Patent Application
20210299272
A critical reason that drugs fail to gain regulatory approval is that off-target side effects limit dosing necessary to achieve therapeutic effect. A long-studied, key strategy to overcome this challenge has been the loading of drugs into nanometer-scale carriers (nanocarriers or nanoparticles) conjugated with ligands (affinity moieties) that specifically direct the nanocarrier to receptors in the target tissue. A broad array of ligand-guided nanocarriers have been developed, beginning with monoclonal antibody-conjugated liposomes (immunoliposomes) over 40 years ago.1 Later iterations of ligand-based targeting have utilized antibody fragments, peptides, nucleic acids, and polysaccharides.2,3 Target organ uptake for these ligand-guided nanocarriers markedly exceeds that of untargeted ones, in some cases by orders of magnitude, showing high specificity of targeting.
Despite the improvement in drug localization by these ligand-guided nanocarriers, in nearly every case the majority of the initial dose is still delivered to off-target locations or cleared from circulation. The efficacy of delivery of ligand-guided nanocarriers in the target site usually does not exceed 5% percent of the initial injected dose (% ID) with an absolute “efficacy ceiling” achieved in animal studies peaking at 15-25% ID.4 A majority of nanocarriers end up outside the target tissue, even in the lungs, which have natural targeting advantages. The lung's capillary network provides a unique test bed for intravenously (IV)-delivered, ligand-targeted nanocarriers, due both to its enormous surface area and that it receives 100% of cardiac output after IV drug injection. Despite this ideal test bed and targeting highly-expressed endothelial cell adhesion molecules (CAMs), approximately 80% of the initial dose is cleared or retained in organs outside the lung.5-8 When the target site is reduced to a sub-organ level, as with a tumor, the delivery efficiency falls even further, with meta-analysis finding ˜0.7% ID reside in tumors and only minimal improvement using antibody targeting.4 Thus, ligand-conjugated nanocarriers show increased organ targeting compared to free drugs, but the target organ still receives only a minority fraction of the injected dose.
A continuing need in the art exists for new and effective compositions and methods for drug delivery using nanocarriers.
Provided herein in one aspect is a composition comprising dual-targeted nanoparticles having a first targeting moiety and a second targeting moiety, wherein the first targeting moiety is a red blood cell (RBC)-targeting moiety. In certain embodiments, the composition further comprises RBCs bound to the nanoparticles ex vivo via said first targeting moiety. In certain embodiments, the nanoparticles are liposomes, nanogels, or polymeric nanoparticles. In certain embodiments, the first and/or second targeting moiety is an antibody or antibody fragment, carbohydrate, carbohydrate-binding compound, peptide, nucleic acid, or aptamer. In a particular embodiment, the first targeting moiety is an antibody specific for a glycophorin, optionally glycophorin A or Band 3, or an Rh antigen.
In certain embodiments, the nanoparticle composition provided herein includes a second targeting moiety that is specific for vascular endothelial cells, intravascular leukocytes, cells of reticuloendothelial system, an immune cell, cells and tissues accessible to RBC under pathological conditions such as hemorrhage and thrombosis, or an infectious microorganism. In certain embodiments, the second targeting moiety is specific for ICAM-1, PECAM-1, VCAM-1, transferrin receptor, or ACE.
In certain embodiments, the compositions provided are characterized by having at least one of (a) about 50 to about 200 bound nanoparticles per RBC; (b) about 5 to about 350 of said first targeting moiety per nanoparticle; and (c) about 2.5% to about 25% of targeting moieties on the nanoparticle surface comprise said first targeting moiety; (d) a total number of first and second targeting moieties per nanoparticle in the range or about 5 to 350; and (e) a particle size diameter of about 10 nm to about 1,000 nm. In certain embodiments, the nanoparticles are loaded with a drug.
Also provided herein are pharmaceutical compositions that include an aqueous suspension comprising dual-targeted nanoparticles.
In yet another embodiment, provided herein is a method for delivering a drug to a mammalian subject having a disease, the method comprising administering to a subject in need thereof a composition or pharmaceutical composition comprising dual-targeted nanoparticles. In certain embodiments, (a) the disease is ARDS, pulmonary arterial hypertension, pneumonia, interstitial lung disease, idiopathic pulmonary fibrosis, post-pulmonary embolism, pulmonary capilliaritis syndrome, stroke, emphysema, lung edema, or a viral or microbial infection; (b) the disease is ARDS and wherein the drug is one of more of albuterol, dexamethasone, and palifermin; and/or (c) the disease involves a selected mammalian organ, and the nanoparticle composition is administered intra-arterially or intravenously. In certain embodiments, the drug is delivered to one or more target organs, including but not limited to the lungs, brain, or heart. In certain embodiments, the dual-targeted nanoparticles are administered intravenously or intraarterially.
In yet a further embodiment, the composition comprising the dual-targeted nanoparticles is administered to a patient and circulating RBCs in the bloodstream of the patient bind to the nanoparticles via the first targeting moiety.
In yet a further embodiment, the method provided includes contacting the dual-targeted nanoparticles with RBCs ex vivo to bind RBCs to the nanoparticles via the first targeting moiety prior to administration to the subject, wherein the RBCs are present in a sample obtained from the patient or an autologous blood donor. In certain embodiments, the method includes separating or enriching RBCs present in the sample prior to binding to the dual-targeted nanoparticles.
Still other aspects and advantages of these compositions and methods are described further in the following detailed description of the preferred embodiments thereof.
A significant number of drugs have serious side effects and toxicities that limit their use. The field of nanomedicine has long promised solutions to this problem, and several nanomedicines are available clinically. However, despite the previously demonstrated drug-carrying capacity of targeted nanocarriers, and accepted clinical use, several challenges remain. These include rapid clearance from circulation by the reticuloendothelial system (RES) and uptake by circulating immune cells. Described herein are targeted nanoparticles (e.g., liposomes) that utilize RBC-hitchhiking to improve drug delivery to target organs.
Red blood cells have been used as drug shuttles and carriers in prior work and in combination with nanocarriers, termed RBC hitchhiking. This first generation of RBC hitchhiking, in which a nanocarrier is associated with an RBC by nonspecific binding, has been shown to successfully evade nanocarrier uptake by the RES. However, these first-generation RBC-nanocarrier complexes had limitations. Hitchhiked particles were non-liposome nanocarriers and thus had reduced potential for clinical development and increased potential to cause damage to the carrier RBC. In some cases, non-liposome nanocarriers were observed to cause toxicity in their RBC shuttles. Additional studies have advanced the theory of RBC-hitchhiking into a reality. RBC were shown to be passively loaded with a variety of nanocarriers including liposomes that enabled targeted delivery to the lungs at an order of magnitude higher than previously obtained with targeted nanocarriers. This lung targeting was shown to be safe in the lung vasculature by using pulmonary artery pressure waveforms. Despite this recent advancement in the field, several critical areas of RBC-hitchhiking need improvement.
Previous RBC-hitchhiking research has relied in some cases on nonspecific adsorption of particles onto RBC. This nonspecific hitchhiking presents several challenges. First, there is no control of binding to the RBC and therefore the association of a particle to RBC is not able to be manipulated for different scenarios. Second, delivery to target organs likely still relies heavily on first pass delivery since the particle can readily separate from the RBC. Third, obtaining nonspecific binding is time consuming and prone to variability. Finally, while new particles may be engineered to nonspecifically adhere to RBC, this technique would exclude all other existing drugs and those in development.
An alternative targeting method that has been previously explored is cell-based delivery of nanocarriers. A popular cell-based delivery methodology has been loading nanocarriers ex vivo into leukocytes, usually via phagocytosis. Upon injection, the nanocarrier-loaded leukocytes travel to destination tissues.9-11 Since leukocytes can behave unpredictably, and are not currently isolated in this manner clinically, red blood cells (RBCs) provide a simpler, cell-based delivery option.
