Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Targeted In Vivo Loading of Red Blood Cells Markedly Prolongs Nanocarrier Circulation.

Engineering drug delivery systems for prolonged pharmacokinetics (PK) has been an ongoing pursuit for nearly 50 years. The gold standard for PK enhancement is the coating of nanoparticles with polymers, namely polyethylene glycol (PEGylation), which has been applied in several clinically used products. In the present work, we utilize the longest circulating and most abundant component of blood─the erythrocyte─to improve the PK behavior of liposomes. Antibody-mediated coupling of liposomes to erythrocytes was tested in vitro to identify a loading dose that did not adversely impact the carrier cells. Injection of erythrocyte targeting liposomes into mice resulted in a ∼2-fold improvement in the area under the blood concentration versus time profile versus PEGylated liposomes and a redistribution from the plasma into the cellular fraction of blood. These results suggest that in vivo targeting of erythrocytes is a viable strategy to improve liposome PK relative to current, clinically viable strategies.

Controlling spatial distribution of functional lipids in a supported lipid bilayer prepared from vesicles.

Conjugating biomolecules, such as antibodies, to bioconjugate moieties on lipid surfaces is a powerful tool for engineering the surface of diverse biomaterials, including cells and nanoparticles. We developed supported lipid bilayers (SLBs) presenting well-defined spatial distributions of functional moieties as models for precisely engineered functional biomolecular-lipid surfaces. We used quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) to determine how vesicles containing a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (DSPE-PEG-N3) form SLBs as a function of the lipid phase transition temperature (Tm). Above the DPPC Tm, DPPC/DSPE-PEG-N3 vesicles form SLBs with functional azide moieties on SiO2 substrates via vesicle fusion. Below this Tm, DPPC/DSPE-PEG-N3 vesicles attach to SiO2 intact. Intact DPPC/DSPE-PEG-N3 vesicles on the SiO2 surfaces fuse and rupture to form SLBs when temperature is brought above the DPPC Tm. AFM studies show uniform and complete DPPC/DSPE-PEG-N3 SLB coverage of SiO2 surfaces for different DSPE-PEG-N3 concentrations. As the DSPE-PEG-N3 concentration increases from 0.01 to 6 mol%, the intermolecular spacing of DSPE-PEG-N3 in the SLBs decreases from 4.6 to 1.0 nm. The PEG moiety undergoes a mushroom to brush transition as DSPE-PEG-N3 concentration varies from 0.1 to 2.0 mol%. Via copper-free click reaction, IgG was conjugated to SLB surfaces with 4.6 nm or 1.3 nm inter-DSPE-PEG-N3 spacing. QCM-D and AFM data show; 1) uniform and complete IgG layers of similar mass and thickness on the two types of SLB; 2) a higher-viscosity/less rigid IgG layer on the SLB with 4.6 nm inter-DSPE-PEG-N3 spacing. Our studies provide a blueprint for SLBs modeling spatial control of functional macromolecules on lipid surfaces, including surfaces of lipid nanoparticles and cells.

Targeting of nanoparticles to the cerebral vasculature after traumatic brain injury.

Traumatic brain injury has faced numerous challenges in drug development, primarily due to the difficulty of effectively delivering drugs to the brain. However, there is a potential solution in targeted drug delivery methods involving antibody-drug conjugates or nanocarriers conjugated with targeting antibodies. Following a TBI, the blood-brain barrier (BBB) becomes permeable, which can last for years and allow the leakage of harmful plasma proteins. Consequently, an appealing approach for TBI treatment involves using drug delivery systems that utilize targeting antibodies and nanocarriers to help restore BBB integrity. In our investigation of this strategy, we examined the efficacy of free antibodies and nanocarriers targeting a specific endothelial surface marker called vascular cell adhesion molecule-1 (VCAM-1), which is known to be upregulated during inflammation. In a mouse model of TBI utilizing central fluid percussion injury, free VCAM-1 antibody did not demonstrate superior targeting when comparing sham vs. TBI brain. However, the administration of VCAM-1-targeted nanocarriers (liposomes) exhibited a 10-fold higher targeting specificity in TBI brain than in sham control. Flow cytometry and confocal microscopy analysis confirmed that VCAM-1 liposomes were primarily taken up by brain endothelial cells post-TBI. Consequently, VCAM-1 liposomes represent a promising platform for the targeted delivery of therapeutics to the brain following traumatic brain injury.

Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Automated measurement of distance-walked as a "sixth vital sign" for detecting infusion reactions during preclinical testing.

