CELL-DERIVED MICROPARTICLE DELIVERY SYSTEM AND USES THEREOF - Patent application

Abstract

The present invention relates to a microparticle comprising a core and a membrane surrounding the core, the membrane comprising a cell membrane component. A method for transporting the microparticle is provided. The method includes administering the microparticle to an endothelium, whereby the microparticle binds to the endothelium; and translocating the microparticle across the endothelium. Also provided is a method for preparing the microparticle.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 275,027, filed November 3, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] FIELD OF THEINVENTION The present invention relates generally to cell-derived microparticles useful as delivery systems for crossing endothelial barriers, and to their use and preparation. [Background technology]

[0003] Targeted drug delivery to many parts of the body, especially to the central nervous system (CNS) and lymph nodes (LNs) due to the selective and restrictive endothelium, remains a central challenge. The endothelium is the tissue that separates the circulating blood and lymph from the body tissues. As such, all fluids, molecules, macromolecules and cells that move from the circulating bloodstream or lymph must cross the endothelial barrier. Dysregulation of the vascular endothelium, which occurs in tumors and other pathological growths, allows passive transport of fluids, molecules and nano-sized aggregates (or nanoparticles), as well as cells. On the other hand, in the absence of pathology, the normal endothelium acts as a selective barrier with local barrier properties in different tissues and different vessel types. For example, in some parts of the body, the endothelium is inherently "leaky" with large fenestrations, e.g. in the bone marrow, or capillary networks in tissues, e.g. in muscle. This architecture allows for the passive transport of fluids, molecules and nano-sized aggregates (or nanoparticles) and also for the active squeezing of cells through open capillary fenestrations, as documented in podocyte formation and active processes from circulating cells. Other sites in the body, such as the blood-brain barrier (BBB) ​​in the central nervous system (CNS) and the high endothelial venules (HEV) in lymph nodes (LNs), have similar features that severely limit the passive transport of most small molecule drugs to these sites. Traditionally, the dogma is that circulating cells can cross these endothelial barriers by actively extravasating through the endothelium, classically depicted in the literature as cell squeezing through the endothelium. Although several mechanisms of cell transport across the endothelium are known, descriptions of these mechanisms rely on active processes from extravasating cells.

[0004] There remains a need for effective delivery systems for transporting a variety of active agents across the endothelial barrier to target sites, for example, in the brain and lymph nodes. Summary of the Invention [Means for solving the problem]

[0005] The present inventors have surprisingly discovered non-naturally occurring microparticles that have the ability to cross the endothelium.The present invention relates to microparticles for delivering active agents across the endothelial barrier to target sites, for example, in the brain and lymph nodes.

[0006] A microparticle is provided, the microparticle comprising a core and a membrane surrounding the core, the membrane comprising a cell membrane component.

[0007] The membrane may further comprise synthetic membrane components.

[0008] The membrane may be from a cell that has been permeabilized (permeabilized cells). The permeabilized cells may have been subjected to cryopermeabilization, detergent, or chemical permeabilization solutions. The permeabilized cells may be permeabilized white blood cells (permeabilized white blood cells).

[0009] The membrane may further comprise a targeting moiety, which may include an integrin, a selectin, a cadherin, an immunoglobulin-like adhesion molecule, an addressin, a chemokine receptor, a chemokine ligand, a growth factor receptor, an immunoglobulin superfamily protein, an ion channel linked receptor, a G protein-coupled receptor, an enzyme-linked receptor, an antibody or fragment thereof, or a binding domain thereof.

[0010] The core may comprise cytoplasm, fluid, polymer, extracellular matrix protein, or combinations thereof. The core may comprise an active agent. The active agent may comprise a biological molecule, a compound, or combinations thereof. The active agent may comprise a nanoparticle, a liposome, a virus, or combinations thereof. The active agent may comprise a therapeutic agent, an imaging agent, a sequestering agent, a prophylactic agent, a diagnostic agent, a prognostic agent, an excipient, or combinations thereof. The core may be prepared from white blood cells.

[0011] The microparticles may be non-immunogenic.

[0012] A method for transporting a microparticle is provided. The microparticle comprises a core and a membrane surrounding the core, the membrane comprising a cell membrane component. The method of transport includes administering the microparticle to an endothelium, whereby the microparticle binds to the endothelium; and translocating the microparticle across the endothelium. The endothelium can be in the brain or a lymph node. The endothelium can be in a subject.

[0013] According to the method of delivery, the membrane may further comprise a targeting moiety. The targeting moiety may comprise an integrin, a selectin, a cadherin, an immunoglobulin-like adhesion molecule, an addressin, a chemokine receptor, a chemokine ligand, a growth factor receptor, an immunoglobulin superfamily protein, an ion channel-linked receptor, a G protein-coupled receptor, an enzyme-linked receptor, an antibody or a fragment thereof, or a binding domain thereof.

[0014] The method of delivery may further include moving the microparticles across the endothelium and then moving the microparticles to a target site. The endothelium may be in a lymph node and the target site may be a lobule in the lymph node. The endothelium may be in the brain and the target site may be in the brain parenchyma or cerebrospinal fluid (CSF). The endothelium and the target site may be in a tumor.

[0015] The core may include an active agent, and the delivery method may further include releasing the active agent at the target site.

[0016] The delivery method may further include sequestering the molecule from the target site with a microparticle.

[0017] The delivery method may further include eliciting a biological response at the target site. The biological response may be selected from the group consisting of an immune interaction, a cancer therapy, a vaccine response, and an immunotherapy.

[0018] Further provided is a method for preparing the microparticles, the method comprising mixing a core with a membrane, the membrane comprising a cell membrane component. The membrane may further comprise a synthetic membrane component.

[0019] The membrane may be a cell membrane of a permeabilized white blood cell, and the preparation method may further include adding a core into the permeabilized white blood cell. The permeabilized white blood cell may be a permeabilized lymphocyte (permeabilized lymphocyte).

[0020] The preparation may further include encasing the core in a membrane. The membrane may be, for example, a cell membrane separated from a permeabilized leukocyte. The permeabilized leukocyte may have been subjected to a freeze permeabilization, detergent, or chemical permeabilization solution. The permeabilized leukocyte may be a permeabilized lymphocyte.

[0021] According to the preparation method, the membrane may further comprise a targeting moiety. The targeting moiety may comprise an integrin, a selectin, a cadherin, an immunoglobulin-like adhesion molecule, an addressin, a chemokine receptor, a chemokine ligand, a growth factor receptor, an immunoglobulin superfamily protein, an ion channel-linked receptor, a G protein-coupled receptor, an enzyme-linked receptor, an antibody or a fragment thereof, or a binding domain thereof.

[0022] The preparation method may further comprise loading the core with an active agent.

