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To unlock the full promise of messenger (mRNA) therapies, expanding the toolkit of lipid nanoparticles is paramount. However, a pivotal component of lipid nanoparticle development that remains a bottleneck is identifying new ionizable lipids. Here we describe an accelerated approach to discovering effective ionizable lipids for mRNA delivery that combines machine learning with advanced combinatorial chemistry tools. Starting from a simple four-component reaction platform, we create a chemically diverse library of 584 ionizable lipids. We screen the mRNA transfection potencies of lipid nanoparticles containing those lipids and use the data as a foundational dataset for training various machine learning models. We choose the best-performing model to probe an expansive virtual library of 40,000 lipids, synthesizing and experimentally evaluating the top 16 lipids flagged. We identify lipid 119-23, which outperforms established benchmark lipids in transfecting muscle and immune cells in several tissues. This approach facilitates the creation and evaluation of versatile ionizable lipid libraries, advancing the formulation of lipid nanoparticles for precise mRNA delivery., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Imidazoquinolines (IMDs), such as resiquimod (R848), are of great interest as potential cancer immunotherapies because of their ability to activate Toll-like receptor 7 (TLR7) and/or TLR8 on innate immune cells. Nevertheless, intravenous administration of IMDs causes severe immune-related toxicities, and attempts to improve their tissue-selective exposure while minimizing acute systemic inflammation have proven difficult. Here, using a library of R848 "bottlebrush prodrugs" (BPDs) that differ only by their R848 release kinetics, we explore how the timing of R848 exposure affects immune stimulation in vitro and in vivo. These studies led to the discovery of R848-BPDs that exhibit optimal activation kinetics to achieve potent stimulation of myeloid cells in tumors and substantial reductions in tumor growth following systemic administration in mouse syngeneic tumor models without any observable systemic toxicity. These results suggest that release kinetics can be tuned at the molecular level to provide safe yet effective systemically administered immunostimulant prodrugs for next-generation cancer immunotherapies.

Endothelial cells play critical roles in circulatory homeostasis and are also the gateway to the major organs of the body. Dysfunction, injury, and gene expression profiles of these cells can cause, or are caused by, prevalent chronic diseases such as diabetes, cardiovascular disease, and cancer. Modulation of gene expression within endothelial cells could therefore be therapeutically strategic in treating longstanding disease challenges. Lipid nanoparticles (LNP) have emerged as potent, scalable, and tunable carrier systems for delivering nucleic acids, making them attractive vehicles for gene delivery to endothelial cells. Here, we discuss the functions of endothelial cells and highlight some receptors that are upregulated during health and disease. Examples and applications of DNA, mRNA, circRNA, saRNA, siRNA, shRNA, miRNA, and ASO delivery to endothelial cells and their targets are reviewed, as well as LNP composition and morphology, formulation strategies, target proteins, and biomechanical factors that modulate endothelial cell targeting. Finally, we discuss FDA-approved LNPs as well as LNPs that have been tested in clinical trials and their challenges, and provide some perspectives as to how to surmount those challenges., (© 2023. The Author(s).)

Differential expression of microRNAs (miRNAs) plays a role in many diseases, including cancer and cardiovascular diseases. Potentially, miRNAs could be targeted with miRNA-therapeutics. Sustained delivery of these therapeutics remains challenging. This study couples miR-mimics to PEG-peptide gold nanoparticles (AuNP) and loads these AuNP-miRNAs in an injectable, shear thinning, self-assembling polymer nanoparticle (PNP) hydrogel drug delivery platform to improve delivery. Spherical AuNPs coated with fluorescently labelled miR-214 are loaded into an HPMC-PEG-b-PLA PNP hydrogel. Release of AuNP/miRNAs is quantified, AuNP-miR-214 functionality is shown in vitro in HEK293 cells, and AuNP-miRNAs are tracked in a 3D bioprinted human model of calcific aortic valve disease (CAVD). Lastly, biodistribution of PNP-AuNP-miR-67 is assessed after subcutaneous injection in C57BL/6 mice. AuNP-miRNA release from the PNP hydrogel in vitro demonstrates a linear pattern over 5 days up to 20%. AuNP-miR-214 transfection in HEK293 results in 33% decrease of Luciferase reporter activity. In the CAVD model, AuNP-miR-214 are tracked into the cytoplasm of human aortic valve interstitial cells. Lastly, 11 days after subcutaneous injection, AuNP-miR-67 predominantly clears via the liver and kidneys, and fluorescence levels are again comparable to control animals. Thus, the PNP-AuNP-miRNA drug delivery platform provides linear release of functional miRNAs in vitro and has potential for in vivo applications.

