and a metal oxide

Particles having a preselected internalization pattern for a particular cell type can be made part of a composition, such as a pharmaceutical composition. Such compositions may be suspensions comprising the above-described particles for administration of a therapeutic or imaging agent. To form a suspension, the particles may be suspended in an aqueous medium at a selected concentration. The optimal concentration may depend on the particle characteristics (e.g., dissolution characteristics), the type of therapeutic application, and the mode of administration. For example, compositions for oral administration may be relatively thick and thus may contain a relatively high concentration (e.g., > 50%) of particles. The solution used for the bolus injection preferably contains a relatively concentrated suspension of particles (e.g., 10-50%), but is concentrated only to the extent that its viscosity is slightly higher than that of saline (to minimize the use of a large bore needle). Solutions for continuous intravenous infusion typically contain lower concentrations (e.g., 2-10% suspension) of particles due to the larger volume of liquid administered.

The particles can be suspended in a variety of suitable aqueous carriers. Suitable pharmaceutical carriers can be those that are non-toxic to recipients at the dosages and concentrations employed and are compatible with the other ingredients of the formulation. Examples of suitable carriers include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. The suspension for injection formulation is preferably isotonic with the blood of the subject. Typically, the carrier will contain minor amounts of additives such as substances which enhance isotonicity and chemical stability, such as buffers and preservatives, as well as low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, sugars including glucose or dextran, chelating agents such as EDTA, or other excipients.

The particle suspension may be sterilized by a suitable sterilization method prior to administration to a subject. The granules made from the heat stable material may be heat sterilized, for example, using an autoclave. Granules made with non-heat stable materials can be sterilized by commercially available sterilization filters. Of course, filter sterilization can only be used when the particles are smaller than the pore size of the sterilization filter.

The particles can be administered to a subject in need of therapeutic intervention by a suitable method of administration. The particular method used for a particular application may be determined by the attending physician. For example, the particles may be administered by one of the following routes: topical, parenteral, inhalation, oral, vaginal and anal routes. Intravascular administration may be particularly preferred, including intravenous (i.v.), intramuscular (i.m.), and subcutaneous (s.c.) injections.

Intravascular administration may be by local or systemic means. Local intravascular delivery can be used to deliver the particles to the vicinity of body sites known to have tumors or inflammation by a guiding catheter system, such as a CAT scan guiding catheter. Common injections, such as bolus injections, i.v. injections or continuous/drip i.v. infusions, are usually systemic.

The particles can be injected into the bloodstream and circulated and localized to their target site. The particles are preferably injected into the blood vessel at the target site.

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***

While specific embodiments are mentioned above, it should be understood that the invention is not limited thereto. It will be appreciated by those of ordinary skill in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to fall within the scope of the present invention.

All publications, patent applications, and patents cited herein are hereby incorporated by reference in their entirety.

Claims (22)

1. A method of formulating a particulate composition having a preselected cellular internalization mode, the method comprising:

a) selecting cells having surface receptors; and

b) obtaining a particle having i) a surface moiety having an affinity for or capable of binding to said receptor, and ii) a shape, wherein the surface distribution of said surface moiety and said shape are effective to achieve a preselected cellular internalization pattern for the selected cell.

2. The method of claim 1, wherein the preselected internalization mode is selected from the group consisting of complete endocytosis, frustrated endocytosis, and non-endocytosis.

3. The method of claim 1, wherein the cell is an endothelial cell.

4. The method of claim 3, wherein the endothelial cells are vascular endothelial cells.

5. The method of claim 4, wherein the receptor is a neovascular receptor, a packed vascular receptor, or a renormalization receptor.

6. The method of claim 1, wherein the cell is a non-phagocytic cell.

7. The method of claim 1, wherein said surface moiety is a ligand capable of binding to said receptor.

8. The method of claim 1, wherein the obtaining comprises making the particles.

9. The method of claim 8, wherein the fabricating comprises fabricating by a top-down method.

10. The method of claim 8, wherein the fabricating comprises disposing the portion on a surface of the particle.

11. The method of claim 1, wherein said obtaining comprises selecting said particles from a population of particles.

12. The method of claim 1, wherein the particles comprise an active agent.

13. The method of claim 12, wherein the active agent comprises an imaging agent or a therapeutic agent.

14. The method of claim 1, wherein the particles are nanoparticles.

15. The method of claim 1, wherein the particles are non-spherical particles.

16. The method of claim 1, wherein the particles are particles that do not have a circular cross-section.

17. The method of claim 1, wherein the particles are elliptical particles.

18. The method of claim 17, wherein the ellipsoidal particles have an aspect ratio effective for the selected cell to achieve a preselected cellular internalization mode.

19. The method of claim 18, further comprising determining the aspect ratio based on a surface density of the surface portion and a surface density of the surface receptor.

20. The method of claim 1, wherein the surface of the particle comprises a convex surface having a curvature and a local surface density of the portion effective to achieve a preselected endocytosis pattern.

21. A method of treating or monitoring a physiological condition, said method comprising administering to a subject in need thereof an effective amount of a particulate composition formulated according to the method of claim 12.

22. A composition formulated according to the method of claim 1.