[0023] The preparation method may further include preparing the cores from permeabilized leukocytes. The permeabilized leukocytes may be those that have been subjected to a freeze permeabilization, detergent, or chemical permeabilization solution. The permeabilized leukocytes may be permeabilized lymphocytes. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 shows a schematic diagram of how the present invention works. [Diagram 2]FIG. 2 shows serial block-face SEM images of HEV cross sections, demonstrating the reorganization of HEV cells that allows transcellular transport of lymphocytes. [Diagram 3] FIG. 3 shows a possible schematic method for producing permeabilized cells as MPs. [Figure 4] Figure 4 shows live / dead viability staining comparing cell death at different freezing rates and cryoprotectant concentrations. Numbers in the bottom left corner indicate cell death. [Diagram 5] FIG. 5 shows that resuspension of MPs without added DNAse (−DNAse) did not show aggregation, whereas resuspension with added DNAse (+DNAse) did show aggregation. [Figure 6] FIG. 6 shows images of live control Jurkat cells (upper panel) and permeabilized CSTL Jurkat cells (MP) (lower panel) under bright field microscopy (left panel), fluorescence microscopy (middle panel), and merged images (right panel). [Figure 7] FIG. 7 shows flow cytometry of (A) live cells, (B) pre-spun MPs, and (C) post-spun MPs, demonstrating that permeabilization of cells (MPs) causes differences in cell size compared to live cells. [Figure 8] FIG. 8 shows the recovery rate of CSTL (MP) under different centrifugation conditions with various spin speeds. [Figure 9] 9A-B show the change in (A) diameter and (B) circularity after spinning at different speeds. [Figure 10] Figures 10A-B show the change in (A) diameter and (B) circularity after successive spins at 300 x g for 5 min. [Figure 11] 11A-B show the change in (A) diameter and (B) circularity after 2 hours of incubation at different temperatures. [Figure 12] Figure 12A-D shows a microfluidic model of blood flow for stability and venipuncture testing (A). Changes in MP number (B), diameter (C) and circularity (D) after a certain number of passes through the vascular mimic. [Figure 13] 13A-C show the release profiles generated using MPs after loading with (A) 70 kDa FITC-dextran, (B) raltegravir and (C) cisplatin, demonstrating the broad potential for drug loading. [Figure 14] FIG. 14 shows images of triple-negative breast cancer cells (4T1-luc2) that were either untreated (control) or treated in vitro with unloaded MPs, free cisplatin, or cisplatin-loaded MPs at a dose equivalent to free cisplatin (cisplatin-MPs). [Figure 15] FIG. 15 shows images of alginate MPs and cell-mimetic membrane-encased alginate MPs (cmMPs), each with a Cy5-labeled core. [Figure 16] FIG. 16 shows images of alginate MPs, T cell-derived plasma membranes (TcPMs), and cell-mimetic membrane-wrapped alginate MPs (cmMPs), each with the plasma membrane labeled with DiD for visualization. [Figure 17] FIG. 17 shows an image of an alginate MP hydrogel core (Alginate core) labeled with AF647 Gydrazide, an image of a T cell-derived plasma membrane (TcPM) labeled with BODIPY TMRC5 Malemide, and a merged image of Alginate care and TcPM (cmMP). [Figure 18] FIG. 18 shows the release curve of passively loaded fluoresceinamine in alginate MP. [Figure 19] Figure 19 shows vibratome sections of mouse LNs with robust uptake of CFSE-labeled MPs within the lobule compared to dye-only controls. Phalloidin counterstaining indicates distinct interaction of MPs with HEV cells and their presence outside the lobule vasculature. [Figure 20] FIG. 20 shows diagrams (left panel) and images (right panel) of MP control injections (upper panel) and FAB+MP injections (lower panel) validating the LN homing ability of MP. [Figure 21]FIG. 21 shows a schematic of the experimental design for brain harvesting. [Figure 22] Figure 22A-F shows images of control MPs from activated or resting T cells in lymph node (A) or brain (D), respectively; targeted MPs from resting or activated T cells in lymph node (LN) (B) or brain (E), respectively; and vascular counterstaining in lymph node (C) or brain (F). MPs from activated T cells migrate inefficiently to LN (A) but efficiently to brain (E). MPs from resting T cells migrate efficiently to LN (B) but inefficiently to brain (D). Vascular counterstaining confirms extravasation of MPs into tissues in both brain and LN (C and E). NHS Cy5.5-labeled MPs were administered 4 hours before sacrifice. [Diagram 23] FIG. 23 shows the biphasic release kinetics of small molecules from MPs. [Figure 24] FIG. 24 shows the PK / PD model of the biodistribution of MP. [Diagram 25] FIG. 25 shows predicted concentrations of MPs and small molecule drugs in plasma and lymph nodes over time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention provides cell-derived microparticles (MPs) as a delivery system across the endothelial barrier. The present invention is based on the inventors' surprising discovery that during the transport of circulating live cells (e.g., leukocytes) across the endothelial barrier, e.g., via extravasation at target sites in the brain and lymph nodes, endothelial cells in the endothelium shuttle circulating live cells across the endothelial barrier after docking of native cells to the apical surface of the endothelial cells. In particular, the inventors unexpectedly discovered that docking of circulating live cells and subsequent trans-endothelial cellular transport (extravasation or diapedesis) is actively controlled by the endothelium, whereas for circulating live cells, this transport is a passive process defined by the composition of their cell membrane. This is evidenced by the finding that dead T cells have the ability to bind to the apical surface of high endothelial venules (HEVs) and extravasate into lymph node lobules or enter the brain parenchyma. Since T cells and other specialized lymphocytes routinely migrate across the endothelial barrier, the inventors further discovered that cell-mimetic microparticles with membrane-encased cores derived from the plasma membrane of cells such as white blood cells (e.g., lymphocytes) have the ability to cross the endothelium. Such functional properties are defined by the composition of the membrane and the size of the core. While nanoparticles are internalized and retained within cells, the microparticles of the present invention are transported across the endothelium.

[0026] The inventors have developed MPs with cell-derived membranes to enable docking and interaction with the endothelium and subsequent transport across the endothelium, e.g., to the tissue parenchyma (Figure 1). Different membrane compositions, e.g., membranes isolated from different cell types, membrane mixtures of cell types, or engineered isolated cell membranes, allow the MPs to bind to distinct sites on the restrictive endothelial barrier of the desired tissue and cross the restrictive endothelial barrier to locally deliver the active agent, also known as a payload (e.g., sequestrant), as a drug depot. In addition, the membrane composition allows direct interaction with live cells at the target site to induce a response. These MPs can circulate systemically in the bloodstream or through lymph of a subject (e.g., human or non-human) and migrate to tissues throughout the body to locally deliver / sequester the agent at the target (e.g., therapeutic) site.

[0027] As used herein, the term "microparticle (MP)" refers to a particle having a size of about 0.1 to 1,000 μm, 0.1 to 900 μm, 0.1 to 800 μm, 0.1 to 700 μm, 0.1 to 600 μm, 0.1 to 500 μm, 0.1 to 400 μm, 0.1 to 300 μm, 0.1 to 200 μm, 0.1 to 100 μm, 0.1 to 50 μm, 0.1 to 10 μm, 0.1 to 1 μm, 0.5 to 1,000 μm, 0.5 to 900 μm, 0.5 to 800 μm, 0.5 to 700 μm, 0.5 to 600 μm, 0.5 to 500 μm, 0.5 to 400 μm, 0.5 to 300μm, 0.5~200μm, 0.5~100μm, 0.6~1,000μm, 0.6~900μm, 0.6~800μm, 0.6~700μm, 0.6~600μm, 0.6~500μm, 0.6~400μm, 0.6~300μm, 0.6~200μm , 0.6~100μm, 0.7~1,000μm, 0.7~900μm, 0.7~800μm, 0.7~700μm, 0.7~60 0μm, 0.7~500μm, 0.7~400μm, 0.7~300μm, 0.7~200μm, 0.7~100μm, 0.8~1 ,000μm, 0.8~900μm, 0.8~800μm, 0.8~700μm, 0.8~600μm, 0.8~500μm, 0 .8~400μm, 0.8~300μm, 0.8~200μm, 0.8~100μm, 0.9~1,000μm, 0.9~900μm m, 0.9~800μm, 0.9~700μm, 0.9~600μm, 0.9~500μm, 0.9~400μm, 0.9~300 μm, 0.9~200μm, 0.9~100μm, 1~1,000μm, 1~900μm, 1~800μm, 1~700μm, 1~ It refers to materials in the ranges 600μm, 1-500μm, 1-400μm, 1-300μm, 1-200μm, 1-100μm, 100-1,000μm, 100-900μm, 100-800μm, 100-700μm, 100-600μm, 100-500μm, 100-400μm, 100-300μm, 100-200μm, 500-1,000μm, 500-900μm, 500-800μm, 500-700μm, 500-600μm, 750-1,000μm, 750-900μm or 750-800μm. For example, MPs can be 0.8-500μm in size.

[0028] As used herein, the term "extravasation" refers to the transport of microparticles (MPs) through a cellular barrier.

[0029] The terms "cell barrier" and "tissue barrier" are used interchangeably herein and refer to one or more layers of cells that separate two biological spaces within a subject. For example, the cell barrier can be an endothelial barrier.

[0030] The terms "endothelial barrier" and "endothelium" are used interchangeably herein and refer to one or more layers of endothelial cells that separate two compartments within a subject. For example, an endothelial barrier may separate a blood vessel from a lymph node lobule.

[0031] As used herein, the term "subject" refers to a mammal, such as a primate or a human. A subject can be human or non-human. A subject can be suffering from or susceptible to a disease or condition.