Infusion reactions are a major risk for advanced therapeutics (e.g., engineered proteins nanoparticles, etc.), which can trigger the complement cascade, anaphylaxis, and other life-threatening immune responses. However, during the early phases of development, it is uncommon to assess for infusion reactions, given the labor involved in measuring multiple physiological parameters in rodents. Therefore, we sought to develop an automated quantification of rodent locomotion to serve as a sensitive screening tool for infusion reactions, with minimal added labor-time for each experiment. Here we present the detailed methods for building a motion tracking cage for mice, requiring ∼$100 of materials, ∼2 h to build and set up completely, and employing freely available software (DeepLabCut). The distance-walked after injection was first shown to have the predicted effects for stimulants (caffeine), sedatives (ketamine), and toxins (lipopolysaccharide). Additionally, the distance-walked more sensitively detected the effects of these compounds than did pulse oximetry-based measurements of the classical vital signs of heart rate, respiratory rate, and blood oxygen saturation. Finally, we examined a nanomedicine formulation that has been in preclinical development, liposomes targeted to the cell adhesion molecule ICAM. While this formulation has been studied across dozens of publications, it has not previously been noted to produce an infusion reaction. However, the automated motion tracking cage showed that ICAM-liposomes markedly reduce the distance-walked, which we confirmed by measuring the other vital signs. Importantly, the motion tracking cage added < 5 min of labor time per 5-mouse condition, while pulse oximetry with a neck cuff (by far the most stable oximetry signal in mice) required âˆ¼ 100 min of labor time. Thus, automated measurement of distance-walked can indeed serve as a "sixth vital sign" for detecting infusion reactions during preclinical testing. Additionally, the device to measure distance-walked is easy and cheap to build and requires negligible labor time for each experiment, enabling distance-walked to be recorded in nearly every infusion experiment.

Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE).

Current nucleoside-modified RNA lipid nanoparticle (modmRNA-LNP) technology has successfully paved the way for the highest clinical efficacy data from next-generation vaccinations against SARS-CoV-2 during the COVID-19 pandemic. However, such modmRNA-LNP technology has not been characterized in common pre-existing inflammatory or immune-challenged conditions, raising the risk of adverse clinical effects when administering modmRNA-LNPs in such cases. Herein, we induce an acute-inflammation model in mice with lipopolysaccharide (LPS) intratracheally (IT), 1 mg kg-1, or intravenously (IV), 2 mg kg-1, and then IV administer modmRNA-LNP, 0.32 mg kg-1, after 4 h, and screen for inflammatory markers, such as pro-inflammatory cytokines. ModmRNA-LNP at this dose caused no significant elevation of cytokine levels in naive mice. In contrast, shortly after LPS immune stimulation, modmRNA-LNP enhanced inflammatory cytokine responses, Interleukin-6 (IL-6) in serum and Macrophage Inflammatory Protein 2 (MIP-2) in liver significantly. Our report identifies this phenomenon as inflammation exacerbation (IE), which was proven to be specific to the LNP, acting independent of mRNA cargo, and was demonstrated to be time- and dose-dependent. Macrophage depletion as well as TLR3 -/- and TLR4-/- knockout mouse studies revealed macrophages were the immune cells involved or responsible for IE. Finally, we show that pretreatment with anti-inflammatory drugs, such as corticosteroids, can partially alleviate IE response in mice. Our findings characterize the importance of LNP-mediated IE phenomena in gram negative bacterial inflammation, however, the generalizability of modmRNA-LNP in other forms of chronic or acute inflammatory and immune contexts needs to be addressed.

Supramolecular arrangement of protein in nanoparticle structures predicts nanoparticle tropism for neutrophils in acute lung inflammation.

This study shows that the supramolecular arrangement of proteins in nanoparticle structures predicts nanoparticle accumulation in neutrophils in acute lung inflammation (ALI). We observed homing to inflamed lungs for a variety of nanoparticles with agglutinated protein (NAPs), defined by arrangement of protein in or on the nanoparticles via hydrophobic interactions, crosslinking and electrostatic interactions. Nanoparticles with symmetric protein arrangement (for example, viral capsids) had no selectivity for inflamed lungs. Flow cytometry and immunohistochemistry showed NAPs have tropism for pulmonary neutrophils. Protein-conjugated liposomes were engineered to recapitulate NAP tropism for pulmonary neutrophils. NAP uptake in neutrophils was shown to depend on complement opsonization. We demonstrate diagnostic imaging of ALI with NAPs; show NAP tropism for inflamed human donor lungs; and show that NAPs can remediate pulmonary oedema in ALI. This work demonstrates that structure-dependent tropism for neutrophils drives NAPs to inflamed lungs and shows NAPs can detect and treat ALI.

Red blood cells: The metamorphosis of a neglected carrier into the natural mothership for artificial nanocarriers.

Drug delivery research pursues many types of carriers including proteins and other macromolecules, natural and synthetic polymeric structures, nanocarriers of diverse compositions and cells. In particular, liposomes and lipid nanoparticles represent arguably the most advanced and popular human-made nanocarriers, already in multiple clinical applications. On the other hand, red blood cells (RBCs) represent attractive natural carriers for the vascular route, featuring at least two distinct compartments for loading pharmacological cargoes, namely inner space enclosed by the plasma membrane and the outer surface of this membrane. Historically, studies of liposomal drug delivery systems (DDS) astronomically outnumbered and surpassed the RBC-based DDS. Nevertheless, these two types of carriers have different profile of advantages and disadvantages. Recent studies showed that RBC-based drug carriers indeed may feature unique pharmacokinetic and biodistribution characteristics favorably changing benefit/risk ratio of some cargo agents. Furthermore, RBC carriage cardinally alters behavior and effect of nanocarriers in the bloodstream, so called RBC hitchhiking (RBC-HH). This article represents an attempt for the comparative analysis of liposomal vs RBC drug delivery, culminating with design of hybrid DDSs enabling mutual collaborative advantages such as RBC-HH and camouflaging nanoparticles by RBC membrane. Finally, we discuss the key current challenges faced by these and other RBC-based DDSs including the issue of potential unintended and adverse effect and contingency measures to ameliorate this and other concerns.