The advances in micro/nanofabrication techniques have enabled miniaturization of printed circuit boards (PCBs) for various applications such as portable devices, smart sensors, and IoTs, to name a few. PCBs provide electrical connectivity between the components as well as mechanical support. Down-scaling of PCBs is crucial for miniaturization of large systems and devices. Currently, microtraces down to 25 μm can be microfabricated with the current microfabrication processes at an industrial scale. In the present work, we report a new approach for microfabrication of PCBs with trace widths down to 3 μm on commercially available PCB substrates. We used electroplating/electroetching, sputtering, and photolithography to achieve these fine trace sizes. The proposed fabrication technique can be used in microelectronics, system on chip, MEMS, and miniaturized circuits and systems in general.

We demonstrate entrapment of the commensal skin bacteria Staphylococcus epidermidis in mats composed of soft nanotubes made by membrane-templated layer-by-layer (LbL) assembly. When cultured in broth, the resulting nanofibrillar patches efficiently delay the escape of bacteria and their planktonic growth, while displaying high steady-state metabolic activity. Additionally, the material properties and metabolic activity can be further tuned by postprocessing the patches with additional polysaccharide LbL layers. These patches offer a promising methodology for the fabrication of bacterial skin dressings for the treatment of skin dysbiosis while preventing adverse effects due to bacterial proliferation.

Commensal skin bacteria such as Staphylococcus epidermidis are currently being considered as possible components in skin-care and skin-health products. However, considering the potentially adverse effects of commensal skin bacteria if left free to proliferate, it is crucial to develop methodologies that are capable of maintaining bacteria viability while controlling their proliferation. Here, we encapsulate S. epidermidis in shells of increasing thickness using layer-by-layer assembly, with either a pair of synthetic polyelectrolytes or a pair of oppositely charged polysaccharides. We study the viability of the cells and their delay of growth depending on the composition of the shell, its thickness, the charge of the last deposited layer, and the degree of aggregation of the bacteria which is varied using different coating procedures-among which is a new scalable process that easily leads to large amounts of nonaggregated bacteria. We demonstrate that the growth of bacteria is not controlled by the mechanical properties of the shell but by the bacteriostatic effect of the polyelectrolyte complex, which depends on the shell thickness and charge of its outmost layer, and involves the diffusion of unpaired amine sites through the shell. The lag times of growth are sufficient to prevent proliferation for daily topical applications.

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5 + population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP + cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5 + ISCs. Considering the key roles Lgr5 + ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This corrects the article DOI: 10.1038/nchem.2857.

The concept of a glucose-responsive insulin (GRI) has been a recent objective of diabetes technology. The idea behind the GRI is to create a therapeutic that modulates its potency, concentration or dosing relative to a patient's dynamic glucose concentration, thereby approximating aspects of a normally functioning pancreas. From the perspective of the medicinal chemist, the GRI is also important as a generalized model of a potentially new generation of therapeutics that adjust potency in response to a critical therapeutic marker. The aim of this Perspective is to highlight emerging concepts, including mathematical modelling and the molecular engineering of insulin itself and its potency, towards a viable GRI. We briefly outline some of the most important recent progress toward this goal and also provide a forward-looking viewpoint, which asks if there are new approaches that could spur innovation in this area as well as to encourage synthetic chemists and chemical engineers to address the challenges and promises offered by this therapeutic approach.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Activating mutations involving the PI3K pathway occur frequently in human cancers. However, PI3K inhibitors primarily induce cell cycle arrest, leaving a significant reservoir of tumor cells that may acquire or exhibit resistance. We searched for genes that are required for the survival of PI3K mutant cancer cells in the presence of PI3K inhibition by conducting a genome scale shRNA-based apoptosis screen in a PIK3CA mutant human breast cancer cell. We identified 5 genes ( PIM2, ZAK, TACC1, ZFR, ZNF565 ) whose suppression induced cell death upon PI3K inhibition. We showed that small molecule inhibitors of the PIM2 and ZAK kinases synergize with PI3K inhibition. In addition, using a microscale implementable device to deliver either siRNAs or small molecule inhibitors in vivo, we showed that suppressing these 5 genes with PI3K inhibition induced tumor regression. These observations identify targets whose inhibition synergizes with PI3K inhibitors and nominate potential combination therapies involving PI3K inhibition.