[0032] The term "membrane" as used herein refers to a lipid-based shell, including monolayer, bilayer, or multilayer. The membrane may include a phospholipid bilayer. The membrane may be about 0.1-200 nm, 0.1-150 nm, 0.1-100 nm, 0.1-50 nm, 0.1-20 nm, 0.1-10 nm, 0.1-1 nm, 0.5-200 nm, 0.5-150 nm, 0.5-100 nm, 0.5-50 nm, 0.5-20 nm, 0.5-10 nm, 0.5-1 nm, 1-200 nm, 1-150 nm, 1-100 nm, 1-50 nm, 1-20 nm, 1-10 nm, 0.1-1 nm, 5-200 nm, 5-150 nm, 5-100 nm, 5-50 nm, 5-20 nm, or 5-10 nm in thickness.

[0033] The term "cell" as used herein refers to any cell from a subject. The cell may be from a subject of the same or of the same genus or species as the subject whose cell barrier is crossed by the MP. The cell may be a blood cell (e.g., red blood cell (RBC), white blood cell (WBC), or platelet). The cell may be an immune cell. The immune cell may be selected from the group consisting of lymphoid progenitor cells and all cells differentiated from the progenitor cells, including all T cells, B cells, and natural killer (NK) cells, NKT cells, plasma cells, and all subsets and subtypes of these cells. The immune cell may be selected from the group consisting of myeloblast progenitor cells and all cells differentiated from the progenitor cells, including granulocytes (eosinophils, basophils, neutrophils, and mast cells), myeloid-derived suppressor cells, and antigen-presenting cells (APCs), including dendritic cells (plasma cells and conventional cell types), monocytes, and macrophages. The immune cells may be selected from innate lymphoid cells, tissue-resident immune cells (e.g., microglial cells), mucosa-associated invariant T (MAIT) cells, and decidual macrophages, decidual natural killer cells. The cells may be placental cells. The placental cells may be selected from the group consisting of trophoblast cells, placental fibroblasts, and placental endothelial cells, extravillous trophoblast cells, and giant cells. The cells may be tumor or cancer cells. The cells may be epithelial cells, endothelial cells, or neuronal cells. The cells may be non-terminally differentiated cells, such as stem cells (e.g., hematopoietic stem cells, bone marrow stem cells, mesenchymal stem cells, cardiac stem cells, or neural stem cells). The cells may be live or dead. The cells may have been modified after isolation from a subject or after being pharmacologically treated live in vitro prior to permeabilization, e.g., by permeabilization or cryo-permeabilization.

[0034] The terms "living cell" or "viable cell" are used interchangeably herein and refer to a cell that has biological activity in metabolism, transcription, translation, or protein synthesis.

[0035] As used herein, the term "dead cell" refers to a cell that has no biological activity in metabolism, transcription, translation, or protein synthesis.

[0036] As used herein, the term "cell membrane component" refers to one or more components in the native cell membrane of a modified or unmodified cell. The cell membrane components may be, for example, about 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-10%, 0.1-1%, 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, or the like, by volume, of the components in the native cell membrane. %, 10~40%, 10~30%, 10~20%, 20~100%, 20~90%, 20~80%, 20~70%, 20~60%, 20~50%, 20~40%, 20~30%, 50~100%, 50~90%, 50~80%, 50~70%, 50~60%, 60~100%, 60~ The cell membrane components may include 90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90% or 90-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20% or 1-10%. The cell membrane components may include receptors in the native cell membrane, which receptors have binding activity with cells of a particular type or in a particular tissue. The cell membrane components may assemble to assume a structure (e.g., a phospholipid bilayer) similar to that in the native cell membrane. The assembly may be self-assembly.

[0037] As used herein, the term "native cell membrane" refers to the naturally occurring cell membrane of a cell. A native cell membrane includes components such as lipids, proteins (e.g., glycoproteins), and combinations thereof.

[0038] As used herein, the term "cell-derived membrane" refers to a membrane that contains the cell membrane components of a native cell membrane that have been modified or have additional components added thereto, the additional components being distinct from the cell membrane components. A cell-derived membrane may contain some or all of the components in a native cell membrane, e.g., about 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-10%, 0.1-1%, 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-10% of the components in a native cell membrane. 40%, 10-30%, 10-20%, 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, 50-100%, 50-90%, 50-80%, 50-70%, 50-60%, 60-100%, 60-90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90% or 90-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20% or 1-10%. The cell membrane components may include receptors that have binding activity to cells of a particular type or to cells in a particular tissue. The cell-derived membrane may be formed by self-assembly of modified cell membrane components or a mixture of modified or unmodified cell membrane components and additional components. The cell-derived membrane may include structures (e.g., phospholipid bilayers) that are similar to structures in native cell membranes. The cell-derived membrane may have biological activity, e.g., binding activity to cells of a particular type or to cells in a particular tissue, and the activity may be, e.g., about 80-120% identical to that of the native cell membrane.

[0039] The term "chimeric membrane" as used herein refers to a cell-derived membrane in which the additional component is an additional cell membrane component of an additional native cell membrane, an intracellular membrane, such as an extracellular vesicle, an exosome, a secretory vesicle, a synaptic vesicle, an endoplasmic reticulum (ER), a Golgi apparatus, a mitochondrion, a vacuole or a nuclear cell membrane, a bacterial membrane, a viral membrane, or a combination thereof. The cell membrane component and the additional cell membrane component may include components (e.g., receptors) of the same native cell membrane or different native cell membranes of the same or different types of cells in the same tissue or different tissues. The weight ratio of the cell membrane component to the additional cell membrane component may be adjusted to adjust the physical and / or biological properties of the chimeric membrane, such as the binding activity to a specific type of cell or to cells in a specific tissue. The chimeric membrane may be formed by self-assembly of a mixture of the cell membrane component and the additional cell membrane component. The cell membrane of red blood cells may be used for the preparation of the chimeric membrane.

[0040] The term "synthetic membrane" as used herein refers to a cell-derived membrane in which the additional component is a synthetic membrane component. The synthetic membrane component may be biocompatible. The synthetic membrane component may be biodegradable. The synthetic membrane component may be produced chemically, recombinantly, or both. The synthetic membrane may be formed by self-assembly of a mixture of cell membrane components and synthetic membrane components. The synthetic membrane may have desirable physical and / or biological properties, such as binding activity to a particular type of cell or to cells in a particular tissue.

[0041] As used herein, the term "targeting moiety" refers to any agent that allows a microparticle to preferentially migrate to one type of cell or tissue over another. A targeting moiety can be a biological molecule (e.g., a peptide or protein), a chemical compound, or a combination thereof.

[0042] The terms "cell cytosol" and "cytoplasm" are used interchangeably herein and refer to the matrix inside a cell.

[0043] As used herein, the term "sequestering agent" refers to any molecule capable of binding an agent via hydrogen bonds, electrostatic interactions, ionic bonds or covalent bonds such that the agent is bound to the microparticle. The agent may be a biological molecule or structure within the subject.

[0044] As used herein, the term "immunogenic" refers to any agent that elicits an immune response when introduced into a subject.

[0045] The present invention provides microparticles (MPs). The MPs are not naturally occurring. The MPs comprise a core and a membrane surrounding the core. The membrane comprises cell membrane components. The MPs of the present invention may have the ability to cross endothelium, and the endothelium may be in a tissue (e.g., the brain or lymph nodes). The tissue may be in a subject (e.g., a human).

[0046] The MP membrane may consist of the native cell membrane of a single cell or a portion thereof. The cell membrane components may be, for example, about 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-10%, 0.1-1%, 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-90%, 10-10 ... 0%, 10~40%, 10~30%, 10~20%, 20~100%, 20~90%, 20~80%, 20~70%, 20~60%, 20~50%, 20~40%, 20~30%, 50~100%, 50~90%, 50~80%, 50~70%, 50~60%, 60~100%, 60 The native cell membrane may comprise ~90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90% or 90-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20% or 1-10%. The native cell membrane may be one obtained without modification. The cell may be a white blood cell. The white blood cell may be a lymphocyte. The lymphocyte may be a T-lymphocyte. The native cell membrane may be from a white blood cell, lymphocyte or T-lymphocyte.

