An engineered bacterial therapeutic lowers urinary oxalate in preclinical models and in silico simulations of enteric hyperoxaluria.

Enteric hyperoxaluria (EH) is a metabolic disease caused by excessive absorption of dietary oxalate leading to the formation of chronic kidney stones and kidney failure. There are no approved pharmaceutical treatments for EH. SYNB8802 is an engineered bacterial therapeutic designed to consume oxalate in the gut and lower urinary oxalate as a potential treatment for EH. Oral administration of SYNB8802 leads to significantly decreased urinary oxalate excretion in healthy mice and non-human primates, demonstrating the strain's ability to consume oxalate in vivo. A mathematical modeling framework was constructed that combines in vitro and in vivo preclinical data to predict the effects of SYNB8802 administration on urinary oxalate excretion in humans. Simulations of SYNB8802 administration predict a clinically meaningful lowering of urinary oxalate excretion in healthy volunteers and EH patients. Together, these findings suggest that SYNB8802 is a promising treatment for EH.

Anisotropic poly(lactic-co-glycolic acid) microparticles enable sustained release of a peptide for long-term inhibition of ocular neovascularization.

Leading causes of vision loss include neovascular age-related macular degeneration (NVAMD) and macular edema (ME), which both require frequent intravitreal injections for treatment. A safe, poly(lactic-co-glycolic acid) (PLGA)-based biodegradable polymeric microparticle (MP) delivery system was developed that encapsulates and protects a biomimetic peptide from degradation, allows sustained intraocular release through polymer hydrolysis, and demonstrates a prolonged anti-angiogenic effect in vivo in three different NVAMD animal models (a laser-induced choroidal neovascularization mouse model, a rhoVEGF transgenic mouse model, and a Tet/opsin/VEGF transgenic mouse model) following intravitreal administration. The role of copolymer composition and microparticle shape was explored and 85:15 lactide-to-glycolide PLGA formed into ellipsoidal microparticles was found to be effective at inhibiting neovascularization for at least 16 weeks in vivo. Treatments were found to not only inhibit the growth of neovascularization, but also to cause regression of the neovasculature, reduce vascular leakage, and prevent exudative retinal detachment. These particulate devices are promising for the sustained release of biologics in the eye and may be useful for treating retinal diseases. STATEMENT OF SIGNIFICANCE: Devastating retinal diseases cause blindness in millions of people around the world. Current protein-based treatments have insufficient efficacy for many patients and also necessitate frequent intravitreal injections. Here, we demonstrate a new treatment consisting of a peptide encapsulated in biodegradable microparticles. We explore the effects of copolymer composition and physical shape of polymeric microparticles and find that both modulate peptide release. Efficacy of the treatment was validated in three different mouse models and the lead formulation was determined to be effective long-term, for at least 16 weeks in vivo, following a single injection. Treatments inhibited and regressed neovascularization as well as reduced vascular leakage. Anisotropic polymeric microparticles are promising for the sustained release of biologics in the eye.

Anisotropic biodegradable lipid coated particles for spatially dynamic protein presentation.

There has been growing interest in the use of particles coated with lipids for applications ranging from drug delivery, gene delivery, and diagnostic imaging to immunoengineering. To date, almost all particles with lipid coatings have been spherical despite emerging evidence that non-spherical shapes can provide important advantages including reduced non-specific elimination and increased target-specific binding. We combine control of core particle geometry with control of particle surface functionality by developing anisotropic, biodegradable ellipsoidal particles with lipid coatings. We demonstrate that these lipid coated ellipsoidal particles maintain advantageous properties of lipid polymer hybrid particles, such as the ability for modular protein conjugation to the particle surface using versatile bioorthogonal ligation reactions. In addition, they exhibit biomimetic membrane fluidity and demonstrate lateral diffusive properties characteristic of natural membrane proteins. These ellipsoidal particles simultaneously provide benefits of non-spherical particles in terms of stability and resistance to non-specific phagocytosis by macrophages as well as enhanced targeted binding. These biomaterials provide a novel and flexible platform for numerous biomedical applications. STATEMENT OF SIGNIFICANCE: The research reported here documents the ability of non-spherical polymeric particles to be coated with lipids to form anisotropic biomimetic particles. In addition, we demonstrate that these lipid-coated biodegradable polymeric particles can be conjugated to a wide variety of biological molecules in a "click-like" fashion. This is of interest due to the multiple types of cellular mimicry enabled by this biomaterial based technology. These features include mimicry of the highly anisotropic shape exhibited by cells, surface presentation of membrane bound protein mimetics, and lateral diffusivity of membrane bound substrates comparable to that of a plasma membrane. This platform is demonstrated to facilitate targeted cell binding while being resistant to non-specific cellular uptake. Such a platform could allow for investigations into how physical parameters of a particle and its surface affect the interface between biomaterials and cells, as well as provide biomimetic technology platforms for drug delivery and cellular engineering.