Nuclear transfection of DNA into mammalian cells is challenging yet critical for many biological and medical studies. Here, by combining cell squeezing and electric-field-driven transport in a device that integrates microfluidic channels with constrictions and microelectrodes, we demonstrate nuclear delivery of plasmid DNA within 1 hour after treatment, the most rapid DNA expression in a high-throughput setting (up to millions of cells per minute per device). Passing cells at high speed through microfluidic constrictions smaller than the cell diameter mechanically disrupts the cell membrane, allowing a subsequent electric field to further disrupt the nuclear envelope and drive DNA molecules into the cytoplasm and nucleus. By tracking the localization of the ESCRT-III (endosomal sorting complexes required for transport) protein CHMP4B, we show that the integrity of the nuclear envelope is recovered within 15 minutes of treatment. We also provide insight into subcellular delivery by comparing the performance of the disruption-and-field-enhanced method with those of conventional chemical, electroporation, and manual-injection systems., Competing Interests: AS, RL, and KFJ have a financial interest in SQZ Biotechnologies.

Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.

Delivery of tissue glues through small-bore needles or trocars is critical for sealing holes, affixing medical devices, or attaching tissues together during minimally invasive surgeries. Inspired by the granule-packaged glue delivery system of sandcastle worms, a nanoparticulate formulation of a viscous hydrophobic light-activated adhesive based on poly(glycerol sebacate)-acrylate is developed. Negatively charged alginate is used to stabilize the nanoparticulate surface to significantly reduce its viscosity and to maximize injectability through small-bore needles. The nanoparticulate glues can be concentrated to ≈30 w/v% dispersions in water that remain localized following injection. With the trigger of a positively charged polymer (e.g., protamine), the nanoparticulate glues can quickly assemble into a viscous glue that exhibits rheological, mechanical, and adhesive properties resembling the native poly(glycerol sebacate)-acrylate based glues. This platform should be useful to enable the delivery of viscous glues to augment or replace sutures and staples during minimally invasive procedures., Competing Interests: J.M.K. and R.L. hold equity in Gecko Biomedical, a company that has an option to license IP generated by J.M.K. and R.L. and that may benefit financially if the IP is licensed and further validated. The interests of J.M.K and R.L. were reviewed and are subject to a management plan overseen by their institutions in accordance with their conflict of interest policies., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Drug design is built on the concept that key molecular targets of disease are isolated in the diseased tissue. Systemic drug administration would be sufficient for targeting in such a case. It is, however, common for enzymes or receptors that are integral to disease to be structurally similar or identical to those that play important biological roles in normal tissues of the body. Additionally, systemic administration may not lead to local drug concentrations high enough to yield disease modification because of rapid systemic metabolism or lack of sufficient partitioning into the diseased tissue compartment. This review focuses on drug delivery methods that physically target drugs to individual compartments of the body. Compartments such as the bladder, peritoneum, brain, eye and skin are often sites of disease and can sometimes be viewed as "privileged," since they intrinsically hinder partitioning of systemically administered agents. These compartments have become the focus of a wide array of procedures and devices for direct administration of drugs. We discuss the rationale behind single compartment drug delivery for each of these compartments, and give an overview of examples at different development stages, from the lab bench to phase III clinical trials to clinical practice. We approach single compartment drug delivery from both a translational and a technological perspective., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Using microarray technologies thousands of biomedical materials can be screened in a rapid, parallel and cost effective fashion to identify the optimum candidate that fulfils a specific biomedical application. High throughput surface characterization (HTSC) of printed microarrays has played a key role in the discovery and development of biomedical materials. This review focuses on the production and HTSC of microarrays, their application in specific biomedical fields and a future perspective on the development of this technology., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The targeted delivery of therapeutic cargos using noninvasive stimuli has the potential to improve efficacy and reduce off-target effects (toxicity). Here, we demonstrate a targeting mechanism that uses a thermoresponsive copolymer to mask a peptide ligand that binds a widely distributed receptor (integrin β1) on the surface of silica core-gold shell nanoparticles. The nanoparticles convert NIR light into heat, which causes the copolymer to collapse, exposing the ligand peptide, allowing cell binding. The use of NIR light could allow targeting of plasmonic nanoparticles deep within tissues. This approach could be extended to a variety of applications including photothermal therapy and drug delivery.

Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions in vivo, leading to potential adverse events and safety concerns. We have developed a synthetic vaccine particle (SVP) technology that enables co-encapsulation of antigen with potent adjuvants. We demonstrate that co-delivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles results in a strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. In contrast, antigen encapsulated into nanoparticles and admixed with free TLR7/8 agonist leads to lower immunogenicity and rapid induction of high levels of inflammatory cytokines in the serum (e.g., TNF-a and IL-6 levels are 50- to 200-fold higher upon injection of free resiquimod (R848) than of nanoparticle-encapsulated R848). Conversely, local immune stimulation as evidenced by cellular infiltration of draining lymph nodes and by intranodal cytokine production was more pronounced and persisted longer when SVP-encapsulated TLR agonists were used. The strong local immune activation achieved using a modular self-assembling nanoparticle platform markedly enhanced immunogenicity and was equally effective whether antigen and adjuvant were co-encapsulated in a single nanoparticle formulation or co-delivered in two separate nanoparticles. Moreover, particle encapsulation enabled the utilization of CpG oligonucleotides with the natural phosphodiester backbone, which are otherwise rapidly hydrolyzed by nucleases in vivo. The use of SVP may enable clinical use of potent TLR agonists as vaccine adjuvants for indications where cellular immunity or robust humoral responses are required., (Copyright © 2014. Published by Elsevier Ltd.)

We report plasmonic gold nanoshells and nanorods coated with reduced graphene oxide that produce an enhanced photothermal effect when stimulated by near-infrared (NIR) light. Electrostatic interactions between nanosized graphene oxide and gold nanoparticles followed by in situ chemical reduction generated reduced graphene oxide-coated nanoparticles; the coating was demonstrated using Raman and HR-TEM. Reduced graphene oxide-coated gold nanoparticles showed enhanced photothermal effect compared to noncoated or nonreduced graphene oxide-coated gold nanoparticles. Reduced graphene oxide-coated gold nanoparticles killed cells more rapidly than did noncoated or nonreduced graphene oxide-coated gold nanoparticles.

Using combinatorial methods, we synthesized a series of new vinyl amide monomers and graft-polymerized them to light-sensitive poly(ether sulfone) (PES) porous films for protein resistance. To increase the discovery rate and statistical confidence, we developed high throughput surface modification methods (HTP) that allow synthesis, screening and selection of desirable monomers from a large library in a relatively short time (days). A series of amide monomers were synthesized by amidation of methacryloyl chloride with amines and grafted onto commercial poly(ether sulfone) (PES) membranes using irradiation from atmospheric pressure plasma (APP). The modified PES membrane surfaces were then tested and screened for static protein adhesion using HTP. Hydroxyl amide monomers N-(3-hydroxypropyl)methacrylamide (A3), N-(4-hydroxybutyl)methacrylamide (A4), and N-(4-hydroxybutyl)methacrylamide (A6), ethylene glycol (EG) monomer N-(3-methoxypropyl)methacrylamide (A7), and N-(2-(dimethylamino)ethyl)-N-methylmethacrylamide (A8), and N-(2-(diethylamino)ethyl)-N-methylmethacrylamide (A9) all terminated with tertiary amines and were shown to have protein resistance. The PES membranes modified with these monomers exhibited both low protein adhesion (i.e. membrane plugging or fouling) and high flux. Their performance is comparable with previously identified best performing PEG and zwitterionic monomers, i.e. the so-called gold-standard for protein resistance. Combining a Hansen solubility parameter (HSP) analysis of the amide monomers and the HTP filtration results, we conclude that monomer solubility in water correlates with protein-resistant surfaces, presumably through its effects on surface-water interactions., (Published by Elsevier Ltd.)

