Publications by authors named "Michael J Mitchell"

86 Publications

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

Scalable mRNA and siRNA Lipid Nanoparticle Production Using a Parallelized Microfluidic Device.

Nano Lett 2021 07 30;21(13):5671-5680. Epub 2021 Jun 30.

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

A major challenge to advance lipid nanoparticles (LNPs) for RNA therapeutics is the development of formulations that can be produced reliably across the various scales of drug development. Microfluidics can generate LNPs with precisely defined properties, but have been limited by challenges in scaling throughput. To address this challenge, we present a scalable, parallelized microfluidic device (PMD) that incorporates an array of 128 mixing channels that operate simultaneously. The PMD achieves a >100× production rate compared to single microfluidic channels, without sacrificing desirable LNP physical properties and potency typical of microfluidic-generated LNPs. In mice, we show superior delivery of LNPs encapsulating either Factor VII siRNA or luciferase-encoding mRNA generated using a PMD compared to conventional mixing, with a 4-fold increase in hepatic gene silencing and 5-fold increase in luciferase expression, respectively. These results suggest that this PMD can generate scalable and reproducible LNP formulations needed for emerging clinical applications, including RNA therapeutics and vaccines.
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http://dx.doi.org/10.1021/acs.nanolett.1c01353DOI Listing
July 2021

Microfluidic formulation of nanoparticles for biomedical applications.

Biomaterials 2021 07 26;274:120826. Epub 2021 Apr 26.

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:

Nanomedicine has made significant advances in clinical applications since the late-20th century, in part due to its distinct advantages in biocompatibility, potency, and novel therapeutic applications. Many nanoparticle (NP) therapies have been approved for clinical use, including as imaging agents or as platforms for drug delivery and gene therapy. However, there are remaining challenges that hinder translation, such as non-scalable production methods and the inefficiency of current NP formulations in delivering their cargo to their target. To address challenges with existing formulation methods that have batch-to-batch variability and produce particles with high dispersity, microfluidics-devices that manipulate fluids on a micrometer scale-have demonstrated enormous potential to generate reproducible NP formulations for therapeutic, diagnostic, and preventative applications. Microfluidic-generated NP formulations have been shown to have enhanced properties for biomedical applications by formulating NPs with more controlled physical properties than is possible with bulk techniques-such as size, size distribution, and loading efficiency. In this review, we highlight advances in microfluidic technologies for the formulation of NPs, with an emphasis on lipid-based NPs, polymeric NPs, and inorganic NPs. We provide a summary of microfluidic devices used for NP formulation with their advantages and respective challenges. Additionally, we provide our analysis for future outlooks in the field of NP formulation and microfluidics, with emerging topics of production scale-independent formulations through device parallelization and multi-step reactions within droplets.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120826DOI Listing
July 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

Delivery technologies to engineer natural killer cells for cancer immunotherapy.

Cancer Gene Ther 2021 09 22;28(9):947-959. Epub 2021 Apr 22.

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

In recent years, immune cell-based cancer therapeutics have been utilized broadly in the clinic. Through advances in cellular engineering, chimeric antigen receptor (CAR) T-cell therapies have demonstrated substantial success in treating hematological tumors and have become the most prominent cell-based therapy with three commercialized products in the market. However, T-cell-based immunotherapies have certain limitations, including a restriction to autologous cell sources to avoid severe side-effects caused by human leukocyte antigen (HLA) mismatch. This necessity for personalized treatment inevitably results in tremendous manufacturing and time costs, reducing accessibility for many patients. As an alternative strategy, natural killer (NK) cells have emerged as potential candidates for improved cell-based immunotherapies. NK cells are capable of killing cancer cells directly without requiring HLA matching. Furthermore, NK cell-based therapies can use various allogeneic cell sources, allowing for the possibility of "off-the-shelf" immunotherapies with reduced side-effects and shortened manufacturing times. Here we provide an overview of the use of NK cells in cancer immunotherapy, their current status in clinical trials, as well as the design and implementation of delivery technologies-including viral, non-viral, and nanoparticle-based approaches-for engineering NK cell-based immunotherapies.
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http://dx.doi.org/10.1038/s41417-021-00336-2DOI Listing
September 2021

Lipid Nanoparticle-Mediated Delivery of mRNA Therapeutics and Vaccines.

