Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles.

The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loading─>40% cargo by volume─and highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.

Therapeutic MK2 inhibition blocks pathological vascular smooth muscle cell phenotype switch.

Vascular procedures, such as stenting, angioplasty, and bypass grafting, often fail due to intimal hyperplasia (IH), wherein contractile vascular smooth muscle cells (VSMCs) dedifferentiate to synthetic VSMCs, which are highly proliferative, migratory, and fibrotic. Previous studies suggest MAPK-activated protein kinase 2 (MK2) inhibition may limit VSMC proliferation and IH, although the molecular mechanism underlying the observation remains unclear. We demonstrated here that MK2 inhibition blocked the molecular program of contractile to synthetic dedifferentiation and mitigated IH development. Molecular markers of the VSMC contractile phenotype were sustained over time in culture in rat primary VSMCs treated with potent, long-lasting MK2 inhibitory peptide nanopolyplexes (MK2i-NPs), a result supported in human saphenous vein specimens cultured ex vivo. RNA-Seq of MK2i-NP-treated primary human VSMCs revealed programmatic switching toward a contractile VSMC gene expression profile, increasing expression of antiinflammatory and contractile-associated genes while lowering expression of proinflammatory, promigratory, and synthetic phenotype-associated genes. Finally, these results were confirmed using an in vivo rabbit vein graft model where brief, intraoperative treatment with MK2i-NPs decreased IH and synthetic phenotype markers while preserving contractile proteins. These results support further development of MK2i-NPs as a therapy for blocking VSMC phenotype switch and IH associated with cardiovascular procedures.

Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides.

Rapid, facile, and noncovalent cell membrane modification with alkyl-grafted anionic polymers was sought as an approach to enhance intracellular delivery and bioactivity of cationic peptides. We synthesized a library of acrylic acid-based copolymers containing varying amounts of an amine-reactive pentafluorophenyl acrylate monomer followed by postpolymerization modification with a series of alkyl amines to afford precise control over the length and density of aliphatic alkyl side chains. This synthetic strategy enabled systematic investigation of the effect of the polymer structure on membrane binding, potentiation of peptide cell uptake, pH-dependent disruption of lipid bilayers for endosome escape, and intracellular bioavailability. A subset of these polymers exhibited pKa of ∼6.8, which facilitated stable membrane association at physiological pH and rapid, pH-dependent endosomal disruption upon endocytosis as quantified in Galectin-8-YFP reporter cells. Cationic cell penetrating peptide (CPP) uptake was enhanced up to 15-fold in vascular smooth muscle cells in vitro when peptide treatment was preceded by a 30-min pretreatment with lead candidate polymers. We also designed and implemented a new and highly sensitive assay for measuring the intracellular bioavailability of CPPs based on the NanoLuciferase (NanoLuc) technology previously developed for measuring intracellular protein-protein interactions. Using this split luciferase class of assay, polymer pretreatment enhanced intracellular delivery of the CPP-modified HiBiT peptide up to 30-fold relative to CPP-HiBiT without polymer pretreatment (p < 0.05). The overall structural analyses show that polymers containing 50:50 or 70:30 molar ratios of carboxyl groups to alkyl side chains of 6-8 carbons maximized peptide uptake, pH-dependent membrane disruption, and intracellular bioavailability and that this potentiation effect was maximized by pairing with CPPs with high cationic charge density. These results demonstrate a rapid, mild method for polymer modification of cell surfaces to potentiate intracellular delivery, endosome escape, and bioactivity of cationic peptides.

An anionic, endosome-escaping polymer to potentiate intracellular delivery of cationic peptides, biomacromolecules, and nanoparticles.

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.

Applied Bioengineering in Tissue Reconstruction, Replacement, and Regeneration.

IMPACT STATEMENT: The use of autologous tissue in the reconstruction of tissue defects has been the gold standard. However, current standards still face many limitations and complications. Improving patient outcomes and quality of life by addressing these barriers remain imperative. This article provides historical perspective, covers the major limitations of current standards of care, and reviews recent advances and future prospects in applied bioengineering in the context of tissue reconstruction, replacement, and regeneration.

Gal8 Visualization of Endosome Disruption Predicts Carrier-Mediated Biologic Drug Intracellular Bioavailability.

