ORCID Profile
0000-0001-5611-4515
Current Organisation
Monash Institute of Pharmaceutical Sciences
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Nanotechnology | Nanobiotechnology | Nanomedicine | Nanotechnology | Biomedical Engineering | Pharmaceutical Sciences | Cellular Interactions (incl. Adhesion, Matrix, Cell Wall) | Pharmacology and Pharmaceutical Sciences | Biomaterials | Colloid And Surface Chemistry | Biomaterials | Nanochemistry and Supramolecular Chemistry | Nanomaterials | Biochemistry and Cell Biology | Colloid and Surface Chemistry | Macromolecular and Materials Chemistry | Synthesis of Materials | Biomechanical Engineering | Physical Chemistry (Incl. Structural) | Biotechnology Not Elsewhere Classified | Immunology not elsewhere classified | Nanoscale Characterisation | Biochemistry and Cell Biology not elsewhere classified | Optical Physics Not Elsewhere Classified | Functional Materials | Cellular Immunology |
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in Engineering | Biological sciences | Expanding Knowledge in the Medical and Health Sciences | Cancer and Related Disorders | Prevention—biologicals (e.g. vaccines) | Plant Production and Plant Primary Products not elsewhere classified | Inherited Diseases (incl. Gene Therapy) | Nervous System and Disorders | Human Biological Preventatives (e.g. Vaccines) | Chemical sciences | Physical sciences | Scientific instrumentation | Human Pharmaceutical Treatments (e.g. Antibiotics) | Diagnostic Methods | Human Pharmaceutical Products not elsewhere classified | Diagnostics | Diagnostics | Immune System and Allergy
Related Links
Publications
Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics
Publisher: Springer Science and Business Media LLC
Date: 03-2021
DOI: 10.1038/S41565-021-00858-8
Abstract: Endocytosis is a critical step in the process by which many therapeutic nanomedicines reach their intracellular targets. Our understanding of cellular uptake mechanisms has developed substantially in the past five years. However, these advances in cell biology have not fully translated to the nanoscience and therapeutics literature. Misconceptions surrounding the role of different endocytic pathways and how to study these pathways are hindering progress in developing improved nanoparticle therapies. Here, we summarize the latest insights into cellular uptake mechanisms and pathways. We highlight limitations of current systems to study endocytosis, particularly problems with non-specific inhibitors. We also summarize alternative genetic approaches to robustly probe these pathways and discuss the need to understand how cells endocytose particles in vivo. We hope that this critical assessment of the current methods used in studying nanoparticle uptake will guide future studies at the interface of cell biology and nanomedicine.
It's what's on the inside that counts: Techniques for investigating the uptake and recycling of nanoparticles and proteins in cells
Publisher: Elsevier BV
Date: 04-2021
Engineering cellular degradation of multilayered capsules through controlled cross-linking
Publisher: American Chemical Society (ACS)
Date: 02-11-2012
DOI: 10.1021/NN3039353
Abstract: We report a versatile approach for controlling the intracellular degradation of polymer capsules by tailoring the degree of cross-linking in the capsules. Poly(2-diisopropylaminoethyl methacrylate) capsules were assembled by the layer-by-layer technique and covalently stabilized with a redox-responsive bisazide cross-linker using click chemistry. The degree of cross-linking, determined using radiation scintillation counting, was tuned from 65% to 98% by adjusting the amount of cross-linker used to stabilize the polymer films. Transmission electron microscopy and fluorescence microscopy studies showed that the pH responsiveness of the capsules was maintained, regardless of the degree of cross-linking. Atomic force microscopy measurements on planar surfaces revealed that increasing the degree of cross-linking decreased the film roughness (from 8.7 to 1.7 nm), hence forming smoother films however the film thicknesses were not significantly altered. Cellular studies showed that the rate of intracellular degradation of the capsules could be controlled between 0 and 6 h by altering the degree of cross-linking in the polymer capsules. These studies also demonstrated that the cellular degradation of highly cross-linked capsules (>90%) was significantly retarded compared to degradation in simulated cellular conditions. This suggests that the naturally occurring cellular reducing environment is rapidly depleted, and there is a significant delay before the cells can replenish the reducing environment. The modular and versatile nature of this approach lends itself to application to a wide range of polymer carriers and thus offers significant potential for the design of polymer-based systems for drug and gene delivery.
Polyelectrolyte functionalization of electrospun fibers
Publisher: American Chemical Society (ACS)
Date: 29-03-2006
DOI: 10.1021/CM052760K
Controlled release of DNA from poly(vinylpyrrolidone) capsules using cleavable linkers
Publisher: Elsevier BV
Date: 09-2011
DOI: 10.1016/J.BIOMATERIALS.2011.05.011
Abstract: The design of polymer carriers with tunable degradation and cargo release is fundamental for applications in drug and gene delivery. In this study, we report low-fouling poly(N-vinyl pyrrolidone) (PVPON) capsules assembled via hydrogen bonding and stabilized using covalent cross-linking. We first investigated the effects of pH and ionic strength to optimize the assembly conditions. A model therapeutic cargo (plasmid DNA) was then loaded in the capsules and used for encapsulation and release studies. Two bisazide cross-linkers that contain a disulfide bond, termed PEG₈ (poly(ethylene glycol)) and PEG(16), were employed to stabilize the multilayer films, and used to tune the degradation and cargo release behavior of the capsules in simulated cytoplasmic conditions. The results suggest that PEG₈-stabilized capsules were more efficiently cross-linked, and hence displayed higher plasmid encapsulation. Consequently, the capsules cross-linked with PEG₈ also showed a two-fold reduction in degradation rate. This ability to achieve controlled carrier degradation and cargo release makes these capsules of potential interest for drug and gene delivery.
Identifying cell-to-cell variability in internalisation using flow cytometry
Publisher: Cold Spring Harbor Laboratory
Date: 25-11-2021
DOI: 10.1101/2021.11.24.469957
Abstract: Biological heterogeneity is a primary contributor to the variation observed in experiments that probe dynamical processes, such as internalisation. Given that internalisation is a critical process by which many therapeutics and viruses reach their intracellular site of action, quantifying cell-to-cell variability in internalisation is of high biological interest. Yet, it is common for studies of internalisation to neglect cell-to-cell variability. We develop a simple mathematical model of internalisation that captures the dynamical behaviour, cell-to-cell variation, and extrinsic noise introduced by flow cytometry. We calibrate our model through a novel distribution-matching approximate Bayesian computation algorithm to flow cytometry data of internalisation of anti-transferrin receptor antibody in a human B-cell lymphoblastoid cell line. Our model reproduces experimental observations, identifies cell-to-cell variability in the internalisation and recycling rates, and, importantly, provides information relating to inferential uncertainty. Given that our approach is agnostic to s le size and signal-to-noise ratio, our modelling framework is broadly applicable to identify biological variability in single-cell data from internalisation assays and similar experiments that probe cellular dynamical processes.
Understanding the Polymer Rearrangement of pH-Responsive Nanoparticles
Publisher: CSIRO Publishing
Date: 23-02-2021
DOI: 10.1071/CH20331
Abstract: The use of self-assembled nanoparticles for drug delivery has received significant attention in recent years. However, the dynamic nature of self-assembled polymeric systems means there is a need to develop greater understanding of the inherent stability of these systems. In particular, understanding if these materials remain as discrete nanoparticles, or if there is dynamic exchange of material between particles is critical. Herein, we labelled pH-responsive nanoparticles with fluorescent dyes and then investigated the change in fluorescence when the particles were mixed with unlabelled nanoparticles in order to investigate their potential for polymer rearrangement. Nanoparticles were formed by the nanoprecipitation of pH-responsive poly(ethylene glycol)-block-poly(2-(diethylamino)ethyl methacrylate) (PEG-b-PDEAEMA) as the shell and poly(2-(diethylamino)ethyl methacrylate)-random-poly(2-(diisopropylamino)ethyl methacrylate) (PDEAEMA-r-PDPAEMA) as the core. The core and shell were labelled by incorporating pentafluorophenyl methacrylate (PFPMA) in core or shell respectively and then coupling with either Sulfo-cyanine5 amine or Cyanine3 amine. Exchange of material between nanoparticles was probed by tracking changes in the self-quenching of fluorescently labelled polymers in the core of the nanoparticles. The fluorescence intensity of the labelled nanoparticles was stable when mixed with unlabelled nanoparticles at physiological pH (pH 7.4), suggesting there is limited migration of polymers between particles in this system. This study provides important insights into the use of non-crosslinked nanoparticles under biologically relevant conditions.
Silicon-Nanotube-Mediated Intracellular Delivery Enables Ex Vivo Gene Editing
Publisher: Wiley
Date: 06-05-2020
Controlling Endosomal Escape Using pH-Responsive Nanoparticles with Tunable Disassembly
Publisher: American Chemical Society (ACS)
Date: 05-06-2018
Assembling DNA into advanced materials: From nanostructured films to biosensing and delivery systems
Publisher: Wiley
Date: 16-10-2007
Probing the permeability of polyelectrolyte multilayer capsules via a molecular beacon approach
Publisher: American Chemical Society (ACS)
Date: 06-03-2007
DOI: 10.1021/LA063674H
Abstract: Application of polyelectrolyte multilayer (PEM) capsules as vehicles for the controlled delivery of substances, such as drugs, genes, pesticides, cosmetics, and foodstuffs, requires a sound understanding of the permeability of the capsules. We report the results of a detailed investigation into probing capsule permeability via a molecular beacon (MB) approach. This method involves preparing MB-functionalized bimodal mesoporous silica (BMSMB) particles, encapsulating the BMSMB particles within the PEM film to be probed, and then incubating the encapsulated BMSMB particles with DNA target sequences of different lengths. Permeation of the DNA targets through the capsule shell causes the immobilized MBs to open due to hybridization of the DNA targets with the complementary loop region of the MBs, resulting in an increase in the MB fluorescence. The assay conditions (BMSMB particle concentration, MB loading within the BMS particles, DNA target concentration, DNA target size, pH, sodium chloride concentration) where the MB-DNA sensing process is effective were first examined. The permeability of DNA through poly(sodium 4-styrenesulfonate) (PSS) oly(allylamine hydrochloride) (PAH) multilayer films, with and without a poly(ethyleneimine) (PEI) precursor layer, was then investigated. The permeation of the DNA targets decreases considerably as the thickness of the PEM film encapsulating the BMSMB particles increases. Furthermore, the presence of a PEI precursor layer gives rise to less permeable PSS/PAH multilayers. The diffusion coefficients calculated for the DNA targets through the PEM capsules range from 10-19 to 10-18 m2 s-1. This investigation demonstrates that the MB approach to measuring permeability is an important new tool for the characterization of PEM capsules and is expected to be applicable for probing the permeability of other systems, such as membranes, liposomes, and emulsions.