[0047] The MP membrane can be a cell-derived membrane, which is a membrane from a native cell membrane. The cell-derived membrane can consist of the native cell membrane of a modified single cell or part thereof. The cell-derived membrane can include the native cell membrane of a modified or unmodified single cell or part thereof, and additional components that are not cellular components. A cell-derived membrane may contain some or all of the components in a native cell membrane, for example, by volume, about 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-10%, 0.1-1%, 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, or the like, of the components in a native cell membrane. %, 10~40%, 10~30%, 10~20%, 20~100%, 20~90%, 20~80%, 20~70%, 20~60%, 20~50%, 20~40%, 20~30%, 50~100%, 50~90%, 50~80%, 50~70%, 50~60%, 60~100%, 60~ It may include 90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90% or 90-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20% or 1-10%.The cell membrane components may be, for example, about 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-10%, 0.1-1%, 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-90%, 10-10 ... 0%, 10~40%, 10~30%, 10~20%, 20~100%, 20~90%, 20~80%, 20~70%, 20~60%, 20~50%, 20~40%, 20~30%, 50~100%, 50~90%, 50~80%, 50~70%, 50~60%, 60~100%, 60 The cell-derived membrane may comprise up to 90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90% or 90-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20% or 1-10%. The cell-derived membrane may include a receptor that has binding activity to a particular type of cell or to cells in a particular tissue. The cell-derived membrane may be a chimeric membrane, where the additional component is an additional cell membrane component, or a synthetic membrane, where the additional component is a synthetic membrane component. Synthetic membrane components can include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, pphingomyelin, dimyristoylphosphatidylglycerol sodium salt, phosphatidic acid, lyosphospholipids, oxidized phospholipids, sterols, proteins, glycoproteins, receptors and transporters.

[0048] In one embodiment, the MP membrane is from a permeabilized cell. The permeabilized cell can be a permeabilized white blood cell, lymphocyte, or T-lymphocyte. The cell-derived membrane can include the cell membrane of a permeabilized cell or a portion thereof. The permeabilized cell can be one that has been subjected to a freeze permeabilization, detergent, or chemical permeabilization solution.

[0049] Permeabilized cell membranes may be prepared by dissolving some or all of the components of the native cell membrane of the corresponding cell used to prepare the permeabilized cell, e.g., by volume, about 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-10%, 0.1-1%, 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-90%, 10-80%, 10-120%, 10-160%, 10-180%, 10-140%, 10-200%, 10-200%, 10-300%, 10-300%, 10-400%, 10-500%, 10-600%, 10-700%, 10-800%, 10-1 ... ~70%, 10~60%, 10~50%, 10~40%, 10~30%, 10~20%, 20~100%, 20~90%, 20~80%, 20~70%, 20~60%, 20~50%, 20~40%, 20~30%, 50~100%, 50~90%, 50~80%, 50~70%, 50~60%, 60 It may include -100%, 60-90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90% or 90-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20% or 1-10%.The cell membrane components in the cell-derived membranes may, for example, be approximately 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-10%, 0.1-1%, 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, or 10-50% by volume of the native cell membrane components of the corresponding cells. %, 10-30%, 10-20%, 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, 50-100%, 50-90%, 50-80%, 50-70%, 50-60%, 60-100%, 60-90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90% or 90-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20% or 1-10%.

[0050] The MP membrane may be a self-assembled cell membrane component and may include additional components. The MP membrane may be prepared by mixing the cell membrane component with additional components. The composition of the MP membrane may be adjusted to adjust the physical and / or biological properties of the MP. The MP membrane may include a structure (e.g., a phospholipid bilayer) similar to that in a native cell membrane. The MP membrane may have biological activity, such as binding activity to a specific type of cell or to cells in a specific tissue, which activity is similar or identical to that of the native cell membrane.

[0051] In MP, the cell membrane component is approximately 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-10%, 0.1-1%, 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-80%, 10-90%, 10-80%, 10-90%, 10-60%, 10-50%, 10-40%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-80%, 10-90%, 10-90%, 10-80%, 10-90%, 10-1 ... It may be present in the range of 0-20%, 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, 50-100%, 50-90%, 50-80%, 50-70%, 50-60%, 60-100%, 60-90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90% or 90-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20% or 1-10%.

[0052] In MPs, the membrane may further comprise a targeting moiety. The targeting moiety may comprise an integrin, a selectin, a cadherin, an immunoglobulin-like adhesion molecule, an addressin, a chemokine receptor, a chemokine ligand, a growth factor receptor, an immunoglobulin superfamily protein (e.g., toll-like receptor (TLRS), a T cell receptor (TCR), a B cell receptor (BCR), a major histocompatibility complex (MHC) molecule), an ion channel-linked receptor, a G protein-coupled receptor, an enzyme-linked receptor, an antibody or a fragment thereof (e.g., a nanobody), or any binding domain of these moieties. The targeting moiety may be on the outer surface of the membrane. The targeting moiety may be a component of the native cell membrane. The targeting moiety may have a specific binding affinity with a particular type of cell or with cells in a particular tissue. The targeting moiety may have specific binding affinity to brain endothelium, examples of which include CCR7, CXCR3, L-selectin, P-selectin glycoprotein ligand 1 (PSGL1), VLA-4, LFA-1, CCR6. The targeting moiety may have specific binding affinity to lymph node endothelium, examples of which include L-selectin, lymphocyte function-associated antigen 1 (LFA-1), chemokine (CC motif) receptor 7 (CCR7), integrin alpha4beta1 (VLA-4), lysophosphatidic acid receptor (LPA2, LPA5, LPA6).

[0053] Where the MP membrane consists of the native cell membrane of a single cell or portion thereof, the targeting moiety may be a component of the native cell membrane. The cell membrane component may comprise the targeting moiety.

[0054] Where the MP membrane is a cell-derived membrane from a native cell membrane, the targeting moiety may be a component of the native cell membrane. The cell membrane component may comprise the targeting moiety.

[0055] In the MP, the core may be in the form of a liquid, solid, or a combination thereof. The core may be biocompatible. The core may be biodegradable. The core may include a cytoplasm, which may be native or modified. The cytoplasm may be of the same cell or the same type of cell as the cell whose native cell membrane is in the MP membrane, or the cell-derived membrane from which is in the MP membrane.

[0056] The liquid core may comprise an aqueous solution, an oil, or a combination thereof. The liquid may be doped with a viscosity modifier, such as dextran and hyaluronic acid, to adjust the liquid viscosity and control the loading and release of the payload from the MP. The liquid core may contain multiple aqueous solutions, multiple oil solutions, or both aqueous and oil solutions. Multiple liquid phases within the MP may be structured, for example, in a core-(multi-)shell arrangement with alternating layers of immiscible phases oriented and / or may exist as a double emulsion with many separate phases, one immiscible liquid inside the other.

[0057] The core may comprise a polymer, which may be natural or synthetic. The core may comprise an extracellular matrix protein, which may be purified, recombinant, or decellularized. The polymer core may comprise synthetic polymers, such as PEG, PLGA, and combinations thereof, natural polymers, such as alginate and collagen, and / or soluble extracellular matrix (ECM) proteins (e.g., Matrigel) isolated from tissues or cell lines. The ECM proteins may be secreted, purified, or recombinant proteins found in or derived from ECM proteins found in tissues from various mammalian species, such as humans, non-human primates, pigs, horses, lampines, and rodents.

[0058] In MP, the cores are of sizes ranging from about 0.1 to 1,000 μm, 0.1 to 900 μm, 0.1 to 800 μm, 0.1 to 700 μm, 0.1 to 600 μm, 0.1 to 500 μm, 0.1 to 400 μm, 0.1 to 300 μm, 0.1 to 200 μm, 0.1 to 100 μm, 0.1 to 50 μm, 0.1 to 10 μm, 0.1 to 1 μm, 0.5 to 1,000 μm, 0.5 to 900 μm, 0.5 to 800 μm, 0.5 to 700 μm, 0.5 to 600 μm, 0.5 to 500 μm, 0.5 to 400 μm, 0.5 to 300 μm, 0.5 to 200μm, 0.5~100μm, 0.6~1,000μm, 0.6~900μm, 0.6~800μm, 0.6~700μm, 0.6~600μm, 0.6~500μm, 0.6~400μm, 0.6~300μm, 0.6~200μm, 0.6~100 μm, 0.7~1,000μm, 0.7~900μm, 0.7~800μm, 0.7~700μm, 0.7~600μm, 0.7~500μm, 0.7~400μm, 0.7~300μm, 0.7~200μm, 0.7~100μm, 0.8~1,000μm , 0.8~900μm, 0.8~800μm, 0.8~700μm, 0.8~600μm, 0.8~500μm, 0.8~400μm, 0.8~300μm, 0.8~200μm, 0.8~100μm, 0.9~1,000μm, 0.9~900μm, 0. 9~800μm, 0.9~700μm, 0.9~600μm, 0.9~500μm, 0.9~400μm, 0.9~300μm, 0.9~200μm, 0.9~100μm, 1~1,000μm, 1~900μm, 1~800μm, 1~700μm, 1~6 00μm, 1-500μm, 1-400μm, 1-300μm, 1-200μm, 1-100μm, 100-1,000μm, 100-900μm, 100-800μm, 100-700μm, 100-600μm, 100-500μm, 100-400μm, 100-300μm, 100-200μm, 500-1,000μm, 500-900μm, 500-800μm, 500-700μm, 500-600μm, 750-1,000μm, 750-900μm or 750-800μm. For example, MPs can be 0.8-500μm in size.