Endolysosome entrapment is one of the key barriers to the therapeutic use of biologic drugs that act intracellularly. The screening of prospective nanoscale endosome-disrupting delivery technologies is currently limited by methods that are indirect and cumbersome. Here, we statistically validate Galectin 8 (Gal8) intracellular tracking as a superior approach that is direct, quantitative, and predictive of therapeutic cargo intracellular bioactivity through in vitro high-throughput screening and in vivo validation. Gal8 is a cytosolically dispersed protein that, when endosomes are disrupted, redistributes by binding to glycosylation moieties selectively located on the inner face of endosomal membranes. The quantitative redistribution of a Gal8 fluorescent fusion protein from the cytosol into endosomes is demonstrated as a real-time, live-cell assessment of endosomal integrity that does not require labeling or modification of either the carrier or the biologic drug and that allows quantitative distinction between closely related, endosome-disruptive drug carriers. Through screening two families of siRNA polymeric carrier compositions at varying dosages, we show that Gal8 endosomal recruitment correlates strongly ( r = 0.95 and p < 10-4) with intracellular siRNA bioactivity. Through this screen, we gathered insights into how composition and molecular weight affect endosome disruption activity of poly[(ethylene glycol)- b-[(2-(dimethylamino)ethyl methacrylate)- co-(butyl methacrylate)]] [PEG-(DMAEMA- co-BMA)] siRNA delivery systems. Additional studies showed that Gal8 recruitment predicts intracellular bioactivity better than current standard methods such as Lysotracker colocalization ( r = 0.35, not significant), pH-dependent hemolysis (not significant), or cellular uptake ( r = 0.73 and p < 10-3). Importantly, the Gal8 recruitment method is also amenable to fully objective high-throughput screening using automated image acquisition and quantitative image analysis, with a robust estimated Z' of 0.6 (whereas assays with Z' > 0 have high-throughput screening utility). Finally, we also provide measurements of in vivo endosomal disruption based on Gal8 visualization ( p < 0.03) of a nanocarrier formulation confirmed to produce significant cytosolic delivery and bioactivity of siRNA within tumors ( p < 0.02). In sum, this report establishes the utility of Gal8 subcellular tracking for the rapid optimization and high-throughput screening of the endosome disruption potency of intracellular delivery technologies.

Nanotechnology Enabled Modulation of Signaling Pathways Affects Physiologic Responses 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.

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.

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. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1022-1033, 2018.

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.

Mechanism of Enhanced Cellular Uptake and Cytosolic Retention of MK2 Inhibitory Peptide Nano-polyplexes.

Electrostatic complexation of a cationic MAPKAP kinase 2 inhibitory (MK2i) peptide with the anionic, pH-responsive polymer poly(propylacrylic acid) (PPAA) yields MK2i nano-polyplexes (MK2i-NPs) that significantly increase peptide uptake and intracellular retention. This study focused on elucidating the mechanism of MK2i-NP cellular uptake and intracellular trafficking in vascular smooth muscle cells. Small molecule inhibition of various endocytic pathways showed that MK2i-NP cellular uptake involves both macropinocytosis and clathrin mediated endocytosis, whereas the free peptide exclusively utilizes clathrin mediated endocytosis for cell entry. Scanning electron microscopy studies revealed that MK2i-NPs, but not free MK2i peptide, induce cellular membrane ruffling consistent with macropinocytosis. TEM confirmed that MK2i-NPs induce macropinosome formation and achieve MK2i endo-lysosomal escape and cytosolic delivery. Finally, a novel technique based on recruitment of Galectin-8-YFP was utilized to demonstrate that MK2i-NPs cause endosomal disruption within 30 minutes of uptake. These new insights on the relationship between NP physicochemical properties and cellular uptake and trafficking can potentially be applied to further optimize the MK2i-NP system and more broadly toward the rational engineering of nano-scale constructs for the intracellular delivery of biologic drugs.

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.

MK2 inhibitory peptide delivered in nanopolyplexes prevents vascular graft intimal hyperplasia.

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.

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.

Delivery of intracellular-acting biologics in pro-apoptotic therapies.

The recent elucidation of molecular regulators of apoptosis and their roles in cellular oncogenesis has motivated the development of biomacromolecular anticancer therapeutics that can activate intracellular apoptotic signaling pathways. Pharmaceutical scientists have employed a variety of classes of biologics toward this goal, including antisense oligodeoxynucleotides, small interfering RNA, proteins, antibodies, and peptides. However, stability in the in vivo environment, tumor-specific biodistribution, cell internalization, and localization to the intracellular microenvironment where the targeted molecule is localized pose significant challenges that limit the ability to directly apply intracellular-acting, pro-apoptotic biologics for therapeutic use. Thus, approaches to improve the pharmaceutical properties of therapeutic biomacromolecules are of great significance and have included chemically modifying the bioactive molecule itself or formulation with auxiliary compounds. Recently, promising advances in delivery of pro-apoptotic biomacromolecular agents have been made using tools such as peptide "stapling", cell penetrating peptides, fusogenic peptides, liposomes, nanoparticles, smart polymers, and synergistic combinations of these components. This review will discuss the molecular mediators of cellular apoptosis, the respective mechanisms by which these mediators are dysregulated in cellular oncogenesis, the history and development of both nucleic-acid and amino-acid based drugs, and techniques to achieve intracellular delivery of these biologics. Finally, recent applications where pro-apoptotic functionality has been achieved through delivery of intracellular-acting biomacromolecular drugs will be highlighted.