Peptide-functionalized, low-biofouling click multilayers for promoting cell adhesion and growth
Publisher: Wiley
Date: 19-02-2009
Tuning particle biodegradation through polymer-peptide blend composition
Publisher: American Chemical Society (ACS)
Date: 31-10-2014
DOI: 10.1021/BM5012272
Abstract: We report the preparation of polymer-peptide blend replica particles via the mesoporous silica (MS) templated assembly of poly(ethylene glycol)-block-poly(2-diisopropylaminoethyl methacrylate-co-2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethyl methacrylate) (PEG45-b-P(DPA55-co-PgTEGMA4)) and poly(l-histidine) (PHis). PEG45-b-P(DPA55-co-PgTEGMA4) was synthesized by atom transfer radical polymerization (ATRP), and was coinfiltrated with PHis into poly(methacrylic acid) (PMA)-coated MS particles assembled from different peptide-to-polymer ratios (1:1, 1:5, 1:10, or 1:15). Subsequent removal of the sacrificial templates and PMA resulted in monodisperse, colloidally stable, noncovalently cross-linked polymer-peptide blend replica particles that were stabilized by a combination of hydrophobic interactions between the PDPA and the PHis, hydrogen bonding between the PEG and PHis backbone, and π-π stacking of the imidazole rings of PHis side chains at physiological pH (pH ∼ 7.4). The synergistic charge-switchable properties of PDPA and PHis, and the enzymatic degradability of PHis, make these particles responsive to pH and enzymes. In vitro studies, in simulated endosomal conditions and inside cells, demonstrated that particle degradation kinetics could be engineered (from 2 to 8 h inside dendritic cells) based on simple adjustment of the peptide-to-polymer ratio used.
Pointing in the Right Direction: Controlling the Orientation of Proteins on Nanoparticles Improves Targeting Efficiency
Publisher: American Chemical Society (ACS)
Date: 18-02-2019
DOI: 10.1021/ACS.NANOLETT.8B04916
Abstract: Protein-conjugated nanoparticles have the potential to precisely deliver therapeutics to target sites in the body by specifically binding to cell surface receptors. To maximize targeting efficiency, the three-dimensional presentation of ligands toward these receptors is crucial. Herein, we demonstrate significantly enhanced targeting of nanoparticles to cancer cells by controlling the protein orientation on the nanoparticle surface. To engineer the point of attachment, we used amber codon reassignment to incorporate a synthetic amino acid, p-azidophenylalanine (azPhe), at specific locations within a single domain antibody (sdAb or nanobody) that recognizes the human epidermal growth factor receptor (EGFR). The azPhe modified sdAb can be tethered to the nanoparticle in a specific orientation using a bioorthogonal click reaction with a strained cyclooctyne. The crystal structure of the sdAb bound to EGFR was used to rationally select sites likely to optimally display the sdAb upon conjugation to a fluorescent nanocrystal (Qdot). Qdots with sdAb attached at the azPhe13 position showed 6 times greater binding affinity to EGFR expressing A549 cells, compared to Qdots with conventionally (succinimidyl ester) conjugated sdAb. As ligand-targeted delivery systems move toward clinical application, this work shows that nanoparticle targeting can be optimized by engineering the site of protein conjugation.
Ultrathin, responsive polymer click capsules
Publisher: American Chemical Society (ACS)
Date: 26-05-2007
DOI: 10.1021/NL070698F
Abstract: We report a general click chemistry approach for the layer-by-layer assembly of ultrathin, polymer films on particles and the subsequent formation of polymer click capsules (CCs). Poly(acrylic acid) copolymers, synthesized with a minor component of either alkyne (PAA-Alk) or azide (PAA-Az) functionality, were alternately assembled on silica particles. The (PAA-Az/PAA-Alk)-coated particles were subsequently functionalized by exploiting the free alkyne click moieties present in the film upon exposure to an azide-modified rhodamine dye. Further, PAA CCs, obtained following removal of the silica particle template, were shown to exhibit pH-responsive behavior. This was demonstrated by reversible size changes of the CCs upon cycling between basic and acidic solutions. Polymer CCs are anticipated to find applications in various fields, including drug delivery and sensing.
Uptake and intracellular fate of disulfide-bonded polymer hydrogel capsules for doxorubicin delivery to colorectal cancer cells
Publisher: American Chemical Society (ACS)
Date: 26-04-2010
DOI: 10.1021/NN100173H
Abstract: Understanding the interactions between drug carriers and cells is of importance to enhance the delivery of therapeutics. The release of therapeutics into different intracellular environments, such as the lysosomes or the cell cytoplasm, will impact their pharmacological activity. Herein, we investigate the intracellular fate of layer-by-layer (LbL)-assembled, submicrometer-sized polymer hydrogel capsules in a human colon cancer derived cell line, LIM1899. The cellular uptake of the disulfide-stabilized poly(methacrylic acid) (PMA(SH)) capsules by colon cancer cells is a time-dependent process. Confocal laser scanning microscopy and transmission electron microscopy reveal that the internalized capsules are deformed in membrane-enclosed compartments, which further mature to late endosomes or lysosomes. We further demonstrate the utility of these redox-responsive PMA(SH) capsules for the delivery of doxorubicin (DOX) to colon cancer cells. The DOX-loaded PMA(SH) capsules demonstrate a 5000-fold enhanced cytotoxicity in cell viability studies compared to free DOX.
Targeting dendritic cells: a promising strategy to improve vaccine effectiveness
Publisher: Wiley
Date: 03-2016
DOI: 10.1038/CTI.2016.6
Stabilization of DNA multilayer films through oligonucleotide crosslinking
Publisher: Wiley
Date: 05-2008
Abstract: Multilayer films and capsules synthesized from DNA are of interest because they are biodegradable, biocompatible, and the structure of the films can be finely controlled by base pairing of the nucleotides. As DNA films are held together through a balance between the attractive hydrogen bonding and the aromatic stacking of the base pairs and the electrostatic repulsion of the negatively charged phosphate backbones, the films can be subject to disintegration at low salt concentrations (<200 mM). Enhancement of the stability of the films is essential if they are to be used in bioapplications. Herein, we describe an approach to form DNA films and capsules that are stable under a variety of buffer conditions, including low salt concentrations (down to 25 mM NaCl). The films are assembled using a triblock oligonucleotide system, in which the two outer blocks facilitate the assembly of the film and the middle block can be used to stabilize the films by hybridizing oligonucleotide sequences that crosslink the films. Additionally, crosslinked DNA capsules are shown to exhibit significantly different shrinkage properties to those of noncrosslinked capsules, thus demonstrating further control over the capsule properties. These DNA capsules are envisaged to find applications as drug-delivery vehicles, in diagnostics, and as microreactors.
Engineering enzyme-cleavable hybrid click capsules with a pH-sheddable coating for intracellular degradation
Publisher: Wiley
Date: 10-07-2014
Abstract: The engineering of layer-by-layer (LbL) hybrid click capsules that are responsive to biological stimuli is reported. The capsules comprise a pH-sheddable, non cross-linked outer coating that protects enzyme-cleavable inner layers. Upon cellular uptake, the outer coating is released and the capsules are enzymatically degraded. In vitro cell degradation results in rapid capsule degradation (10 min) upon cellular internalization.
In Vivo Quantification of Nanoparticle Association with Immune Cell Subsets in Blood
Publisher: Wiley
Date: 28-02-2021
Targeting and uptake of multilayered particles to colorectal cancer cells
Publisher: Wiley
Date: 06-07-2006
Exploiting the directionality of DNA: Controlled shrinkage of engineered oligonucleotide capsules
Publisher: Wiley
Date: 23-03-2007
SNAPSwitch: A Molecular Sensor to Quantify the Localization of Proteins, DNA and Nanoparticles in Cells
Publisher: Cold Spring Harbor Laboratory
Date: 17-12-2019
DOI: 10.1101/2019.12.16.877043
Abstract: Intracellular trafficking governs receptor signalling, pathogenesis, immune responses and the cellular fate of nanomedicines. These processes are typically tracked by confocal microscopy, where colocalization of fluorescent markers implies an interaction or co-compartmentalization. However, this type of analysis is inherently low-throughput, is limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we have developed a localization sensor composed of a quenched and attachable SNAP-tag substrate (SNAP Switch ). SNAP Switch enables quantitative detection of protein, nucleic acid and nanoparticle trafficking to locations of interest within live cells using flow cytometry. Using this approach, we followed the trafficking of DNA complexes travelling from endosomes into the cytosol and to the nucleus. We also show that antibody targeted to the transferrin (CD71) or hyaluronan (CD44) receptor is initially sorted into different compartments following endocytosis. These results demonstrate SNAP Switch is a high-throughput and broadly applicable tool to quantitatively track the localization of materials in cells.
