Publications by authors named "Margaret M Billingsley"

14 Publications

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Amniotic fluid stabilized lipid nanoparticles for in utero intra-amniotic mRNA delivery.

J Control Release 2021 Nov 3. Epub 2021 Nov 3.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA; Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Congenital disorders resulting in pathological protein deficiencies are most often treated postnatally with protein or enzyme replacement therapies. However, treatment of these disorders in utero before irreversible disease onset could significantly minimize disease burden, morbidity, and mortality. One possible strategy for the prenatal treatment of congenital disorders is the in utero delivery of messenger RNA (mRNA). mRNA is a gene therapeutic that has previously been investigated for protein replacement therapies and gene editing technologies. While viral vectors have been explored to induce intracellular expression of mRNA, they are limited in their clinical application due to concerns of immunogenicity and genomic integration. As an alternative to viral vectors, safe and efficient in utero mRNA delivery can be achieved using ionizable lipid nanoparticles (LNPs). While LNPs have demonstrated potent in vivo mRNA delivery to the liver following intravenous administration, intra-amniotic delivery has the potential to deliver mRNA to cells and tissues beyond those in the liver, such as in the skin, lung, and digestive tract. However, LNP stability in fetal amniotic fluid and how this stability affects mRNA delivery has not been previously investigated. Here, we engineered a library of LNPs using orthogonal design of experiments (DOE) to evaluate how LNP structure affects ex utero stability in amniotic fluid, and whether a lead candidate identified from these stability measurements enables intra-amniotic mRNA delivery in utero. We used a combination of techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM), and chromatography followed by protein content quantification to screen ex vivo LNP stability in amniotic fluids. These results identified multiple lead LNP formulations that are highly stable in amniotic fluids ranging from small animals to humans, including mouse, sheep, pig, and human amniotic fluid samples. We then demonstrate that stable LNPs from the ex utero screen in mouse amniotic fluid enabled potent mRNA delivery in vitro in fetal lung fibroblasts and in utero following intra-amniotic injection in a murine model. This exploration of ex utero stability in amniotic fluids demonstrates a means by which to identify novel LNP formulations for prenatal treatment of congenital disorders via in utero mRNA delivery.
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http://dx.doi.org/10.1016/j.jconrel.2021.10.031DOI Listing
November 2021

Orthogonal Design of Experiments for Optimization of Lipid Nanoparticles for mRNA Engineering of CAR T Cells.

Nano Lett 2021 Oct 20. Epub 2021 Oct 20.

Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.

Viral engineered chimeric antigen receptor (CAR) T cell therapies are potent, targeted cancer immunotherapies, but their permanent CAR expression can lead to severe adverse effects. Nonviral messenger RNA (mRNA) CAR T cells are being explored to overcome these drawbacks, but electroporation, the most common T cell transfection method, is limited by cytotoxicity. As a potentially safer nonviral delivery strategy, here, sequential libraries of ionizable lipid nanoparticle (LNP) formulations with varied excipient compositions were screened in comparison to a standard formulation for improved mRNA delivery to T cells with low cytotoxicity, revealing B10 as the top formulation with a 3-fold increase in mRNA delivery. When compared to electroporation in primary human T cells, B10 LNPs induced comparable CAR expression with reduced cytotoxicity while demonstrating potent cancer cell killing. These results demonstrate the impact of excipient optimization on LNP performance and support B10 LNPs as a potent mRNA delivery platform for T cell engineering.
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http://dx.doi.org/10.1021/acs.nanolett.1c02503DOI Listing
October 2021

One-Component Multifunctional Sequence-Defined Ionizable Amphiphilic Janus Dendrimer Delivery Systems for mRNA.

J Am Chem Soc 2021 08 29;143(31):12315-12327. Epub 2021 Jul 29.

Roy & Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, United States.