Dual-targeted RBC hitchhiking (DTRH) actively targets nanoparticles to RBC to overcome many of the limitations of passive RBC-hitchhiking. For example, liposomes are engineered to display RBC-targeting and organ-specific moieties on their surfaces, allowing precise control of the association between liposomes and RBC. This controlled loading does not cause harm to the carrier RBC. In some cases, the surface of dual-targeted liposomes replaces 10% of the tissue targeting moieties on a liposome with RBC-targeting moieties, which was shown to yield a 2.5 times greater dose delivery to the lungs compared to a single-targeted particle, and 70% of total injected dose is delivered to the lungs. In addition to unprecedented dose delivery to the lungs, dual-targeted liposomes have been shown to transfer from carrier RBCs in vivo. Thus, while antibody targeting has been demonstrated previously, dual-targeted liposomes can sequentially target two different cell types. Such two-cell targeting exploits the myriad interactions that different cell types undergo together, potentially enabling nanocarrier transfer from one cell to another with precise timing and specificity.
RBCs possess multiple advantages as cellular carriers, and their transporting function can be co-opted to shuttle nanocarriers throughout the circulation. They are standardly isolated clinically, have a long shelf-life, and have well understood donor-recipient compatibility. RBCs collectively comprise the largest cellular surface area, and have a months-long blood half-life.
Thus, the compositions and methods provided herein take advantage of the improved drug delivery obtained using RH, and further enhance specificity and flexibility by using ligand-mediated targeting. Termed “dual-targeted RBC hitchhiking” (DTRH), nanocarriers are conjugated with two distinct ligands that bind two different cell types: RBCs and target endothelial cells (
Provided herein are compositions comprising dual-targeted nanoparticles and uses therefor, wherein the dual-targeted nanoparticles have a first targeting moiety specific for a red blood cell (RBC) and a second-targeting moiety. In certain embodiments, provided are compositions having dual-targeted nanoparticles having a first targeting moiety bound to RBCs ex vivo and a second-targeting moiety for administration to a subject. In certain embodiments, the first and/or second targeting moiety is an antibody or an antibody fragment, a carbohydrate, or a carbohydrate-binding compound.
As a prototype for DTRH, the inventors focused on the largest cell-cell interface in the body, RBCs with pulmonary endothelial cells. Specifically, carrier RBCs loaded with liposomes shuttle through the circulation until contact with, and transfer to, the pulmonary endothelium (
By “nanoparticle” or “NP” (also referred to as “nanocarrier” or “NC”) as used herein is meant a particle having diameter of between about 1 to about 1000 nm. In one embodiment the NP is globular. Inclusive in this definition are particles with a diameter of at least 1, at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300 nm in diameter. In other embodiments, also included are particles having diameters of at least 320, at least 340, at least 360, at least 380, at least 400, at least 420, at least 440, at least 460, at least 480, at least 500, at least 520, at least 540, at least 560, at least 580, at least 600, at least 620, at least 640, at least 660, at least 680, or at least 700 nm. In yet other embodiment, also included are particles having diameters of at least 720, at least 740, at least 760, at least 780, at least 800, at least 820, at least 840, at least 860, at least 880, at least 900, at least 920, at least 940, at least 960, at least 980, or up to about 1000 nm. All numbers and fractions between any two of these numbers are also included. In certain embodiments, a dual-targeted NP is provided having a diameter of about 10 nm to about 1000 nm. In yet a further embodiment a dual-targeted NP is provided having a diameter of about 145 nm to about 150 nm.
In one embodiment, the nanoparticle (NP) is a liposome. By “liposome” as used herein is meant a material a microscopic spherical particle formed by a lipid bilayer enclosing an aqueous compartment. In certain embodiments, the nanoparticle is a hydrogel NP (also referred to as a nanogel or NG). In certain embodiments, the NP is a dendrimer. In yet other embodiments, the NP is a polymersome, a hybrid carrier combining artificial and natural components, or a protein multimolecular composition (e.g., ferritin or albumin aggregates).
By “hydrogel nanoparticle” or “nanogel” as used herein is mean a polymeric material having a hydrophilic structure which renders it capable of holding amounts of selected drug compounds in their three-dimensional networks, the resulting particle having nanoparticle dimensions. Macroscopic dextran hydrogels have shown Young's moduli of ˜10-50 kPa in the literature. See, e.g., Hwang M R, Kim J O, Lee J H, Kim Y I, Kim J H, Chang S W, Jin S G, Kim J A, Lyoo W S, Han S S, Ku S K, Yong C S, Choi H G. Gentamicin-Loaded Wound Dressing with Polyvinyl Alcohol/Dextran Hydrogel: Gel Characterization and In vivo Healing Evaluation. AAPS PharmSciTech 2010; 11(3):1092-1103. In certain embodiments, the NG is a lysozyme-dextran nanogel (also referred to as LDNG). In certain embodiments, the LDNG is a synthetic construct of lysozyme and dextran. In other embodiments, the nanogel is made of chitosan or chitin, pullulan, hyaluronic acid, PEG, pluronics (e.g. F127), poly(acrylic acid) or poly(acrylate), poly(oligo(ethylene glycol)methyl ether methacrylate), poly(ethylene oxide), polyethylenimine, poly(caprolactone), and poly(N-isopropylacrylamide), among other options encompassing a wide range of hydrophilic polymers capable of chemical modifications enabling incorporation in a nanoparticle. See, e.g., Eckmann D M, Composto R J, Tsourkas A, Muzykantov V R. Nanogel Carrier Design for Targeted Drug Delivery. J Mater Chem B Mater Biol Med 2015; 2(46):8085-8097; and Ahmed E M, March 2015, “Hydrogel: Preparation, Characterization, and Applications: A Review”, J. Adv. Res., 6(2):105-121, among other publications in the art. In certain embodiments described herein, nanogel particles containing a drug and associated with an RBC behave best in this form of drug delivery.
In certain embodiments, the nanogel or NP has a carbohydrate surface which aids with RBC and endothelial glycocalyx interaction. In another embodiment, the protein coating, e.g., IgG or albumin, prevents RBC toxicity. In yet another embodiment, the protein-coated nanogels are cross-linked or lyophilized. In still another embodiment, the nanogel or flexible NP has a capacity sufficient for large drugs or imaging agents. In certain embodiments, the nanoparticle has a polyethylene glycol (PEG) coating. In yet a further embodiment, the nanoparticle is a biological particle (e.g. an exosome or LDL).
The dual-targeted nanoparticles for use in the compositions and methods described herein have at least one targeting moiety that specifically binds a RBC surface antigen. In certain embodiments, the nanoparticles have a targeting moiety specific for a RBC target antigen that is a glycophorin, optionally glycophorin A (GPA) or Band 3. In certain embodiments, the nanoparticles have a targeting moiety specific for an Rh antigen. In certain embodiments, the nanoparticles have a targeting moiety specific for RhCE. Iin certain embodiments, the targeting moiety that specifically binds a RBC surface antigen is the CD235a monoclonal antibody or the Bric69 monoclonal antibody.
By the term “targeting moiety” as used herein, is meant a molecule, including an antibody, a fragment thereof or an antibody fusion protein, which is capable of specifically binding to another molecule. A targeting moiety may be an antibody, an aptamer, a nucleic acid, a peptide, a carbohydrate (sugar), a lipid, a vitamin, a toxin, a component of a microorganism, a hormone, a receptor ligand, and or any derivative thereof. If the targeting moiety is an antibody, it may be a monoclonal antibody, a humanized antibody, a synthetic antibody, a heavy chain antibody, and a biologically active fragment of an antibody, wherein the biologically active fragment is a Fab fragment, a F(ab′)2 fragment, and a Fv fragment.
As provided herein, a number of parameters contribute to improved delivery of nanoparticles using the compositions provided herein, including, but not limited to the total number of targeting moieties and the relative abundance of first and second targeting moieties. In certain embodiments, the dual-targeted nanoparticles are characterized by having about 5 to about 350 of a first targeting moiety per nanoparticle. In certain embodiments, the dual-targeted nanoparticles are characterized by having at least 5, at least 20, at least 50, at least 75, at least 100, at least 100, at least 150, at least 200, at least 250, at least 300, or at least 350 first targeting moieties per nanoparticle. In yet another embodiment, the dual-targeted nanoparticle is characterized by having less than 5, less than 20, less than 50, less than 75, less than 100, less than 100, less than 150, less than 200, less than 250, less than 300, or less than 350 first targeting moieties per nanoparticle. In yet another embodiment, the total number of first and second targeting moieties is about 5 to about 350 per nanoparticle. In certain embodiments, the dual-targeted nanoparticles are characterized by having a total of at least 5, at least 20, at least 50, at least 75, at least 100, at least 100, at least 150, at least 200, at least 250, at least 300, or at least 350 first and second targeting moieties per nanoparticle. In yet another embodiment, the dual-targeted nanoparticles are characterized by having a total of less than 5, less than 20, less than 50, less than 75, less than 100, less than 100, less than 150, less than 200, less than 250, less than 300, or less than 350 first and second targeting moieties per nanoparticle.