Antibody-mediated targeting of antigen to C-type lectin-like receptors Clec9A and Clec12A elicits different vaccination outcomes
Publisher: Elsevier BV
Date: 2017
DOI: 10.1016/J.MOLIMM.2016.12.010
Abstract: Targeting antigen (Ag) to dendritic cell (DC) surface receptors is a potential new mode of vaccination. C-type lectin-like receptors Clec9A and Clec12A are attractive receptor targets however their targeting in vivo elicits significantly different outcomes for unknown reasons. To gain insight into the mechanisms responsible, we have examined the intrinsic capacity of Clec9A and Clec12A to elicit MHC I and MHC II Ag presentation following ex vivo targeting with primary murine DC. Both receptors exhibited high rates of internalization by CD8
Nanoescapology: progress toward understanding the endosomal escape of polymeric nanoparticles
Publisher: Wiley
Date: 03-02-2017
DOI: 10.1002/WNAN.1452
Abstract: Using nanoparticles to deliver drugs to cells has the potential to revolutionize the treatment of many diseases, including HIV , cancer, and diabetes. One of the major challenges facing this field is controlling where the drug is trafficked once the nanoparticle is taken up into the cell. In particular, if drugs remain localized in an endosomal or lysosomal compartment, the therapeutic can be rendered completely ineffective. To ensure the design of more effective delivery systems we must first develop a better understanding of how nanoparticles and their cargo are trafficked inside cells. This needs to be combined with an understanding of what characteristics are required for nanoparticles to achieve endosomal escape, along with methods to detect endosomal escape effectively. This review is focused into three sections: first, an introduction to the mechanisms governing internalization and trafficking in cells, second, a discussion of methods to detect endosomal escape, and finally, recent advances in controlling endosomal escape from polymer‐ and lipid‐based nanoparticles, with a focus on engineering materials to promote endosomal escape. WIREs Nanomed Nanobiotechnol 2017, 9:e1452. doi: 10.1002/wnan.1452 This article is categorized under: Nanotechnology Approaches to Biology Nanoscale Systems in Biology
Antibody-dependent phagocytosis (ADP) responses following trivalent inactivated influenza vaccination of younger and older adults
Publisher: Elsevier BV
Date: 11-2017
DOI: 10.1016/J.VACCINE.2017.09.062
Abstract: Globally the most commonly utilised immunisation against influenza is the trivalent inactivated influenza vaccine (TIV) derived from an A/H1N1, an A/H3N2 and aB type influenza virus. Vaccine effectiveness of TIV varies year to year, depending on how well antigenically matched the strains in the vaccine are compared to circulating strains [1,2]. Moreover, vaccine effectiveness can vary within certain subpopulations such as HIV-positive, young children and the elderly. Decreased vaccine effectiveness in the elderly is associated with impaired Ab production, as measured by standard hemagglutination inhibition (HAI) assays. We investigated the level of Antibody Dependent Phagocytosis (ADP)-mediating Abs induced by the 2008-TIV in healthy Australian adults aged over and under 60years to determine if this immune function was also reduced in the elderly. We utilised an ADP assay that measures the uptake of IgG-opsonised HA-coated fluorescent microspheres by a monocytic cell line. We also measured HA-specific Abs that are close enough to bind to dimeric FcγRIIa ectodomains in an ELISA-based assay. Furthermore, we compared the extent of cross-reactive recognition of erse influenza strains by ADP-mediating Abs found in pre- and post-vaccination sera in both of these groups. We found that young adults and older adults mounted similar ADP activity against HAs contained in the 2008-TIV, despite older adults have diminished HI responses. The level of cross-reactive antibodies against other HAs was limited in both groups. We conclude that seasonal influenza vaccination elicits limited cross-reactive ADP to HA in both young and older adults. New influenza vaccination strategies that elicit cross-reactive and polyfunctional antibodies are needed.
A programmable sensor to probe the internalization of proteins and nanoparticles in live cells
Publisher: Wiley
Date: 19-04-2013
Synthesis and functionalization of nanoengineered materials using click chemistry
Publisher: Elsevier BV
Date: 07-2012
Self-assembling dual component nanoparticles with endosomal escape capability
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5SM00082C
Abstract: This work reports a modular nanoparticle system engineered to undergo disassembly within a biologically relevant pH range. These nanoparticles demonstrate endosomal escape, making them attractive for biomedical applications.
Clickable Cubosomes for Antibody-Free Drug Targeting and Imaging Applications
Publisher: American Chemical Society (ACS)
Date: 20-12-2017
DOI: 10.1021/ACS.BIOCONJCHEM.7B00659
Abstract: The combination of copper-free click chemistry with metabolic labeling offers new opportunities in drug delivery. The objective of this study was to determine whether cubosomes functionalized with azide or dibenzocyclooctyne (DBCO) groups are able to undergo copper-free click chemistry with a strained cyclooctyne or azide, respectively. Phytantriol-based cubosomes were functionalized using phospholipids bearing an azide or DBCO group. The modified cubosome dispersions were characterized using dynamic light scattering, cryo-TEM, and small-angle X-ray scattering. The efficiency of "clickability" was assessed by reacting the cubosomes with a complementary dye and determining bound and unbound dye via size exclusion chromatography. The clickable cubosomes reacted specifically and efficiently with a click-Cy5 dye with minor changes to the size, shape, and structure of the cubosomes. This indicates that cubosomes can retain their unique internal structure while participating in copper-free click chemistry. This proof of concept study paves the way for the use of copper-free click chemistry and metabolic labeling with cubosomes for targeted drug delivery and imaging.
HD Flow Cytometry: An Improved Way to Quantify Cellular Interactions with Nanoparticles
Publisher: Wiley
Date: 05-07-2016
Abstract: Histogram deconvolution flow cytometry enables improved quantification of nanomaterial-cell interactions. The algorithm identifies the positive cells in highly overlapped populations and calculates the fluorescence intensity of the positive population. This technique performs better than commercially available methods with the additional benefit of visualizing the output.
Journey to the Centre of the cell (JTCC)∗: A 3D VR experience derived from migratory breast cancer cell image data
Publisher: ACM
Date: 28-11-2016
pH-Responsive Polymer Nanoparticles for Drug Delivery
Publisher: Wiley
Date: 05-03-2019
Abstract: Stimuli-responsive nanoparticles have the potential to improve the delivery of therapeutics to a specific cell or region within the body. There are many stimuli that have shown potential for specific release of cargo, including variation of pH, redox potential, or the presence of enzymes. pH variation has generated significant interest for the synthesis of stimuli-responsive nanoparticles because nanoparticles are internalized into cells via vesicles that are acidified. Additionally, the tumor microenvironment is known to have a lower pH than the surrounding tissue. In this review, different strategies to design pH-responsive nanoparticles are discussed, focusing on the use of charge-shifting polymers, acid labile linkages, and crosslinking.
Quantifying the Endosomal Escape of pH-Responsive Nanoparticles Using the Split Luciferase Endosomal Escape Quantification Assay
Publisher: American Chemical Society (ACS)
Date: 29-12-2021
Abstract: All nanoparticles have the potential to revolutionize the delivery of therapeutic cargo such as peptides, proteins, and RNA. However, effective cytosolic delivery of cargo from nanoparticles represents a significant challenge in the design of more efficient drug delivery vehicles. Recently, research has centered on designing nanoparticles with the capacity to escape endosomes by responding to biological stimuli such as changes in pH, which occur when nanoparticles are internalized into the endo-/lysosomal pathway. Current endosomal escape assays rely on indirect measurements and yield little quantitative information, which hinders the design of more efficient drug delivery vehicles. Therefore, we adapted the highly sensitive split luciferase endosomal escape quantification (SLEEQ) assay to better understand nanoparticle-induced endosomal escape. We applied SLEEQ to evaluate the endosomal escape behavior of two pH-responsive nanoparticles: the first with a poly(2-diisopropylamino ethyl methacrylate) (PDPAEMA) core and the second with 1:1 ratio of poly(2-diethylamino ethyl methacrylate) (PDEAEMA) and PDPAEMA. SLEEQ directly measured the cytosolic delivery and showed that engineering the nanoparticle disassembly pH could improve the endosomal escape efficiency by fivefold. SLEEQ is a versatile assay that can be used for a wide range of nanomaterials and will improve the development of drug delivery vehicles in the future.
Controlling endosomal escape using nanoparticle composition: Current progress and future perspectives
Publisher: Future Medicine Ltd
Date: 2019
Abstract: Polymer nanoparticles offer significant benefits for improving delivery of biological therapeutics such as DNA and proteins, as they allow the cargo to be protected until it is delivered to a target cell. However, there are still challenges with achieving efficient delivery to the optimal cellular region. One significant roadblock is escape of nanoparticles from within the endosomal/lysosomal compartments into the cytosol. Here, we review the recent advances in understanding endosomal escape of polymer nanoparticles. We also discuss the current progress on investigating how nanoparticle structure can control endosomal escape. It is important to understand the fundamental biological processes that govern endosomal escape in order to design more effective therapeutic delivery systems.
Inducing immune tolerance with dendritic cell-targeting nanomedicines
Publisher: Springer Science and Business Media LLC
Date: 21-12-2021
Targeting of cancer cells using click-functionalized polymer capsules
Publisher: American Chemical Society (ACS)
Date: 26-10-2010
DOI: 10.1021/JA106405C
Abstract: Targeted delivery of drugs to specific cells allows a high therapeutic dose to be delivered to the target site with minimal harmful side effects. Combining targeting molecules with nanoengineered drug carriers, such as polymer capsules, micelles and polymersomes, has significant potential to improve the therapeutic delivery and index of a range of drugs. We present a general approach for functionalization of low-fouling, nanoengineered polymer capsules with antibodies using click chemistry. We demonstrate that antibody (Ab)-functionalized capsules specifically bind to colorectal cancer cells even when the target cells constitute less than 0.1% of the total cell population. This precise targeting offers promise for drug delivery applications.
Interfacing materials science and biology for drug carrier design
Publisher: Wiley
Date: 26-02-2015
Abstract: Over the last ten years, there has been considerable research interest in the development of polymeric carriers for biomedicine. Such delivery systems have the potential to significantly reduce side effects and increase the bioavailability of poorly soluble therapeutics. The design of carriers has relied on harnessing specific variations in biological conditions, such as pH or redox potential, and more recently, by incorporating specific peptide cleavage sites for enzymatic hydrolysis. Although much progress has been made in this field, the specificity of polymeric carriers is still limited when compared with their biological counterparts. To synthesize the next generation of carriers, it is important to consider the biological rationale for materials design. This requires a detailed understanding of the cellular microenvironments and how these can be harnessed for specific applications. In this review, several important physiological cues in the cellular microenvironments are outlined, with a focus on changes in pH, redox potential, and the types of enzymes present in specific regions. Furthermore, recent studies that use such biologically inspired triggers to design polymeric carriers are highlighted, focusing on applications in the field of therapeutic delivery.
