(12) United States Patent (1o) Patent No.: US 8,173,115 B2 Decuzzi et al. (45) Date of Patent: *May 8, 2012
(54) PARTICLE COMPOSITIONS WITH A OTHER PUBLICATIONS PRE-SELECTED CELL INTERNALIZATION International Search Report and Written Opinion mailed Jun. 3, MODE 2009, in PCT/US2008/071470, 15 pages. (75) Inventors: Paolo Decuzzi, Bari (IT); Mauro U.S. Appl. No. 12/110,515, filed Apr. 28, 2008, Ferrari et al. Ferrari, Houston, TX (US) Champion et al., "Making polymeric micro- and nanoparticles of complex shapes," PHAS, Jul. 17, 2007, 104(29):11901-11904. (73) Assignee: The Board of Regents of The Champion et al., "Role of target geometry in phagocytosis," PHAS, University of Texas System, Austin, TX Mar. 28, 2006, 103(13):4930-4934. Cohen et al., "Microfabrication of Silicon-Based Nanoporous Par- (US) ticulates for Medical Applications," Biomedical Microdevices, 2003, 5(3):253-259. (*) Notice: Subject to any disclaimer, the term of this Conner et al., "Regulated portals of entry into the cell," Nature, Mar. patent is extended or adjusted under 35 6, 2003, 422:37-44. U.S.C. 154(b) by 390 days. Decuzzi et al,. "The role of specific and non-specific interactions in This patent is subject to a terminal dis- receptor-mediated endocytosis of nanoparticles," Biomaterials, claimer. 2007, 28:2915-2922. Decuzzi et al., "A Theoretical Model for the Margination of Particles (21) Appl. No.: 12/181,759 within Blood Vessels," Annals of Biomedical Engineering, Feb. 2005, 33(2):179-190. (22) Filed: Jul. 29, 2008 Decuzzi et al., "Adhesion of Microfabricated Particles of Vascular Endothelium: A Parametric Analysis," Annals of Biomedical Engi- (65) Prior Publication Data neering, Jun. 2004, 32(6):793-802. Decuzzi et al., "The adhesive strength of non-spherical particles US 2010/0029785 Al Feb. 4, 2010 mediated by specific interactions," Biomaterials, 2006, 27:5307- 5314. (51) Int. Cl. Decuzzi et al., "The Effective Dispersion of Nanovectors Within the A61K47148 (2006.01) Tumor Microvasculature," Annals of Biomedical Engineering, 2006, (52) U.S. Cl ...... 424/78.17; 525/54.1; 977/904; 34(4):633-641. 977/962 Decuzzi et al., "The Receptor-Mediated Endocytosis of Nonspheri- (58) Field of Classification Search ...... None cal Particles," Biophysical Journal, May 2008, 94:3790-3797. See application file for complete search history. Ferrari, Mauro, "Cancer Nanotechnology: Opportunities and Chal- lenges," Nature Reviews, Mar. 2005, 5:1-11. (56) References Cited Ferrari, Mauro,"Nanovector therapeutics," Current Opinion in Chemical Biology, 2005, 9:343-346. U.S. PATENT DOCUMENTS Freund et al., "The role of binder mobility in spontaneous adhesive contact and implications for cell adhesion," Journal of the Mechanics 4,520,110 A 5/1985 Stryer et al. and Physics of Solids, 2004, 52:2455-2472. 4,888,176 A 12/1989 Langer et al. Ganong, William F., Review of Medical Physiology, 21st ed. New 4,933,185 A 6/1990 Wheatley et al. York: Lange Medical Books/McGraw-Hill Medical Publishing Divi- 5,010,167 A 4/1991 Ron etal. 5,651,900 A 7/1997 Keller et al. sion, 2003, Table of Contents, 9 pages. 5,770,076 A 6/1998 Chu et al. Gao et al., "Mechanics of receptor-mediated endocytosis," PNAS, 5,798,042 A 8/1998 Chu et al. Jul. 5, 2005, 102(27):9469-9474. 5,893,974 A 4/1999 Keller et al. Hanahan et al., "The Hallmarks of Cancer," Cell, Jan. 7, 2000, 5,938,923 A 8/1999 Tu et al. 100:57-70. 6,107,102 A 8/2000 Ferrari 6,858,184 B2 2/2005 Pelrine et al. (Continued) 7,091,041 B2 8/2006 Monahan et al. 2003/0114366 Al 6/2003 Martin et al. Primary Examiner Daniel C Garnett 2003/0205552 Al 11/2003 Hansford et al. (74) Attorney, Agent, or Firm Winstead PC 2004/0064050 Al 4/2004 Liu et al. 2005/0053590 Al 3/2005 Meininger 2005/0095174 Al 5/2005 Wolf (57) ABSTRACT 2006/0105032 Al 5/2006 Lynch et al. A method of formulating a particle composition having a 2007/0066138 Al 3/2007 Ferrari et al. 2007/0299340 Al 12/2007 Liu et al. pre-selected cell internalization mode involves selecting a 2008/0102030 Al 5/2008 Decuzzi et al. target cell having surface receptors and obtaining particles 2008/0206344 Al 8/2008 Decuzzi et al. that have i) surface moieties, that have an affinity for or are FOREIGN PATENT DOCUMENTS capable of binding to the surface receptors of the cell and ii) a preselected shape, where a surface distribution of the sur- WO WO 03/029403 A3 4/2003 face moieties on the particles and the shape of the particles are WO WO 2007/120248 A2 10/2007 WO WO 2008/021908 A2 2/2008 effective for the pre-selected cell internalization mode. WO WO 2008/041970 A2 4/2008 WO WO 2008/067049 A2 6/2008 13 Claims, 4 Drawing Sheets US 8,173,115 B2 Page 2