Injectable drug depot engineered to release multiple ophthalmic therapeutic agents with precise time profiles for postoperative treatment following ocular surgery.

A multi-drug delivery platform is developed to address current shortcomings of post-operative ocular drug delivery. The sustained biodegradable drug release system is composed of biodegradable polymeric microparticles (MPs) incorporated into a bulk biodegradable hydrogel made from triblock copolymers with poly(ethylene glycol) (PEG) center blocks and hydrophobic biodegradable polyester blocks such as poly(lactide-co-glycolide) (PLGA), Poly(lactic acid) (PLA), or Poly(lactide-co-caprolactone) (PLCL) blocks. This system is engineered to flow as a liquid solution at room temperature for facile injection into the eye and then quickly gel as it warms to physiological body temperatures (approximately 37 °C). The hydrogel acts as an ocular depot that can release three different drug molecules at programmed rates and times to provide optimal release of each species. In this manuscript, the hydrogel is configured to release a broad-spectrum antibiotic, a potent corticosteroid, and an ocular hypotensive, three ophthalmic therapeutic agents that are essential for post-operative management after ocular surgery, each drug released at its own timescale. The delivery platform is designed to mimic current topical application of postoperative ocular formulations, releasing the antibiotic for up to a week, and the corticosteroid and the ocular hypotensive agents for at least a month. Hydrophobic blocks, such as PLCL, were utilized to prolong the release duration of the biomolecules. This system also enables customization by being able to vary the initial drug loading to linearly tune the drug dose released, while maintaining a constant drug release profile over time. This minimally invasive biodegradable multi-drug delivery system is capable of replacing a complex ocular treatment regimen with a simple injection. Such a depot system has the potential to increase patient medication compliance and reduce both the immediate and late term complications following ophthalmic surgery. STATEMENT OF SIGNIFICANCE: After ocular surgery, patients routinely receive multiple medications including antibiotics, steroids and ocular hypotensives to ensure optimal surgical outcomes. The current standard of care for postoperative treatment after ocular surgery involves using eye drops daily, which has limited effectiveness mainly due to poor patient adherence. To improve patient experience and outcomes, this article presents the first thermoresponsive hydrogel able to release multiple drug molecules for the application of post-operative treatment following ocular surgery. By varying the parameters such as hydrogel type and polymer hydrophobicity, the drug release profile, duration and dosage can finely be tuned. The approach presented in this article can readily be applied to other applications by simply changing the drug loaded in the drug delivery system.

Biomimetic peptide display from a polymeric nanoparticle surface for targeting and antitumor activity to human triple-negative breast cancer cells.

While poly(lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) can encapsulate drug cargos and prolong circulation times, they show nonspecific accumulation in off-target tissues. Targeted delivery of drugs to tumor tissue and tumor vasculature is a promising approach for treating solid tumors while enhancing specificity and reducing systemic toxicity. AXT050, a collagen-IV derived peptide with both antitumor and antiangiogenic properties, is shown to bind to tumor-associated integrins with high affinity, which leads to targeted accumulation in tumor tissue. AXT050 conjugated to PLGA-PEG NPs at precisely controlled surface density functions both as a targeting agent to human tumor cells and demonstrates potential for simultaneous antitumorigenic and antiangiogenic activity. These targeted NPs cause inhibition of adhesion and proliferation in vitro when added to human triple-negative breast cancer cells and microvascular endothelial cells through binding to integrin αV ß3 . Furthermore, we find an in vivo biphasic relationship between tumor targeting and surface coating density of NPs coated with AXT050. NPs with an intermediate level of 10% peptide surface coating show approximately twofold greater accumulation in tumors and lower accumulation in the liver compared to nontargeted PLGA-PEG NPs in a murine biodistribution model. Display of biomimetic peptides from NP surfaces to both target and inhibit cancer cells has the potential to enhance the activity of cancer nanomedicines. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1753-1764, 2018.