Resolving subcellular pH with a quantitative fluorescent lifetime biosensor
Publisher: Cold Spring Harbor Laboratory
Date: 13-04-2022
DOI: 10.1101/2022.04.13.488146
Abstract: Changes in sub-cellular pH play a key role in metabolism, cell growth, membrane transport, and can also be exploited to control cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however these measurements are h ered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, we have developed a method to accurately quantify sub-cellular pH in in idual vesicles using fluorescent lifetime imaging microscopy (pHLIM). pHLIM exploits the linear pH dependant lifetime of the fluorescent protein mApple and uses deep learning models to automatically identify and measure the pH of subcellular compartments. We have engineered mApple fusion proteins to measure the pH of the cytosol, endosomes, lysosomes and demonstrated the utility of pHLIM by measuring pH changes induced by drugs (bafilomycin A1) and polyethylenimine (a common transfection reagent). pHLIM is a simple and quantitative method to measure sub-cellular pH that has the potential to help with the design of the next generation of controlled drug release systems and to understand drug action and disease progression.
Tailoring the interfaces between nematic liquid crystal emulsions and aqueous phases via layer-by-layer assembly
Publisher: American Chemical Society (ACS)
Date: 12-09-2006
DOI: 10.1021/NL061604P
Abstract: We report the assembly of polyelectrolyte multilayer (PEM) films at the interfaces of thermotropic liquid crystal (LC) droplets dispersed in an aqueous phase. Exposure of PEM-coated droplets to surfactant slowed the bipolar-to-radial ordering transition of the LCs by 2 orders of magnitude relative to naked droplets. This shows that PEMs can be used to influence the interactions of analytes with the LC cores of the droplets, allowing tuning of the LC emulsion sensing properties.
Introduction to the Special Issue: Nanomaterials in Immunology
Publisher: Elsevier BV
Date: 06-2018
Impact of the Core Architecture of Dual pH-Responsive Polymer Nanoparticles on Intracellular Delivery of Doxorubicin
Publisher: American Chemical Society (ACS)
Date: 05-06-2023
Probing Endosomal Escape Using pHlexi Nanoparticles
Publisher: Wiley
Date: 27-10-2017
Abstract: The effective escape of nanocarriers from endosomal compartments of the cell remains a major hurdle in nanomedicine. The endosomal escape of pH-responsive, self-assembled, dual component particles based on poly[2-(diethylamino)ethyl methacrylate)(PDEAEMA) and poly(ethylene glycol)-b-poly[2-(diethylamino)ethyl methacrylate) (PEG-b-PDEAEMA) has been recently reported. Herein, we report that polymer molecular weight (M
Minimum information reporting in bio-nano experimental literature
Publisher: Springer Science and Business Media LLC
Date: 09-2018
Organic arsenicals as efficient and highly specific linkers for protein/peptide-polymer conjugation
Publisher: American Chemical Society (ACS)
Date: 20-03-2015
DOI: 10.1021/JACS.5B01140
Abstract: The entropy-driven affinity of trivalent (in)organic arsenicals for closely spaced dithiols has been exploited to develop a novel route to peptide rotein-polymer conjugation. A trivalent arsenous acid (As(III)) derivative (1) obtained from p-arsanilic acid (As(V)) was shown to readily undergo conjugation to the therapeutic peptide salmon calcitonin (sCT) via bridging of the Cys(1)-Cys(7) disulfide, which was verified by RP-HPLC and MALDI-ToF-MS. Conjugation was shown to proceed rapidly (t < 2 min) in situ and stoichiometrically through sequential reduction-conjugation protocols, therefore exhibiting conjugation efficiencies equivalent to those reported for the current leading disulfide-bond targeting strategies. Furthermore, using bovine serum albumin as a model protein, the trivalent organic arsenical 1 was found to demonstrate enhanced specificity for disulfide-bond bridging in the presence of free cysteine residues relative to established maleimide functional reagents. This specificity represents a shift toward potential orthogonality, by clearly distinguishing between the reactivity of mono- and disulfide-derived (vicinal or neighbors-through-space) dithiols. Finally, p-arsanilic acid was transformed into an initiator for aqueous single electron-transfer living radical polymerization, allowing the synthesis of hydrophilic arsenic-functional polymers which were shown to exhibit negligible cytotoxicity relative to a small molecule organic arsenical, and an unfunctionalized polymer control. Poly(poly[ethylene glycol] methyl ether acrylate) (PPEGA480, DPn = 10, Mn,NMR = 4900 g·mol(-1), Đ = 1.07) possessing a pentavalent arsenic acid (As(V)) α-chain end was transformed into trivalent As(III) post-polymerization via initial reduction by biological reducing agent glutathione (GSH), followed by binding of GSH. Conjugation of the resulting As(III)-functional polymer to sCT was realized within 35 min as indicated by RP-HPLC and verified later by thermodynamically driven release of sCT, from the conjugate, in the presence of strong chelating reagent ethanedithiol.
Fundamental studies of hybrid poly(2-(diisopropylamino)ethyl methacrylate)/poly(N-vinylpyrrolidone) films and capsules
Publisher: American Chemical Society (ACS)
Date: 27-06-2014
DOI: 10.1021/BM500640T
Abstract: Hybrid and multicompartment carriers are of significant interest for the development of next-generation therapeutic drug carriers. Herein, fundamental investigations on layer-by-layer (LbL) capsules consisting of two different polymers are presented. The hybrid systems were designed to have pH-responsive, charge-shifting poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) inner layers and low-fouling poly(N-vinylpyrrolidone) (PVPON) outer layers. Planar hybrid films with different layer ratios were studied by quartz crystal microgravimetry (QCM) and atomic force microscopy (AFM). The information obtained was translated to particulate templates to prepare hybrid capsules, which were stabilized by click chemistry. The charge-shifting behavior of PDPA improved the cargo encapsulation and initial retention of a model CpG cargo, while outer layers of PVPON improved biofouling properties compared to single-component PDPA capsules. The results demonstrate the need to understand and design multifunctional systems that can successfully embody different functionalities in a single, stable construct for the fabrication of next-generation drug and gene delivery carriers aimed at overcoming the challenges encountered in biological systems.
Role of monocytes in mediating HIV-specific antibody-dependent cellular cytotoxicity
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.JIM.2012.07.006
Abstract: Antibodies (Abs) that mediate antibody-dependent cellular cytotoxicity (ADCC) activity against HIV-1 are of major interest. A widely used method to measure ADCC Abs is the rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay. Antibody-dependent killing of a labelled target cell line by PBMC is assessed by loss of intracellular CFSE but retention of membrane dye PKH26 (CFSE-PKH26+). Cells of this phenotype are assumed to be derived from CFSE+PKH26+ target cells killed by NK cells. We assessed the effector cells that mediate ADCC in this assay. Backgating analysis and phenotyping of CFSE-PKH26+ revealed that the RFADCC assay's readout mainly represents CD3-CD14+ monocytes taking up the PKH26 dye. This was confirmed for 53 HIV+plasma-purified IgG s les when co-cultured with PBMC from three separate healthy donors. Emergence of the CFSE-PKH26+ monocyte population was observed upon co-culture of targets with purified monocytes but not with purified NK cells. Image flow cytometry and microscopy showed a monocyte-specific interaction with target cells without typical morphological changes associated with phagocytosis, suggesting a monocyte-mediated ADCC process. We conclude that the RFADCC assay primarily reflects Ab-mediated monocyte function. Further studies on the immunological importance of HIV-specific monocyte-mediated ADCC are warranted.
Targeting cancer cells: Controlling the binding and internalization of antibody-functionalized capsules
Publisher: American Chemical Society (ACS)
Date: 07-08-2012
DOI: 10.1021/NN3010476
Abstract: The development of nanoengineered particles, such as polymersomes, liposomes, and polymer capsules, has the potential to offer significant advances in vaccine and cancer therapy. However, the effectiveness of these carriers has the potential to be greatly improved if they can be specifically delivered to target cells. We describe a general method for functionalizing nanoengineered polymer capsules with antibodies using click chemistry and investigate their interaction with cancer cells in vitro. The binding efficiency to cells was found to be dependent on both the capsule-to-cell ratio and the density of antibody on the capsule surface. In mixed cell populations, more than 90% of target cells bound capsules when the capsule-to-target cell ratio was 1:1. Strikingly, greater than 50% of target cells exhibited capsules on the cell surface even when the target cells were present as less than 0.1% of the total cell population. Imaging flow cytometry was used to quantify the internalization of the capsules, and the target cells were found to internalize capsules efficiently. However, the role of the antibody in this process was determined to enhance accumulation of capsules on the cell surface rather than promote endocytosis. This represents a significant finding, as this is the first study into the role antibodies play in internalization of such capsules. It also opens up the possibility of targeting these capsules to cancer cells using targeting molecules that do not trigger an endocytic pathway. We envisage that this approach will be generally applicable to the specific targeting of a variety of nanoengineered materials to cells.
A molecular beacon approach to measuring the DNA permeability of thin films
Publisher: American Chemical Society (ACS)
Date: 23-06-2005
DOI: 10.1021/JA0527166
Abstract: A new method for determining the permeability of thin films has been developed. A molecular beacon immobilized inside a porous silica particle that is subsequently encapsulated within a thin film can be used to determine the size of DNA that can permeate through the film. Using this technique, it has been determined that over 3 h, molecules larger than 4.7 nm do not permeate 15-nm thick polyelectrolyte multilayers and after 75 h molecules larger than 6 nm were excluded. This technique has applications for determining the permeability of films used for controlled drug and gene delivery.