OTHER PUBLICATIONS Panes et al., "Regional differences in constitutive and induced ICAM-1 expression in vivo," Am. J. Physiol., 1995, Harris et al., "Influenza virus pleiomorphy characterized by 269(6Pt2):H1955-H1964. cryoelectron tomography," PNAS, Dec. 12, 2006, 103(50):19123- Prodan et al., "Low-frequency, low-field dielectric spectroscopy of 19127. living cell suspensions," Journal of Applied Physics, Apr. 1, 2004, Herant et al., "Mechanics of neutrophil phagocytosis experiments 95(7):3754-3756. and quantitative models," Journal of Cell Science, 2006, 119:1903- 1913. Rolland et al., "Direct Fabrication and Harvesting of Monodisperse, Herantetal., "Mechanics ofneutrophilphagocytosis: behavior ofthe Shape-Specific Nanobiomaterials," JACS, Jun. 21,2005,127:10096- cortical tension," Journal of Cell Science, 2006, 118:1789-1797. 10100. Hill, Terrell L., "An Introcution to Statistical Thermodynamics," Simson et al., "Membrane Bending Modulus andAdhesion Energy of 1960, Table of Contents, 5 pages. Wild-Type and Mutant Cells of Dictyostelium Lacking Talin or Hochmuth, Robert M., "Micropipette aspiration of living cells," Jour- Cortexillins," Biophysical Journal, Jan. 1998, 74:514-522. nal of Biomechanics, 2000, 33:15-22. Smith et al., "How Viruses Enter Animal Cells," Science, Apr. 9, Marsh et al., "Virus Entry: Open Sesame," Cell, Feb. 24, 2006, 2004,304:237-242. 124:729-740. Smythe et al., "Actin regulation in endocytosis," Journal of Cell May et al., "Phagocytosis and the actin cytoskeleton," Journal of Cell Science, 2006, 119(22):4589-4598. Science, Mar. 2001, 114(6):1061-1077. Sun et al., "Mechanics of Enveloped Virus Entry into Host Cells," Mukherjee et al., "Endocytosis," Physiol. Rev., Jul. 1997, 77(3):759- Biophysical Journal: Biophsical Letters, 2006, 90:L10-L 12. 803. Tasciotti et al., "Mesoporous silicon particles as amultistage delivery Oberd6rster et el., "Nanotoxicology: An Emerging Discipline Evolv- system for imaging and therapeutic applications," Nature ing from Studies of Ultrafine Particles," Environmental Health Per- Nanotechnology, Mar. 2008, 3:151-157. spectives, Jul. 2005, 113(7):823-839. Taylor, Richard F., Ed., "Protein Immobilization Fundamentals and Paik et al., "Micromachining of mesoporous oxide films for Applications," 1991, 109-110. microelectromechanical system structures," J. Mater. Res., Aug. Vasir et al., "Polymeric nanoparticles for gene delivery," Expert Opin. 2002,17(8):2121-2129. Drug Delivery, 2006, 3(3):325-344 U.S. Patent May 8, 2012 Sheet 1 of 4 US 8,173,115 B2
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US 8,173,115 B2 1 2 PARTICLE COMPOSITIONS WITH A mode, which comprises a) selecting a cell having surface PRE-SELECTED CELL INTERNALIZATION receptors; and b) obtaining particles that have i) surface moi- MODE eties, that have an affinity for or are capable of binding to the receptors, and ii) a shape, wherein a surface distribution of the STATEMENT FOR FEDERALLY FUNDED 5 surface moieties and the shape are effective for the pre-se- RESEARCH lected cell internalization mode for the selected cell. Another embodiment is a method of treating or monitoring This invention was made with government support under a physiological condition comprising administering to a sub- Grant No. W311`4Q-07-1-0008, awarded by Defense ject in need thereof an effective amount of a particle compo- Advanced Research Projects Agency (DARPA); and under 10 sition having a pre-selected cell internalization mode. Grant No. NNJ06HE06A, awarded by NASA. The U.S. gov- Yet another embodiment is a particle composition having a ernment has certain rights in the invention. pre-selected cell internalization mode.