In certain embodiments, the dual-targeted nanoparticles have a specific proportion of first and second targeting moieties. As provided herein, the first targeting moiety (RBC-specific) may range from about 2.5% to about 50% of total targeting moieties of the dual-targeted nanoparticle. In certain embodiments, the first target moiety is at least 2%, at least 2.5%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of total targeting moieties of the dual-targeted nanoparticle. In yet another embodiment, the first targeting moiety is less than 2%, less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90% of total targeting moieties of the dual-targeted nanoparticle. All numbers and fractions or ratios between any two of these percentages are also included, as well as endpoints.
As described herein, the number of nanoparticles bound to an RBC can alter the preparation of a composition (e.g. reduce agglutination ex vivo, limit disruption of RBC membranes, and improve loading of RBCs with nanoparticles) and levels of drug delivery to one or more target organs. Accordingly, in certain embodiments, provided herein are compositions comprising dual-targeted nanoparticles having RBCs bound ex vivo, wherein the composition has about 50 to about 200 bound nanoparticles per RBC. In certain embodiments, the composition has at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 bound nanoparticles per RBC. In yet another embodiment, the composition includes of less than 5, less than 10, less than 20, less than 30, less than 40, less than 50, less than 70, less than 80, less than 90, less than 100, or less than 110, less than 120, less than 130, less than 140, less than 150, less than 160, less than 170, less than 180, less than 190, or less than 200 bound nanoparticles per RBC. All numbers and ranges between any two of these numbers are also included, as well as endpoints.
In certain embodiments, a nanoparticle composition is provided which comprises dual-targeted nanoparticles having about 10% to about 25% of a first RBC-targeting moiety and about 75% to about 90% of a second targeting moiety that binds, e.g., an endothelial target.
In certain embodiments, a nanoparticle composition is provided which comprises dual-targeted nanoparticles having about 2.5% to about 5% of a first RBC-targeting moiety and about 95% to about 97.5% of a second targeting moiety that binds, e.g., an endothelial target.
In certain embodiments, a nanoparticle composition is provided which comprises dual-targeted nanoparticles having about 2.5% to about 10% of a first RBC-targeting moiety and about 90% to about 97.5% of a second targeting moiety that binds, e.g., an endothelial target.
All of the ranges described herein include endpoints.
By the term “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. As used herein, antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of an immunoglobulin molecule, and fusions of immunologically active portions of an immunoglobulin molecule.
The antibody may be a naturally occurring antibody or may be a synthetic or modified antibody (e.g., a recombinantly generated antibody; a chimeric antibody; a bispecific antibody; a humanized antibody; a camelid antibody; and the like). The antibody may comprise at least one purification tag. In a particular embodiment, the framework antibody is an antibody fragment. The term “antibody fragment” includes a portion of an antibody that is an antigen binding fragment or single chains thereof. An antibody fragment can be a synthetically or genetically engineered polypeptide. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those in the art, and the fragments can be screened for utility in the same manner as whole antibodies. Antibody fragments include, without limitation, immunoglobulin fragments including, without limitation: single domain (Dab; e.g., single variable light or heavy chain domain), Fab, Fab′, F(ab′)2, and F(v); and fusions (e.g., via a linker) of these immunoglobulin fragments including, without limitation: scFv, scFv2, scFv-Fc, minibody, diabody, triabody, and tetrabody. The antibody may also be a protein (e.g., a fusion protein) comprising at least one antibody or antibody fragment.
As used herein, “specifically binding,” “binds specifically to,” “specific binding” refer, for example, to an antibody selectively or preferentially binding to an antigen. For example, with respect to a targeting moiety (such as an antibody), specifically binding refers to preferential binding refers to the ability of the antibody to bind one or more epitopes of an antigen or binding partner of interest without substantially recognizing and binding other molecules in a sample or environment containing a mixed population of antigens. Specific binding interactions are mediated by one or, typically, more noncovalent bonds between the binding molecules or binding partners.
The antibodies utilized herein may be further modified. For example, the antibodies may be humanized. In a particular embodiment, the antibodies (or a portion thereof) are inserted into the backbone of an antibody or antibody fragment construct. For example, the variable light domain and/or variable heavy domain of the antibodies of the instant invention may be inserted into another antibody construct. Methods for recombinantly producing antibodies are well-known in the art. Indeed, commercial vectors for certain antibody and antibody fragment constructs are available.
“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a nonhuman species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for FR substitution(s) as noted above. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For further details, see Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol 2:593-596 (1992).
The term “antibody fragment” as used herein for the described methods and compositions refers to less than an intact antibody structure having antigen-binding ability. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as e.g. single chain Fab, scFv, and multispecific antibodies formed from antibody fragments. The “single chain Fab” format is described, e.g., in Hust M. et al. BMC Biotechnol. 2007 March 8; 7:14. scFvV constructs include complementary scFvs produced as a single chain (tandem scFvs) or bispecific tandem scFvs.
In certain embodiments, the nanoparticle compositions provided herein include a second targeting moiety that is selected for delivery to, e.g, a target organ or tissue, such as antibody that bind to the endothelium (e.g., antibodies targeting endothelial proteins including PECAM-1, ICAM-1, VCAM, ACE, transferrin receptor, tissue factor/platelet tissue factor/factor III, and others) or antibodies that bind to other targeted cells. The targeting moiety may bind to at least one of a cell surface protein, carbohydrate, or lipid. In some embodiments, the targeting moiety binds to a cell adhesion molecule (CAM). The CAM may be intercellular adhesion molecule (ICAM), platelet-endothelial cell adhesion molecule (PECAM), activated leukocyte cell adhesion molecule (ALCAM), B-lymphocyte cell adhesion molecule (BL-CAM), vascular cell adhesion molecule (VCAM), mucosal vascular addressin cell adhesion molecule (MAdCAM), CD44, LFA-2, LFA-3, P-selectin, and basigin. In certain embodiments, the targeting moiety binds specifically to a CAM selected from ICAM, PECAM, or VECAM. In certain embodiments, the nanoparticles include a second targeting moiety specific for ICAM-1 and are targeted to pulmonary endothelial cells and white blood cells in the lungs (since ICAM-1 is expressed on both cell types). In yet other embodiments, the targeting moiety binds specifically to ACE, transferrin receptor, or a suitable endothelial target known in the art (See e.g., Simone et al. Cell Tissue Res. 2009 January; 335(1): 283-300, which is incorporated herein by reference). In other embodiments, the targeting moiety binds to a cell surface molecule associated with classical endocytosis. Preferably, the cell surface molecule associated with classical endocytosis is one of mannose-6-phosphate receptor and transferrin receptor. In certain embodiments, the second targeting moiety is specific for one or more of vascular endothelial cells, intravascular leukocytes, cells of reticuloendothelial system, an immune cell (e.g., B cell, T cell, NK cell, dendritic cell, macrophage), an infectious microorganism (including, but not limited to, a viral particle or bacterium), or targets tissues accessible to RBC under pathological conditions such as hemorrhage and thrombosis.