[0059] The core may include an active agent, also known as a payload. The active agent may include a biological molecule, a compound, or a combination thereof. The active agent may include a nanoparticle (e.g., a metal particle, a polymer particle, a dendrimer particle, or an inorganic particle), a liposome, a virus, or a combination thereof. The active agent may have a biological activity, e.g., a therapeutic effect. The active agent may include a therapeutic agent, an imaging agent, a sequestering agent, a preventive agent, a diagnostic agent, a prognostic agent, an additive, or a combination thereof.

[0060] Cores can be prepared from cells. Such cores can be prepared by dissolving some or all of the cytoplasm of a cell, for example, by volume, about 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-10%, 0.1-1%, 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-80%, 10-90%, 10-60%, 10-50%, 10-40%, 10-50%, 10-60%, 10-50%, 10-60%, 10-70%, 10-80%, 10-80%, 10-90%, 10-90%, 10-90%, 10-1 ... %, 10-30%, 10-20%, 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, 50-100%, 50-90%, 50-80%, 50-70%, 50-60%, 60-100%, 60-90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90% or 90-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20% or 1-10%. The cell can be a white blood cell (eg, a lymphocyte).

[0061] The MP may be biocompatible and biodegradable. The MP may be non-immunogenic.

[0062] For each MP of the invention, a method is provided for delivering the MP. The delivery method comprises administering the MP to the endothelium, whereby the microparticles bind to the endothelium. The delivery method further comprises translocating the microparticles across the endothelium.

[0063] According to the method of delivery, the MP comprises a core and a membrane surrounding the core, the membrane comprising a cell membrane component. The membrane may further comprise additional components. The membrane may consist of a native cell membrane. The membrane may comprise a cell-derived membrane. The cell-derived membrane may be a chimeric or synthetic membrane. The membrane may comprise a targeting moiety. The targeting moiety may comprise an integrin, a selectin, a cadherin, an immunoglobulin-like adhesion molecule, an addressin, a chemokine receptor, a chemokine ligand, a growth factor receptor, an immunoglobulin superfamily protein (e.g., toll-like receptor (TLRS), T cell receptor (TCR), B cell receptor (BCR), major histocompatibility complex (MHC) molecule), an ion channel-linked receptor, a G protein-coupled receptor, an enzyme-linked receptor, an antibody or a fragment thereof (e.g., a nanobody), or any binding domain of these moieties. The endothelium may be within a subject. The subject may be human or non-human. The MP may be administered intravenously to the subject.

[0064] The transport method may further include translocating the MPs across the endothelium and then translocating the MPs to a target site. The target site is a site in a tissue or organ to which the MPs migrate. The endothelium of such tissue or organ may have a unique receptor profile that interacts with the MPs to translocate them across the endothelium. The unique endothelial profile may vary depending on the area in the subject's body due to disease. The target site may be in a tissue on the other side of the endothelium. The target site may be in a lymph node (LN) lobule, brain parenchyma, tissue interstitium or tissue parenchyma. The target site may be in a lymph node (LN), central nervous system (CNS), gut-associated lymphoid tissue, testes, lung, tumor site (e.g., tumor-associated macrophages (TAMS) or tumor-associated lymphocytes (TAL)), or inflammation site.

[0065] Depending on the method of transport, MPs can circulate from the bloodstream or lymph across the endothelium to the surrounding interstitium or tissue. The endothelium can be in the brain or lymph node. For example, the endothelium can be in a lymph node and the target site can be a lymph node lobule. The endothelium can be in the brain and the target site can be in the brain parenchyma or cerebrospinal fluid (CSF). Both the endothelium and the target site can be in a tumor.

[0066] The core may include an active agent, and the delivery method may further include releasing the active agent at the target site. The active agent may include a biological molecule, a compound, or a combination thereof. The active agent may include a nanoparticle (e.g., a metal particle, a polymer particle, a dendrimer particle, or an inorganic particle), a liposome, a virus, or a combination thereof. The active agent may have a biological activity, e.g., a therapeutic effect. The active agent may include a therapeutic agent, an imaging agent, a sequestering agent, a preventive agent, a diagnostic agent, a prognostic agent, an additive, or a combination thereof.

[0067] The transport method may further include sequestering the molecule from the target site by the MP particle. The MP may include a sequestering agent. The sequestering agent may be in the core, the membrane, or both.

[0068] The method of transport may further include inducing a biological response at the target site. The biological response may be initiated in the endothelium and may involve translocating the MP across the endothelium. The biological response may be initiated in the tissue or stroma or parenchyma on the other side of the endothelium. The biological response may be selected from the group consisting of immune interactions, cancer therapy, vaccine responses, and immunotherapy.

[0069] For each MP of the invention, a method for preparing the MP is provided. The preparation method includes mixing a core with a membrane. The membrane includes a cell membrane component. The prepared MP includes a core and a membrane surrounding the core, the membrane including a cell membrane component. The preparation method may further include mixing the cell membrane component with the core. The cell membrane components are approximately 0.1-100%, 0.1-90%, 0.1-80%, 0.1-70%, 0.1-60%, 0.1-50%, 0.1-40%, 0.1-30%, 0.1-20%, 0.1-10%, 0.1-1%, 1-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-100%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-30%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-80%, 10-90%, 10-80%, 10-90%, 10-10 ... It may be present at 0%, 20-100%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, 50-100%, 50-90%, 50-80%, 50-70%, 50-60%, 60-100%, 60-90%, 60-80%, 60-70%, 70-100%, 70-90%, 70-80%, 80-100%, 80-90% or 90-100%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20% or 1-10%.

[0070] The membrane may further comprise an additional component, and the preparation may further comprise mixing the cell membrane component, the core and the additional component. The membrane may be a chimeric membrane, where the additional component is an additional cell membrane component. The membrane may be a synthetic membrane, where the additional component is a synthetic membrane component.

[0071] In one embodiment, the membrane is a cell membrane of a permeabilized cell, and the preparation method comprises adding a core to the permeabilized cell.The permeabilized cell can be a permeabilized white blood cell, lymphocyte or T-lymphocyte.The permeabilized cell can be subjected to a freeze permeabilization, a detergent, or a chemical permeabilization solution.

[0072] In another embodiment, the membrane is a cell membrane of a permeabilized white blood cell and the preparation method further comprises diffusing the material into the permeabilized white blood cell by injection or using a concentration gradient. The material can be further engineered to undergo a sol-gel transition using chemical or photoactivation methods.

[0073] In yet another embodiment, the preparation method further comprises enveloping the core with a membrane. The membrane can be the cell membrane of a permeabilized cell. The permeabilized cell can be a permeabilized white blood cell, lymphocyte or T-lymphocyte. The permeabilized cell can be subjected to a freeze permeabilization, a detergent, or a chemical permeabilization solution.

[0074] The preparation method may further include loading the core with an active agent. The active agent may include a biological molecule, a compound, or a combination thereof. The active agent may include a nanoparticle (e.g., a metal particle, a polymer particle, a dendrimer particle, or an inorganic particle), a liposome, a virus, or a combination thereof. The active agent may have a biological activity, for example, a therapeutic effect. The active agent may include a therapeutic agent, an imaging agent, a sequestering agent, a preventive agent, a diagnostic agent, a prognostic agent, an additive, or a combination thereof.

[0075] The preparation method may further include preparing cores from permeabilized cells. The permeabilized cells may be permeabilized leukocytes, lymphocytes or T-lymphocytes. The permeabilized cells may be subjected to a freeze permeabilization, detergent or chemical permeabilization solution.

