Brian C. Evans

Scaling and systems biology for integrating multiple organs-on-a-chip.

Coupled systems of in vitro microfabricated organs-on-a-chip containing small populations of human cells are being developed to address the formidable pharmacological and physiological gaps between monolayer cell cultures, animal models, and humans that severely limit the speed and efficiency of drug development. These gaps present challenges not only in tissue and microfluidic engineering, but also in systems biology: how does one model, test, and learn about the communication and control of biological systems with individual organs-on-chips that are one-thousandth or one-millionth of the size of adult organs, or even smaller, i.e., organs for a milliHuman (mHu) or microHuman (μHu)? Allometric scaling that describes inter-species variation of organ size and properties provides some guidance, but given the desire to utilize these systems to extend and validate human pharmacokinetic and pharmacodynamic (PK/PD) models in support of drug discovery and development, it is more appropriate to scale each organ functionally to ensure that it makes the suitable physiological contribution to the coupled system. The desire to recapitulate the complex organ-organ interactions that result from factors in the blood and lymph places a severe constraint on the total circulating fluid (~5 mL for a mHu and ~5 μL for a μHu) and hence on the pumps, valves, and analytical instruments required to maintain and study these systems. Scaling arguments also provide guidance on the design of a universal cell-culture medium, typically without red blood cells. This review presents several examples of scaling arguments and discusses steps that should ensure the success of this endeavour.

Ex vivo red blood cell hemolysis assay for the evaluation of pH-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs.

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

MK2 inhibitory peptide delivered in nanopolyplexes prevents vascular graft intimal hyperplasia

Nanopolyplexes formulated from a pH-responsive, endosomolytic polymer with a peptide inhibitor of MAPKAP kinase 2 block inflammatory and migratory signaling in vascular smooth muscle cells and prevent intimal hyperplasia in human saphenous vein grafts. Nano keeps MK2 inhibitor intact, on-target A peptide, currently in clinical trials, that can penetrate cells and block the activity of MAPKAP kinase 2 (MK2) may be able to stop inflammation and fibrosis after vein grafting, but it has low bioavailability and is degraded easily once inside the cell. To more effectively translate this inhibitory peptide, called MK2i, Evans et al. formulated it in electrostatically complexed nanoparticles—nanopolyplexes—for delivery to vascular cells and tissues. The MK2i nanopolyplexes were taken up readily by vascular smooth muscle cells and endothelial cells in human saphenous veins and significantly inhibited neointima formation ex vivo. In rabbit vein grafts, treatment with the MK2 nanopolyplexes prevented intimal hyperplasia for 1 month after transplant; by contrast, free MK2i peptide had no effect. Thus, complexing the MK2 inhibitor peptide with an endosomolytic polymer could improve long-term graft patency. Both treatments were able to block macrophage recruitment and/or signaling in vivo, possibly leading to less inflammation. In human saphenous veins, the MK2i nanopolyplexes similarly reduced proinflammatory cytokines and were also shown to reduce vascular smooth muscle cell migration. Such new insights into the effects of MK2i on intimal hyperplasia could open doors to new therapeutic options in this multifactorial disease. Furthermore, this nanoencapsulation approach could be broadly applied to other therapeutic cell-penetrating peptides to prolong bioavailability and enhance stability in vivo. Autologous vein grafts are commonly used for coronary and peripheral artery bypass but have a high incidence of intimal hyperplasia (IH) and failure. We present a nanopolyplex (NP) approach that efficiently delivers a mitogen-activated protein kinase (MAPK)–activated protein (MAPKAP) kinase 2 inhibitory peptide (MK2i) to graft tissue to improve long-term patency by inhibiting pathways that initiate IH. In vitro testing in human vascular smooth muscle cells revealed that formulation into MK2i-NPs increased cell internalization, endosomal escape, and intracellular half-life of MK2i. This efficient delivery mechanism enabled MK2i-NPs to sustain potent inhibition of inflammatory cytokine production and migration in vascular cells. In intact human saphenous vein, MK2i-NPs blocked inflammatory and migratory signaling, as confirmed by reduced phosphorylation of the posttranscriptional gene regulator heterogeneous nuclear ribonucleoprotein A0, the transcription factor cAMP (adenosine 3′,5′-monophosphate) element–binding protein, and the chaperone heat shock protein 27. The molecular effects of MK2i-NPs caused functional inhibition of IH in human saphenous vein cultured ex vivo. In a rabbit vein transplant model, a 30-min intraoperative graft treatment with MK2i-NPs significantly reduced in vivo IH 28 days posttransplant compared with untreated or free MK2i–treated grafts. The decrease in IH in MK2i-NP–treated grafts in the rabbit model also corresponded with decreased cellular proliferation and maintenance of the vascular wall smooth muscle cells in a more contractile phenotype. These data indicate that nanoformulated MK2 inhibitors are a promising strategy for preventing graft failure.

Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction

A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.

Hydrolytic Charge‐reversal of PEGylated Polyplexes Enhances Intracellular Un‐packaging and Activity of siRNA

Hydrolytically degrading nano-polyplexes (HDG-NPs) that reverse charge through conversion of tertiary amines to carboxylic acids were investigated to improve intracellular un-packaging of siRNA and target gene silencing compared to a non-degradable analog (non-HDG-NPs). Both NP types comprised reversible addition-fragmentation chain-transfer (RAFT) synthesized diblock copolymers of a poly(ethylene glycol) (PEG) corona-forming block and a cationic block for nucleic acid packaging that incorporated butyl methacrylate (BMA) and either dimethylaminoethyl methacrylate (DMAEMA, non-HDG-NPs) or dimethylaminoethyl acrylate (DMAEA, HDG-NPs). HDG-NPs decreased significantly in size and released significantly more siRNA (∼40%) than non-HDG-NPs after 24 h in aqueous solution. While both HDG-NPs and non-HDG-NPs had comparable uptake and cytotoxicity up to 150 nM siRNA doses, HDG-NPs achieved significantly higher target gene silencing of the model gene luciferase in vitro. High resolution FRET confocal microscopy was used to monitor the intracellular un-packaging of siRNA. Non-HDG-NPs had significantly higher FRET efficiency than HDG-NPs, indicating that siRNA delivered from HDG-NPs was more fully un-packaged and therefore had improved intracellular bioavailability.

Nanotechnology Enabled Modulation of Signaling Pathways Affects Physiology in Intact Vascular Tissue

IMPACT STATEMENT Subarachnoid hemorrhage (SAH) is associated with vasospasm that is refractory to traditional vasodilators, and inhibition of vasospasm after SAH remains a large unmet clinical need. SAH causes changes in the phosphorylation state of the small heat shock proteins (HSPs), HSP20 and HSP27, in the vasospastic vessels. In this study, the levels of HSP27 and HSP20 were manipulated using nanotechnology to mimic the intracellular phenotype of SAH-induced vasospasm, and the effect of this manipulation was tested on vasomotor responses in intact tissues. This work provides insight into potential therapeutic targets for the development of more effective treatments for SAH induced vasospasm.

Excipients for the lyoprotection of MAPKAP kinase 2 inhibitory peptide nano-polyplexes