The surface of the NP may be modified to facilitate insertion, conjugation, absorption, or otherwise coupling of a moiety, either directly or via a spacer. The first and/or second targeting moieties of the invention may be linked noncovalently or covalently to the nanoparticles. Covalent linkages include linkages susceptible to cleavage once internalized in a cell. Such linkages include pH-labile, photo-labile and radio-labile bonds and are well known in the art. A targeting moiety may be directly or indirectly linked to a nanoparticle, e.g. via an avidin- or streptavidin-biotin interaction. In certain embodiments, the compositions include nanoparticles (e.g. liposomes) having a polyethylene glycol coating that includes or mediates attachment of a targeting moiety to the nanoparticle. In certain embodiments, the nanoparticles are chemically conjugated to targeting moieties using molecular cross-linkers, spacers, and bridges. By cross-linkers, spacer and bridges are meant any moiety used to attach or associate the NP to the targeting moiety. Thus, in one embodiment, the cross-linker is a covalent bond. In another embodiment, the linker is a non-covalent bond. In still other embodiments, the linker can be a larger compound or two or more compounds that associate covalently or non-covalently. In still other embodiment, the linker can be a combination of the linkers, e.g., chemical compounds, nucleotides, amino acids, proteins, etc. In one embodiment, the cross-linker is biotin-avidin or biotin-streptavidin. In this embodiment, interconnecting molecule(s) such as streptavidin can be coupled to a targeting moiety or an RBC either directly via chemical modification, or via biotin derivatives conjugated to the functional groups on the targeting moiety. In one embodiment a spacer is positioned between biotin and a reactive group, such as succinimide ester group. In certain embodiments, a spacer version is a polyethylene glycol (PEG) chain with MW from ˜100 Daltons (D) to up to 10,000 D. In another embodiment, the spacer is an aliphatic chain —CH2—CH2—CH2— with size varying from 1 angstroms (A) up to 5, 10, 15, 20, 25 or 30 Angstroms. Longer spacer arms give more flexibility for interactions. In another embodiment the linker is composed of at least one to about 25 atoms. In still another embodiment, the cross-linker is formed of a sequence of at least 2 to 60 nucleic acids. In yet another embodiment, the cross-linker refers to at least one to at least 2, up to about 30 amino acids or 1 or more proteins within that size. In certain embodiments, the linkage of the first and/or second moiety to the nanoparticles is reversible (e.g. following administration to the subject and binding of the second moiety to a target antigen).
In certain embodiments, the NPs for use in this invention are pre-loaded with a cargo (e.g. before coupling to a RBC the NP is loaded with one or more selected drugs). The drug may be encapsulated in the inner volume and/or bound to the surface of nanoparticles. In one embodiment, the NP loading is high capacity, e.g., the mass of the drug is >5% the mass of the NP. In one embodiment the selected NP contains a single drug component. In another embodiment, the selected NP is loaded with multiple drug components. By “drug” as used herein is meant any therapeutic, prophylactic, or diagnostic compound or reagent that is contained within the flexible nanoparticles described herein. In one embodiment, the drug is a water-miscible compound. In another embodiment, the drug is an anti-rejection drug. Anti-rejection drugs include agents such as alemtuzumab, tacrolimus, and other drugs currently delivered systemically post-transplant to prevent rejection. In another embodiment, the drug is an anti-inflammatory agent. Anti-inflammatory agents include corticosteroids, methotrexate, mycophenolate mofetil, azathioprine and other agents intended to limit inflammation. In another embodiment, the drug is a pro-angiogenic factor, such as VEGF-receptor agonists and other known such factors. In another embodiment, the drug is an anti-edema agent, e.g., albuterol. In still another embodiment the drug is a compound that prevent ischemia-reperfusion injury, such as N-acetylcysteine, allopurinol, L-arginine, among known agents.
In a specific example, where the composition is designed for the improvement of ARDS, in one embodiment, multiple drugs are employed in the NPs. These drugs include combinations that act on multiple cell types, such as albuterol (acting on epithelial ENac to pump out alveolar fluid), dexamethasone (enhances endothelial barrier function and decreases neutrophil activity), and palifermin (enhances repair and regeneration of alveoli). Other drug combinations with possible applications in other diseases include but are not limited to: Imatinib Mesylate (trade name Gleevec, a tyrosine kinase inhibitor approved for treatment of a variety of cancers), EUK-134 (a superoxide dismutase and catalase mimetic capable of mediating oxidative stress injury), MJ33 (an NADPH oxidase inhibitor used for antioxidant protection). Still other drug(s) or combinations for loading in the NPs of these compositions may be selected from among libraries of drugs for a variety of diseases.
In still another embodiment, the drug is an imaging agent. An “imaging agent” as used herein is a compound that has one or more properties that permit its presence and/or location to be detected directly or indirectly. Examples of such imaging agents include proteins and small molecule compounds incorporating a labeled entity that permits detection. Among suitable imaging agents are molecules containing radionuclides that are amenable to SPECT or PET imaging (e.g., Indium-111 for SPECT imaging); molecules containing moieties that provide contrast for CT imaging (e.g., gold nanoparticles or iodinated contrast agents); molecules containing moieties that provide contrast for MRI imaging (e.g., gadolinium); nano- or micro-scale complexes that provide contrast for ultrasound imaging (e.g., microbubbles filled with gas). A “detectable label” is a marker used for detection or imaging. Examples of such labels include: a radiolabel, a fluorophore, a chromophore, or an affinity tag. In one embodiment, the label is a radiolabel used for medical imaging, for example tc99m or iodine-123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, iron, etc.
In another aspect, a suitable nanoparticle for use in the methods herein is miscible with endothelial glycocalyx (e.g., carbohydrates on nanogel interdigitate with endothelial cells).
In another aspect, a suitable nanoparticle for use in the methods herein is characterized by having a more efficient or effective delivery than previously demonstrated using delivery passive RBC—hitchhiking. For example, lung targeting may be as much as 400% (injected dose/gram (ID/g) at 30 minutes) greater than delivery previously obtained by passive RBC-hitchhiking of targeted particles. In certain embodiments, administration of dual-targeted nanoparticles results in at least 2× greater delivery (e.g. to the lungs) than a passive RBC-hitchhiked particle targeted only to the lungs. In certain embodiments, administration of dual-targeted nanoparticles results in at least 2.5× increased delivery (e.g. to the lungs) compared to direct injection of free dual-targeted particles (i.e. not coupled to RBC ex vivo).
The biocompatible and/or non-toxic coupling of NP to the targeting moieties in these compositions allows ready transfer of the NP from the RBC to the end target, e.g., vascular endothelium, white blood cells, or other cells of the end organ. In certain embodiments, the target organ is the lungs, brain, or heart. In certain embodiments, the target is a blood clot (e.g. for treatment of ischemic stroke). In a method using dual-targeted NPs, i.e., in which the NP is associated with an antibody to RBC and an antibody to an antigen on a target cell, the delivery of a drug in the NP may be aided by covalent binding of the NPs to RBCs and then further targeting by the covalent binding of the antibody to the target antigen to the resulting target. Without wishing to be bound by theory, the method operates in the following manner: an RBC to which NPs are coupled ex vivo via a targeting moiety are administered to a patient and transfers the NPs to the surface of a vascular vessel, e.g., a capillary, due to a second targeting moiety specific for an endothelial antigen. In certain embodiments, the NPs are thereafter taken up by the target cell and the RBC is released. In certain embodiments, the NPs are nanogels carrying one or more selected drugs for delivery to the vessel or target organ.
In certain embodiments, the selected NPs can be optionally coated with a protein that does not induce an immunological reaction to the nanoparticle in a mammalian subject. By “mammalian subject” is meant primarily a human, but also domestic animals, e.g., dogs, cats; and livestock, such as cattle, pigs, etc.; common laboratory mammals, such as primates, rabbits, and rodents; and pest or wild animals, such as deer, rodents, rabbits, squirrels, etc. The selected coated or non-coated NP is then loaded with a suitable drug or multiple drugs generally by incubation at about 37° C. in a buffer. Desirable buffers are those that are RBC-compatible, such as phosphate buffered saline or the like. Other methods for drug loading in drug carriers include osmotic loading of a variety of small molecule drugs in protein, nanogel, or nanoparticle carriers either before or after linking the NPs and RBC. Drug loading and release from nanogels on RBCs may be modified by using crosslinkers incorporated in the nanogel to prolong or enhance encapsulation of loaded drugs and performing the crosslinking after drug loading. Crosslinkers can include responsive moieties (e.g. enzyme-cleavable crosslinkers that allow stimulated drug release in response to protease activity). In another embodiment, the drug is kept in the solution or in the wash buffer during all drug loading steps (except the last resuspension).