FIELD DRAWINGS 15 The inventions relate generally to micro and nanosized FIG.1 schematically depicts a particle that has an elliptical particle compositions and their applications and, more par- cross-section. The particle has ligand molecules on its surface ticularly, to micro and nanosized particle compositions hav- that can interact with receptor molecules on a surface of cell's ing a particular pre-selected cell internalization mode. 20 membrane. BACKGROUND FIG. 2 illustrates evolution of the elliptical cylindrical par- ticle presenting the half-wrapping time 0.5ti w and the wrap- Endocytosis is a general term defining processes, by which ping length ratio x_,,/R, as a function of the aspect ratio F, a cell can import and/or export selected extracellular species, ranging from 0.9 to 4, for Rz fixed at 50 mu. such as molecules, viruses, particles and microorganisms and 25 FIG. 3 presents a graph of F_,,, which is a value of E for a target them to specific organelles within a cytoplasm. fixed Rz corresponding to the minimum for the half-wrapping Endocytosis can occur through a variety of pathways includ- time, as a function of R2. FIG. 3 also presents a graph of the
ing clathrin-mediated and caveole-mediated endocytosis; half-wrapping time for F_,,„ as a function of R2 . phagocytosis; clathrin- and caveole-independent endocyto- FIG. 4 illustrates evolution of the elliptical cylindrical par- sis. A particular endocytosis pathway may depend on the size 30 ticle presenting the half-wrapping time 0.5ti w and the wrap- and nature of the extracellular cargo, see e.g. Conner S. D. and ping length ratio x_,,/R, as a function of the aspect ratio F, S. L. Schmid. 2003. Nature 422: 37-44. For instance, caveole- ranging from 0.9 to 4, for a fixed volume (R, -50 mu). mediated endocytosis is assisted by the activation of caveole, plasma membrane invaginations, with a characteristic size of DETAILED DESCRIPTION 50-60 mu; clathrin-mediated endocytosis requires the con- 35 centration of transmembrane receptors and their bound Related Documents ligands on the plasma membrane leading to the formation of vesicular cages with a characteristic size up to few hundreds The following research articles and patent documents, microns (100-500 mu); phagocytosis involves specific cell- which are all incorporated herein by reference in their surface receptors and signaling cascades with the formation 40 entirety, may be useful for understanding the present inven- of cell membrane protrusions that eventually envelope the tions: external micrometer cargo (>1 µm). Clathrin- and caveole- independent endocytosis can be associated with the forma- 1) PCT publication no. WO 2007/120248 published Oct. 25, tion of invaginating vesicles smaller than 100 mu. 2007; Particle endocytosis can be of fundamental importance in 45 2) PCT publication no. WO 2008/041970 published Apr. 10, several fields, such as virology, drug and gene delivery and in 2008; nanotoxicology, see e.g. Marsh M. and A. Helenius. 2006. 3) PCT publication no. WO 2008/021908 published Feb. 21, Cell 124:729-40; Vasir J. K., and V. Labhasetwar. 2006. 2008; Expert Opin. Drug Deliv. 3:325-344; Oberdorster G., E. 4) US Patent Application Publication no. 2008/0102030 pub- Oberdorster, J. Oberdorster. 2005. 113:823-39. 50 lished May 1, 2008; For nanosized particles, both natural, such as enveloped 5) US Patent Application Publication no. 2003/0114366; viruses, or artificial, such as biomimetic particulates, the most 6) U.S. patent application Ser. No. 12/034,259 filed Feb. 20, effective internalization mechanism can be a receptor-medi- 2008; ated endocytosis, in which molecules (ligands) distributed 7) U.S. patent application Ser. No. 12/110,515 filed Apr. 28, over the particle surface bind to countermolecules (receptors) 55 2008; expressed over the cell membrane, which can eventually bend 8) Tasciotti et al., Nature Nanotechnology, vol. 3, 151-158, to invaginate the foreign cargo, see e.g. Marsh M. and A. 2008; Helenius. 2006. Cell 124:729-40; Smith A. E., A. Helenius. 9) Decuzzi and Ferrari, Biomaterials 28, 2007, 2915-2922; 2004. Science 304:237-42. These receptors can be collected 10) Decuzzi and Ferrari, Biophysical Journal, 94, 2008, at the site of invagination by surface diffusion, without which 60 3790-3797. endocytosis would not occur or would occur over a much longer time scale. Definitions