Thiol-Reactive Star Polymers Display Enhanced Association with Distinct Human Blood Components
Publisher: American Chemical Society (ACS)
Date: 03-04-2017
Abstract: Directing nanoparticles to specific cell types using nonantibody-based methods is of increasing interest. Thiol-reactive nanoparticles can enhance the efficiency of cargo delivery into specific cells through interactions with cell-surface proteins. However, studies to date using this technique have been largely limited to immortalized cell lines or rodents, and the utility of this technology on primary human cells is unknown. Herein, we used RAFT polymerization to prepare pyridyl disulfide (PDS)-functionalized star polymers with a methoxy-poly(ethylene glycol) brush corona and a fluorescently labeled cross-linked core using an arm-first method. PDS star polymers were examined for their interaction with primary human blood components: six separate white blood cell subsets, as well as red blood cells and platelets. Compared with control star polymers, thiol-reactive nanoparticles displayed enhanced association with white blood cells at 37 °C, particularly the phagocytic monocyte, granulocyte, and dendritic cell subsets. Platelets associated with more PDS than control nanoparticles at both 37 °C and on ice, but they were not activated in the duration examined. Association with red blood cells was minor but still enhanced with PDS nanoparticles. Thiol-reactive nanoparticles represent a useful strategy to target primary human immune cell subsets for improved nanoparticle delivery.
Journey to the centre of the cell: Virtual reality immersion into scientific data
Publisher: Wiley
Date: 21-11-2018
DOI: 10.1111/TRA.12538
Abstract: Visualization of scientific data is crucial not only for scientific discovery but also to communicate science and medicine to both experts and a general audience. Until recently, we have been limited to visualizing the three-dimensional (3D) world of biology in 2 dimensions. Renderings of 3D cells are still traditionally displayed using two-dimensional (2D) media, such as on a computer screen or paper. However, the advent of consumer grade virtual reality (VR) headsets such as Oculus Rift and HTC Vive means it is now possible to visualize and interact with scientific data in a 3D virtual world. In addition, new microscopic methods provide an unprecedented opportunity to obtain new 3D data sets. In this perspective article, we highlight how we have used cutting edge imaging techniques to build a 3D virtual model of a cell from serial block-face scanning electron microscope (SBEM) imaging data. This model allows scientists, students and members of the public to explore and interact with a "real" cell. Early testing of this immersive environment indicates a significant improvement in students' understanding of cellular processes and points to a new future of learning and public engagement. In addition, we speculate that VR can become a new tool for researchers studying cellular architecture and processes by populating VR models with molecular data.
Optical barcoding of colloidal suspensions: applications in genomics, proteomics and drug discovery
Publisher: Royal Society of Chemistry (RSC)
Date: 24-04-2002
DOI: 10.1039/B200038P
Abstract: The enormous amount of information generated through sequencing of the human genome has increased demands for more economical and flexible alternatives in genomics, proteomics and drug discovery. Many companies and institutions have recognised the potential of increasing the size and complexity of chemical libraries by producing large chemical libraries on colloidal support beads. Since colloid-based compounds in a suspension are randomly located, an encoding system such as optical barcoding is required to permit rapid elucidation of the compound structures. We describe in this article innovative methods for optical barcoding of colloids for use as support beads in both combinatorial and non-combinatorial libraries. We focus in particular on the difficult problem of barcoding extremely large libraries, which if solved, will transform the manner in which genomics, proteomics and drug discovery research is currently performed.
High-Density Lipoprotein Composition Influences Lymphatic Transport after Subcutaneous Administration
Publisher: American Chemical Society (ACS)
Date: 16-06-2020
Triggering release of encapsulated cargo
Publisher: Wiley
Date: 26-03-2010
How to do things with metaphors: Engineering life as hodgepodge Carmen McLeod, Brigitte Nerlich
Publisher: Springer Science and Business Media LLC
Date: 17-09-2018
Engineered Layer-by-Layer Assembled Capsules for Biomedical Applications
Publisher: Wiley
Date: 09-05-2012
Tuning the properties of pH responsive nanoparticles to control cellular interactions in vitro and ex vivo
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6PY01332E
Abstract: Engineering the properties of nanoparticles to limit non-specific cellular interactions is critical for developing effective drug delivery systems. Differences between interactions with cultured cells and human blood highlights the need for appropriate assays.
Binding, internalization, and antigen presentation of vaccine-loaded nanoengineered capsules in blood
Publisher: Wiley
Date: 16-12-2008
Polymersome-loaded capsules for controlled release of DNA
Publisher: Wiley
Date: 04-07-2011
Abstract: The formation of a novel drug-delivery carrier for the controlled release of plasmid DNA that comprises layer-by-layer polymer capsules subcompartmentalized with pH-sensitive nanometer-sized polymersomes is reported. The hiphilic diblock copolymer poly(oligoethylene glycol methacrylate)-block-poly(2-(diisopropylamino)ethyl methacrylate) forms polymersomes at physiological pH, but transitions to unimeric polymer chains upon acidification to cellular endocytic pH. These polymersomes can thus release an encapsulated payload in response to a change in pH from physiological to endocytic conditions. Multicomponent layer-by-layer capsules are formed by exploiting the ability of tannic acid to act as an efficient hydrogen-bond donor for both the polymersomes and poly(N-vinyl pyrrolidone) at physiological pH. These capsules show release of a plasmid DNA payload encapsulated within the polymersome subcompartments in response to changes in pH between physiological and endocytic conditions.
Click-engineered, bioresponsive, drug-loaded PEG spheres
Publisher: Wiley
Date: 20-11-2009
Abstract: A click-chemistry approach to synthesize bioresponsive poly(ethylene glycol acrylate) particles is described. The particles are loaded with a model anticancer drug (doxorubicin, DOX), and undergo simultaneous particle deconstruction and DOX release upon specific activation by the simulated environment of the cellular cytoplasm.
Endocytic pH-triggered degradation of nanoengineered multilayer capsules
Publisher: Wiley
Date: 27-12-2014
Abstract: The synthesis of cross-linker free layer-by-layer (LbL) capsules that solely utilize cellular pH variations as a trigger to specifically deconstruct and subsequently release cargo in cells is reported. These capsules demonstrate retention of water-soluble therapeutic molecules as small as 500 Da at extracellular pH. Triggered capsule degradation and release of cargo is observed within 30 min of cell uptake.
HIV-specific antibody-dependent phagocytosis matures during HIV infection
Publisher: Wiley
Date: 10-06-2014
DOI: 10.1038/ICB.2014.42
Abstract: Antibody-dependent phagocytosis (ADP) is a potentially important immune mechanism to clear HIV. How HIV-specific ADP responses mature during HIV infection or in response to vaccinations administered, including the partially successful RV144 HIV vaccine, is not known. We established a modified ADP assay to measure internalisation of HIV antibody (Ab)-opsonised targets using a specific hybridisation internalisation probe. Labelled beads were coated with both biotinylated HIV gp140 envelope protein and a fluorescent internalisation probe, opsonised with Abs and incubated with a monocytic cell line. The fluorescence derived from the fluorescent internalisation probe on surface-bound beads, but not from internalised beads, was quenched by the addition of a complementary quencher probe. HIV Env-specific ADP was measured in 31 subjects during primary infection and early chronic HIV infection. Although ADP responses were present early during HIV infection, a significant increase in ADP responses in all 31 subjects studied was detected (P<0.001). However, when we tested 30 HIV-negative human subjects immunised with the Canarypox/gp120 vaccine regimen (subjects from the RV144 trial) we did not detect HIV-specific ADP activity. In conclusion, a modified assay was developed to measure HIV-specific ADP. Enhanced ADP responses early in the course of HIV infection were observed but no ADP activity was detected following the vaccinations administered in the RV144 trial. Improved vaccine regimens may be needed to capitalise on ADP-mediated immunity against HIV.
Assembly and degradation of low-fouling click-functionalized poly(ethylene glycol)-based multilayer films and capsules
Publisher: Wiley
Date: 21-03-2011
Abstract: Nano-/micrometer-scaled films and capsules made of low-fouling materials such as poly(ethylene glycol) (PEG) are of interest for drug delivery and tissue engineering applications. Herein, the assembly and degradation of low-fouling, alkyne-functionalized PEG (PEG(Alk) ) multilayer films and capsules, which are prepared by combining layer-by-layer (LbL) assembly and click chemistry, are reported. A nonlinear, temperature-responsive PEG(Alk) is synthesized, and is then used to form hydrogen-bonded multilayers with poly(methacrylic acid) (PMA) at pH 5. The thermoresponsive behavior of PEG(Alk) is exploited to tailor film buildup by adjusting the assembly conditions. Using alkyne-azide click chemistry, PEG(Alk)/PMA multilayers are crosslinked with a bisazide linker that contains a disulfide bond, rendering these films and capsules redox-responsive. At pH 7, by disrupting the hydrogen bonding between the polymers, PEG(Alk) LbL films and PEG(Alk) -based capsules are obtained. These films exhibit specific deconstruction properties under simulated intracellular reducing conditions, but remain stable at physiological pH, suggesting potential applications in controlled drug release. The low-fouling properties of the PEG films are confirmed by incubation with human serum and a blood clot. Additionally, these capsules showed negligible toxicity to human cells.
Secondary Self-Assembly of Supramolecular Nanotubes into Tubisomes and Their Activity on Cells
Publisher: Wiley
Date: 21-11-2018
Abstract: The properties and structures of viruses are directly related to the three-dimensional structure of their capsid proteins, which arises from a combination of hydrophobic and supramolecular interactions, such as hydrogen bonds. The design of synthetic materials demonstrating similar synergistic interactions still remains a challenge. Herein, we report the synthesis of a polymer/cyclic peptide conjugate that combines the capability to form supramolecular nanotubes via hydrogen bonds with the properties of an hiphilic block copolymer. The analysis of aqueous solutions by scattering and imaging techniques revealed a barrel-shaped alignment of single peptide nanotubes into a large tubisome (length: 260 nm (from SANS)) with a hydrophobic core (diameter: 16 nm) and a hydrophilic shell. These systems, which have a structure that is similar to those of viruses, were tested in vitro to elucidate their activity on cells. Remarkably, the rigid tubisomes are able to perforate the lysosomal membrane in cells and release a small molecule into the cytosol.