Also provided herein are methods for generating dual-targeted nanoparticle compositions. For example, dual-targeted nanoparticles having a first targeting moiety and a second targeting moiety, wherein the first targeting moiety is a red blood cell(RBC)-targeting moiety, is contacted with RBC, resulting in the dual-targeted nanoparticles being bound to the RBC. In certain embodiments, the NPs are bound to RBCs via a second targeting moiety prior to delivery in vivo and following drug loading. In certain embodiments, the RBCs are present in a sample of enriched or isolated (i.e., separated from one or more other components of whole blood) RBCs. In one embodiment, RBCs are in a sample obtained from a mammalian subject (e.g., an autologous donor or the subject to be treated) and bound to NPs before or after leukoreduction. In one embodiment, the RBCs are present in a whole blood sample isolated from the donor (e.g., an autologous donor or the subject to be treated). In another embodiment, the RBCs are isolated or enriched from a sample obtained from the donor (e.g., an autologous donor or the subject to be treated). In another embodiment, no leukoreduction is performed. In one embodiment, the source of the RBCs is a typed and crossmatched unit of peripheral RBCs. In another embodiment, the source of the RBCs is a universal donor (O-neg). In another embodiment, the source of the RBCs is the subject to which the dual-targeted nanoparticle composition is to be administered.
In yet another embodiment, a composition comprising dual-targeted nanoparticles is administered to a subject and the nanoparticles bind RBCs in vivo via a first targeting moiety.
Similarly, the methods may employ other adjunctive steps or devices to prepare the compositions described herein including microfluidic devices to put RBCs and NPs into small volume to increase adsorption efficiency or storage of NPs in bags that are RBC compatible, or use of bags that do not adsorb NPs, use of wide bore infusion tubing or tubing that doesn't adsorb nanoparticles, short-tubing to avoid adsorption to tubes, or use of transfer pipettes (large bore) during wash steps.
In certain embodiments, where the nanoparticles are hydrogels, the preparative method can involve retaining NPs in a solution containing a high concentration of a drug and centrifuging the NPs and resuspending same in a solution with a low concentration of said drug within 60 minutes before in vivo delivery. In another method, the binding step can occur by adding nanoparticles to a unit of red blood cells without excipients. In still another preparative method step, the binding can involve extracting blood from a mammalian subject and agitating a mixture of RBCs and loaded nanoparticles ex vivo within 60 minutes before in vivo delivery. In still a further modification, the nanoparticles are present in a syringe and binding of the NPs to RBCs via a first targeting moiety occurs when a mammalian subject's blood is withdrawn into the syringe. In another embodiment, if a small volume of RBCs is used, the container may be pre-coated with RBCs before NPs are introduced.
In another aspect, the compositions describe herein comprising dual-targeted NPs are useful in therapeutic treatment of disease, diagnosis of disease, or prophylactic treatments to prevent disease, depending upon the identities of the drugs with which the NPs are loaded.
By “disease” as used herein is meant, without any limitation, any disease in which small arterioles, capillaries, venules and/or the endothelial barrier plays an important role. Without limitation, such diseases and disorders include those involving the lung, including ARDS, IPF (idiopathic pulmonary fibrosis), pulmonary arterial hypertension, post-pulmonary embolism to prevent reactive vasoconstriction, pulmonary capilliaritis syndromes, such as the vasculitidies of granulomatosis and polyangitis (GPA) and Goodpasture's syndrome, among others. Still other diseases suitable for such treatment include those involving the heart, such as heart attack, ischemia-reperfusion injury, stroke or other diseases involving the heart; diabetic retinopathy or macular degeneration and other disorders involving the eye; hyperthyroidism or hypothyroidism and other diseases involving the thyroid; autoimmune hepatitis, alcoholic hepatitis, NAFLD/NASH and other diseases involving the liver; pancreatitis or other diseases of the pancreas; immunological disorders or other diseases of the spleen; inflammatory bowel disease and other diseases of the intestines; benign prostatic hypertrophy (BPH), prostate cancer, and other disease of the prostate; disorders of the brain and cancers of any organ or tissue. Still other diseases or conditions suitable for treatment, prophylaxis or diagnosis with compositions described herein include ischemic stroke, prevention of ischemia reperfusion injury (IRI), prevention of post-myocardial infarction, also for prevention of IRI, prevention or treatment of PAD (peripheral artery disease, especially the legs), prevention or treatment of cancer, especially head/neck cancer after subarachnoid hemorrhage, and encephalitis and meningitis. In still another embodiment, the disease or disorder may be a need for a transplanted organ, and the transplanted organ itself may be treated ex vivo with compositions described herein, e.g., to prevent IRI. This includes all solid organ transplants.
As used herein, the term “treatment,” and variations thereof such as “treat” or “treating,” refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing or reducing the occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, effectors described herein are used to delay development of a disease or to slow the progression of a disease. In certain embodiments, the drug is released or transferred from the nanoparticles within 5, 10, 15, 20, 25, or 30 minutes of administration in vivo to a target organ. In certain embodiment, the composition contains a single drug or multiple drugs. In another embodiment, multiple drugs are administered simultaneously in the same or multiple RBC-coupled NP compositions. In certain embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the administered dose of a drug is delivered a target organ. In a certain embodiment, the target organ is the lung and at least 70% of the injected dose of the drug is delivered. In a further embodiment, where the target organ is the lung, delivery of dual-targeted nanoparticles results in enhanced delivery to the lung, reduced reticuloendothelial system (RES) clearance, plasma-half life and/or reduced toxicity or off target effects as compared to drug delivery using non-targeted NPs, single-targeted NPs, or RBCs with physisorbed NP or passive RBC hitchhiking.
By “administering” or “route of administration” is meant delivery of composition described herein, with or without a pharmaceutical carrier or excipient, to the subject. Routes of administration may be combined, if desired. In certain embodiments, the method includes intravenous delivery of a composition described herein. In certain the method includes intraarterial delivery of a composition described herein.
In certain embodiments, administration of the compositions described herein can be intravenous (iv) for delivery of the drug to the lungs. For example, where the disease is ARDS, pneumonia, interstitial lung disease, idiopathic pulmonary fibrosis, post-pulmonary embolism, pulmonary capilliaritis syndrome, emphysema, or a viral infection (such as SARS, influenza, or COVID). the compositions may be administered intravenously. In certain embodiments, the methods employ injecting iv the compositions described herein carrying one or more of albuterol, dexamethasone, and palifermin for the treatment of ARDS. In certain embodiments, where the disease involves any other selected mammalian organ, the composition is administered in vivo intra-arterially immediately upstream of the organ for delivery of effective doses of the drug. Additionally, for treatment of disease involving a selected mammalian organ designated for transplantation (other than the lung), the composition is administered ex vivo via feeding arterial opening into the organ prior to reperfusion and transplantation. In certain embodiments, the composition is administered via an arterial conduit of a selected organ using an arterial catheter. In any intra-arterial administration, the composition can be administered via an intra-arterial catheter. Such organs include, without limitation, the heart, brain, eyes, thyroid, kidney, liver, pancreas, spleen, intestines, or prostate.
Certain adjunctive steps for administering the compositions described herein include rotating or agitating or diffusing the RBC-coupled NPs before administering to prevent settling and aggregation of RBCs. In another embodiment, the method may employ a syringe that removes the RBCs from a patient's own blood and then reinjects after RBC-NP coupling. The syringe may be pre-loaded with anti-coagulant (that reverses upon re-injection to patient; e.g. drug marketed as Eloquis®), or a leukoreduction filter. An intra-arterial catheter may be employed that uses ultrasound to scan for the presence of arterial plaque prior to intracarotid injection, preventing embolization of atheromatous plaques. Similarly, a device to allow intracarotid (or other arterial injection) with simultaneous closure of the injection hole can be employed in these methods.
In still a further aspect, the compositions and methods can also be employed for imaging, such as to map capillary structure or pathology, wherein the drug is an imaging reagent. In one embodiment, a method of imaging a mammalian organ comprises injecting intravenously or intraarterially in vivo or ex vivo a composition comprising dual-targeted nanoparticles described herein, wherein the nanoparticles contain an imaging drug, and wherein the nanoparticles are transferred from the dual-targeted nanoparticles to the vascular bed of a target organ of the subject and release the imaging drug to the selected target organ or vascular tissue.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
Methods and agents well known in the art for making formulations are described, for example, in “Remington's Pharmaceutical Sciences,” Mack Publishing Company, Easton, Pa. Formulations may, for example, contain excipients, carriers, stabilizers, or diluents such as sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes, preservatives (such as octadecyldimethylbenzyl, ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
All scientific and technical terms used herein have their known and normal meaning to a person of skill in the fields of biology, biotechnology and molecular biology and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. However, for clarity, certain terms are defined as provided herein.
