Makan Khoshnejad

Size and targeting to PECAM vs ICAM control endothelial delivery, internalization and protective effect of multimolecular SOD conjugates.

Controlled endothelial delivery of SOD may alleviate abnormal local surplus of superoxide involved in ischemia-reperfusion, inflammation and other disease conditions. Targeting SOD to endothelial surface vs. intracellular compartments is desirable to prevent pathological effects of external vs. endogenous superoxide, respectively. Thus, SOD conjugated with antibodies to cell adhesion molecule PECAM (Ab/SOD) inhibits pro-inflammatory signaling mediated by endogenous superoxide produced in the endothelial endosomes in response to cytokines. Here we defined control of surface vs. endosomal delivery and effect of Ab/SOD, focusing on conjugate size and targeting to PECAM vs. ICAM. Ab/SOD enlargement from about 100 to 300nm enhanced amount of cell-bound SOD and protection against extracellular superoxide. In contrast, enlargement inhibited endocytosis of Ab/SOD and diminished mitigation of inflammatory signaling of endothelial superoxide. In addition to size, shape is important: endocytosis of antibody-coated spheres was more effective than that of polymorphous antibody conjugates. Further, targeting to ICAM provides higher endocytic efficacy than targeting to PECAM. ICAM-targeted Ab/SOD more effectively mitigated inflammatory signaling by intracellular superoxide in vitro and in animal models, although total uptake was inferior to that of PECAM-targeted Ab/SOD. Therefore, both geometry and targeting features of Ab/SOD conjugates control delivery to cell surface vs. endosomes for optimal protection against extracellular vs. endosomal oxidative stress, respectively.

Vascular Accessibility of Endothelial Targeted Ferritin Nanoparticles

Targeting nanocarriers to the endothelium, using affinity ligands to cell adhesion molecules such as ICAM-1 and PECAM-1, holds promise to improve the pharmacotherapy of many disease conditions. This approach capitalizes on the observation that antibody-targeted carriers of 100 nm and above accumulate in the pulmonary vasculature more effectively than free antibodies. Targeting of prospective nanocarriers in the 10-50 nm range, however, has not been studied. To address this intriguing issue, we conjugated monoclonal antibodies (Ab) to ICAM-1 and PECAM-1 or their single chain antigen-binding fragments (scFv) to ferritin nanoparticles (FNPs, size 12 nm), thereby producing Ab/FNPs and scFv/FNPs. Targeted FNPs retained their typical symmetric core-shell structure with sizes of 20-25 nm and ∼4-5 Ab (or ∼7-9 scFv) per particle. Ab/FNPs and scFv/FNPs, but not control IgG/FNPs, bound specifically to cells expressing target molecules and accumulated in the lungs after intravenous injection, with pulmonary targeting an order of magnitude higher than free Ab. Most intriguing, the targeting of Ab/FNPs to ICAM-1, but not PECAM-1, surpassed that of larger Ab/carriers targeted by the same ligand. These results indicate that (i) FNPs may provide a platform for targeting endothelial adhesion molecules with carriers in the 20 nm size range, which has not been previously reported; and (ii) ICAM-1 and PECAM-1 (known to localize in different domains of endothelial plasmalemma) differ in their accessibility to circulating objects of this size, common for blood components and nanocarriers.

Ferritin nanocages with biologically orthogonal conjugation for vascular targeting and imaging