Understanding Cell Interactions Using Modular Nanoparticle Libraries
Publisher: CSIRO Publishing
Date: 2019
DOI: 10.1071/CH19269
Abstract: Nanoparticle delivery systems have significant potential to facilitate the delivery of novel therapeutics, such as proteins, DNA or small molecules. However, there are multiple biological barriers that need to be overcome to deliver the cargo in an active form. These challenges include evading clearance by the reticuloendothelial system, minimising adverse immune responses, targeting specific cells and tissues, and trafficking into the right compartment of the cell. In this account, we will discuss how nanoparticle structure can be tuned to optimise biological interactions and thus improve the ability of nanoparticles to overcome these barriers. The focus of this article will be on controlling cell targeting and trafficking within a cell, e.g. endosomal escape.
Differential use of autophagy by primary dendritic cells specialized in cross-presentation
Publisher: Informa UK Limited
Date: 07-05-2015
Engineered bacterially expressed polypeptides: Assembly into polymer particles with tailored degradation profiles
Publisher: Wiley
Date: 23-11-2012
Abstract: In nature, the sequence of amino acids in a protein is determined by the genetic code. Biosynthesis of polypeptides by bacteria can be used to exploit this natural process to afford exact control over properties such as molecular weight, chemical functionality, and structure. It is demonstrated how control over the positioning of functional groups can be used to tune the degradation of assembled polypeptide particles (see scheme).
Converging layer-by-layer polyelectrolyte microcapsule and cubic lyotropic liquid crystalline nanoparticle approaches for molecular encapsulation
Publisher: Royal Society of Chemistry (RSC)
Date: 2011
DOI: 10.1039/C0SM01410A
Influence of size, surface, cell line, and kinetic properties on the specific binding of A33 antigen-targeted multilayered particles and capsules to colorectal cancer cells
Publisher: American Chemical Society (ACS)
Date: 14-08-2007
DOI: 10.1021/NN700060M
Abstract: There has been increased interest in the use of polymer capsules formed by the layer-by-layer (LbL) technique as therapeutic carriers to cancer cells due to their versatility and ease of surface modification. We have investigated the influence of size, surface properties, cell line, and kinetic parameters such as dosage (particle concentration) and incubation time on the specific binding of humanized A33 monoclonal antibody (huA33 mAb)-coated LbL particles and capsules to colorectal cancer cells. HuA33 mAb binds to the A33 antigen present on almost all colorectal cancer cells and has demonstrated great promise in clinical trials as an immunotherapeutic agent for cancer therapy. Flow cytometry experiments showed the cell binding specificity of huA33 mAb-coated particles to be size-dependent, with the optimal size for enhanced selectivity at approximately 500 nm. The specific binding was improved by increasing the dosage of particles incubated with the cells. The level of specific versus nonspecific binding was compared for particles terminated with various polyelectrolytes to examine the surface dependency of antibody attachment and subsequent cell binding ability. The specific binding of huA33 mAb-coated particles is also reported for two colorectal cancer cell lines, with an enhanced binding ratio between 4 and 10 obtained for the huA33 mAb-functionalized particles. This investigation aims to improve the level of specific targeting of LbL particles, which is important in targeted drug and gene delivery applications.
Nanoparticles for vaccine and gene therapy: Overcoming the barriers to nucleic acid delivery
Publisher: Wiley
Date: 11-2022
DOI: 10.1002/WNAN.1809
Abstract: Nucleic acid therapeutics can be used to control virtually every aspect of cell behavior and therefore have significant potential to treat genetic disorders, infectious diseases, and cancer. However, while clinically approved to treat a small number of diseases, the full potential of nucleic acid therapeutics is h ered by inefficient delivery. Nucleic acids are large, highly charged biomolecules that are sensitive to degradation and so the approaches to deliver these molecules differ significantly from traditional small molecule drugs. Current studies suggest less than 1% of the injected nucleic acid dose is delivered to the target cell in an active form. This inefficient delivery increases costs and limits their use to applications where a small amount of nucleic acid is sufficient. In this review, we focus on two of the major barriers to efficient nucleic acid delivery: (1) delivery to the target cell and (2) transport to the subcellular compartment where the nucleic acids are therapeutically active. We explore how nanoparticles can be modified with targeting ligands to increase accumulation in specific cells, and how the composition of the nanoparticle can be engineered to manipulate or disrupt cellular membranes and facilitate delivery to the optimal subcellular compartments. Finally, we highlight how with intelligent material design, nanoparticle delivery systems have been developed to deliver nucleic acids that silence aberrant genes, correct genetic mutations, and act as both therapeutic and prophylactic vaccines. This article is categorized under: Nanotechnology Approaches to Biology Cells at the Nanoscale Therapeutic Approaches and Drug Discovery Nanomedicine for Infectious Disease Biology‐Inspired Nanomaterials Lipid‐Based Structures
Life under the Microscope: Quantifying Live Cell Interactions to Improve Nanoscale Drug Delivery
Publisher: American Chemical Society (ACS)
Date: 05-01-2017
DOI: 10.1021/ACSSENSORS.6B00725
Abstract: The behavior of cells and how they react to stimuli is critically important for drug development, drug delivery, and understanding the molecular basis of many diseases. However, we still lack a comprehensive understanding of these interactions, particularly in relation to drug delivery from nanoparticles. This Sensors Issues article discusses the importance of quantifying these interactions and highlights some key areas where advances in sensor technology have the potential to transform our understanding of drug delivery and cell biology.
Challenges facing colloidal delivery systems: From synthesis to the clinic
Publisher: Elsevier BV
Date: 06-2011
Bio-click chemistry: enzymatic functionalization of PEGylated capsules for targeting applications
Publisher: Wiley
Date: 28-06-2012
Abstract: All sorted: The enzyme Sortase A was used to catalyze functionalization of PEGylated capsules with an activation-specific anti-platelet single-chain antibody (scFv). This enzymatic method allows fast, covalent, and site-directed functionalization of delivery vehicles under mild conditions. Activation-specific anti-platelet scFv-coated PEGylated capsules exhibited a high level of selective binding to thrombi, thus suggesting their potential for thrombosis therapy.
Carboxylated Cy5-Labeled Comb Polymers Passively Diffuse the Cell Membrane and Target Mitochondria
Publisher: American Chemical Society (ACS)
Date: 08-2019
Abstract: A detailed understanding of the cellular uptake and trafficking of nanomaterials is essential for the design of "smart" intracellular drug delivery vehicles. Typically, cellular interactions can be tailored by endowing materials with specific properties, for ex le, through the introduction of charges or targeting groups. In this study, water-soluble carboxylated
Elucidating the Influences of Size, Surface Chemistry, and Dynamic Flow on Cellular Association of Nanoparticles Made by Polymerization-Induced Self-Assembly
Publisher: Wiley
Date: 25-07-2018
Abstract: The size and surface chemistry of nanoparticles dictate their interactions with biological systems. However, it remains unclear how these key physicochemical properties affect the cellular association of nanoparticles under dynamic flow conditions encountered in human vascular networks. Here, the facile synthesis of novel fluorescent nanoparticles with tunable sizes and surface chemistries and their association with primary human umbilical vein endothelial cells (HUVECs) is reported. First, a one-pot polymerization-induced self-assembly (PISA) methodology is developed to covalently incorporate a commercially available fluorescent dye into the nanoparticle core and tune nanoparticle size and surface chemistry. To characterize cellular association under flow, HUVECs are cultured onto the surface of a synthetic microvascular network embedded in a microfluidic device (SynVivo, INC). Interestingly, increasing the size of carboxylic acid-functionalized nanoparticles leads to higher cellular association under static conditions but lower cellular association under flow conditions, whereas increasing the size of tertiary amine-decorated nanoparticles results in a higher level of cellular association, under both static and flow conditions. These findings provide new insights into the interactions between polymeric nanomaterials and endothelial cells. Altogether, this work establishes innovative methods for the facile synthesis and biological characterization of polymeric nanomaterials for various potential applications.
The Endosomal Escape of Nanoparticles: Toward More Efficient Cellular Delivery
Publisher: American Chemical Society (ACS)
Date: 19-11-2019
DOI: 10.1021/ACS.BIOCONJCHEM.8B00732
Abstract: Many emerging therapies rely on the delivery of biological cargo into the cytosol. Nanoparticle delivery systems hold great potential to deliver these therapeutics but are hindered by entrapment and subsequent degradation in acidic compartments of the endo/lysosomal pathway. Engineering polymeric delivery systems that are able to escape the endosome has significant potential to address this issue. However, the development of safe and effective delivery systems that can reliably deliver cargo to the cytosol is still a challenge. Greater understanding of the properties that govern endosomal escape and how it can be quantified is important for the development of more efficient nanoparticle delivery systems. This Topical Review highlights the current understanding of the mechanisms by which nanoparticles escape the endosome, and the emerging techniques to improve the quantification of endosomal escape.
Diaminomaleonitrile-functionalised schiff bases: Synthesis, solvatochromism, and lysosome-specific imaging
Publisher: CSIRO Publishing
Date: 2020
DOI: 10.1071/CH19536
Influenza-specific antibody-dependent phagocytosis
Publisher: Public Library of Science (PLoS)
Date: 28-04-2016
Engineering particles for therapeutic delivery: Prospects and challenges
Publisher: American Chemical Society (ACS)
Date: 30-04-2012
DOI: 10.1021/NN3016162
Abstract: Nanoengineered particles that can facilitate drug formulation and passively target tumors have reached the clinic in recent years. These early successes have driven a new wave of significant innovation in the generation of advanced particles. Recent developments in enabling technologies and chemistries have led to control over key particle properties, including surface functionality, size, shape, and rigidity. Combining these advances with the rapid developments in the discovery of many disease-related characteristics now offers new opportunities for improving particle specificity for targeted therapy. In this Perspective, we summarize recent progress in particle-based therapeutic delivery and discuss important concepts in particle design and biological barriers for developing the next generation of particles.