The terms “a” or “an” refers to one or more, for example, “an assay” is understood to represent one or more assays. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.
The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to ±10% from the specified value; as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.”
Various embodiments in the specification are presented using “comprising” language, which is inclusive of other components or method steps. When “comprising” is used, it is to be understood that related embodiments include descriptions using the “consisting of” terminology, which excludes other components or method steps, and “consisting essentially of” terminology, which excludes any components or method steps that substantially change the nature of the embodiment or invention.
The following examples disclose specific embodiments of preparation of compositions of this invention, their characteristics, and methods of use thereof. These examples should be construed to encompass any and all variations that become evident as a result of the teachings provided herein.
Azide functionalized PEG liposomes were prepared as described previously. 37 Briefly, lipids DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), cholesterol, and azide PEG2000 DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000]) (Avanti Polar Lipids, Alabaster, Ala.) were combined with the phospholipid to cholesterol ratio at 3:1. Liposomes requiring 111In radiolabeling include 0.2 mol % DTPA-PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid), and those requiring fluorescence include 0.5 mol % Top FL-PC (1-palmitoyl-2-(dipyrrometheneboron difluoride) undecanoyl-sn-glycero-3-phosphocholine) or [TopFl-PE, Rhodamine-PE. Lipid solutions were subjected to a constant stream of nitrogen gas until visibly dried, then lyophilized for 1-2 h to remove residual solvent. Dried lipid films were rehydrated with buffer, either sterile PBS or 0.3N metal free citrate at pH 4. This lipid solution underwent 3 cycles of freeze thaw between liquid N2 and a 50° C. water bath, followed by 10× extrusion cycles through 200 nm polycarbonate filters using an Avanti Mini Extruder (Avanti Polar Lipids). At each stage of particle synthesis and modification, particle size, distribution, and polydispersity index (PDI) were taken at 1:125 dilution in PBS using a Zetasizer Nano ZSP. Particle concentration in #/ml was measured using a NanoSight NS300 at a dilution in ultrapure DI water of ˜104. (Both instruments by Malvern Panalytical, Malvern UK.)
As described previously51, highly stable and homogeneous immunoliposomes were synthesized using copper free click chemistry methods. All monoclonal antibodies and control IgG were modified with dibenzylcyclooctyne-PEG4-NHS ester (Jena Bioscience; Thuringia, Germany). The proteins, buffered in PBS and adjusted to pH 8.3 with 1 M NaHCO3 buffer, were reacted for 1 h at room temperature (RT) at a ratio of 1:5 antibody/NHS ester PEG4 DBCO. Post reaction, the mixture was buffer exchanged with an Amicon 10 k MWCO centrifugal filter (MilliporeSigma, Burlington Mass.) to remove unreacted NHS ester PEG4 DBCO by 30 vol washes. The efficiency of DBCO-IgG reaction was determined optically, with absorbance at 280 nm indicating IgG concentration and absorbance at 309 nm indicating DBCO concentration. Spectral overlap of DBCO and IgG absorbance was noted by correcting absorbance at 280 nm. Molar IgG concentration was determined using Beer's Law calculation, with an IgG extinction coefficient of 204,000 L mol−1cm−1 at 280 nm. Likewise, the molar DBCO concentration was determined using the DBCO extinction coefficient at 309 nm, 12,000 L mol−1 cm−1. The number of DBCO per IgG was determined as the ratio. All Ab-DBCO used in these studies had between 2-5 DBCO/Ab.
Monoclonal antibodies modified included those against endothelial targets intracellular adhesion molecule (ICAM-1) and platelet-endothelial cell adhesion molecule (PECAM-1) for both mouse (YN1 and Mec13, respectively) and human (R6.5 and Ab62) and those against RBC target GPA both mouse (Ter119) and human (CD235), and Rh in human (Bric69). Whole molecule rat IgG was included for controls, and as a non-immune vehicle for 125I to quantify particle localization. Radiolabeling of IgG-DBCO with Na-125I was done using the iodogen method as already described.52 For quantification of conjugation individual antibodies in dual preparations, Ab-PEG4-DBCO were further modified with either NHS-Alexafluor 488 or 594 as directed by the manufacturer (ThermoFisher, US), and purified using Amicon filters as described.
Liposomes were isotope traced either by inclusion of 125I-IgG/DBCO on the surface of the particle at no more than 10% of total antibody coating or by surface chelation of 111In to DTPA-PE on the particle surface as already described.37 IgG-DBCO underwent radio-iodination with Na-125I using the iodogen method. Surface chelation of 111In was done using metal free conditions to reduce reaction inefficiencies due to metal contamination. 111In Cl3 (Nuclear Diagnostic Products, Cherry Hill, N.J.) was diluted in citrate buffer and added to preformed azide 0.2% DTPA liposomes, hydrated with metal-free pH 4 citrate buffer, and reacted for 1 h at 37° C. The reaction mixture was quenched with 50 mM DTPA to 1 mM final concentration to chelate unincorporated 111In. The radiochemical purity and yield quantified using thin film chromatography (TLC) with mobile phase EDTA 10 μM gamma counting of the aluminum silica strips (Sigma Chemical, St Louis Mo.). For biodistributions liposomes were labeled at 50-100 μCi/μmol. Liposome samples were buffer exchanged with sterile PBS using Amicon centrifugal filters, followed by targeting ligand conjugation.
Antibodies were conjugated to liposomes using copper-free click chemistry as previously described.37 DBCO-functionalized monoclonal antibodies described earlier were incubated with azide-bearing liposomes from 4 h to overnight at 37° C. with rotation. Post incubation mixtures were purified using size exclusion chromatography using Sepharose 4B-Cl (GE Healthcare, Pittsburgh Pa.) packed in a 20 mL Biorad polyprep column taking 1.0 mL fractions for 25 mL, quantification of binding was done via tracing ligand fluorescence. Dual antibody formulations were characterized individually using different fluorophores conjugated to the proteins directly as described, e.g. Alexafluor 488 for YN1 and Alexafluor 594 for Ter119, with fractions read on a plate reader (Spectramax M2; Molecular Devices, San Jose, Calif.) or radioactivity (fractions measured on a gamma counter). Efficiency of conjugation reaction is quantitatively defined as the ratio of the area under the curve of the ligand signal in the liposome peak (4.0-6 mL) over the integration of the entire 25 mL elution plotted by signal over elution volume (
Murine RBC were obtained from male C57BL/6 mice (The Jackson Laboratory, Bar Harbor, Me.) by inferior vena cava puncture after anesthesia with ketamine/xylazine (100/10 mg/kg). Human RBC were obtained by sterile venipuncture from healthy adult donors. For ex vivo lung perfusion (EVLP) experiments, donor RBC blood type was matched to the blood type for donor lung tissue. Murine and human RBC were treated and washed identically after blood draw. To prevent coagulation, syringes and collection tubes were pre-treated with ethylenediaminetetraacetic acid (EDTA, Sigma Aldrich, St Louis, Mo.), in DPBS (Corning, Manassas, Va.). RBC were purified from WBC, platelets, and serum by centrifugation and washing 2× with DPBS. RBC were either used immediately or resuspended in 5 mMol glucose in DPBS (Sigma Aldrich, St Louis, Mo.) for storage up to 24 h at 4° C.
When RBC tracing or labeling was required, RBC were resuspended at 10% hematocrit (hct) in 5 mM glucose and incubated with chromium-51 radionuclide (51Cr, sodium chromate in normal saline, Perkin Elmer Life & Analytical Sciences) for up to 12 h at 4 C. RBC were washed 2× with DPBS to remove free 51Cr and either used immediately or stored as previously described.