Genetic incorporation of biologically orthogonal functional groups into macromolecules has the potential to yield efficient, controlled, reproducible, site-specific conjugation of affinity ligands, contrast agents, or therapeutic cargoes. Here, we applied this approach to ferritin, a ubiquitous iron-storage protein that self-assembles into multimeric nanocages with remarkable stability, size uniformity (12 nm), and endogenous capacity for loading and transport of a variety of inorganic and organic cargoes. The unnatural amino acid, 4-azidophenylalanine (4-AzF), was incorporated at different sites in the human ferritin light chain (hFTL) to allow site-specific conjugation of alkyne-containing small molecules or affinity ligands to the exterior surface of the nanocage. The optimal positioning of the 4-AzF residue was evaluated by screening a library of variants for the efficiency of copper-free click conjugation. One of the engineered ferritins, hFTL-5X, was found to accommodate ∼14 small-molecule fluorophores (AlexaFluor 488) and 3-4 IgG molecules per nanocage. Intravascular injection in mice of radiolabeled hFTL-5X carrying antibody to cell adhesion molecule ICAM-1, but not control IgG, enabled specific targeting to the lung due to high basal expression of ICAM-1 (43.3 ± 6.99 vs 3.48 ± 0.14%ID/g for Ab vs IgG). Treatment of mice with endotoxin known to stimulate inflammatory ICAM-1 overexpression resulted in 2-fold enhancement of pulmonary targeting (84.4 ± 12.89 vs 43.3 ± 6.99%ID/g). Likewise, injection of fluorescent, ICAM-targeted hFTL-5X nanocages revealed the effect of endotoxin by enhancement of near-infrared signal, indicating potential utility of this approach for both vascular targeting and imaging.

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo in vitro and in vivo.

Molecular engineering of antibodies for site-specific covalent conjugation using CRISPR/Cas9

Site-specific modification of antibodies has become a critical aspect in the development of next-generation immunoconjugates meeting criteria of clinically acceptable homogeneity, reproducibility, efficacy, ease of manufacturability, and cost-effectiveness. Using CRISPR/Cas9 genomic editing, we developed a simple and novel approach to produce site-specifically modified antibodies. A sortase tag was genetically incorporated into the C-terminal end of the third immunoglobulin heavy chain constant region (CH3) within a hybridoma cell line to manufacture antibodies capable of site-specific conjugation. This enabled an effective enzymatic site-controlled conjugation of fluorescent and radioactive cargoes to a genetically tagged mAb without impairment of antigen binding activity. After injection in mice, these immunoconjugates showed almost doubled specific targeting in the lung vs. chemically conjugated maternal mAb, and concomitant reduction in uptake in the liver and spleen. The approach outlined in this work provides a facile method for the development of more homogeneous, reproducible, effective, and scalable antibody conjugates for use as therapeutic and diagnostic tools.

Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude

Drug delivery by nanocarriers (NCs) has long been stymied by dominant liver uptake and limited target organ deposition, even when NCs are targeted using affinity moieties. Here we report a universal solution: red blood cell (RBC)-hitchhiking (RH), in which NCs adsorbed onto the RBCs transfer from RBCs to the first organ downstream of the intravascular injection. RH improves delivery for a wide range of NCs and even viral vectors. For example, RH injected intravenously increases liposome uptake in the first downstream organ, lungs, by ~40-fold compared with free NCs. Intra-carotid artery injection of RH NCs delivers >10% of the injected NC dose to the brain, ~10× higher than that achieved with affinity moieties. Further, RH works in mice, pigs, and ex vivo human lungs without causing RBC or end-organ toxicities. Thus, RH is a clinically translatable platform technology poised to augment drug delivery in acute lung disease, stroke, and several other diseases.Unwanted uptake in the liver and limited accumulation in target organs is a major obstacle to targeted drug delivery. Here, the authors report on the hitchhiking of nanocarriers on red blood cells and the targeted upstream delivery to different target organs in mice, pigs and ex vivo human lungs.

Ferritin-based drug delivery systems: Hybrid nanocarriers for vascular immunotargeting

ABSTRACT Ferritin subunits of heavy and light polypeptide chains self‐assemble into a spherical nanocage that serves as a natural transport vehicle for metals but can include diverse cargoes. Ferritin nanoparticles are characterized by remarkable stability, small and uniform size. Chemical modifications and molecular re‐engineering of ferritin yield a versatile platform of nanocarriers capable of delivering a broad range of therapeutic and imaging agents. Targeting moieties conjugated to the ferritin external surface provide multivalent anchoring of biological targets. Here, we highlight some of the current work on ferritin as well as examine potential strategies that could be used to functionalize ferritin via chemical and genetic means to enable its utility in vascular drug delivery. Graphical abstract Figure. No Caption available.