SUMMARY Unless otherwise specified "a" or "an" means one or more. 65 Unless otherwise specified the terms "endocytosis" and One embodiment provides a method of formulating a par- "endocytotic" mean receptor mediated endocytosis and ticle composition having a pre-selected cell internalization receptor mediated endocytotic respectively. US 8,173,115 B2 3 4 "Microparticle" refers to a particle having a maximum A particular selected internalization mode may depend on dimension from 1 micrometer to 1000 micrometers, or, in the application intended for the particles. For example, the some embodiments from 1 micron to 100 microns as speci- endocytosis mode may be preferred when the particle con- fied. tains a cargo, such as an imaging or a therapeutic agent, which "Nanoparticle" refers to a particle having a maximum 5 is desired to be delivered inside the cell. On the other hand, no dimension of less than 1 micron. endocytosis or frustrated endocytosis modes may be pre- "Phagocytosis" refers to an uptake of large (characteristic ferred when the particle contains a cargo, which is not size of more than 2 microns) particles by specialized phago- intended for delivery inside the cell. One example of such cyte cells, which include macrophages, monocytes and neu- situation can be a multistage delivery vehicle disclosed in trophils. Phagocytosis involves the formation of protrusions 10 PCT publication WO 2008/021908, where a first stage par- in the cell membrane that eventually envelope the external ticle of the vehicle, which contains inside second stage par- particle. ticles, is intended to recognize and adhere to a target site in "Receptor mediated endocytosis" or RME refers to an endothelium, without being internalized by the endothelial internalization by a cell of aparticle, which has moieties, such 15 cells. After adherence, the first stage particle can release the as ligands, distributed over its surface, which can bind to countermoieties (receptors) expressed over the cell's mem- second stage particles. brane. RME involves bending of the cell's membrane, which In many embodiments, the selected cell can be a non- results in a full wrapping of the particle by the membrane and phagocyte cell, i.e. a cell that cannot perform phagocytosis. eventual internalization of the particle by the cell. Particles Examples of phagocyte cells, i.e. cells that perform phagocy- that are internalized via RME can have smaller characteristic 20 tosis include neutrophills, monocytes and macrophages. size than particles that undergo internalization via phagocy- In some embodiments, the selected cell may be an endot- tosis. RME is not limited to phagocyte cells. helial cell, such as an endothelial vasculature cell, and the "Biodegradable" refers to a material that can dissolve or target site may be a vasculature site, such as a coopted vas- degrade in a physiological medium or a biocompatible poly- culature, an angiotensic vasculature or a renormalized vascu- meric material that can be degraded under physiological con- 25 lature. For cells of coopted vasculature, the surface receptors ditions by physiological enzymes and/or chemical condi- may be angiopoietin 2 receptors; for cells of angiogenic vas- tions. culature, the surface receptors may be vascular endothelial growth factors (VEGF), basic fibroblast growth factors or Disclosure endothelial markers such as 04 3 integrins; for cells of renor- 30 malized vasculature, the surface receptors may be carcino- The present inventors recognized an importance of a par- embionic-related cell adhesion molecules 1 (CEACAMI), ticle shape for a receptor mediated endocytosis. Accordingly, endothelin-B receptor (ET-B), vascular endothelial growth the present invention provides a method of formulating a factor inhibitors gravin/AKAP12, scaffolding proteins for particle composition with a pre-selected cell internalization protein kinase A and protein kinase C. mode, which can involve selecting a target cell and obtaining 35 The surface moieties of the particle's surface can be com- particles that have on their surfaces surface moieties, that plimentary to the receptors on the selected cell's surface. The have affinity for or are capable of binding to surface receptors surface moieties maybe, for example, antibodies, aptamers or of the surface of the target cell. The surface distribution of the ligands with affinity for or capable of binding to the receptors surface moieties on the particles and a shape of the particles on the selected cell's membrane surface. In some embodi- are such that they are effective for the pre-selected cell inter- 40 ments, the surface moieties may include specific moieties to nalization mode for the selected cell. the receptors on the selected cell's membrane surface. In The pre-selected cell internalization mode may be selected some embodiments, the surface moieties may include non- from an "endocytosis" or "no-endocytosis" mode. "Endocy- specific moieties to the receptors on the selected cell's mem- tosis" refers to a mode, when a particle can be fully wrapped brane surface. Yet in other embodiments, the surface moieties by the cell's membrane via a receptor mediated endocytosis 45 may include both specific and non-specific moieties. and eventually internalized, "no-endocytosis" refers to a Particles with a pre-selected internalization mode may be a mode, when the particle can be at most partially wrapped by part of a composition that can also