Efficient viral or nonviral delivery of nucleic acids is the key step of genetic nanomedicine. Both viral and synthetic vectors have been successfully employed for genetic delivery with recent examples being DNA, adenoviral, and mRNA-based Covid-19 vaccines. Viral vectors can be target specific and very efficient but can also mediate severe immune response, cell toxicity, and mutations. Four-component lipid nanoparticles (LNPs) containing ionizable lipids, phospholipids, cholesterol for mechanical properties, and PEG-conjugated lipid for stability represent the current leading nonviral vectors for mRNA. However, the segregation of the neutral ionizable lipid as droplets in the core of the LNP, the "PEG dilemma", and the stability at only very low temperatures limit their efficiency. Here, we report the development of a one-component multifunctional ionizable amphiphilic Janus dendrimer (IAJD) delivery system for mRNA that exhibits high activity at a low concentration of ionizable amines organized in a sequence-defined arrangement. Six libraries containing 54 sequence-defined IAJDs were synthesized by an accelerated modular-orthogonal methodology and coassembled with mRNA into dendrimersome nanoparticles (DNPs) by a simple injection method rather than by the complex microfluidic technology often used for LNPs. Forty four (81%) showed activity and 31 (57%) . Some, exhibiting organ specificity, are stable at 5 °C and demonstrated higher transfection efficiency than positive control experiments and . Aside from practical applications, this proof of concept will help elucidate the mechanisms of packaging and release of mRNA from DNPs as a function of ionizable amine concentration, their sequence, and constitutional isomerism of IAJDs.
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http://dx.doi.org/10.1021/jacs.1c05813DOI Listing
August 2021

Delivery technologies for T cell gene editing: Applications in cancer immunotherapy.

EBioMedicine 2021 May 25;67:103354. Epub 2021 Apr 25.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA; Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

While initial approaches to adoptive T cell therapy relied on the identification and expansion of rare tumour-reactive T cells, genetic engineering has transformed cancer immunotherapy by enabling the modification of primary T cells to increase their therapeutic potential. Specifically, gene editing technologies have been utilized to create T cell populations with improved responses to antigens, lower rates of exhaustion, and potential for use in allogeneic applications. In this review, we provide an overview of T cell therapy gene editing strategies and the delivery technologies utilized to genetically engineer T cells. We also discuss recent investigations and clinical trials that have utilized gene editing to enhance the efficacy of T cells and broaden the application of cancer immunotherapies.
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http://dx.doi.org/10.1016/j.ebiom.2021.103354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099660PMC
May 2021

Ionizable lipid nanoparticles for in utero mRNA delivery.

Sci Adv 2021 Jan 13;7(3). Epub 2021 Jan 13.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

Clinical advances enable the prenatal diagnosis of genetic diseases that are candidates for gene and enzyme therapies such as messenger RNA (mRNA)-mediated protein replacement. Prenatal mRNA therapies can treat disease before the onset of irreversible pathology with high therapeutic efficacy and safety due to the small fetal size, immature immune system, and abundance of progenitor cells. However, the development of nonviral platforms for prenatal delivery is nascent. We developed a library of ionizable lipid nanoparticles (LNPs) for in utero mRNA delivery to mouse fetuses. We screened LNPs for luciferase mRNA delivery and identified formulations that accumulate within fetal livers, lungs, and intestines with higher efficiency and safety compared to benchmark delivery systems, DLin-MC3-DMA and jetPEI. We demonstrate that LNPs can deliver mRNAs to induce hepatic production of therapeutic secreted proteins. These LNPs may provide a platform for in utero mRNA delivery for protein replacement and gene editing.
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http://dx.doi.org/10.1126/sciadv.aba1028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806221PMC
January 2021

Nanomaterials for T-cell cancer immunotherapy.

Nat Nanotechnol 2021 01 12;16(1):25-36. Epub 2021 Jan 12.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.

T-cell-based immunotherapies hold promise for the treatment of many types of cancer, with three approved products for B-cell malignancies and a large pipeline of treatments in clinical trials. However, there are several challenges to their broad implementation. These include insufficient expansion of adoptively transferred T cells, inefficient trafficking of T cells into solid tumours, decreased T-cell activity due to a hostile tumour microenvironment and the loss of target antigen expression. Together, these factors restrict the number of therapeutically active T cells engaging with tumours. Nanomaterials are uniquely suited to overcome these challenges, as they can be rationally designed to enhance T-cell expansion, navigate complex physical barriers and modulate tumour microenvironments. Here, we present an overview of nanomaterials that have been used to overcome clinical barriers to T-cell-based immunotherapies and provide our outlook of this emerging field at the interface of cancer immunotherapy and nanomaterial design.
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http://dx.doi.org/10.1038/s41565-020-00822-yDOI Listing
January 2021

Helper lipid structure influences protein adsorption and delivery of lipid nanoparticles to spleen and liver.