PECAM-1 directed re-targeting of exogenous mRNA providing two orders of magnitude enhancement of vascular delivery and expression in lungs independent of apolipoprotein E-mediated uptake

ABSTRACT Systemic administration of lipid nanoparticle (LNP)‐encapsulated messenger RNA (mRNA) leads predominantly to hepatic uptake and expression. Here, we conjugated nucleoside‐modified mRNA‐LNPs with antibodies (Abs) specific to vascular cell adhesion molecule, PECAM‐1. Systemic (intravenous) administration of Ab/LNP‐mRNAs resulted in profound inhibition of hepatic uptake concomitantly with ˜200‐fold and 25‐fold elevation of mRNA delivery and protein expression in the lungs compared to non‐targeted counterparts. Unlike hepatic delivery of LNP‐mRNA, Ab/LNP‐mRNA is independent of apolipoprotein E. Vascular re‐targeting of mRNA represents a promising, powerful, and unique approach for novel experimental and clinical interventions in organs of interest other than liver. HIGHLIGHTSRobust pulmonary targeting of mRNA was achieved by endothelial targeted anti‐PECAM/LNP‐mRNA nanoparticles.Delivery of targeted Anti‐PECAM/LNP‐mRNA nanoparticles is independent of apolipoprotein E pathway.Around 200‐fold elevation in pulmonary mRNA expression was reached upon IV administration of targeted nanoparticles.Rapid, transient, and specific protein expression from reporter mRNA was observed, with limited off‐target biodistribution.

Spatially controlled assembly of affinity ligand and enzyme cargo enables targeting ferritin nanocarriers to caveolae

One of the goals of nanomedicine is targeted delivery of therapeutic enzymes to the sub-cellular compartments where their action is needed. Endothelial caveolae-derived endosomes represent an important yet challenging destination for targeting, in part due to smaller size of the entry aperture of caveolae (ca. 30-50 nm). Here, we designed modular, multi-molecular, ferritin-based nanocarriers with uniform size (20 nm diameter) for easy drug-loading and targeted delivery of enzymatic cargo to these specific vesicles. These nanocarriers targeted to caveolar Plasmalemmal Vesicle-Associated Protein (Plvap) deliver superoxide dismutase (SOD) into endosomes in endothelial cells, the specific site of influx of superoxide mediating by such pro-inflammatory signaling as some cytokines and lipopolysaccharide (LPS). Cell studies showed efficient internalization of Plvap-targeted SOD-loaded nanocarriers followed by dissociation from caveolin-containing vesicles and intracellular transport to endosomes. The nanocarriers had a profound protective anti-inflammatory effect in an animal model of LPS-induced inflammation, in agreement with the characteristics of their endothelial uptake and intracellular transport, indicating that these novel, targeted nanocarriers provide an advantageous platform for caveolae-dependent delivery of biotherapeutics.

Unintended effects of drug carriers: Big issues of small particles

ABSTRACT Humoral and cellular host defense mechanisms including diverse phagocytes, leukocytes, and immune cells have evolved over millions of years to protect the body from microbes and other external and internal threats. These policing forces recognize engineered sub‐micron drug delivery systems (DDS) as such a threat, and react accordingly. This leads to impediment of the therapeutic action, extensively studied and discussed in the literature. Here, we focus on side effects of DDS interactions with host defenses. We argue that for nanomedicine to reach its clinical potential, the field must redouble its efforts in understanding the interaction between drug delivery systems and the host defenses, so that we can engineer safer interventions with the greatest potential for clinical success.