include particles without the cell's membrane. the pre-selected internalization mode. The particles with the In some embodiments, the pre-selected cell internalization pre-selected internalization mode may constitute at least 10% mode may a frustrated or partial endocytosis. "Frustrated 50 or at least 25% or at least 50% or at least 75% or at least 90% endocytosis" refers to a mode, when the particle gets only by number of the total number of particles in the composition. partially wrapped by the cell's membrane and does not get In many embodiments, the particle can be a non-spherical internalized by the cell. particle. In some embodiments, the particle can be a particle The particles with a pre-selected cell internalization mode with a circular cross section. In some embodiments, the par- may be used for treating and/or monitoring a physiological 55 ticle can be an ellipsoidal particle. Yet in other embodiments, condition. In such a case, one can select a target site, which is the particle can be a cylindrical particle with an elliptical affected with the physiological condition and has cells of cross section in a direction perpendicular to the axis of the which have surface receptors on its surface, in a body of a cylinder. subject, such as a mammal, preferably a human, and admin- In some embodiments, the particle's largest characteristic istering to the subject an effective amount of a composition 60 size, such as a length of its major axis for a particle with an that comprises particles with a pre-selected internalization elliptical cross section, canbe less than 2 microns, or less than mode for the cells of the target site. The physiological condi- 1 micron or less than 800 nm or from 5 nm to 500 nm or from tion can be for example a disease, such as cancer or an 5 nm to 800 nm from 5 nm to 1 micron or from 10 nm to 1 inflammation. micron or from 10 nm to 800 nm or from 10 nm to 500 nm or In general, the selected cell may be any type of cell that has 65 from 20 nm to 1 micron or from 20 nm to 800 nm or from 20 surface receptors on its surface. In many embodiments, the nm to 500 nm or from 50 nm to 1 micron or from 50 nm to 800 selected cells can be a mammal cell, such as a human cell. nm or from 50 nm to 500 nm. US 8,173,115 B2 5 6 Preferably, the particle's largest characteristic size is sub- The maximum endocytosis curvature K_ can be defined stantially smaller than a characteristic size of the selected cell. as an inverse minimum endocytotic radius calculated for the Theparticle's largest characteristic size can be at least times selected cell for a spherical particle or a particle with a circu- smaller or at least 5 times smaller or at least 10 times smaller lar cross section that has the same ligands on its surface as the or at least 20 times smaller or at least 30 times smaller or at 5 non-spherical particle. least 50 times smaller or at least 100 times smaller or at least The minimum endocytotic radius and thus the maximum 200 times smaller or at least 300 times smaller or at least 500 endocytotic curvature can be evaluated using the formula (1) times smaller or at least 100 times smaller than the charac- teristic size of the selected cell. A characteristic size of the selected cell may vary from about 5 microns to about 40 10 B m' m, K_,— R.i,,_ C--+log—+1 microns or from about 10 microns to about 30 microns. m 1 m 1 In some embodiments, the obtained particles may be par- Fm ticles that have a convex subsurface with a local curvature and a local surface density of the moieties, such as ligands, that In the equation above, C is the ligand-receptor binding are effective for internalization of the particles via a receptor 15 energy relative to kBT, where kB is the Bolzmann's constant mediated endocytosis, which means that the local curvature K and T is a temperature of the cell or the target site expressed is smaller than a maximum endocytosis curvature K_ for the in Kelvins (absolute temperature). C depends on a particular local surface density of the moieties. Maximum endocytosis ligand-receptor pair. In particular, C= log[Kd2D] Kd2D is an curvature can depend on a binding energy between moieties equilibrium dissociation constant for the ligand-receptor on the surface of the particle and receptors on the surface of 20 interacting at the cell/particle interface. Kd2D can be esti- the selected cell's membrane, a bending energy factor of the mated from the following relation Kd' Kd/h, where Kd i s an selected cell's membrane and surface densities of the moi- equilibrium dissociation constant for the same ligand-recep- eties of the particle and of the receptors on the selected cell's tor pair determined, for example, experimentally in solution membrane. Methods of evaluating maximum endocytosis and h is a thickness of a confinement region, to which the curvature are disclosed below. 