RBC were isolated and purified as described. Loading was found to have the highest efficiency when performed at higher RBC concentration (hematocrit) and given at least 90 minutes for binding (supplemental Fi) so liposomes were highly concentrated to maintain a RBC hematocrit of approximately 50% after mixing. Liposome/RBC mixtures were incubated at 4° C., rotating, for 90 minutes in Axygen maximum recovery microtubes (Corning, Mexico). After incubation with liposomes, RBC were washed 2× with DPBS to remove unbound liposomes then the washes and remaining pellet were measured for radioactivity using a Wallac 1470 Wizard gamma counter (Perkin Elmer Life and Analytical Sciences-Wallac Oy, Turku, Finland). Liposome loading efficiency was calculated from radioactivity remaining in the RBC pellet after washing divided by radioactivity in the pellet plus washes.
A standard agglutination assay was performed as is done clinically.53 The assay was performed at 2% hct: 20 uL of pRBC with a varied number of liposomes (
Naming conventions used hereafter in Example 1 and Example 2 are diagrammed in
Naïve C57BL/6 male mice (The Jackson Laboratory, Bar Harbor, Me.) anesthetized with ketamine/xylazine (100/10 mg/kg) were injected intravascularly with 1 μmol (0.75 mg) total radioimmunoliposome dual conjugated with targeting ligand against ICAM or PECAM antibody, Ter119 against GPA on the RBC, and 125I-IgG). Animals were euthanized at designated times after injections; the organs of interest harvested, rinsed with saline, blotted dry, and weighed. Blood samples (˜200 ul) were spun down at 500 rcf in a microcentrifuge tube with RBCs separated from plasma. Radioactivity in organs and separated blood components were measured with a Wallac 1470 Wizard gamma counter (PerkinElmer Life and Analytical Sciences-Wallac Oy, Turku, Finland). The gamma data of the 125I and 51Cr (or 111In) measurements and organ weights were used to calculate the tissue biodistribution injected dose per gram. The total injected dose was measured prior to injections, corrected for tube and syringe residuals, and verified to be ≥75% of the sum of the individual measures.
Following intravenous administration of dual-targeted liposomes that were either injected freely (direct injection) or loaded ex vivo onto RBC, lung tissue was prepared for flow cytometry to determine which cell types liposomes were delivered to. At 30 minutes, a tracheostomy and cannulation were performed then animals were sacrificed. The right ventricle was cannulated and perfused with cold PBS at 20 cm H2O to flush RBC from the pulmonary capillary bed. Lungs were re-inflated with 0.8 mL digestive enzyme solution (collagenase type 1 (Life tech), dispase (Collaborative), DNase1 (Roche) with PBS) and removed from the chest cavity. Harvested lungs were prepared into single cell suspension first by manual chopping with addition of additional digestive enzyme. Samples were incubated in 37° C. water intermittently vortexed then mixed with fetal bovine serum (Sigma, Pa.). Homogenate was strained through a 100-micron filter, centrifuged, and resuspended in ACK lysing buffer (Gibco) to remove RBC, then strained through a 40-micron filter on ice, centrifuged, and resuspended in FACS buffer (1% FBS, 1 mM EDTA in PBS, reagents already specified). Cells were fixed then centrifuged and resuspended in FACS buffer for flow cytometry. This single cell suspension was stained for CD45 (Anti-mouse CD45-brilliant violet 421, BioLegend) and PECAM (Anti-Mouse-CD31-APC, Invitrogen, CA). Final resuspension in 2:2000 DAPI was used to exclude dead cells. Flow cytometry was performed on an LSR Fortessa (BD Biosciences) then gated for viability and singlets and analyzed with FlowJo software (FlowJo LLC).
For in vitro analysis of RBC binding with FITC-labeled TER119-coated liposomes RBC were incubated with the liposomes, washed by centrifugation, adsorbed on glass slides, washed and mounted. In in vivo studies animals were sacrificed; lungs were harvested, immersed in OCT, and frozen by liquid N2. Frozen tissues were cut using Cryostat with 10-20 μm/slice. Samples were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.3% Triton X-100 for 15 min prior staining with antibodies. Leukocytes were stained with rabbit anti-mouse CD45 antibody (Abcam, #ab10558) followed by Alexa Fluor 647 labeled anti-rabbit IgG. Liposomes were stained with Alexa Fluor 594 goat anti-rat IgG (Invitrogen). Microscopy studies were performed on a confocal laser scanning microscope Leica TCS-SP8 (Leica, Germany) using HC PL APO CS2 63x/1.40 Oil objective and 488/552/638 lasers. Image analysis was performed using Volocity 6.3 Cellular Imaging & Analysis.
Human lungs were obtained following organ harvest from transplant donors after lung tissue was deemed unsuitable for transplantation. The lungs were perfused and harvested by the organ procurement team and kept submerged in PBS at 4° C. until use in the lab, within 24 hours of harvest. The lungs were accepted for research if oxygenation, cause of death, and visual assessment was all consistent with normal lung function. We used a modified ex vivo lung perfusion (EVLP) protocol.54 The airway was cannulated and inflated with low pressure oxygen; oxygen flow was continued at approximately 0.8 L/min to maintain gentle inflation. A subsegmental branch of the pulmonary artery was cannulated and perfused with Steen solution for 5 minutes at 20 cm H2O. Green tissue dye was used to test for retrograde flow and identify efflux from the pulmonary vein. Using RBC labeled with 51Cr and DT liposome labeled with 125I, a 3 mL DTRH sample was perfused by slow push into the arterial cannulation. This was chased with 3 mL of tissue dye to achieve bright staining of the perfused area of tissue. Finally, Steen solution was perfused for 10 mg at 20 cm H2O. All efflux was collected from the pulmonary vein. The lung tissue was then dissected and areas perfused by green tissue dye were measured for retention of liposomes and RBC using 51Cr and 125I signal measured by gamma counter.
Although the primary focus of targeted drug delivery is to concentrate drugs to a single cell type or organ, the majority of systemically administered drug nanocarriers never reach their target. Here, we combined a nanocarrier targeting approach of ligand (antibody) targeting with cell-based delivery to localize a majority of injected nanocarriers to their target. In dual-targeted RBC-hitchhiking (DTRH), liposomes are conjugated with two antibody types: one targeting red blood cells (RBCs) and a second targeting epitopes expressed in target vasculature. We demonstrated that DTRH localizes nanocarriers to the lungs at as high as 65% of the injected dose, >2.5 times that of comparable single antibody targeting. DTRH also improved delivery to specific cell populations (e.g., alveolar endothelial cells) within the target organ by >6.4-fold. Finally, we confirmed that DTRH enabled specific delivery in fresh ex vivo human lungs. Thus, DTRH shows great potential to improve organ- and cellular-level localization of therapeutics.
Design of Liposomes with Dual Avidity to RBC and Endothelial Cells (EC)
Dual-targeted (DT) liposomes (
The components of DT liposomes are depicted in
While DTRH is a fairly straightforward concept, its realization relies on balancing several challenging dualisms. For example, the surface of RBC contains abundant GPA. This is favorable for liposome loading and delivery, but unfavorable if multivalent anchoring occurs and prevents liposomes from transferring to EC. We varied the ratio between anti-GPA and anti-PECAM on the liposome surface, keeping a constant 200 monoclonal antibodies per liposome.