[0076] As used herein, the term "about" when referring to a measurable value, e.g., an amount, a percentage, and the like, is meant to encompass a variation of ±20% or ±10%, more preferably ±5%, still more preferably ±1%, and even more preferably ±0.1% from the stated value, as such variations are appropriate. EXAMPLES

[0077] Example 1. Trafficking of T cells through high endothelial venules in lymph nodes To determine the mechanism of cell migration across the endothelial barrier, mouse lymph nodes were harvested, sectioned at 200 μm on a vibratome, and fixed using 2% paraformaldehyde, 2% glutaraldehyde with 2 mM CaCl2 in 0.1 M Na-cacodylate buffer. After a multi-step process in which 2% osmium tetroxide was added to the samples for contrast, the samples were embedded in Durcupan resin blocks and imaged using serial block-face scanning electron microscopy (SBF-SEM). For SBF-SEM imaging, samples were mounted and serially sectioned and imaged at a Z-step of 10 nm. This collected image stack was analyzed for T-cell crossing interactions into the lobule, which showed clear engulfment of T cells by the endothelium (Figure 2).

[0078] Example 2. Creation of permeabilized dead cells as MPs for drug release MPs (herein referred to as CSTLs) can be produced from killed T lymphocytes (or any other cell line). These CSTLs can be loaded by diffusion with any dye for visualization, loaded with hydrogels, or loaded with any other payload (e.g., drugs). CSTLs can be delivered intravenously to mice or human individuals for targeted uptake in many organs and can cross endothelial barriers.

[0079] As shown in Figure 3, freshly isolated cultured cells (live cells) with cell membranes bearing targeted receptors can be treated with cytoskeleton stabilization buffer (CSK) to produce permeabilized dead cells with preserved cell membranes bearing targeted receptors. For example, after isolation, the sample was rinsed with 25 mL of warm phosphate-buffered saline with 0.01% Tween 20 (PBS-T) and immediately immersed in modified ice-cold cytoskeleton stabilization buffer (CSK) (10 mM HEPES, 0.5% Triton X-100, 300 mM sucrose, 3 mM MgCl2, and 50 mM NaCl in DI H2O) for 1 min. The sample was removed from CSK and immediately immersed in ice-cold 4% paraformaldehyde in PBS-T and placed in a 37 °C water bath for 10 min. The sample was then rinsed with 25 mL of warm PBS-T. For cryo-permeabilization, freshly isolated cultured cells were cultured at 1x10 7 mL -1 The cells were resuspended in a vial containing 5% DMSO in PBS at a cell density of 100 μg / mL and the cell vial was immersed in liquid nitrogen (LN2) for 12 h before use. Alternatively, freshly isolated cultured cells were resuspended in a vial containing 1% DMSO in PBS and the cell vial was frozen at -80 °C for 12 to 18 h before use.

[0080] To test the effect of permeabilization on primary cells, various freezing temperatures (-20, -80, LN2) and cryoprotectant concentrations (DMSO 0.1–10%) were used and cell death was assessed by using Live / Dead viability staining and trypan blue permeability assay (Figure 4). Freezing in LN2 for 12 h in 5% DMSO resulted in >99% cell death with the most optimal stability. In principle, any method that permeabilizes the cell membrane to induce death while preserving the overall structure of the cell, defined by maintaining the approximate diameter, circularity, and morphology, is compatible with these methods. In addition, hydrogel prepolymers, e.g., alginate, can diffuse into the permeabilized cells and, after solvent exchange, undergo a sol-gel transition by adding CaCl2 to the bath to form an inner hydrogel core.

[0081] Stability and process testing: DNAse concentrations (0.1-25 μg / ml), temperatures (4-37 °C), and repeated centrifugation spins (2-3 times) were evaluated. Effects on the vehicle were measured using fluorescence microscopy, hemacytometer, and flow cytometry. Addition of DNAse was essential for successful resuspension of MPs (Figure 5). After treatment, cells maintained their individual morphology and structure, while the optical and fluorescent properties of the cells changed compared to live cells (Figure 6). Finally, combining the imaging details with the flow cytometry data (Figure 7) reveals that live cells are changed and / or modified throughout the permeabilization process, but remain intact as individual MPs. These cells can be used in this state or can be filled with cores (e.g., oil, viscous aqueous solutions, and / or hydrogel solutions that are subsequently induced to undergo a sol-gel transition by thermal, chemical or photochemical methods).

[0082] Centrifugation and flow stability assessment: To test the handling and recovery of these MPs with respect to MP stability, various centrifugation speeds (e.g., 100–1000 × g for 10 s–10 min) were tested. As shown in the centrifugation experiments, centrifugation speeds between 300–500 × g for 2–3 min resulted in the greatest retention of MPs (Figure 8) and the least change in diameter and circularity (Figure 9). No change in size or circularity was observed upon repeated 3 min spins at 500 × g (up to 3 consecutive spins) (Figure 10). Following this, the diameter and circularity of MPs stored for 2 h at different temperatures were measured to determine the refrigerated or room temperature storage conditions that minimized MP variability / stability (Figure 11). Finally, the effect of fluid flow on vehicular disruption was tested at 0.52 mL / min, at 37 °C, by a rocker or a microfluidic vascular mimic (Figure 12). After testing MPs under flow, 30 passes through the 0.61 meter vascular mimic, a reduction in MP numbers of approximately 40% was observed (Figure 8).

[0083] MP Conservation: Experiments were performed by creating MPs from permeabilized cells and storing them for various periods of time. MPs can be stored in LN2 for up to 6 weeks and thawed for use without significant degradation of MP structure and function. This long-term storage potential of permeabilized MPs enables translational capabilities.

[0084] MP Drug Loading: MPs were incubated with various concentrations (0.05-0.5 mg / ml) of cisplatin for 1 h at 37 °C to allow passive uptake of the drug. After incubation, half of the sample was centrifuged and the pellet resuspended in RIPA buffer. This pellet fraction was analyzed by mass spectrometry (MS) to determine the theoretical maximum drug loading per MP. The remainder of the sample was centrifuged and resuspended in fresh PBS. This sample was added to the top of a transwell insert, and ICP-MS was performed on the bottom solution taken after t = 0, 0.5, 1, 2, 4, 8, 12, and 24 h to perform longitudinal release kinetics studies. Similar studies by MS or fluorescence measurements were performed with raltegravir and FITC-dextran. As shown in these studies, permeabilized cell MPs successfully released many payloads with predictable release kinetics similar to synthetic drug carrier systems. For small molecules, e.g., cisplatin and raltegravir, approximately 80-90% of the drug is released after 4 hours (Figure 13). For larger molecules, e.g., 70 kDa FITC-dextran, the release kinetics is slower, with approximately 35% of the total cargo released after 8 hours.

[0085] In vitro validation of released drug efficacy: Using the release profiles discussed above, 4T1-luc2 cells were treated with cisplatin-loaded MPs and compared to free drug treatment. The cumulative release of cisplatin was measured after 4 hours and added to the cells to calculate the corresponding free drug dose. Additional controls included unloaded MPs and untreated cells. Free cisplatin and unloaded MPs induced little to no cell death compared to controls after 4 hours (Figure 14). Cisplatin-loaded MPs induced significant cell death as evidenced by changes in morphology and clear areas of cell detachment. As shown by these experiments, localized release of high levels of payload can lead to significant improvements in the efficiency of treatment of diseased cells.

[0086] Example 3. Preparation of membrane-encapsulated T-lymphocyte-derived sodium alginate MPs A general approach to prepare membrane-encapsulated alginate microparticles (MPs): A preparative method to synthesize biomembrane-covered microparticles (MPs), also known as cell-mimetic microparticles (cmMPs), consists of membrane-wrapped hydrogel "cores" that were independently fabricated and subsequently isolated from T-lymphocytes. Micron-sized sodium alginate hydrogels were fabricated using a microfluidic droplet generator using a flow-focusing configuration to achieve high monodispersity while finely controlling the size of the hydrogels. Sodium alginate crosslinks in the presence of divalent cations, undergoing a sol-gel transition. Monodisperse liquid alginate droplets were produced by modulating the flow rate of the continuous phase (oil) relative to the dispersed phase (sodium alginate and Ca-EDTA) in a single microfluidic device at rates of thousands to tens of thousands per second. 0.2% acetic acid was added to the oil phase to enable the sol-gel transition. The subsequent alginate hydrogel microsphere "cores" were solvent exchanged to remove the oil phase (Figure 15). Separately, membrane extraction of T lymphocytes was achieved using standard techniques by osmotically-based mild hypotonic cell lysis with physical homogenization followed by differential centrifugation and ultracentrifugation steps to obtain enriched isolated plasma membranes. Purified membranes and alginate cores were mixed and co-extruded through polycarbonate membranes with pore sizes ranging from 8 μm to 20 μm depending on the desired MP size. Encapsulated MPs were subsequently characterized for size (monodispersity), zeta potential, and specific membrane protein composition using DLS, Zetasizer, and flow cytometry (Figure 16).