25 ligand-receptor sites are restricted. In many cases, h can be In some embodiments, the local surface density of the equal approximately to 10 mu. moieties at the convex surface may be greater than a surface B is a bending energy factor of the cell's membrane, which density of the moieties at other parts of the surface of the can be determined as detailed in Hochmuth, R. M., J. Bio- particle, thus, making a surface distribution of the surface mech., 33:15-22, 2000. moieties on the surface of the particle non-uniform. 30 m,,, which is an average surface density on the receptors, For particles with an elliptical cross section, obtaining when the particle is not interacting with the cell, can be particles with a pre-selected cell internalization mode may determined using methods known to those of ordinary skill in involve obtaining particles that have an aspect ratio corre- the art. For example, one can determine m,, in vivo by using sponding to the selected mode. radiolabeled monoclonal antibodies complimentary to the For example, FIG. 1 illustrates a elliptical cross section of 35 receptors as detailed for intercellular adhesion molecule 1 an elliptical cylindrical particle that interacts with a mem- receptors in Panes J., et al. Am. J. Physiol. 1995; 269(6Pt2): brane via specific ligand receptor bonds. A surface density of H1955-64. Alternatively, m,, can be determined using fluores- the ligands on the surface of the particle is rn , while the cently labeled monoclonal antibodies complementary to the surface density of the receptors on a surface of the cell's receptors. Such fluorescently labeled monoclonal antibodies membrane is m, 40 can be antibodies labeled with phycoerythrin disclosed in The elliptical cross section can be characterized R 1 and Rz, U.S. Pat. No. 4,520,110. which are semi-lengths of the axes of the ellipse. An aspect m, is a local surface density of ligands rn, which can be ratio of the ellipse, which can be defined as F=R,/R 2 . The varied by controlling surface functionalization conditions for cross sectional area of the particle isA=7iR 1 R2 7iFR22-7iRs2, the particle and/or by varying a size of the ligand molecule. where RS is a radius of a particle with a circular cross-section 45 The actual surface density of ligands on the particle can be that has the same are as the elliptical particle. verified experimentally using citofluometry or radiolabeled The geometry of the particle can be fully defined by a countermolecules in radioassays. number of pairs of parameters such R 1 and R2 ; F and R 1 ; F For a particle with a uniform surface distribution of and R21 F and RS or F and V, where V is the volume of the ligands, the local surface density and the average surface particle. 50 density can be the same. In a case, when the geometry of the particle is defined In certain embodiments, the minimum endocytotic radius through F and R2, a shape parameter determining a pre- and thus the maximum endocytotic curvature can be evalu- selected internalization mode can be an aspect ratio F for a ated as disclosed in U.S. patent application Ser. No. 12/034, particular R2 . 259 "Endocytotic particles" filed Feb. 20, 2008, which is The particle with an elliptical cross section can be in a 55 incorporated herein by reference in its entirety. "no-endocytosis" mode if the aspect ratio of the particle for a FIG. 2 illustrates evolution of the elliptical cylindrical par- particular semilength-112 of a minor axis is less than a first critical ticle presenting the half-wrapping time 0.5ti w and the wrap- value F'=(R2Km,,) , where K_ is a maximum endocytosis ping length ratio x_,,/R1 as a function of the aspect ratio F, curvature, which is defined below. ranging from 0.9 to 4, for R2-50 nm evaluated using a theo- The particle can be in a frustrated endocytosis mode if the 60 retical model disclosed in Decuzzi and Ferrari, Biophysical aspect ratio of the particle for a particular semilength of its Journal, 94, 2008, 3790-3797, which is incorporated herein minor axis is no greater than a second critical value, by reference in its entirety. xm ,, is a projection of the parti- F—R 2 Km_ cle's wrapping length over the x-axis in FIG. 1. For xm,,/ The particle can be in an endocytosis mode if the aspect R1=1 complete wrapping occurs at time tiw . In FIG. 2, for ratio of the particle for a particular semilength of its minor 65 F