Trends Mol Med 2021 06 6;27(6):616-617. Epub 2021 Apr 6.

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

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http://dx.doi.org/10.1016/j.molmed.2021.03.003DOI Listing
June 2021

Peptide functionalized liposomes for receptor targeted cancer therapy.

APL Bioeng 2021 Mar 25;5(1):011501. Epub 2021 Jan 25.

Most clinically approved cancer therapies are potent and toxic small molecules that are limited by severe off-target toxicities and poor tumor-specific localization. Over the past few decades, attempts have been made to load chemotherapies into liposomes, which act to deliver the therapeutic agent directly to the tumor. Although liposomal encapsulation has been shown to decrease toxicity in human patients, reliance on passive targeting via the enhanced permeability and retention (EPR) effect has left some of these issues unresolved. Recently, investigations into modifying the surface of liposomes via covalent and/or electrostatic functionalization have offered mechanisms for tumor homing and subsequently controlled chemotherapeutic delivery. A wide variety of biomolecules can be utilized to functionalize liposomes such as proteins, carbohydrates, and nucleic acids, which enable multiple directions for cancer cell localization. Importantly, when nanoparticles are modified with such molecules, care must be taken as not to inactivate or denature the ligand. Peptides, which are small proteins with <30 amino acids, have demonstrated the exceptional ability to act as ligands for transmembrane protein receptors overexpressed in many tumor phenotypes. Exploring this strategy offers a method in tumor targeting for cancers such as glioblastoma multiforme, pancreatic, lung, and breast based on the manifold of receptors overexpressed on various tumor cell populations. In this review, we offer a comprehensive summary of peptide-functionalized liposomes for receptor-targeted cancer therapy.
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http://dx.doi.org/10.1063/5.0029860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837755PMC
March 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

Delivery technologies for in utero gene therapy.

Adv Drug Deliv Rev 2021 02 9;169:51-62. Epub 2020 Nov 9.

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:

Advances in prenatal imaging, molecular diagnostic tools, and genetic screening have unlocked the possibility to treat congenital diseases in utero prior to the onset of clinical symptoms. While fetal surgery and in utero stem cell transplantation can be harnessed to treat specific structural birth defects and congenital hematological disorders, respectively, in utero gene therapy allows for phenotype correction of a wide range of genetic disorders within the womb. However, key challenges to realizing the broad potential of in utero gene therapy are biocompatibility and efficiency of intracellular delivery of transgenes. In this review, we outline the unique considerations to delivery of in utero gene therapy components and highlight advances in viral and non-viral delivery platforms that meet these challenges. We also discuss specialized delivery technologies for in utero gene editing and provide future directions to engineer novel delivery modalities for clinical translation of this promising therapeutic approach.
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http://dx.doi.org/10.1016/j.addr.2020.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855052PMC
February 2021

Proton-driven transformable nanovaccine for cancer immunotherapy.

Nat Nanotechnol 2020 12 26;15(12):1053-1064. Epub 2020 Oct 26.

Laboratory of Controllable Nanopharmaceuticals, Chinese Academy of Sciences (CAS) Center for Excellence in Nanoscience and CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, China.