Biomater Sci 2021 Feb 6;9(4):1449-1463. Epub 2021 Jan 6.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

Nucleic acids, such as messenger RNAs, antisense oligonucleotides, and short interfering RNAs, hold great promise for treating previously 'undruggable' diseases. However, there are numerous biological barriers that hinder nucleic acid delivery to target cells and tissues. While lipid nanoparticles (LNPs) have been developed to protect nucleic acids from degradation and mediate their intracellular delivery, it is challenging to predict how alterations in LNP formulation parameters influence delivery to different organs. In this study, we utilized high-throughput in vivo screening to probe for structure-function relationships of intravenously administered LNPs along with quartz crystal microbalance with dissipation monitoring (QCM-D) to measure the binding affinity of LNPs to apolipoprotein E (ApoE), a protein implicated in the clearance and uptake of lipoproteins by the liver. High-throughput in vivo screening of a library consisting of 96 LNPs identified several formulations containing the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) that preferentially accumulated in the liver, while identical LNPs that substituted DOPE with the helper lipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) preferentially accumulated in the spleen. Using QCM-D, it was found that one DOPE-containing LNP formulation (LNP 42) had stronger interactions with ApoE than an identical LNP formulation that substituted DOPE with DSPC (LNP 90). In order to further validate our findings, we formulated LNP 42 and LNP 90 to encapsulate Cy3-siRNA or mRNA encoding for firefly luciferase. The DSPC-containing LNP (LNP 90) was found to increase delivery to the spleen for both siRNA (two-fold) and mRNA (five-fold). In terms of liver delivery, the DOPE-containing LNP (LNP 42) enhanced mRNA delivery to the liver by two-fold and improved liver transfection by three-fold. Understanding the role of the helper lipid in LNP biodistribution and ApoE adsorption may aid in the future design of LNPs for nucleic acid therapeutics.
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http://dx.doi.org/10.1039/d0bm01609hDOI Listing
February 2021

Engineering precision nanoparticles for drug delivery.

Nat Rev Drug Discov 2021 02 4;20(2):101-124. Epub 2020 Dec 4.

Department of Chemical Engineering and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

In recent years, the development of nanoparticles has expanded into a broad range of clinical applications. Nanoparticles have been developed to overcome the limitations of free therapeutics and navigate biological barriers - systemic, microenvironmental and cellular - that are heterogeneous across patient populations and diseases. Overcoming this patient heterogeneity has also been accomplished through precision therapeutics, in which personalized interventions have enhanced therapeutic efficacy. However, nanoparticle development continues to focus on optimizing delivery platforms with a one-size-fits-all solution. As lipid-based, polymeric and inorganic nanoparticles are engineered in increasingly specified ways, they can begin to be optimized for drug delivery in a more personalized manner, entering the era of precision medicine. In this Review, we discuss advanced nanoparticle designs utilized in both non-personalized and precision applications that could be applied to improve precision therapies. We focus on advances in nanoparticle design that overcome heterogeneous barriers to delivery, arguing that intelligent nanoparticle design can improve efficacy in general delivery applications while enabling tailored designs for precision applications, thereby ultimately improving patient outcome overall.
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http://dx.doi.org/10.1038/s41573-020-0090-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717100PMC
February 2021

Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering.

Nano Lett 2020 03 5;20(3):1578-1589. Epub 2020 Feb 5.

Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.

Chimeric antigen receptor (CAR) T cell therapy relies on the manipulation of patient T cells to create potent, cancer-targeting therapies, shown to be capable of inducing remission in patients with acute lymphoblastic leukemia and large B cell lymphoma. However, current CAR T cell engineering methods use viral delivery vectors, which induce permanent CAR expression and could lead to severe adverse effects. Messenger RNA (mRNA) has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate the adverse effects associated with viral vectors, but it most commonly requires electroporation for T cell mRNA delivery, which can be cytotoxic. Here, ionizable lipid nanoparticles (LNPs) were designed for mRNA delivery to human T cells. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for luciferase mRNA delivery to Jurkat cells, revealing seven formulations capable of enhanced mRNA delivery over lipofectamine. The top-performing LNP formulation, C14-4, was selected for CAR mRNA delivery to primary human T cells. This platform induced CAR expression at levels equivalent to electroporation, with substantially reduced cytotoxicity. CAR T cells engineered via C14-4 LNP treatment were then compared to electroporated CAR T cells in a coculture assay with Nalm-6 acute lymphoblastic leukemia cells, and both CAR T cell engineering methods elicited potent cancer-killing activity. These results demonstrate the ability of LNPs to deliver mRNA to primary human T cells to induce functional protein expression, and indicate the potential of LNPs to enhance mRNA-based CAR T cell engineering methods.
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http://dx.doi.org/10.1021/acs.nanolett.9b04246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313236PMC
March 2020

Ionizable lipid nanoparticles encapsulating barcoded mRNA for accelerated in vivo delivery screening.