[0087] Importantly, this platform is easily adaptable through simple chemistry. For visualization of MPs in in vitro and in vivo experiments, sodium alginate has been covalently coupled to different fluorescent dyes, e.g., FITC, TRITC, and DAPI, via carbodiimide cross-linking. Similarly, visualization of MP membranes has been achieved using bioreactive dyes, e.g., Cy5.5 NHS ester and BODIPY TMR C5 maleimide. 85This is achieved by covalent conjugation with ligands such as EDTA or membrane dyes such as DiD and DiO (Figures 16 and 17). For drug payloads consisting of macromolecules such as antibodies, the therapeutic agent was mixed with the sodium alginate solution prior to forming the hydrogel core. For diffusible small molecules, the MPs were soaked in the drug solution until fully saturated prior to administration or testing.

[0088] Modulation of release kinetics and MP degradation to enable sustained release formulations: The in vitro release kinetics of two separate classes of representative therapeutic compounds (payloads) were quantified using small molecule fluorophores, HIV antiretrovirals (ARVs) such as tenofovir disoproxil fumarate and darunavir, gold nanoparticles, and antibodies. For small molecules (fluorophores and ARVs), MPs were soaked in a solution of payload until saturated. For macromolecules (antibodies and 80 nm gold nanoparticles (NPs)), the payload was added to the alginate solution prior to the formation of the MP core. The weight percentage of the MP alginate core was varied to quantify drug release kinetics. MPs were placed in the top reservoir of a transwell in water, and the bottom compartment was sampled over time in an incubator rocker. As expected, the release kinetics from the membrane-encased MPs follows the expected release kinetics, with higher mass fractions resulting in longer release half-lives (Figure 18).

[0089] MP membranes and formulations can be varied independently to engineer payload loading and release. Sterol concentration is known to affect liposome and membrane stability and drug release, with higher concentrations increasing stability and slowing release kinetics. Sterols can be added to purified membranes. Similarly, MP core formulations can be varied by using a number of standard approaches to modulate the resulting pore size, for example, by changing the mass fraction of sodium alginate and varying the degree of crosslinking within the resulting hydrogel core. In addition, by creating MP cores as described above and performing a secondary crosslinking procedure in a CaCl2 bath, a spatial gradient of pore size along the radius can be induced to form a core-shell organization within a single MP "core". This wave of secondary diffusive crosslinking from the core surface into the core allows for reshaping the release kinetics into multiple phases, particularly allowing for a delayed initial release of the payload. Sequential crosslinking procedures can be used to create multiple "shells" of reduced porosity within the MP core. These modifications can be used to delay phase 1 and 2 release kinetics, which, based on PK modeling analysis, are the most impactful parameters in reducing off-target loss of drug payload and prolonging release of drug in target tissues compared to the performance of current formulations.

[0090] Conclusion: The physicochemical properties of the alginate core, such as the degree of cross-linking and the weight fraction of alginate, govern the pore size and degradation of the alginate, which in turn influences important properties of the formulation, such as cargo loading efficiency and release kinetics. For small molecule drugs, the alginate core acts as a sponge and the payload elutes through a diffusion-like process. Our data show that this mechanism allows for payload elution half-lives on the order of weeks. For larger molecules, such as antibodies, the payload can be trapped within the hydrogel matrix of the core and undergo prolonged release over a month or more as the core disintegrates. Smaller molecules chemically bound to the core can also have such prolonged release kinetics. Our data show that both the dissociation rate of the MP and the elution rate of its incorporated payload can be tunable core material properties. Thus, the particles can be optimized using several approaches, detailed below, to achieve sustained release of drugs to target tissues over extended periods of time.

[0091] Example 4. Transport and tissue localization of MPs in mouse lymph nodes MP injection and LN homing in a mouse model: T lymphocytes were isolated from the spleens of CD1 mice using a commercial kit. The cells were permeabilized by freezing. To label MPs for visualization in tissues, MPs were thawed at 4°C and incubated with 78 μM NHS Cy5.5 in 1×PBS for 20 min. This dye labels the amino groups remaining in cells after freezing and permeabilization. CD-1 mice were injected with 1.5×10 6 Cells were injected intravenously at a dose of 0.01 mg / mL and euthanized at distinct time points (t=0, 1, 2, 4, 8, 16, and 24 hours). Blood samples were collected by intracardiac puncture at each time point to quantify CSTL remaining in the circulation. Brachial, inguinal, and popliteal LNs were isolated, fixed overnight in 4% paraformaldehyde (PFA), and embedded in 6% agarose gel for vibratome sectioning. 200 μm sections were then imaged to assess CSTL uptake.

[0092] Evidence that membrane composition controls specific processes for MP accumulation in tissues by crossing the endothelial barrier: To verify that MP targeting and uptake are specific, functional LN extravasation was confirmed using L-selectin antibody blockade. L-selectin is a glycoprotein expressed on the membrane of T lymphocytes and is involved in their initial tethering and rolling on high endothelial venules (HEVs). Therefore, blocking this interaction is expected to prevent extravasation into the lobules. In the experimental group, mice were injected with 0.4 mg / kg L-selectin antibody and MPs. In the control group, mice received only MPs. Mice were euthanized 2 h after cell injection. Vibratome sections are prepared as previously described. CSTL uptake in both sections is compared by counting lobule ingression.

[0093] Conclusions for LN targeting: From the experiments, it is clear that delivery of permeabilized T cell MPs was a viable approach to target and enter lymph node lobules. MPs were able to survive the bloodstream and bind to HEVs in lymph nodes (Figure 19). Although transendothelial migration of T lymphocytes is considered an active process, and there is evidence supporting lymphocyte motility, other leukocyte-endothelial cell interactions may also act as possible modes of crossing biological barriers. In addition, recent studies have identified “HEV pockets,” temporary retention sites in the HEV lumen for transient lymphocyte migration inhibition. This interaction may control the rate at which lymphocytes enter to balance their exit through lymphatic vessels. Furthermore, this process is specific to membrane composition, as functional antibody blockade of L-selectin prevented MP extravasation into tissues similar to live T cells (Figure 20). It is clear that HEV endothelial cells play a major role in lymphocyte passage across the barrier, and therefore, their role in MP entry into lymph nodes may be paramount. Overall, permeabilized T cell MPs have the ability to load and release drugs and enter lymph node lobules, providing a powerful tool for LN-targeted drug delivery.

[0094] Example 5. Transport and tissue localization of MPs in mouse brain MPs cross the blood-brain barrier and enter the brain parenchyma: Utilizing MPs to deliver therapeutic cargo to pharmacological sites in the body is an innovative and unexplored drug delivery system. The brain provides an interesting target site. A mouse model was used to track the distribution of cell-mediated drug delivery vehicles within the brain and across the blood-brain barrier (BBB). Experiments were performed with labeled syngeneic transplanted live cells and compared with engineered syngeneic MPs (Figure 21).

[0095] Preparation of live T lymphocyte injections: Injections containing live T cells were prepared immediately before the start of the experiment. Spleens were removed from healthy CD1 mice. The spleens were mechanically and enzymatically digested. Once a cell suspension free of red blood cells was obtained, T lymphocytes were separated from other splenic cells using a magnetic antibody-coupled bead system. Pure T lymphocytes were incubated with fluorescent CSFE solution. This cell suspension was injected intravenously into the lateral tail vein of another healthy mouse.

[0096] Preparation of MP injections: To prepare MP injections, live T cells were obtained from mouse spleens as previously described. To produce MPs with a brain-targeting phenotype, isolated T cell pools were activated in vitro prior to freeze-permeabilization. To induce general activation, isolated cells were incubated with 25 ng / ml phorbol myristate acetate (PMA) and 1 μg / ml ionomycin for 6 h to bypass the T cell membrane receptor complex and induce downstream membrane receptor changes. After freeze injury, these MPs were thawed and labeled with Cy5 as previously described for administration.