Cancer vaccines hold great promise for improved cancer treatment. However, endosomal trapping and low immunogenicity of tumour antigens usually limit the efficiency of vaccination strategies. Here, we present a proton-driven nanotransformer-based vaccine, comprising a polymer-peptide conjugate-based nanotransformer and loaded antigenic peptide. The nanotransformer-based vaccine induces a strong immune response without substantial systemic toxicity. In the acidic endosomal environment, the nanotransformer-based vaccine undergoes a dramatic morphological change from nanospheres (about 100 nanometres in diameter) into nanosheets (several micrometres in length or width), which mechanically disrupts the endosomal membrane and directly delivers the antigenic peptide into the cytoplasm. The re-assembled nanosheets also boost tumour immunity via activation of specific inflammation pathways. The nanotransformer-based vaccine effectively inhibits tumour growth in the B16F10-OVA and human papilloma virus-E6/E7 tumour models in mice. Moreover, combining the nanotransformer-based vaccine with anti-PD-L1 antibodies results in over 83 days of survival and in about half of the mice produces complete tumour regression in the B16F10 model. This proton-driven transformable nanovaccine offers a robust and safe strategy for cancer immunotherapy.
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http://dx.doi.org/10.1038/s41565-020-00782-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719078PMC
December 2020

Nanoparticle-encapsulated siRNAs for gene silencing in the haematopoietic stem-cell niche.

Nat Biomed Eng 2020 11 5;4(11):1076-1089. Epub 2020 Oct 5.

Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

Bone-marrow endothelial cells in the haematopoietic stem-cell niche form a network of blood vessels that regulates blood-cell traffic as well as the maintenance and function of haematopoietic stem and progenitor cells. Here, we report the design and in vivo performance of systemically injected lipid-polymer nanoparticles encapsulating small interfering RNA (siRNA), for the silencing of genes in bone-marrow endothelial cells. In mice, nanoparticles encapsulating siRNA sequences targeting the proteins stromal-derived factor 1 (Sdf1) or monocyte chemotactic protein 1 (Mcp1) enhanced (when silencing Sdf1) or inhibited (when silencing Mcp1) the release of stem and progenitor cells and of leukocytes from the bone marrow. In a mouse model of myocardial infarction, nanoparticle-mediated inhibition of cell release from the haematopoietic niche via Mcp1 silencing reduced leukocytes in the diseased heart, improved healing after infarction and attenuated heart failure. Nanoparticle-mediated RNA interference in the haematopoietic niche could be used to investigate haematopoietic processes for therapeutic applications in cancer, infection and cardiovascular disease.
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http://dx.doi.org/10.1038/s41551-020-00623-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655681PMC
November 2020

Exploiting the placenta for nanoparticle-mediated drug delivery during pregnancy.

Adv Drug Deliv Rev 2020 18;160:244-261. Epub 2020 Sep 18.

Department of Bioengineering, University of Pennsylvania, 210 South 33rd Street, Philadelphia, PA 19104, USA; Department of Biomedical Engineering, Rowan University, 201 Mullica Hill Road, Glassboro, NJ 08028, USA. Electronic address:

A major challenge to treating diseases during pregnancy is that small molecule therapeutics are transported through the placenta and incur toxicities to the developing fetus. The placenta is responsible for providing nutrients, removing waste, and protecting the fetus from toxic substances. Thus, the placenta acts as a biological barrier between the mother and fetus that can be exploited for drug delivery. Nanoparticle technologies provide the opportunity for safe drug delivery during pregnancy by controlling how therapeutics interact with the placenta. In this Review, we present nanoparticle drug delivery technologies specifically designed to exploit the placenta as a biological barrier to treat maternal, placental, or fetal diseases exclusively, while minimizing off-target toxicities. Further, we discuss opportunities, challenges, and future directions for implementing drug delivery technologies during pregnancy.
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http://dx.doi.org/10.1016/j.addr.2020.09.006DOI Listing
September 2021

Summary From the First Kidney Cancer Research Summit, September 12-13, 2019: A Focus on Translational Research.