Mechlorethamine, Melphalan, Mercaptopurine, Methotrex- 50 inhibiting 4(5)-imidazoles, 4 Hydroxytamoxifen, Trioxifene, ate, Mitomycin, Mitotane, Mitoxantrone, Oxaliplatin, Pacli- Keoxifene, Onapristone, And Toremifene (Fareston); and taxel, Pamidronate, Pentostatin, Plicamycin, Procarbazine, anti-androgens such as Flutamide, Nilutamide, Bicalutamide, Rituximab, Steroids, Streptozocin, STI-571, Streptozocin, Leuprolide, and Goserelin; and pharmaceutically acceptable Tamoxifen, Temozolomide, Teniposide, Tetrazine, Thiogua- salts, acids or derivatives of any of the above. nine, Thiotepa, Tomudex, Topotecan, Treosulphan, Trimetr- 55 Cytokines may be also used as the therapeutic agent. exate, Vinblastine, Vincristine, Vindesine, Vinorelbine, Examples of such cytokines are lymphokines, monokines, VP-16, and Xeloda. and traditional polypeptide hormones. Included among the Useful cancer chemotherapy drugs also include alkylating cytokines are growth hormones, such as human growth hor- agents, such as Thiotepa and cyclosphosphamide; alkyl sul- mone, N-methionyl human growth hormone, and bovine fonates, such as Busulfan, Improsulfan and Piposulfan; aziri- 60 growth hormone; parathyroid hormone; thyroxine; insulin; dines, such as Benzodopa, Carboquone, Meturedopa, and proinsulin; relaxin; prorelaxin; glycoprotein hormones, such Uredopa; ethylenimines and methylamelamines, including as follicle stimulating hormone (FSH), thyroid stimulating altretamine, triethylenemelamine, trietylenephosphoramide, hormone (TSH), and luteinizing hormone (LH); hepatic triethylenethiophosphaoramide and trimethylolomelamine; growth factor; fibroblast growth factor; prolactin; placental nitrogen mustards, such as Chlorambucil, Chlornaphazine, 65 lactogen; tumor necrosis factor-a and -(3; mullerian-inhibit- Cholophosphamide, Estramustine, Ifosfamide, mechlore- ing substance; mouse gonadotropin-associated peptide; thamine, mechlorethamine oxide hydrochloride, Melphalan, inhibin; activin; vascular endothelial growth factor; integrin; US 8,173,115 B2 11 12 thrombopoietin (TPO); nerve growth factors, such as NGF-(3; not heat-stable material may be sterilized by passage through platelet growth factor; transforming growth factors (TGFs), a commercially-available sterilization filter. Of course, filtra- such as TGF-a and TGF-(3; insulin-like growth factor-I and tion may be used only in cases where the particles is smaller -II; erythropoietin (EPO); osteoinductive factors; interferons than the pores of the sterilizing filter. such as interferon-a, -(3 and -y; colony stimulating factors 5 The particles may be administered to a subject in need of (CSFs), such as macrophage-CSF (M-CSF); granulocyte- therapeutic intervention via a number of suitable administra- macrophage-CSF (GM-CSF); and granulocyte-CSF (GCSF); tion methods. The particular method employed for a specific interleukins (ILs), such as IL-1, IL-I a, IL-2, IL-3, IL-4, IL-5, application can be determined by the attending physician. For IL-6, IL-7, IL-8, IL-9, IL-I1, IL-12, IL-15; a tumor necrosis example, the particles may be administered by one of the factor, such as TNF-a or TNF-(3; and other polypeptide fac- io following routes: topical, parenteral, inhalation, oral, vaginal tors including LIE and kit ligand (KL). As used herein, the and anal. Intravascular administration, which includes intra- tern cytokine includes proteins from natural sources or from venous (i.v.), intramuscular (i.m.) and subcutaneous (s.c.) recombinant cell culture and biologically active equivalents injection, may be particularly preferred. of the native sequence cytokines. Intravascular administration may be either local or sys- The imaging agent may be a substance, that can provide 15 temic. Local intravascular delivery may be used to bring the imaging information about a targeted site in a body of an particles in the vicinity of a body site having a known tumor animal, such as a mammal or a human being. The imaging or inflammation by use of guided catheter system, such as a CAT-scan guided catheter. General injection, such as a bolus agent can comprise magnetic material, such as iron oxide, for magnetic resonance imaging. For optical imaging, the active i.v. injection or continuous/trickle-feed i.v. infusion are typi- agent can be, for example, a semiconductor nanocrystal or 20 cally systemic. quantum dot. For optical coherence tomography imaging, the The particles may be injected into the blood stream and imaging agent can be a metal, e.g. gold or silver, nanocage allowed to circulate and localize to their target site. Prefer- particle. The imaging agent can be also an ultrasound contrast ably, the particles are injected to a vasculature of the target agent, such as a micro or nanobubble or iron oxide micro or site. nanoparticle. 25 References Compositions 1. Gao H, et al. 2005. Proc. Natl. Acad. Sci. USA 102:9469- The particles having a pre-selected internalization cell 74. mode for particular type of cells can be part of a composition, 30 2. Sun S. X. and D. Wirtz. 2006. Biophysical Journal 90:LI0- such as a pharmaceutical composition. Such a composition 2. may be a suspension comprising the particles described above 3. Champion J. A., et al. 2007. Proc. Natl. Acad. Sci. USA for use in administering a therapeutic or imaging agent. To 104:11901-4. form the suspension, the particles may be suspended in an 4. Champion J. A., S. Mitragotri. 