Tyrosine kinase blocking collagen IV-derived peptide suppresses ocular neovascularization and vascular leakage.

Vascular endothelial growth factor (VEGF)-neutralizing proteins provide benefit in several retinal and choroidal vascular diseases, but some patients still experience suboptimal outcomes, and the need for frequent intraocular injections is a barrier to good outcomes. A mimetic peptide derived from collagen IV, AXT107, suppressed subretinal neovascularization (NV) in two mouse models predictive of effects in neovascular age-related macular degeneration (NVAMD) and inhibited retinal NV in a model predictive of effects in ischemic retinopathies. A combination of AXT107 and the current treatment aflibercept suppressed subretinal NV better than either agent alone. Furthermore, AXT107 caused regression of choroidal NV. AXT107 reduced the VEGF-induced vascular leakage that underlies macular edema in ischemic retinopathies and NVAMD. In rabbit eyes, which are closer to the size of human eyes, intraocular injection of AXT107 significantly reduced VEGF-induced vascular leakage by 86% at 1 month and 70% at 2 months; aflibercept significantly reduced leakage by 69% at 1 month and did not reduce leakage at 2 months, demonstrating the longer effectiveness of AXT107. AXT107 reduced ligand-induced phosphorylation of multiple receptors: VEGFR2, c-Met, and PDGFRß. Optimal signaling through these receptors requires complex formation with ß3 integrin, which was reduced by AXT107 binding to αvß3 AXT107 also reduced total VEGFR2 levels by increasing internalization, ubiquitination, and degradation. This biomimetic peptide is a sustained, multitargeted therapy that may provide advantages over intraocular injections of specific VEGF-neutralizing proteins.

Long-term suppression of ocular neovascularization by intraocular injection of biodegradable polymeric particles containing a serpin-derived peptide.

Aberrant angiogenesis can cause or contribute to a number of diseases such as neovascular age-related macular degeneration (NVAMD). While current NVAMD treatments target angiogenesis, these treatments are not effective for all patients and also require frequent intravitreal injections. New agents and delivery systems to treat NVAMD could be beneficial to many patients. We have recently developed a serpin-derived peptide as an anti-angiogenic agent. Here, this peptide is investigated for activity in human retinal endothelial cells in vitro and for reducing angiogenesis in a laser-induced choroidal neovascularization mouse model of NVAMD in vivo. While frequent intravitreal injections can be tolerated clinically, reducing the number of injections can improve patient compliance, safety, and outcomes. To achieve this goal, and to maximize the in vivo activity of injected peptide, we have developed biodegradable polymers and controlled release particle formulations to extend anti-angiogenic therapy. To create these devices, the anionic peptides are first self-assembled into nanoparticles using a biodegradable cationic polymer and then as a second step, these nanoparticles are encapsulated into biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles. In situ, these particles show approximately zero-order, linear release of the anionic peptide over 200 days. These particles are made of safe, hydrolytically degradable polymers and have low endotoxin. Long-term in vivo experiments in the laser-induced neovascularization model for NVAMD show that these peptide-releasing particles decrease angiogenesis for at least fourteen weeks in vivo following a single particle dose and therefore are a promising treatment strategy for NVAMD.

Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

Poly(ß-amino ester) nanoparticle delivery of TP53 has activity against small cell lung cancer in vitro and in vivo.