J Natl Cancer Inst 2021 03;113(3):234-243

Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Kidney cancer is one of the 10 most common cancers both in the United States and worldwide. Until this year, there had not previously been a conference focused on translational studies in the broad and heterogeneous group of kidney cancers. Therefore, a group of researchers, clinicians, and patient advocates dedicated to renal cell carcinoma launched the Kidney Cancer Research Summit (KCRS) to spur collaboration and further therapeutic advances in these tumors. This commentary aims to summarize the oral presentations and serve as a record for future iterations of this meeting. The KCRS sessions addressed the tumor microenvironment, novel methods of drug delivery, single cell sequencing strategies, novel immune checkpoint blockade and cellular therapies, predictive biomarkers, and rare variants of kidney cancers. In addition, the meeting included 2 sessions to promote scientific mentoring and kidney cancer research collaborations. A subsequent KCRS will be planned for the fall of 2020.
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http://dx.doi.org/10.1093/jnci/djaa064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7936057PMC
March 2021

Cyclodextrins in drug delivery: applications in gene and combination therapy.

Drug Deliv Transl Res 2020 06;10(3):661-677

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

Gene therapy is a powerful tool against genetic disorders and cancer, targeting the source of the disease rather than just treating the symptoms. While much of the initial success of gene delivery relied on viral vectors, non-viral vectors are emerging as promising gene delivery systems for efficacious treatment with decreased toxicity concerns. However, the delivery of genetic material is still challenging, and there is a need for vectors with enhanced targeting, reduced toxicity, and controlled release. In this article, we highlight current work in gene therapy which utilizes the cyclic oligosaccharide molecule cyclodextrin (CD). With a number of unique abilities, such as hosting small molecule drugs, acting as a linker or modular component, reducing immunogenicity, and disrupting membranes, CD is a valuable constituent in many delivery systems. These carriers also demonstrate great promise in combination therapies, due to the ease of assembling macromolecular structures and wide variety of chemical derivatives, which allow for customizable delivery systems and co-delivery of therapeutics. The use of combination and personalized therapies can result in improved patient health-modular systems, such as those which incorporate CD, are more conducive to these therapy types. Graphical abstract.
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http://dx.doi.org/10.1007/s13346-020-00724-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524011PMC
June 2020

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

Chiral Supraparticles for Controllable Nanomedicine.

Adv Mater 2020 Jan 5;32(1):e1903878. Epub 2019 Nov 5.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Chirality is ubiquitous in nature and hard-wired into every biological system. Despite the prevalence of chirality in biological systems, controlling biomaterial chirality to influence interactions with cells has only recently been explored. Chiral-engineered supraparticles (SPs) that interact differentially with cells and proteins depending on their handedness are presented. SPs coordinated with d-chirality demonstrate greater than threefold enhanced cell membrane penetration in breast, cervical, and multiple myeloma cancer cells. Quartz crystal microbalance with dissipation and isothermal titration calorimetry measurements reveal the mechanism of these chiral-specific interactions. Thermodynamically, d-SPs show more stable adhesion to lipid layers composed of phospholipids and cholesterol compared to l-SPs. In vivo, d-SPs exhibit superior stability and longer biological half-lives likely due to opposite chirality and thus protection from endogenous proteins including proteases. This work shows that incorporating d-chirality into nanosystems enhances uptake by cancer cells and prolonged in vivo stability in circulation, providing support for the importance of chirality in biomaterials. Thus, chiral nanosystems may have the potential to provide a new level of control for drug delivery systems, tumor detection markers, biosensors, and other biomaterial-based devices.
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http://dx.doi.org/10.1002/adma.201903878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986383PMC
January 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

Nanomaterial Interactions with Human Neutrophils.