Diabetes mellitus, a disorder of glucose regulation, is a global burden affecting 366 million people across the world. An artificial "closed-loop" system able to mimic pancreas activity and release insulin in response to glucose level changes has the potential to improve patient compliance and health. Herein we develop a glucose-mediated release strategy for the self-regulated delivery of insulin using an injectable and acid-degradable polymeric network. Formed by electrostatic interaction between oppositely charged dextran nanoparticles loaded with insulin and glucose-specific enzymes, the nanocomposite-based porous architecture can be dissociated and subsequently release insulin in a hyperglycemic state through the catalytic conversion of glucose into gluconic acid. In vitro insulin release can be modulated in a pulsatile profile in response to glucose concentrations. In vivo studies validated that these formulations provided improved glucose control in type 1 diabetic mice subcutaneously administered with a degradable nano-network. A single injection of the developed nano-network facilitated stabilization of the blood glucose levels in the normoglycemic state (<200 mg/dL) for up to 10 days.

Colorectal cancer is a significant source of morbidity and mortality in the United States and other Western countries. Oral delivery of therapeutics remains the most patient accepted form of medication. The development of an oral delivery formulation for local delivery of chemotherapeutics in the gastrointestinal tract can potentially alleviate the adverse side effects including systemic cytotoxicity, as well as focus therapy to the lesions. Here we develop an oral formulation of the chemotherapeutic drug oxaliplatin for the treatment of colorectal cancer. Oxaliplatin was encapsulated in pH sensitive, mucoadhesive chitosan-coated alginate microspheres. The microparticles were formulated to release the chemotherapeutics after passing through the acidic gastric environment thus targeting the intestinal tract. In vivo, these particles substantially reduced the tumor burden in an orthotopic mouse model of colorectal cancer, and reduced mortality., (Copyright © 2012. Published by Elsevier Ltd.)

Despite the promise of RNA interference (RNAi) therapeutics, progress toward the clinic has been slowed by the difficulty of delivering short interfering RNA (siRNA) into cellular targets within the body. Nearly all siRNA delivery vehicles developed to date employ a single cationic or ionizable material. In order to increase the material space available for development of siRNA delivery therapeutics, this study examined the possibility of using binary combinations of ionizable lipid-like materials to synergistically achieve gene silencing. Interestingly, it was found that ineffective single lipid-like materials could be formulated together in a single delivery vehicle to induce near-complete knockdown of firefly luciferase and factor VII in HeLa cells and in mice, respectively. Microscopy experiments suggested that synergistic action resulted when combining materials that respectively mediated cellular uptake and endosomal escape, two important steps in the delivery process. Together, the data indicate that formulating lipid-like materials in combination can significantly improve siRNA delivery outcomes while increasing the material space available for therapeutic development. It is anticipated that this binary formulation strategy could be applicable to any siRNA delivery material in any target cell population that utilizes the two-step endosomal delivery pathway.

The development of tools and processes used to fabricate, measure, and image nanoscale objects has lead to a wide range of work devoted to producing sensors that interact with extremely small numbers (or an extremely small concentration) of analyte molecules. These advances are particularly exciting in the context of biosensing, where the demands for low concentration detection and high specificity are great. Nanoscale biosensors, or nanobiosensors, provide researchers with an unprecedented level of sensitivity, often to the single molecule level. The use of biomolecule-functionalized surfaces can dramatically boost the specificity of the detection system, but can also yield reproducibility problems and increased complexity. Several nanobiosensor architectures based on mechanical devices, optical resonators, functionalized nanoparticles, nanowires, nanotubes, and nanofibers have been demonstrated in the lab. As nanobiosensor technology becomes more refined and reliable, it is likely it will eventually make its way from the lab to the clinic, where future lab-on-a-chip devices incorporating an array of nanobiosensors could be used for rapid screening of a wide variety of analytes at low cost using small samples of patient material., (Copyright © 2011 John Wiley & Sons, Inc.)