Small cell lung cancer (SCLC) is an aggressive disease with one of the highest case-fatality rates among cancer. The recommended therapy for SCLCs has not changed significantly over the past 30 years; new therapeutic approaches are a critical need. TP53 is mutated in the majority of SCLC cases and its loss is required in transgenic mouse models of the disease. We synthesized an array of biodegradable poly(ß-amino ester) (PBAE) polymers that self-assemble with DNA and assayed for transfection efficiency in the p53-mutant H446 SCLC cell line using high-throughput methodologies. Two of the top candidates were selected for further characterization and TP53 delivery in vitro and in vivo. Nanoparticle delivery of TP53 resulted in expression of exogenous p53, induction of p21, induction of apoptosis, and accumulation of cells in sub-G1 consistent with functional p53 activity. Intratumoral injection of subcutaneous H446 xenografts with polymers carrying TP53 caused marked tumor growth inhibition. This is the first demonstration of TP53 gene therapy in SCLC using nonviral polymeric nanoparticles. This technology may have general applicability as a novel anticancer strategy based on restoration of tumor suppressor gene function.

Gene delivery nanoparticles specific for human microvasculature and macrovasculature.

Endothelial cell dysfunction is a critical component of ocular diseases such as age-related macular degeneration and diabetic retinopathy. An important limitation in endothelial cell research is the difficulty in achieving efficient transfection of these cells. A new polymer library was here synthesized and utilized to find polymeric nanoparticles that can transfect macrovascular (human umbilical vein, HUVECs) and microvascular (human retinal, HRECs) endothelial cells. Nanoparticles were synthesized that can achieve transfection efficiency of up to 85% for HRECs and 65% for HUVECs. These nanoparticle systems enable high levels of expression while avoiding problems associated with viral gene delivery. The polymeric nanoparticles also show cell-specific behavior, with a high correlation between microvascular and macrovascular transfection (R(2) = 0.81) but low correlation between retinal endothelial and retinal epithelial transfection (R(2) = 0.21). These polymeric nanoparticles can be used in vitro as experimental tools and potentially in vivo to target and treat vascular-specific diseases. FROM THE CLINICAL EDITOR: Polymeric nanoparticles were synthesized with the goal of transfecting endothelial cells, which are commonly considered difficult targets. The authors report excellent transfection efficiency of up to 85% for human retinal and 65% for human umbilical vein endothelial cells. These NPs can be used in vitro as experimental tools and potentially in vivo to target and treat vascular-specific diseases.

Drug delivery strategies for therapeutic angiogenesis and antiangiogenesis.

INTRODUCTION: Angiogenesis is essential to human biology and of great clinical significance. Excessive or reduced angiogenesis can result in, or exacerbate, several disease states, including tumor formation, exudative age-related macular degeneration (AMD) and ischemia. Innovative drug delivery systems can increase the effectiveness of therapies used to treat angiogenesis-related diseases. AREAS COVERED: This paper reviews the basic biology of angiogenesis, including current knowledge about its disruption in diseases, with the focus on cancer and AMD. Anti- and proangiogenic drugs available for clinical use or in development are also discussed, as well as experimental drug delivery systems that can potentially improve these therapies to enhance or reduce angiogenesis in a more controlled manner. EXPERT OPINION: Laboratory and clinical results have shown pro- or antiangiogenic drug delivery strategies to be effective in drastically slowing disease progression. Further research in this area will increase the efficacy, specificity and duration of these therapies. Future directions with composite drug delivery systems may make possible targeting of multiple factors for synergistic effects.

Electrostatic surface modifications to improve gene delivery.

IMPORTANCE OF THE FIELD: Gene therapy has the potential to treat a wide variety of diseases, including genetic diseases and cancer. AREAS COVERED IN THIS REVIEW: This review introduces biomaterials used for gene delivery and then focuses on the use of electrostatic surface modifications to improve gene delivery materials. These modifications have been used to stabilize therapeutics in vivo, add cell-specific targeting ligands, and promote controlled release. Coatings of nanoparticles and microparticles as well as non-particulate surface coatings are covered in this review. Electrostatic principles are crucial for the development of multilayer delivery structures fabricated by the layer-by-layer method. WHAT THE READER WILL GAIN: The reader will gain knowledge about the composition of biomaterials used for surface modifications and how these coatings and multilayers can be utilized to improve spatial control and efficiency of delivery. Examples are shown for the delivery of nucleic acids, including DNA and siRNA, to in vitro and in vivo systems. TAKE HOME MESSAGE: The versatile and powerful approach of electrostatic coatings and multilayers will lead to the development of enhanced gene therapies.