[0097] Brain Imaging: After different time points (4, 6, and 8 hours), mice were sacrificed according to IACUC guidelines. Brains were washed and then fixed in 4% PFA supplemented with 0.1% Triton X-100 for 8–12 hours at 4°C. Tissue samples were embedded in 6% agarose gel and 300 micron thick sections were cut using a vibratome. Tissues were blocked for 12 hours at 4°C on a rocker and then incubated with rat PECAM1 and rat Endomucin for 48 hours at 4°C on a rocker to label endothelial cells of the vasculature. After primary antibodies, the tissues were washed three times for 5 minutes with 1x PBS and labeled with goat anti-rat Alexa Fluor 488.

[0098] Conclusion: Following administration of live T cells, untreated MPs, and activated MPs, it is clear that MP membrane phenotype plays a critical role in tissue targeting (Figure 21). There is significant evidence that T cell activation is a critical step for entry into CNS tissues. In vitro "programming" of live cells with activation cocktails to alter membrane composition prior to MP creation has been demonstrated as a viable strategy to produce targeted MPs. Changes in cell phenotype, such as increased cell size, cell elongation, and increased cell-cell aggregation, were observed prior to MP production. From animal administration and confocal microscopy imaging, it is clear that activated MPs result in a complete change in targeting profile such that LN uptake is decreased and brain uptake is greatly increased. Finally, as in lymph nodes, counterstaining of blood vessels indicates escape of MPs from blood vessels and transport into the brain parenchyma.

[0099] Example 6: Initial PK / PD Modeling of MP Microparticles have kinetics similar to live T cells, being taken up from the blood into target tissues with a mean residence time in blood of 1 h and mean tissue residence time of approximately 24 h. On the other hand, unlike live T cells, microparticles cannot usually leave tissues. Once MPs enter tissue, they remain in the tissue. We assume that the membrane dissociates from the particle over 3–5 days and the hydrogel core slowly disintegrates over approximately 30 days, releasing their payload into the tissue. Our studies of small molecule fluorophores showed a biphasic release kinetics from the core, with a longer second phase half-lifetime for denser gel formulations (Figure 23). The first phase release kinetics has a half-lifetime on the order of 1 h, and the second phase release kinetics has a half-lifetime of approximately 4 days. We simulated conditions in which all drug was released from the core, and the exhausted particles remained in the tissue for approximately 30 days before disintegrating. These kinetics were incorporated into a PK / PD model (Figure 24) and the predicted concentrations of particles and delivered drug are shown in Figure 25. These results indicate that the particles are capable of achieving tissue-specific sustained release of payload over a period of several weeks.

[0100] All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and / or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and examples of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

1. 1. A method for transporting a microparticle, the microparticle comprising a core and a membrane surrounding the core, the membrane comprising a cell membrane component, the method comprising: (a) administering microparticles to the endothelium, whereby the microparticles bind to the endothelium; and (b) Translocating the microparticles across the endothelium The method comprising:

2. The method of claim 1 , wherein the endothelium is in the brain or a lymph node.

3. The method of claim 1 or 2, wherein the endothelium is in a subject.

4. The method of any one of claims 1 to 3, wherein the membrane further comprises a targeting moiety.

5. 5. The method of claim 4, wherein the targeting moiety comprises an integrin, a selectin, a cadherin, an immunoglobulin-like adhesion molecule, an addressin, a chemokine receptor, a chemokine ligand, a growth factor receptor, an immunoglobulin superfamily protein, an ion channel-linked receptor, a G protein-coupled receptor, an enzyme-linked receptor, an antibody or fragment thereof, or a binding domain thereof.

6. The method of any one of claims 1 to 5, further comprising translocating the microparticles to a target site after translocating the microparticles across the endothelium.

7. 7. The method of claim 6, wherein the endothelium is within a lymph node and the target site is a lobule within the lymph node.

8. 7. The method of claim 6, wherein the endothelium is in the brain and the target site is in the brain parenchyma or cerebrospinal fluid (CSF).

9. The method of claim 6 , wherein the endothelium and the target site are within a tumor.

10. The method of any one of claims 6 to 9, wherein the core comprises an active agent, and further comprising releasing the active agent at a target site.

11. The method of any one of claims 6 to 10, further comprising sequestering the molecule from the target site with a microparticle.

12. The method of any one of claims 6 to 11, further comprising eliciting a biological response at the target site.

13. 13. The method of claim 12, wherein the biological response is selected from the group consisting of an immune interaction, a cancer treatment, a vaccine response, and an immunotherapy.

14. A microparticle comprising a core and a membrane surrounding the core, the membrane comprising cell membrane components.

15. 15. The microparticle of claim 14, wherein the membrane further comprises a synthetic membrane component.

16. 16. The microparticle of claim 14 or 15, wherein the membrane is from a permeabilized cell.

17. 17. The microparticle of claim 16, wherein the permeabilized cells have been subjected to a freeze permeabilization, detergent, or chemical permeabilization solution.

18. 18. The microparticle of claim 16 or 17, wherein the permeabilized cells are permeabilized white blood cells.

19. The microparticle of any one of claims 14 to 18, wherein the membrane further comprises a targeting moiety.

20. 20. The microparticle of claim 19, wherein the targeting moiety comprises an integrin, a selectin, a cadherin, an immunoglobulin-like adhesion molecule, an addressin, a chemokine receptor, a chemokine ligand, a growth factor receptor, an immunoglobulin superfamily protein, an ion channel-linked receptor, a G protein-coupled receptor, an enzyme-linked receptor, an antibody or fragment thereof, or a binding domain thereof.

21. The microparticle of any one of claims 14 to 20, wherein the core comprises cytoplasm, liquid, polymer, extracellular matrix protein, or a combination thereof.

22. The microparticle of any one of claims 14 to 21, wherein the core comprises an active agent.

23. 23. The microparticle of claim 22, wherein the active agent comprises a biological molecule, a chemical compound, or a combination thereof.

24. 24. The microparticle of claim 22 or 23, wherein the active agent comprises a nanoparticle, a liposome, a virus, or a combination thereof.

25. 25. The microparticle of any one of claims 22-24, wherein the active agent comprises a therapeutic agent, an imaging agent, a sequestering agent, a prophylactic agent, a diagnostic agent, a prognostic agent, an additive, or a combination thereof.

26. The microparticle of any one of claims 14 to 25, wherein the core is prepared from white blood cells.

27. The microparticle of any one of claims 14 to 26, wherein the microparticle is not immunogenic.

28. 11. A method for preparing microparticles comprising mixing a core with a membrane, the membrane comprising a cell membrane component.

29. 30. The method of claim 28, wherein the membrane further comprises a synthetic membrane component.

30. 30. The method of claim 28 or 29, wherein the membrane is a cell membrane of a permeabilized leukocyte, and the method further comprises adding a core within the permeabilized leukocyte.

31. 30. The method of claim 28 or 29, further comprising encasing the core in a membrane.

32. 32. The method of claim 31, wherein the membrane is a permeabilized leukocyte membrane from permeabilized leukocytes.

33. 33. The method of claim 30 or 32, wherein the permeabilized leukocytes have been subjected to a freeze permeabilization, detergent, or chemical permeabilization solution.

34. 34. The method of claim 30, 32 or 33, wherein the permeabilized leukocytes are permeabilized lymphocytes.

35. The method of any one of claims 28 to 34, wherein the membrane further comprises a targeting moiety.

36. 37. The method of claim 36, wherein the targeting moiety comprises an integrin, a selectin, a cadherin, an immunoglobulin-like adhesion molecule, an addressin, a chemokine receptor, a chemokine ligand, a growth factor receptor, an immunoglobulin superfamily protein, an ion channel-linked receptor, a G protein-coupled receptor, an enzyme-linked receptor, an antibody or fragment thereof, or a binding domain thereof.

37. The method of any one of claims 28 to 36, further comprising loading the core with an active agent.

38. 38. The method of any one of claims 28 to 37, further comprising preparing cores from the permeabilized leukocytes.

39. 40. The method of claim 38, wherein the permeabilized leukocytes have been subjected to a freeze permeabilization, detergent, or chemical permeabilization solution.

40. 31. The method of claim 38 or 30, wherein the permeabilized leukocytes are permeabilized lymphocytes.

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