Dual-Targeted Liposome Interaction with RBC In Vitro
The DT liposome variants described in
Continuing to define DT liposome formulations' interaction with RBC in vitro, the loading characteristics of DT liposomes onto RBC were determined by immunoreactivity and flow cytometry. DT liposomes isotope labeled with 125I were tested against a vast excess (>1000×) of GPA binding sites. As the % RBC antibody (with anti-ICAM antibody filling in the remainder) reaches between 10-25%, the fraction of particles bound to the RBC approaches an asymptote (
Having established the above parameters, two formulations of DT liposomes were chosen to quantify the effective ex vivo loading of liposomes onto RBCs for injection in vivo. Shown in
Lastly, the loading characteristics onto RBC were quantified using flow cytometry and visualized with confocal microscopy. Based on the results described above, DT liposomes were formulated with 200 antibodies on their surface; 10% anti-RBC and 90% anti-EC. To simulate untargeted RH, ET liposomes were composed 100% of anti-EC. After loading, DT liposomes coated 99.7% of the RBC population (
Dual-Targeted Liposomes are Precisely Formulated to Maintain Organ-Specificity and Improve Targeting Efficacy without RBC Retention in the Lungs
The in vivo behavior of 125I-labeled liposomes loaded onto 51Cr-labeled RBC was characterized by comparing the following formulations, based on in vitro data (
At first glance, this result may appear to contradict the following logic. Pulmonary vascular targeting by ET liposomes is known to be mediated by endothelial avidity provided by anti-PECAM; however, DT liposomes actually have a portion of their anti-PECAM replaced by anti-GPA. Therefore, it would follow that the overall pulmonary endothelial avidity of DT liposomes should be lower than that of ET liposomes. The fact that RT liposomes coated with anti-GPA and IgG (
Having demonstrated high efficacy in lung targeting and transfer ratio without deleterious effect on RBC in vivo, the same DT liposome formulation was used to study kinetics over time. RBC-loaded DT liposomes were again IV injected into mice; 125I-DT liposomes but not 51Cr-RBC rapidly accumulate in the lungs with an immediate peak at 2 min post-injection (
The original approach for delivering nanocarriers to the vasculature using RBC hitchhiking (RH) did not involve specific ligand-mediated binding to RBC or to EC. Instead, accumulation in the target was predominantly mediated by the first pass phenomenon. In contrast, DTRH is based on specific binding of liposomes mediated by conjugated ligands. For example,
In case of true DTRH as demonstrated here (where the liposome surface is composed of anti-GPA plus anti-EC), the avidity of DT liposomes to RBC, in addition to total Fc burden, may contribute to a reduction of DT liposome biocompatibility. For example, multivalent binding of DT liposomes to GPA on the RBC surface may rigidify the carrier RBC. We determined conditions for the number of surface antibodies, GPA targeting antibodies, and ratio of GPA:EC targeting antibodies for DT liposome formulations and RBC loading that do not cause RBC pathology (
This concept is again demonstrated in
As presented, DT liposomes were developed in a murine model and shown to successfully deliver to the lungs with high efficacy and efficient transfer from carrier RBC. To determine the feasible utility in human therapeutics, DT liposomes were modified to target human RBC and EC, and were tested for proof-of-concept ex vivo in human lungs. DT liposomes were coated with antibodies against either GPA or RhCE surface antigens on human RBC. GPA was chosen to mimic the murine model testing, knowing that binding to GPA may induce RBC membrane rigidification.35 Therefore RhCE, a ubiquitous membrane protein expressed on human RBC that does not induce rigidification, was also evaluated. An anti-human-PECAM antibody was used to target ECs, as human pulmonary EC express PECAM similarly to murine pulmonary endothelium. We used the human PECAM clone Ab62, and one of two RBC-binding antibodies, either the GPA clone CD235a, or the RhCE clone Bric69. Each liposome was coated with 100 antibodies for all experiments targeting human cells. The percent of RBC antibodies was 0, 5, 10, 25, or 100%, with the remainder directed against EC for binding studies (
These humanized DT liposomes were tested for binding and agglutination of human RBCs to anticipate their behavior in vivo, using the same techniques shown in
Using humanized DT liposomes that can safely and effectively load onto human RBC, we next endeavored to show that DTRH can deliver to the human pulmonary vasculature. We have previously published on testing nanoparticle binding in an ex vivo human lung model.18 Briefly, we used whole lobes from fresh human lungs that were rejected for transplantation. The lungs are perfused, ventilated, and endovascularly cannulated for infusion of nanocarriers (
The preceding lung uptake studies, in both murine and human lungs, were completed with DT liposomes targeting the endothelial adhesion molecule PECAM. One goal of DTRH is that it be a generalizable method for effective, specific drug delivery. We therefore tested DTRH with the EC adhesion molecule target, ICAM. ICAM is an advantageous endothelial target since the adhesion molecule's expression, robustly expressed normally, is further enhanced by inflammation. 8,42,44-47 To construct DT liposomes, we were guided by the in vitro RBC binding experiments of
To test these DT liposomes and compare them to their cognate ET and RT versions, we first IV injected them as free liposomes, without RBC hitchhiking, and harvested organs 30 minutes later to create biodistribution plots (
As RBC loading is now shown to be required for the DTRH delivery effect, both formulations were first bound to RBC ex vivo before IV injection and 30 min biodistribution. Both 51Cr-RBC and 125I-DT liposomes were traced. Neither DTRH formulation caused RBC retention in the lungs (
Having observed that the 10%:90% DTRH formulation yielded ˜400% ID/g in the lung, higher than any ICAM-targeted formulation previously studied, we next investigated the kinetics of this formulation. We aimed to determine not only DT liposome association with tissue, but also the kinetics of dissociation between the liposomes and RBCs. In the table in
Analyzing the data of
The experiments above show that DTRH can increase organ-targeting, but give no insight into precisely where or to what cell subtypes the liposomes localize. DT liposomes were reformulated to include a fluorescent lipid for histology and flow cytometry analysis. We compared these DTRH liposomes versus identical “free” DT liposomes (not loaded onto RBC) using the same dual targeting ratio of 10%:90% (RBC-binding vs ICAM-binding antibody) used in
Lung histology (
The goal of nanomedicine has long been to localize drug-loaded nanocarriers to a specific organ and/or cell type. The field has made tremendous progress towards this goal, in large part from conjugating ligands onto the surface of nanocarriers. However, in nearly every case, far less than half the nanocarrier ends up in the target organ (unless the target organ is the liver), with values of <1% being common for many target tissues such as brain and tumor.
DTRH provides synergy between dual ligand targeting and cell-mediated delivery and has advantages over predicate technologies:
First, compared to passive RH, DTRH dramatically increased the efficiency of adsorbing nanocarriers onto the RBC surface. As shown in
Second, DTRH markedly improved organ-targeting.
Third, DTRH also dramatically improved cell-type-targeting within the target organ.
Fourth, DTRH can increase the types of nanocarriers that work with RBC-hitchhiking. In previous work18, while passive RH improved lung uptake at least to some extent on seven tested types of nanocarriers, only two of those produced lung localization comparable to anti-CAM nanocarriers, with the others displaying at least 5-fold lower uptake. The mechanism underlying passive RH's variability between nanocarriers is still unknown. By using a more defined binding system for RH, namely the two-antibody system of DTRH, we could convert a nanocarrier that does not work for passive RH into one that does benefit from RH's several advantages. Indeed, the nanocarriers employed in this Example do not work with passive RH, but do work with DTRH. The nanocarriers employed were as close to clinical application as possible. For the carrier itself, we chose liposomes, since they are the most clinically employed nanocarrier. These liposomes were conjugated to IgG molecules (the most common ligand employed clinically) via copper-free “click chemistry”, chosen because of its advantages for scale-up manufacturing (near 100% efficiency, stoichiometric addition, with no toxins to purify after). When adsorbed passively onto RBCs, these liposomes did not show a significant RH-effect or lung uptake (
In addition to the above advantages, another potential benefit of DTRH is that it can be rationally engineered, rather than relying on unknown mechanisms like passive RH. DTRH is composed of multiple components with easily quantifiable properties, namely one ligand that binds the mobile cell and another that binds the target cell. There is tremendous design flexibility, as the ligands can be changed in terms of: target epitopes (e.g., here we showed DTRH works with both anti-ICAM and -PECAM antibodies), absolute number, ratio of the two ligands, specific affinity (e.g., changing to a different antibody clone), and type of ligand (e.g., changing from monoclonal antibody to the single chain variable (scFv) format).
In summary, DTRH combines ligand-targeted and cell-mediated nanocarrier delivery that provides significant advantages over prior technologies such as passive RBC-hitchhiking and single-antibody targeting with anti-CAM antibodies. First, DTRH improves the adsorption efficiency compared to passive RH. Second, DTRH improves organ-targeting by >2-fold over passive RH and single-antibody-targeting, with delivering as much as 65% of the injected dose to the target organ. Third, DTRH improves cell-type-targeting, >6-fold over single-targeting. Fourth, DTRH can work with nanocarriers that do not work with passive RH.
Each and every patent, patent application, and publication, including websites and other publications cited throughout the specification, is incorporated herein by reference. While the invention has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.
This invention was made with government support under grant numbers HL143806 and HL138269 awarded by the National Institutes of Health. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 63002996 | Mar 2020 | US |