Controlled-release drug delivery systems are capable of treating debilitating diseases, including cancer. Brain cancer, in particular glioblastoma multiforme (GBM), is an extremely invasive cancer with a dismal prognosis. The use of drugs capable of crossing the blood-brain barrier has shown modest prolongation in patient survival, but not without unsatisfactory systemic, dose-limiting toxicity. Among the reasons for this improvement include a better understanding of the challenges of delivery of effective agents directly to the brain tumor site. The combination of carmustine delivered by biodegradable polyanhydride wafers (Gliadel(®)), with the systemic alkylating agent, temozolomide, allows much higher effective doses of the drug while minimizing the systemic toxicity. We have previously shown that locally delivering these two drugs leads to further improvement in survival in experimental models. We postulated that microcapsule devices capable of releasing temozolomide would increase the therapeutic capability of this approach. A biocompatible drug delivery microcapsule device for the intracranial delivery of temozolomide is described. Drug release profiles from these microcapsules can be modulated based on the physical chemistry of the drug and the dimensions of the release orifices in these devices. The drug released from the microcapsules in these experiments was the clinically utilized chemotherapeutic agent, temozolomide. In vitro studies were performed in order to test the function, reliability, and drug release kinetics of the devices. The efficacy of the temozolomide-filled microcapsules was tested in an intracranial experimental rodent gliosarcoma model. Immunohistochemical analysis of tissue for evidence of DNA strand breaks via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed. The experimental release curves showed mass flow rates of 36 μg/h for single-orifice devices and an 88 μg/h mass flow rate for multiple-orifice devices loaded with temozolomide. In vivo efficacy results showed that localized intracranial delivery of temozolomide from microcapsule devices was capable of prolonging animal survival and may offer a novel form of treatment for brain tumors., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Surface wrinkles are interesting since they form spontaneously into well-defined patterns. The mechanism of formation is well-studied and is associated with the development of a critical compressive stress that induces the elastic instability. In this work, we demonstrate surface wrinkles that dynamically change in response to a stimulus can improve interfacial adhesion with a hydrogel surface through the dynamic evolution of the wrinkle morphology. We observe that this control is related to the local pinning of the crack separation pathway facilitated by the surface wrinkles during debonding, which is dependent on the contact time with the hydrogel.

Since its inception more than a decade ago, the field of short interfering RNA (siRNA) therapeutics has demonstrated potential in the treatment of a wide variety of diseases. The power behind RNA interference (RNAi) therapy lies in its ability to specifically silence target genes of interest. As more biological data have become available, it has become evident that, in addition to mediating RNAi, siRNA molecules have the potential to potently induce the innate immune system. One of the significant challenges facing the field today is the differentiation between therapeutic effects caused by target-specific, RNAi-mediated gene silencing and those caused by nonspecific stimulation of the innate immune system. Unless appropriate experimental measures are taken to control for RNA-induced immunostimulation, genetic manipulation can be confused with immune activation. This review attempts to provide an accessible background in siRNA-relevant immunology and to highlight the ways in which siRNA can be engineered to avoid or provoke an innate immune response.

In retinal transplantation experiments it is hypothesized that remaining diseased photoreceptor cells in the host retina and inner retinal cells in transplants physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). We also coated PGS membranes with electrospun nanofibers, composed of laminin and poly(epsilon-caprolactone) (PCL), to promote attachment of embryonic retinal explants, allowing the resulting composites to be handled surgically as a single entity. Here, we report subretinal transplantation of these composites into adult porcine eyes. In hematoxylin and eosin stained sections of composite explants after 5-7 days in vitro, excellent fusion of retinas and biomaterial membranes was noted, with the immature retinal components showing laminated as well as folded and rosetted areas. The composite grafts could be transplanted in all cases and, 3 months after surgery, eyes displayed clear media, attached retinas and the grafts located subretinally. Histological examination revealed that the biomaterial membrane had degraded without any signs of inflammation. Transplanted retinas displayed areas of rosettes as well as normal lamination. In most cases inner retinal layers were present in the grafts. Laminated areas displayed well-developed photoreceptors adjacent to an intact host retinal pigment epithelium and degeneration of the host outer nuclear layer (ONL) was often observed together with occasional fusion of graft and host inner layers., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Biodegradable polymers with high mechanical strength, flexibility and optical transparency, optimal degradation properties and biocompatibility are critical to the success of tissue engineered devices and drug delivery systems. Most biodegradable polymers suffer from a short half life due to rapid degradation upon implantation, exceedingly high stiffness, and limited ability to functionalize the surface with chemical moieties. This work describes the fabrication of microfluidic networks from poly(ester amide), poly(1,3-diamino-2-hydroxypropane-co-polyol sebacate) (APS), a recently developed biodegradable elastomeric poly(ester amide). Microfluidic scaffolds constructed from APS exhibit a much lower Young's Modulus and a significantly longer degradation half-life than those of previously reported systems. The device is fabricated using a modified replica-molding technique, which is rapid, inexpensive, reproducible, and scalable, making the approach ideal for both rapid prototyping and manufacturing of tissue engineering scaffolds.