ACS Biomater Sci Eng 2018 Dec 5;4(12):4255-4265. Epub 2018 Nov 5.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

Neutrophils are the most abundant circulating leukocyte and the first point of contact between many drug delivery formulations and human cells. Despite their prevalence and implication in a range of immune functions, little is known about how human neutrophils respond to synthetic particulates. Here, we describe how human neutrophils respond to particles which vary in both size (5 nm to 2 m) and chemistry (lipids, poly(styrene), poly(lactic--glycolic acid), and gold). In particular, we show that (i) particle uptake is rapid, typically plateauing within 15 min; (ii) for a given particle chemistry, neutrophils preferentially take up larger particles at the nanoscale, up to 200 nm in size; (iii) uptake of nanoscale poly(styrene) and liposomal particles at concentrations of up to 5 g/mL does not enhance apoptosis, activation, or cell death; (iv) particle-laden neutrophils retain the ability to degranulate normally in response to chemical stimulation; and (v) ingested particles reside in intracellular compartments that are retained during activation and degranulation. Aside from the implications for design of intravenously delivered particulate formulations in general, we expect these observations to be of particular use for targeting nanoparticles to circulating neutrophils, their clearance site (bone marrow), or distal sites of active inflammation.
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http://dx.doi.org/10.1021/acsbiomaterials.8b01062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731026PMC
December 2018

Recommendations for clinical laboratory testing of activated protein C resistance; communication from the SSC of the ISTH.

J Thromb Haemost 2019 09 17;17(9):1555-1561. Epub 2019 Jul 17.

Colorado Coagulation, Laboratory Corporation of America Holdings, Englewood, Colorado.

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http://dx.doi.org/10.1111/jth.14532DOI Listing
September 2019

Nanoparticles for nucleic acid delivery: Applications in cancer immunotherapy.

Cancer Lett 2019 08 14;458:102-112. Epub 2019 May 14.

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

Immunotherapy has recently emerged as a powerful tool for cancer treatment. Early clinical successes from cancer immunotherapy have led to a growing list of FDA approvals, and many new therapies are in clinical and preclinical development. Nucleic acid therapeutics, including DNA, mRNA, and genome editing systems, hold significant potential as a form of immunotherapy due to its robust use in cancer vaccination, adoptive T-cell therapy, and gene regulation. However, these therapeutics must overcome numerous delivery obstacles to be successful, including rapid in vivo degradation, poor uptake into target cells, required nuclear entry, and potential in vivo toxicity in healthy cells and tissues. Nanoparticle delivery systems have been engineered to overcome several of these barriers as a means to safely and effectively deliver nucleic acid therapeutics to immune cells. In this Review, we discuss the applications of nucleic acid therapeutics in cancer immunotherapy, and we detail how nanoparticle platforms have been designed to deliver mRNA, DNA, and genome editing systems to enhance the potency and safety of these therapeutics.
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http://dx.doi.org/10.1016/j.canlet.2019.04.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613653PMC
August 2019

Delivery technologies for cancer immunotherapy.

Nat Rev Drug Discov 2019 03;18(3):175-196

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

Immunotherapy has become a powerful clinical strategy for treating cancer. The number of immunotherapy drug approvals has been increasing, with numerous treatments in clinical and preclinical development. However, a key challenge in the broad implementation of immunotherapies for cancer remains the controlled modulation of the immune system, as these therapeutics have serious adverse effects including autoimmunity and nonspecific inflammation. Understanding how to increase the response rates to various classes of immunotherapy is key to improving efficacy and controlling these adverse effects. Advanced biomaterials and drug delivery systems, such as nanoparticles and the use of T cells to deliver therapies, could effectively harness immunotherapies and improve their potency while reducing toxic side effects. Here, we discuss these research advances, as well as the opportunities and challenges for integrating delivery technologies into cancer immunotherapy, and we critically analyse the outlook for these emerging areas.
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http://dx.doi.org/10.1038/s41573-018-0006-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410566PMC
March 2019

Publisher Correction: Engineering and physical sciences in oncology: challenges and opportunities.

Nat Rev Cancer 2018 11;18(11):720

Department of Chemical Engineering, David H. Koch Institute for Integrated Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts, 02139, USA.

The article as originally published incorrectly cited reference 31 in the legend for figure 7. The correct reference should have been reference 132 (Angelo, M. et al. Multiplexed ion beam imaging of human breast tumors. Nat. Med. 20, 436-442 (2014)).
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http://dx.doi.org/10.1038/s41568-018-0072-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357229PMC
November 2018

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

Potent in vivo lung cancer Wnt signaling inhibition via cyclodextrin-LGK974 inclusion complexes.