Toward therapeutic delivery with layer-by-layer engineered particles
Publisher: American Chemical Society (ACS)
Date: 25-05-2011
DOI: 10.1021/NN201793F
Abstract: Layer-by-layer (LbL)-engineered particles have recently emerged as a promising class of materials for applications in biomedicine, with studies progressing from in vitro to in vivo. The versatility of LbL assembly coupled with particle templating has led to engineered particles with specific properties (e.g., stimuli-responsive, high cargo encapsulation efficiency, targeting), thus offering new opportunities in targeted and triggered therapeutic release. This Perspective highlights an important development by Poon et al. on tumor targeting in vivo using LbL-engineered nanoparticles containing a pH-responsive poly(ethylene glycol) (PEG) surface layer. Further, we summarize recent progress in the application of LbL particles in the fields of drug, gene, and vaccine delivery and cancer imaging. Finally, we explore future directions in this field, focusing on the biological processing of LbL-assembled particles.
Human immune cell targeting of protein nanoparticles-caveospheres
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6NR00506C
Abstract: Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.
Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay
Publisher: Springer Science and Business Media LLC
Date: 17-06-2021
DOI: 10.1038/S41467-021-23997-X
Abstract: Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.
Resolving subcellular pH with a quantitative fluorescent lifetime biosensor
Publisher: Springer Science and Business Media LLC
Date: 12-10-2022
DOI: 10.1038/S41467-022-33348-Z
Abstract: Changes in sub-cellular pH play a key role in metabolism, membrane transport, and triggering cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however, these measurements are h ered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, here we combine pH dependant fluorescent lifetime imaging microscopy (pHLIM) with deep learning to accurately quantify sub-cellular pH in in idual vesicles. We engineer the pH sensitive protein mApple to localise in the cytosol, endosomes, and lysosomes, and demonstrate that pHLIM can rapidly detect pH changes induced by drugs such as bafilomycin A1 and chloroquine. We also demonstrate that polyethylenimine (a common transfection reagent) does not exhibit a proton sponge effect and had no measurable impact on the pH of endocytic vesicles. pHLIM is a simple and quantitative method that will help to understand drug action and disease progression.
Self-Immolative Polymer Nanoparticles with Precise and Controllable pH-Dependent Degradation
Publisher: American Chemical Society (ACS)
Date: 14-09-2023
Engineering the Orientation, Density, and Flexibility of Single-Domain Antibodies on Nanoparticles to Improve Cell Targeting
Publisher: American Chemical Society (ACS)
Date: 09-01-2020
Abstract: Nanoparticles targeted to specific cells have the potential to improve the delivery of therapeutics. The effectiveness of cell targeting can be significantly improved by optimizing how the targeting ligands are displayed on the nanoparticle surface. Crucial to optimizing the cell binding are the orientation, density, and flexibility of the targeting ligand on the nanoparticle surface. In this paper, we used an anti-EGFR single-domain antibody (sdAb or nanobody) to target fluorescent nanocrystals (Qdots) to epidermal growth factor receptor (EGFR)-positive cells. The sdAbs were expressed with a synthetic amino acid (azPhe), enabling site-specific conjugation to Qdots in an improved orientation. To optimize the targeting efficiency, we engineered the point of attachment (orientation), controlled the density of targeting groups on the surface of the Qdot, and optimized the length of the poly(ethylene glycol) linker used to couple the sdAb to the Qdot surface. By optimizing orientation, density, and flexibility, we improved cell targeting by more than an order of magnitude. This work highlights the importance of understanding the structure of the nanoparticle surface to achieve the optimal interactions with the intended receptors and how engineering the nanoparticle surface can significantly improve cell targeting.
Unravelling cytosolic delivery of endosomal escape peptides with a quantitative endosomal escape assay (SLEEQ)
Publisher: Cold Spring Harbor Laboratory
Date: 20-08-2020
DOI: 10.1101/2020.08.20.258350
Abstract: Endosomal escape is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of endosomal escape mechanisms remains limited due to significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we developed a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We applied SLEEQ to evaluate the endosomal escape of a range of widely studied putative endosomal escape peptides (EEPs). We demonstrated that positively-charged EEPs enhanced cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how EEPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.
A molecular sensor to quantify the localization of proteins, DNA and nanoparticles in cells
Publisher: Springer Science and Business Media LLC
Date: 08-09-2020
DOI: 10.1038/S41467-020-18082-8
Abstract: Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAP Switch ) that can be conjugated to biomolecules using click chemistry. SNAP Switch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAP Switch , we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAP Switch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.
Porous functionalised silica particles: a potential platform for biomolecular screening
Publisher: Royal Society of Chemistry (RSC)
Date: 2005
DOI: 10.1039/B415376F
Abstract: Novel, porous, functionalised silica particles have been developed with controlled morphology, which promote covalent attachment of fluorescent dyes which can act as an optical barcode.
Distribution of therapeutic proteins into thoracic lymph after intravenous administration is protein size-dependent and primarily occurs within the liver and mesentery
Publisher: Elsevier BV
Date: 02-2018
DOI: 10.1016/J.JCONREL.2017.12.031
Abstract: Therapeutic proteins can facilitate the targeting and treatment of lymphatic diseases (such as cancer metastases, infections and inflammatory diseases) since they are cleared via the lymphatics following interstitial (SC or IM) administration. However, therapeutic proteins are often administered intravenously (IV). Recently therapeutic proteins have been found to access the thoracic lymph in surprisingly high quantities after IV administration. The aim of this study was to determine, for the first time, the major sites of thoracic lymph access of therapeutic proteins, and the protein properties that enhance lymph access, after IV administration. In order to achieve this, novel methods were developed or optimized to collect hepatic, mesenteric or thoracic lymph from male SD rats. Four different sized PEGylated or non-PEGylated therapeutic proteins (native interferon α2b (IFN, 19kDa), PEGylated interferon α2b (IFN-PEG12, 31kDa), PEGylated interferon α2a (IFN-PEG40, 60kDa) or trastuzumab (150kDa)) were then administered via short IV infusion, and plasma and lymph concentrations of the proteins determined via ELISA. The recovery of the therapeutic proteins in the thoracic lymph duct, which collects lymph from most of the body, was significantly greater for trastuzumab, IFN-PEG40 and IFN-PEG12 (all >3% dose over 8h) when compared to native IFN (0.9% dose). Conversely, the thoracic lymph lasma (L/P) concentration ratio and thus efficiency of extravasation and transport through the interstitium to lymph was highest for the smaller proteins IFN and IFN-PEG12 (at 90-100% vs 15-30% for trastuzumab and IFN-PEG40). The lower total recovery of IFN and IFN-PEG12 in thoracic lymph reflected more rapid systemic clearance and thus lower systemic exposure. For all therapeutic proteins, the majority (>80%) of lymph access occurred via the hepatic and mesenteric lymphatics. This lymphatic distribution pattern was supported by quantitative imaging of the lymph node distribution of IV administered Cy5 labelled trastuzumab. Optimizing the properties of IV administered therapeutic proteins represents a viable approach to better target and treat pathological states involving the lymphatics, particularly in the liver and mesentery. This includes cancer metastases, infections and inflammatory diseases. Successful development of the novel technique to collect hepatic lymph will also enable future work to evaluate tissue-specific lymph transport in health and disease.
A Mechanism for Forming Large Fluorescent Organo-Silica Particles: Potential Supports for Combinatorial Synthesis
Publisher: American Chemical Society (ACS)
Date: 12-2006
DOI: 10.1021/CM0526260
Particle generation, functionalization and sortase A-mediated modification with targeting of single-chain antibodies for diagnostic and therapeutic use
Publisher: Springer Science and Business Media LLC
Date: 11-12-2015
Abstract: Antibody fusion to nonprotein materials such as contrast agents or radio-tracers, nano- or microparticles or small-molecule drugs is attracting major interest for molecular imaging and drug delivery. Nondirected bioconjugation techniques may impair antibody affinity, result in lower amounts of functional antibodies and generate multicomponent mixtures. We present a detailed protocol for the enzymatic bioconjugation of small recombinant antibodies to imaging particles, and we also describe the generation of and conjugation to a low-fouling capsule assembled for drug delivery from PEG and PVPON (poly(N-vinylpyrrolidone) by a layer-by-layer (LbL) technique. The single-chain variable fragment (scFv) is equipped with a short C-terminal LPETG tag and the fusion partners are functionalized with an N-terminal GGG nucleophilic group for sortase A conjugation. The LbL capsules are assembled through hydrogen bonding by depositing alkyne-modified poly(vinylpyrrolidone) and poly(methacrylic acid) layers on silica particles, followed by depositing alkyne-modified PEG. The generation of the antibodies and LbL capsules takes ∼1-2 weeks each. The conjugation and functional testing takes another 3-4 d.
DNA-based probes for flow cytometry analysis of endocytosis and recycling
Publisher: Wiley
Date: 07-02-2017
DOI: 10.1111/TRA.12466
Abstract: The internalization of proteins plays a key role in cell development, cell signaling and immunity. We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane-bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo-TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor-antibody complex behaves differently to the receptor-ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands.
Mechanically tunable, self-adjuvanting nanoengineered polypeptide particles
Publisher: Wiley
Date: 10-05-2013
Abstract: DNA-loaded polypeptide particles are prepared via templated assembly of mesoporous silica for the delivery of adjuvants. The elasticity and cargo-loading capacity of the obtained particles can be tuned by the amount of cross-linker used to stabilize the polypeptide particles. The use of polypeptide particles as biocarriers provides a promising method for vaccine delivery.
The potential of nanoparticle vaccines as a treatment for cancer
Publisher: Elsevier BV
Date: 06-2018
DOI: 10.1016/J.MOLIMM.2017.12.022
Abstract: A complex and multifaceted relationship exists between cancer and the immune system. Advances in our understanding of this relationship have resulted in significant clinical attention in the possibilities of cancer immunotherapy. Harnessing the immune system's potent and selective destructive capability is a major focus of attempts to treat cancer. Despite significant progress in the field, cancer therapy still remains significantly deficient, with cancer being one of the largest contributors to morbidity and mortality in the developed world. It is evident that the design of new treatment regimes is required to exploit cancer immunotherapy. Herein we review the potential for nanotechnology to overcome the challenges that have limited the more widespread implementation of immunotherapy to cancer treatment.