ABSTRACT Herein, excipients are investigated to ameliorate the deleterious effects of lyophilization on peptide‐polymer nano‐polyplex (NP) morphology, cellular uptake, and bioactivity. The NPs are a previously‐described platform technology for intracellular peptide delivery and are formulated from a cationic therapeutic peptide and the anionic, pH‐responsive, endosomolytic polymer poly(propylacrylic acid) (PPAA). These NPs are effective when formulated and immediately used for delivery into cells and tissue, but they are not amenable to reconstitution following storage as a lyophilized powder due to aggregation. To develop a lyophilized NP format that facilitates longer‐term storage and ease of use, MAPKAP kinase 2 inhibitory peptide‐based NPs (MK2i‐NPs) were prepared in the presence of a range of concentrations of the excipients sucrose, trehalose, and lactosucrose prior to lyophilization and storage. All excipients improved particle morphology post‐lyophilization and significantly improved MK2i‐NP uptake in human coronary artery smooth muscle cells relative to lyophilized NPs without excipient. In particular, MK2i‐NPs lyophilized with 300 mM lactosucrose as an excipient demonstrated a 5.23 fold increase in cellular uptake (p < 0.001), a 2.52 fold increase in endosomal disruption (p < 0.05), and a 2.39 fold increase in ex vivo bioactivity (p < 0.01) compared to MK2i‐NPs lyophilized without excipients. In sum, these data suggest that addition of excipients, particularly lactosucrose, maintains and even improves the uptake and therapeutic efficacy of peptide‐polymer NPs post‐lyophilization relative to freshly‐made formulations. Thus, the use of excipients as lyoprotectants is a promising approach for the long‐term storage of biotherapeutic NPs and poises this NP platform for clinical translation. Graphical abstract Figure. No Caption available. HighlightsNano‐Polyplexes (NPs) aggregate and lose bioactivity post‐lyophilization.Excipients preserve NP size and morphology post‐lyophilization.NPs lyophilized with lactosucrose have higher cellular uptake than even fresh NPs.NPs lyophilized with lactosucrose retain effective endosomal disruption ability.NPs lyophilized with lactosucrose have potent bioactivity in intact human vein.

Formulation and characterization of poly(propylacrylic acid)/poly(lactic-co-glycolic acid) blend microparticles for pH-dependent membrane disruption and cytosolic delivery

Poly(lactic-co-glycolic acid) (PLGA) is widely used as a vehicle for delivery of pharmaceutically relevant payloads. PLGA is readily fabricated as a nano- or microparticle (MP) matrix to load both hydrophobic and hydrophilic small molecular drugs as well as biomacromolecules such as nucleic acids and proteins. However, targeting such payloads to the cell cytosol is often limited by MP entrapment and degradation within acidic endolysosomes. Poly(propylacrylic acid) (PPAA) is a polyelectrolyte polymer with the membrane disruptive capability triggered at low pH. PPAA has been previously formulated in various carrier configurations to enable cytosolic payload delivery, but requires sophisticated carrier design. Taking advantage of PPAA functionality, we have incorporated PPAA into PLGA MPs as a simple polymer mixture to enhance cytosolic delivery of PLGA-encapsulated payloads. Rhodamine loaded PLGA and PPAA/PLGA blend MPs were prepared by a modified nanoprecipitation method. Incorporation of PPAA into PLGA MPs had little to no effect on the size, shape, or loading efficiency, and evidenced no toxicity in Chinese hamster ovary epithelial cells. Notably, incorporation of PPAA into PLGA MPs enabled pH-dependent membrane disruption in a hemolysis assay, and a three-fold increased endosomal escape and cytosolic delivery in dendritic cells after 2 h of MP uptake. These results demonstrate that a simple PLGA/PPAA polymer blend is readily fabricated into composite MPs, enabling cytosolic delivery of an encapsulated payload.

Unregulated saphenous vein graft distension decreases tissue viscoelasticity

Objectives: Unregulated intraoperative distension of human saphenous vein (SV) graft leads to supraphysiologic luminal pressures and causes acute physiologic and cellular injury to the conduit. The effect of distension on tissue viscoelasticity, a biophysical property critical to a successful graft, is not well described. In this investigation, we quantify the loss of viscoelasticity in SV deformed by distension and compare the results to tissue distended in a pressure-controlled fashion. Materials and Methods: Unmanipulated porcine SV was used as a control or distended without regulation and distended with an in-line pressure release valve (PRV). Rings were cut from these tissues and suspended on a muscle bath. Force versus time tracings of tissue constricted with KCl (110 mM) and relaxed with sodium nitroprusside (SNP) were fit to the Hill model of viscoelasticity, using mean absolute error (MAE) and r2-goodness of fit as measures of conformity. Results: One-way ANOVA analysis demonstrated that, in tissue distended manually, the MAE was significantly greater and the r2-goodness of fit was significantly lower than both undistended tissues and tissues distended with a PRV (p<0.05) in KCl-induced vasoconstriction and SNP-induced vasodilation. Conclusions: Unregulated manual distension of SV graft causes loss of viscoelasticity and such loss may be mitigated with the use of an in-line PRV.