J Control Release 2018 11 2;290:75-87. Epub 2018 Oct 2.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Activation of the Wnt signaling pathway promotes lung cancer progression and contributes to poor patient prognosis. The porcupine inhibitor LGK974, a novel orally bioavailable cancer therapeutic in Phase I clinical trials, induces potent Wnt signaling inhibition and leads to suppressed growth and progression of multiple types of cancers. The clinical use of LGK974, however, is limited in part due to its low solubility and high toxicity in tissues that rely on Wnt signaling for normal homeostasis. Here, we report the use of host-guest chemistry to enhance the solubility and bioavailability of LGK974 in mice through complexation with cyclodextrins (CD). We assessed the effects of these complexes to inhibit Wnt signaling in lung adenocarcinomas that are typically driven by overactive Wnt signaling. 2D H NMR confirmed host-guest complexation of CDs with LGK974. CD:LGK974 complexes significantly decreased the expression of Wnt target genes in lung cancer organoids and in lung cancer allografts in mice. Further, CD:LGK974 complexes increased the bioavailability upon oral administration in mice compared to free LGK974. In a mouse lung cancer allograft model, CD:LGK974 complexes induced potent Wnt signaling inhibition with reduced intestinal toxicity compared to treatment with free drug. Collectively, the development of these complexes enables safer and repeated oral or parenteral administration of Wnt signaling inhibitors, which hold promise for the treatment of multiple types of malignancies.
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http://dx.doi.org/10.1016/j.jconrel.2018.09.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441337PMC
November 2018

Loss of Calmodulin- and Radial-Spoke-Associated Complex Protein CFAP251 Leads to Immotile Spermatozoa Lacking Mitochondria and Infertility in Men.

Am J Hum Genet 2018 09 16;103(3):413-420. Epub 2018 Aug 16.

Aix Marseille Université, INSERM, Marseille Medical Genetics, U 1251, Marseille, France. Electronic address:

Flagella and motile cilia share a 9 + 2 microtubule-doublet axoneme structure, and asthenozoospermia (reduced spermatozoa motility) is found in 76% of men with primary ciliary dyskinesia (PCD). Nevertheless, causal genetic variants in a conserved axonemal component have been found in cases of isolated asthenozoospermia: 30% of men with multiple morphological anomalies of sperm flagella (MMAF) carry bi-allelic mutations in DNAH1, encoding one of the seven inner-arm dynein heavy chains of the 9 + 2 axoneme. To further understand the basis for isolated asthenozoospermia, we used whole-exome and Sanger sequencing to study two brothers and two independent men with MMAF. In three men, we found bi-allelic loss-of-function mutations in WDR66, encoding cilia- and flagella-associated protein 251 (CFAP251): the two brothers were homozygous for the frameshift chr12: g.122359334delA (p.Asp42Metfs4), and the third individual was compound heterozygous for chr12: g.122359542G>T (p.Glu111) and chr12: g.122395032_122395033delCT (p.Leu530Valfs4). We show that CFAP251 is normally located along the flagellum but is absent in men carrying WDR66 mutations and reveal a spermatozoa-specific isoform probably generated during spermatozoon maturation. CFAP251 is a component of the calmodulin- and radial-spoke- associated complex, located adjacent to DNAH1, on the inner surface of the peripheral microtubule doublets of the axoneme. In Tetrahymena, the CFAP251 ortholog is necessary for efficient coordinated ciliary beating. Using immunofluorescent and transmission electron microscopy, we provide evidence that loss of CFAP251 affects the formation of the mitochondrial sheath. We propose that CFAP251 plays a structural role during biogenesis of the spermatozoon flagellum in vertebrates.
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http://dx.doi.org/10.1016/j.ajhg.2018.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128251PMC
September 2018
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