Given the involvement of post-mitotic neurons, long axonal tracts and incompletely elucidated injury and repair pathways, spinal cord injury (SCI) presents a particular challenge for the creation of preclinical models to robustly evaluate longitudinal changes in neuromotor function in the setting in the presence and absence of intervention. While rodent models exhibit high degrees of spontaneous recovery from SCI injury, animal care concerns preclude complete cord transections in non-human primates and other larger vertebrate models. To overcome such limitations a segmental thoracic (T9-T10) spinal cord hemisection was created and characterized in the African green monkey. Physiological tolerance of the model permitted behavioral analyses for a prolonged period post-injury, extending to predefined study termination points at which histological and immunohistochemical analyses were performed. Four monkeys were evaluated (one receiving no implant at the lesion site, one receiving a poly(lactide-co-glycolide) (PLGA) scaffold, and two receiving PLGA scaffolds seeded with human neural stem cells (hNSC)). All subjects exhibited Brown-Séquard syndrome 2 days post-injury consisting of ipsilateral hindlimb paralysis and contralateral hindlimb hypesthesia with preservation of bowel and bladder function. A 20-point observational behavioral scoring system allowed quantitative characterization of the levels of functional recovery. Histological endpoints including silver degenerative staining and Iba1 immunohistochemistry, for microglial and macrophage activation, were determined to reliably define lesion extent and correlate with neurobehavioral data, and justify invasive telemetered electromyographic and kinematic studies to more definitively address efficacy and mechanism., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Target-activatable fluorogenic probes based on gold nanoparticles (AuNPs) functionalized with self-assembled heterogeneous monolayers of dye-labeled peptides and poly(ethylene glycol) have been developed to visualize proteolytic activity in vivo. A one-step synthesis strategy that allows simple generation of surface-defined AuNP probe libraries is presented as a means of tailoring and evaluating probe characteristics for maximal fluorescence enhancement after protease activation. Optimal AuNP probes targeted to trypsin and urokinase-type plasminogen activator required the incorporation of a dark quencher to achieve 5- to 8-fold signal amplification. These probes exhibited extended circulation time in vivo and high image contrast in a mouse tumor model.

Retinal transplantation experiments have advanced considerably during recent years, but remaining diseased photoreceptor cells in the host retina and inner retinal cells in the transplant physically obstruct the development of graft-host neuronal contacts which are required for vision. Recently, we developed methods for the isolation of donor photoreceptor layers in vitro, and the selective removal of host photoreceptors in vivo using biodegradable elastomeric membranes composed of poly(glycerol-co-sebacic acid) (PGS). Here, we report the surface modification of PGS membranes to promote the attachment of photoreceptor layers, allowing the resulting composite to be handled surgically as a single entity. PGS membranes were chemically modified with peptides containing an arginine-glycine-aspartic acid (RGD) extracellular matrix ligand sequence. PGS membranes were also coated with electrospun nanofiber meshes, containing laminin and poly(epsilon-caprolactone) (PCL). Following in vitro co-culture of biomaterial membranes with isolated embryonic retinal tissue, composites were tested for surgical handling and examined with hematoxylin and eosin staining and immunohistochemical markers. Electrospun nanofibers composed of laminin and PCL promoted sufficient cell adhesion for simultaneous transplantation of isolated photoreceptor layers and PGS membranes. Composites developed large populations of recoverin and rhodopsin labeled photoreceptors. Furthermore, ganglion cells, rod bipolar cells and AII amacrine cells were absent in co-cultured retinas as observed by neurofilament, PKC and parvalbumin labeling respectively. These results facilitate retinal transplantation experiments in which a composite graft composed of a biodegradable membrane adhered to an immature retina dominated by photoreceptor cells may be delivered in a single surgery, with the possibility of improving graft-host neuronal connections., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Adipose-derived stem cells (ASCs) may provide a clinical option for rebuilding damaged superficial lamina propria of the vocal fold. We investigated the effects of five hydrogels (hyaluronic acid [HA], collagen, fibrin, and cogels of fibrin-collagen and fibrin-HA) on the differentiation of ASCs, with the long-term goal of establishing the conditions necessary for controlling the differentiation of ASC into the functional equivalent of superficial lamina propria fibroblasts. Human ASCs were isolated and characterized by fluorescence-activated cell sorting and real-time polymerase chain reaction. According to fluorescence-activated cell sorting and gene analysis, over 90% of isolated ASCs expressed adult stem cell surface markers and expressed adult stem cell genes. Scaffold-specific gene expression and morphology were assessed by culturing the ASCs in three-dimensional hydrogels. Twofold higher amounts of total DNA were detected in fibrin and cogel cultures than in collagen and HA cultures. Elastin expression was significantly higher in cells grown in fibrin-based gels than in cells grown in other gels. Cells grown in the cogels showed elongated morphology, expressed decorin marker, and exhibited glycosaminoglycan synthesis, which indicate ASC differentiation. Our data suggest that it may be possible to control the differentiation of ASCs using scaffolds appropriate for vocal fold tissue engineering applications. In particular, cogels of HA or collagen with fibrin enhanced proliferation, differentiation, and elastin expression.