2006. Proc. Natl. Acad. Sci. aqueous medium at a selected concentration. The optimal 35 USA 103:4930-4. concentration can depend on the characteristics (e.g., solubi- 5. Rolland J. P., et al. 2005. J. Am. Chem. Soc. 127:10096- lization properties) of the particle, type of therapeutic appli- 100. 6. Cohen M. H, et al. 2001. Biomedical Microdevices 3:253- cation and mode of administration. For example, composi- tions for oral administration can be relatively viscous, and 259. may therefore contain a higher concentration (e.g., >50%) of 40 7. Harris A, G. et al. 2006. Proc. Natl. Acad. Sci. USA 103: the particle. Solutions for bolus injections preferably contain 19123-7 a relatively concentrated suspension of the particles (e.g., 8. Freund L. B. and Y. Lin. 2004. Journal of the Mechanics 10-50%), but not so concentrated that it has an appreciably and Physics of Solids 52:2455-2472 higher viscosity than saline (to minimize need for large-bore 9. Hill T L. 1960. An Introduction to Statistical Thermody- needles). The solution used for continuous intravenous infu- 45 namics. Dover Publications, Inc. New York. sion typically can contain a relatively low concentration (e.g., 10. Herant M., et al. 2006. J. Cell. Sci. 119: 1903-13. 2-10% suspension) of the particles, due to the relatively large 11. Smythe E., K. R. Ayscough. 2006. J. Cell Sci. 119:4589- volumes of fluid that are administered. 98. The particles may be suspended in a number of suitable 12. May R. C., L. M. Machesky. 2001. J. Cell Sci. 114: aqueous carrier vehicles. A suitable pharmaceutical carrier 50 1061-77. can be a carrier that is non-toxic to the recipient at the dosages 13. Simson R, et al. 1998. Biophysical Journal. 74: 514-522. and concentrations employed and is compatible with other 14. Herant M, et al. 2005. J. Cell Sci. 118:1789-97. ingredients in the formulation. Examples of suitable carrier Although the foregoing refers to particular preferred vehicles include but are not limited to water, saline, Ringer's embodiments, it will be understood that the present invention solution, dextrose solution, and 5% human serum albumin. 55 is not so limited. It will occur to those of ordinary skill in the Suspensions for use in injectable formulations are preferably art that various modifications may be made to the disclosed isotonic with the subject's blood. Generally, the carrier can embodiments and that such modifications are intended to be contain minor amounts of additives, such as substances that within the scope of the present invention. enhance isotonicity and chemical stability, e.g., buffers and All of the publications, patent applications and patents preservatives, as well as low molecular weight (less than 60 cited in this specification are incorporated herein by reference about 10 residues) polypeptides, proteins, amino acids, car- in their entirety. bohydrates including glucose or dextrans, chelating agents What is claimed is: such as EDTA, or other excipients. 1. A method of formulating a particle composition having Prior to administration to the subject, the suspension of a preselected cell internalization mode, comprising: particles may be sterilized by a suitable sterilization method. 65 a) selecting a cell having surface receptors; and Particles fabricated from a heat-stable material may be heat- b) fabricating ellipsoidal particles that have i) surface moi- sterilized, e.g., using an autoclave. Particles fabricated from a eties that have an affinity for or are capable of binding to US 8,173,115 B2 13 14 the receptors, and ii) an aspect ratio that is effective for 7. The method of claim 1, wherein the fabricating com- the preselected cell internalization mode for the selected prises fabricating by a top-down process. cell, wherein the pre-selected internalization mode is 8. The method of claim 1, wherein the fabricating com- selected from a full endocytosis, a frustrated endocytosis prises disposing the moieties onto a surface of the particles. and no endocytosis. 5 9. The method of claim 1, wherein the obtaining comprises 2. The method of claim 1, wherein the cell is an endothelial selecting the particles from a particle population. cell. 10. The method of claim 1, wherein the particles comprise 3. The method of claim 2, wherein the endothelial cell is an an active agent. endothelial vasculature cell. 11. The method of claim 10, wherein the active agent io comprises an imaging agent or a therapeutic agent. 4. The method of claim 3, wherein the receptors are angio- 12. The method of claim 1, wherein said particles are genesis vasculature receptors, coopted vasculature receptors nanoparticles. or renormalized receptors. 13. The method of claim 1, further comprising determining 5. The method of claim 1, wherein the cell is a non-phago- the aspect ratio based on a surface density of the surface cyte cell. 15 moieties and a surface density of the surface receptors. 6. The method of claim 1, wherein the surface moieties are ligands that are capable of binding to the receptors.