J Control Release 2019 12 31;316:404-417. Epub 2019 Oct 31.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, United States; Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Messenger RNA (mRNA) has recently emerged as a promising class of nucleic acid therapy, with the potential to induce protein production to treat and prevent a range of diseases. However, the widespread use of mRNA as a therapeutic requires safe and effective in vivo delivery technologies. Libraries of ionizable lipid nanoparticles (LNPs) have been designed to encapsulate mRNA, prevent its degradation, and mediate intracellular delivery. However, these LNPs are typically characterized and screened in an in vitro setting, which may not fully replicate the biological barriers that they encounter in vivo. Here, we designed and evaluated a library of engineered LNPs containing barcoded mRNA (b-mRNA) to accelerate the screening of mRNA delivery platforms in vivo. These b-mRNA are similar in structure and function to regular mRNA, and contain barcodes that enable their delivery to be quantified via deep sequencing. Using a mini-library of b-mRNA LNPs formulated via microfluidic mixing, we show that these different formulations can be pooled together, administered intravenously into mice as a single pool, and their delivery to multiple organs (liver, spleen, brain, lung, heart, kidney, pancreas, and muscle) can be quantified simultaneously using deep sequencing. In the context of liver and spleen delivery, LNPs that exhibited high b-mRNA delivery also yielded high luciferase expression, indicating that this platform can identify lead LNP candidates as well as optimal formulation parameters for in vivo mRNA delivery. Interestingly, LNPs with identical formulation parameters that encapsulated different types of nucleic acid barcodes (b-mRNA versus a DNA barcode) altered in vivo delivery, suggesting that the structure of the barcoded nucleic acid affects LNP in vivo delivery. This platform, which enables direct barcoding and subsequent quantification of a functional mRNA, can accelerate the in vivo screening and design of LNPs for mRNA therapeutic applications such as CRISPR-Cas9 gene editing, mRNA vaccination, and other mRNA-based regenerative medicine and protein replacement therapies.
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http://dx.doi.org/10.1016/j.jconrel.2019.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032071PMC
December 2019

Biomaterials for vaccine-based cancer immunotherapy.

J Control Release 2018 12 9;292:256-276. Epub 2018 Oct 9.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, United States; Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States. Electronic address:

The development of therapeutic cancer vaccines as a means to generate immune reactivity against tumors has been explored since the early discovery of tumor-specific antigens by Georg Klein in the 1960s. However, challenges including weak immunogenicity, systemic toxicity, and off-target effects of cancer vaccines remain as barriers to their broad clinical translation. Advances in the design and implementation of biomaterials are now enabling enhanced efficacy and reduced toxicity of cancer vaccines by controlling the presentation and release of vaccine components to immune cells and their microenvironment. Here, we discuss the rational design and clinical status of several classes of cancer vaccines (including DNA, mRNA, peptide/protein, and cell-based vaccines) along with novel biomaterial-based delivery technologies that improve their safety and efficacy. Further, strategies for designing new platforms for personalized cancer vaccines are also considered.
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http://dx.doi.org/10.1016/j.jconrel.2018.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355332PMC
December 2018

Evaluating the Mechanisms of Light-Triggered siRNA Release from Nanoshells for Temporal Control Over Gene Regulation.

Nano Lett 2018 06 2;18(6):3565-3570. Epub 2018 May 2.

Helen F. Graham Cancer Center & Research Institute , Newark , Delaware 19713 , United States.

The ability to regulate intracellular gene expression with exogenous nucleic acids such as small interfering RNAs (siRNAs) has substantial potential to improve the study and treatment of disease. However, most transfection agents and nanoparticle-based carriers that are used for the intracellular delivery of nucleic acids cannot distinguish between diseased and healthy cells, which may cause them to yield unintended widespread gene regulation. An ideal delivery system would only silence targeted proteins in diseased tissue in response to an external stimulus. To enable spatiotemporal control over gene silencing, researchers have begun to develop nucleic acid-nanoparticle conjugates that keep their nucleic acid cargo inactive until it is released from the nanoparticle on-demand by externally applied near-infrared laser light. This strategy can overcome several limitations of other nucleic acid delivery systems, but the mechanisms by which these platforms operate remain ill understood. Here, we perform a detailed investigation of the mechanisms by which silica core/gold shell nanoshells (NSs) release conjugated siRNA upon excitation with either pulsed or continuous wave (CW) near-infrared (NIR) light, with the goal of providing insight into how these nanoconjugates can enable on-demand gene regulation. We demonstrate that siRNA release from NSs upon pulsed laser irradiation is a temperature-independent process that is substantially more efficient than siRNA release triggered by CW irradiation. Contrary to literature, which suggests that only pulsed irradiation releases siRNA duplexes, we found that both modes of irradiation release a mixture of siRNA duplexes and single-stranded oligonucleotides, but that pulsed irradiation results in a higher percentage of released duplexes. To demonstrate that the siRNA released from NSs upon pulsed irradiation remains functional, we evaluated the use of NSs coated with green fluorescent protein (GFP)-targeted siRNA (siGFP-NS) for on-demand knockdown of GFP in cells. We found that GFP-expressing cells treated with siGFP-NS and irradiated with a pulsed laser experienced a 33% decrease in GFP expression compared to cells treated with no laser. Further, we observed that light-triggered gene silencing mediated by siGFP-NS is more potent than using commercial transfection agents to deliver siRNA into cells. This work provides unprecedented insight into the mechanisms by which plasmonic NSs release siRNA upon light irradiation and demonstrates the importance of thoroughly characterizing photoresponsive nanosystems for applications in triggered gene regulation.
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http://dx.doi.org/10.1021/acs.nanolett.8b00681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450696PMC
June 2018

Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods.

PLoS One 2017 11;12(5):e0177592. Epub 2017 May 11.

Department of Biomedical Engineering, University of Delaware, Newark, Delaware, United States of America.

Accurate antigen detection is imperative for clinicians to diagnose disease, assess treatment success, and predict patient prognosis. The most common technique used for the detection of disease-associated biomarkers is the enzyme linked immunosorbent assay (ELISA). In an ELISA, primary antibodies are incubated with biological samples containing the biomarker of interest. Then, detectible secondary antibodies conjugated with horseradish peroxidase (HRP) bind the primary antibodies. Upon addition of a color-changing substrate, the samples provide a colorimetric signal that directly correlates to the targeted biomarker concentration. While ELISAs are effective for analyzing samples with high biomarker content, they lack the sensitivity required to analyze samples with low antigen levels. We hypothesized that the sensitivity of ELISAs could be enhanced by replacing freely delivered primary antibodies with antibody-nanoparticle conjugates that provide excess binding sites for detectible secondary antibodies, ultimately leading to increased signal. Here, we investigated the use of nanoshells (NS) decorated with antibodies specific to epidermal growth factor receptor (EGFR) as a model system (EGFR-NS). We incubated one healthy and two breast cancer cell lines, each expressing different levels of EGFR, with EGFR-NS, untargeted NS, or unconjugated EGFR antibodies, as well as detectable secondary antibodies. We found that EGFR-NS consistently increased signal intensity relative to unconjugated EGFR antibodies, with a substantial 13-fold enhancement from cells expressing high levels of EGFR. Additionally, 40x more unconjugated antibodies were required to detect EGFR compared to those conjugated to NS. Our results demonstrate that antibody-nanoparticle conjugates lower the detection limit of traditional ELISAs and support further investigation of this strategy with other antibodies and nanoparticles. Owing to their enhanced sensitivity, we anticipate that nanoparticle-modified ELISAs can be used to detect low levels of biomarkers found in various diseases, such as cancers, tuberculosis, and rheumatoid arthritis, and may ultimately enable earlier diagnosis, better prognostication, and improved treatment monitoring.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177592PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426781PMC
September 2017

Quantification of siRNA Duplexes Bound to Gold Nanoparticle Surfaces.

Methods Mol Biol 2017 ;1570:1-15

Department of Biomedical Engineering, University of Delaware, 161 Colburn Lab, Newark, DE, 19716, USA.

RNA interference (RNAi)-based gene regulation has recently emerged as a promising strategy to silence genes that drive disease progression. RNAi is typically mediated by small interfering ribonucleic acids (siRNAs), which, upon delivery into the cell cytoplasm, trigger degradation of complementary messenger RNA molecules to halt production of their encoded proteins. While RNAi has enormous clinical potential, its in vivo utility has been hindered because siRNAs are rapidly degraded by nucleases, cannot passively enter cells, and are quickly cleared from the bloodstream. To overcome these delivery barriers, siRNAs can be conjugated to nanoparticles (NPs), which increase their stability and circulation time to enable in vivo gene regulation. Here, we present methods to conjugate siRNA duplexes to NPs with gold surfaces. Further, we describe how to quantify the resultant amount of siRNA sense and antisense strands loaded onto the NPs using a fluorescence-based assay. This method focuses on the attachment of siRNAs to 13 nm gold NPs, but it is adaptable to other types of nucleic acids and nanoparticles as discussed throughout the protocol.
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http://dx.doi.org/10.1007/978-1-4939-6840-4_1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555579PMC
February 2018
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