Maintenance of ocular viability is one of the major impediments to successful whole-eye transplantation. This review provides a comprehensive understanding of the current literature to help guide future studies in order to overcome this hurdle. A systematic multistage review of published literature was performed. Three specific questions were addressed: (1) Is recovery of visual function following eye transplantation greater in cold-blooded vertebrates when compared with mammals? (2) Is outer retina function following enucleation and reperfusion improved compared with enucleation alone? (3) Following optic-nerve transection, is there a correlation between retinal ganglion cell (RGC) survival and either time after transection or proximity of the transection to the globe? In a majority of the studies performed in the literature, recovery of visual function can occur after whole-eye transplantation in cold-blooded vertebrates. Following enucleation (and reperfusion), outer retinal function is maintained from 4 to 9 h. RGC survival following optic-nerve transection is inversely related to both the time since transection and the proximity of transection to the globe. Lastly, neurotrophins can increase RGC survival following optic-nerve transection. This review of the literature suggests that the use of a donor eye is feasible for whole-eye transplantation.

Background: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany., Methods: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine., Results: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine., Conclusions: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system., (Copyright 2008 Massachusetts Medical Society.)

Poly(orthoester) (POE) microspheres have been previously shown to possess certain advantages for the in vivo delivery of DNA vaccines. In particular, timing of DNA release from POE microspheres in response to acidic phagosomal pH was shown to be an important factor in determining immunogenicity, which was hypothesized to be linked to the natural progression of antigen-presenting cell uptake, transfection, maturation, and antigen presentation. Here we report in vitro characterization of the enhanced efficacy of POE microspheres by blending poly(ethylenimine) (PEI), a well-characterized cationic transfection agent, into the POE matrix. Blending of a tiny amount of PEI (approximately 0.04 wt%) with POE caused large alterations in POE microsphere properties. PEI provided greater control over the rate of pH-triggered DNA release by doubling the total release time of plasmid DNA and enhanced gene transfection efficiency of the microspheres up to 50-fold without any significant cytotoxicity. Confocal microscopy results of labeled PEI and DNA plasmids revealed that PEI caused a surface-localizing distribution of DNA and PEI within the POE microsphere as well as focal co-localization of PEI with DNA. We provide evidence that upon degradation, the microspheres of POE-PEI blends released electrostatic complexes of DNA and PEI, which are responsible for the enhanced gene transfection. Furthermore, blending PEI into the POE microsphere induced 50-60% greater phenotypic maturation and activation of bone marrow-derived dendritic cells in vitro, judged by the up-regulation of co-stimulatory markers on the cell surface. Physically blending PEI with POE is a simple approach for modulating the properties of biodegradable microspheres in terms of gene transfection efficiency and DNA release kinetics. Combined with the ability to induce maturation of antigen-presenting cells, POE-PEI blended microspheres may be excellent carriers for DNA vaccines.