Combining vascular targeting and the local first pass provides 100-fold higher uptake of ICAM-1-targeted vs untargeted nanocarriers in the inflamed brain.

New advances in intra-arterial (IA) catheters offer clinically proven local interventions in the brain. Here we tested the effect of combining local IA delivery and vascular immunotargeting. Microinjection of tumor necrosis factor alpha (TNFα) in the brain parenchyma causes cerebral overexpression of Inter-Cellular Adhesion Molecule-1 (ICAM-1) in mice. Systemic intravenous injection of ICAM-1 antibody (anti-ICAM-1) and anti-ICAM-1/liposomes provided nearly an order of magnitude higher uptake in the inflamed vs normal brain (from ~0.1 to 0.8%ID/g for liposomes). Local injection of anti-ICAM-1 and anti-ICAM-1/liposomes via carotid artery catheter provided an additional respective 2-fold and 5-fold elevation of uptake in the inflamed brain vs levels attained by IV injection. The uptake in the inflamed brain of respective untargeted IgG counterparts was markedly lower (e.g., uptake of anti-ICAM-1/liposomes was 100-fold higher vs IgG/liposomes). These data affirm the specificity of the combined effect of the first pass and immunotargeting. Intravital real-time microscopy via cranial window revealed that anti-ICAM-1/liposomes, but not IgG/liposomes bind to the lumen of blood vessels in the inflamed brain within minutes after injection. This straightforward framework provides the basis for translational efforts towards local vascular drug targeting to the brain.

Nanoparticle Properties Modulate Their Attachment and Effect on Carrier Red Blood Cells.

Attachment of nanoparticles (NPs) to the surface of carrier red blood cells (RBCs) profoundly alters their interactions with the host organism, decelerating NP clearance from the bloodstream while enabling NP transfer from the RBC surface to the vascular cells. These changes in pharmacokinetics of NPs imposed by carrier RBCs are favorable for many drug delivery purposes. On the other hand, understanding effects of NPs on the carrier RBCs is vital for successful translation of this novel drug delivery paradigm. Here, using two types of distinct nanoparticles (polystyrene (PSNP) and lysozyme-dextran nanogels (LDNG)) we assessed potential adverse and sensitizing effects of surface adsorption of NPs on mouse and human RBCs. At similar NP loadings (approx. 50 particles per RBC), adsorption of PSNPs, but not LDNGs, induces RBCs agglutination and sensitizes RBCs to damage by osmotic, mechanical and oxidative stress. PSNPs, but not LDNGs, increase RBC stiffening and surface exposure of phosphatidylserine, both known to accelerate RBC clearance in vivo. Therefore, NP properties and loading amounts have a profound impact on RBCs. Furthermore, LDNGs appear conducive to nanoparticle drug delivery using carrier RBCs.

Nanomedicine for the Treatment of Acute Respiratory Distress Syndrome. The 2016 ATS Bear Cage Award-winning Proposal.

After dozens of clinical trials, there are still no Food and Drug Administration-approved drugs that improve mortality in acute respiratory distress syndrome (ARDS). These poor results may be caused in part by three unique pharmacological challenges presented by ARDS: (1) Patients with ARDS are fragile because of concomitant multiple organ dysfunction, so they do not tolerate off-target side effects of drugs; (2) inhaled drug delivery is impeded by the column of proteinaceous fluid covering the injured alveoli; and (3) ARDS is heterogeneous in its underlying pathophysiology, so targeting one pathway is unlikely to improve most patients. To address these three pharmacological problems, I present the development of pulmonary endothelium-targeted liposomes (PELs). PELs are approximately 100-nm drug carriers coated with antibodies that bind to the pulmonary capillary endothelium. In model organisms, intravenously injected PELs strongly concentrate drugs in alveoli, even in animal models displaying severe, spatially heterogeneous pathology similar to severe ARDS. By concentrating drugs in inflamed alveoli, PELs solve pharmacological challenge (1) above. By being obligate intravenous medications, they solve challenge (2). Finally, because PELs can be loaded with at least three drugs, they can solve challenge (3) with combination drug therapy. My colleagues and I are currently testing PELs loaded with numerous candidate drugs in mouse models of ARDS, and we are testing drug distribution in live pigs and ex vivo human lungs. We aim to use such preclinical validation to move PELs into a partnership with industry, and then to patients.