Toward colloid-based biosensors for SNP genotyping and personalised medicine applications
Publisher: Inderscience Publishers
Date: 2008
Engineered hydrogen-bonded polymer multilayers: From assembly to biomedical applications
Publisher: Royal Society of Chemistry (RSC)
Date: 2011
DOI: 10.1039/C0CS00001A
Abstract: In this tutorial review , developments in hydrogen-bonded LbL materials are discussed, with an emphasis on loading and release of cargo for biomedical applications.
Opportunities for innovation: Building on the success of lipid nanoparticle vaccines
Publisher: Elsevier BV
Date: 10-2011
Organ-specific lymphatics play distinct roles in regulating HDL trafficking and composition
Publisher: American Physiological Society
Date: 04-2020
Abstract: Recently, peripheral lymphatic vessels were found to transport high-density lipoprotein (HDL) from interstitial tissues to the blood circulation during reverse cholesterol transport. This function is thought to be critical to the clearance of cholesterol from atherosclerotic plaques. The role of organ-specific lymphatics in modulating HDL transport and composition is, however, incompletely understood. This study aimed to 1) determine the contribution of the lymphatics draining the intestine and liver (which are major sites of HDL synthesis) to total (thoracic) lymph HDL transport and 2) verify whether the HDLs in lymph are derived from specific organs and are modified during trafficking in lymph. The mesenteric, hepatic, or thoracic lymph duct was cannulated in nonfasted Sprague-Dawley rats, and lymph was collected over 5 h under anesthesia. Whole lymph and specific lymph lipoproteins (isolated by ultracentrifugation) were analyzed for protein and lipid composition. The majority of thoracic lymph fluid, protein, and lipid mass was sourced from the mesenteric, and to a lesser extent, hepatic lymph. Mesenteric and thoracic lymph were both rich in chylomicrons and very low-density lipoprotein, whereas hepatic lymph and plasma were HDL-rich. The protein and lipid mass in thoracic lymph HDL was mostly sourced from mesenteric lymph, whereas the cholesterol mass was equally sourced from mesenteric and hepatic lymph. HDLs were compositionally distinct across the lymph sources and plasma. The composition of HDL also appeared to be modified during passage from the mesenteric and hepatic to the thoracic lymph duct. Overall, this study demonstrates that the lipoproteins in lymph are organ specific in composition, and the intestine and liver appear to be the main source of HDL in the lymph. NEW & NOTEWORTHY High-density lipoprotein in lymph are organ-specific in composition and derive mostly from the intestine and liver. High-density lipoprotein also appears to be remodeled during transport through the lymphatics. These findings have implications to cardiometabolic diseases that involve perturbations in lipoprotein distribution and metabolism.
Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus
Publisher: MDPI AG
Date: 24-03-2021
DOI: 10.3390/IJMS22073310
Abstract: Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5′end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP–host interactions further, we expressed TP in Escherichia coli (E. coli). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp β, and a complex of Imp α/β but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp β or Imp α/β. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.
Analysis of intracellular trafficking of dendritic cell receptors for antigen targeting
Publisher: Springer New York
Date: 2016
DOI: 10.1007/978-1-4939-3606-9_15
Abstract: Antibody-targeted vaccination aims to efficiently deliver antigen to dendritic cells by targeting specific receptors at the cell surface. The choice of receptor depends on different factors, including their capacity to induce internalization of the delivered antigen/adjuvant cargo. Assays currently used to monitor internalization in dendritic cells have several limitations. We have developed a novel DNA-based probe that allows for simple and robust high-throughput analysis of internalization. Designed for flow cytometry, the probe can also be used for fluorescence microscopy to clearly distinguish internalized from surface-bound material. Here, we describe the steps for modifying material (antibodies, proteins) with the probe, undertaking the assay, and analyzing the data obtained from flow cytometry.
Layer-by-Layer Assembled Capsules for Biomedical Applications
Publisher: Elsevier
Date: 2011
DNA multilayer films on planar and colloidal supports: Sequential assembly of like-charged polyelectrolytes
Publisher: American Chemical Society (ACS)
Date: 20-04-2005
DOI: 10.1021/NL050608B
Abstract: Multilayer films comprising solely negatively charged polyelectrolytes were sequentially assembled based on DNA hybridization. Films prepared from alternating layers of two-block homopolymeric nucleotides (polyA(20)G(20) olyT(20)C(20)) grew linearly with increasing layer number, as verified by quartz crystal microgravimetry, UV-vis spectrophotometry and optical microscopy. Urea treatment of the films induced morphological changes, while exposure to low ionic strength solutions resulted in film disassembly. DNA multilayer films were also formed on silica particles, and DNA hollow capsules were obtained following dissolution of the template core.
A protective vaccine delivery system for in vivo T cell stimulation using nanoengineered polymer hydrogel capsules
Publisher: American Chemical Society (ACS)
Date: 13-10-2009
DOI: 10.1021/NN900715G
Abstract: Successful delivery of labile vaccine antigens, such as peptides and proteins, to stimulate CD4 and CD8 T cell immunity could improve vaccine strategies against chronic infections such as HIV and Hepatitis C. Layer-by-layer (LbL)-assembled nanoengineered hydrogel capsules represent a novel and promising technology for the protection and delivery of labile vaccine candidates to antigen-presenting cells (APCs). Here we report on the in vitro and in vivo immunostimulatory capabilities of LbL-assembled disulfide cross-linked poly(methacrylic acid) (PMA(SH)) hydrogel capsules as a delivery strategy for protein and peptide vaccines using robust transgenic mice models and ovalbumin (OVA) as a model vaccine. We demonstrate that OVA protein as well as multiple OVA peptides can be successfully encapsulated within nanoengineered PMA(SH) hydrogel capsules. OVA-containing PMA(SH) capsules are internalized by mouse APCs, resulting in presentation of OVA epitopes and subsequent activation of OVA-specific CD4 and CD8 T cells in vitro. OVA-specific CD4 and CD8 T cells are also activated to proliferate in vivo following intravenous vaccination of mice with OVA protein- and OVA peptide-loaded PMA(SH) hydrogel capsules. Furthermore, we show that OVA encapsulated within the PMA(SH) capsules resulted in at least 6-fold greater proliferation of OVA-specific CD8 T cells and 70-fold greater proliferation of OVA-specific CD4 T cells in vivo compared to the equivalent amount of OVA protein administered alone. These results highlight the potential of nanoengineered hydrogel capsules for vaccine delivery.
Fluorescent nanosensors for intracellular measurements: Synthesis, characterization, calibration, and measurement
Publisher: Frontiers Media SA
Date: 2014
Compositional and structural engineering of DNA multilayer films
Publisher: American Chemical Society (ACS)
Date: 03-02-2006
DOI: 10.1021/LA052581H
Abstract: We report the layer-by-layer (LbL) preparation of multilayered thin films that consist solely of DNA. The properties of the films were varied by assembling the layers from different oligonucleotide building blocks, which are composed of repeating homopolymeric units of nucleotides [adenosine (A), cytosine (C), guanine (G), and thymidine (T)] or "random" sequences. Films assembled from oligonucleotides with a single complementary unit did not show continual layer buildup. To form a repeating multilayer system, it was necessary for single-stranded DNA to be available for subsequent layers to hybridize. By using oligonucleotides with multiple nucleotide units, multilayer films were successfully assembled. We demonstrate that the thickness and swellability of the films can be controlled by the extent of hydrogen bonding (the G/C content of the oligonucleotide) and orientation of the oligomers. We have examined the stability and swellability of the films in solutions of varying salt concentration as well as in a denaturing urea solution. Stable, hollow DNA capsules were also formed by preparing the films on sacrificial colloidal templates, followed by removal of the core. The assembly of propagating structures through DNA hybridization paves the way for the engineering of DNA films with tailored composition, structure, and permeability, making them likely to find application in drug/gene delivery and biomolecular sensing.
Compositional engineering of polyelectrolyte blend capsules
Publisher: American Chemical Society (ACS)
Date: 14-09-2007
DOI: 10.1021/MA071372W
Layer-by-layer engineered capsules and their applications
Publisher: Elsevier BV
Date: 10-2006
Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack
Publisher: American Society of Hematology
Date: 04-04-2013
DOI: 10.1182/BLOOD-2012-07-446146
Abstract: Granzymes diffuse through perforin pores on the target cell plasma membrane.
PH-Responsive Transferrin-pHlexi particles capable of targeting cells in vitro
Publisher: American Chemical Society (ACS)
Date: 09-03-2017
Targeting dendritic cells: The role of specific receptors in the internalization of polymer capsules
Publisher: Wiley
Date: 20-01-2013
Quantifying Nanoparticle Internalization Using a High Throughput Internalization Assay
Publisher: Springer Science and Business Media LLC
Date: 05-07-2016
DOI: 10.1007/S11095-016-1984-3
Abstract: The internalization of nanoparticles into cells is critical for effective nanoparticle mediated drug delivery. To investigate the kinetics and mechanism of internalization of nanoparticles into cells we have developed a DNA molecular sensor, termed the Specific Hybridization Internalization Probe - SHIP. Self-assembling polymeric 'pHlexi' nanoparticles were functionalized with a Fluorescent Internalization Probe (FIP) and the interactions with two different cell lines (3T3 and CEM cells) were studied. The kinetics of internalization were quantified and chemical inhibitors that inhibited energy dependent endocytosis (sodium azide), dynamin dependent endocytosis (Dyngo-4a) and macropinocytosis (5-(N-ethyl-N-isopropyl) amiloride (EIPA)) were used to study the mechanism of internalization. Nanoparticle internalization kinetics were significantly faster in 3T3 cells than CEM cells. We have shown that ~90% of the nanoparticles associated with 3T3 cells were internalized, compared to only 20% of the nanoparticles associated with CEM cells. Nanoparticle uptake was via a dynamin-dependent pathway, and the nanoparticles were trafficked to lysosomal compartments once internalized. SHIP is able to distinguish between nanoparticles that are associated on the outer cell membrane from nanoparticles that are internalized. This study demonstrates the assay can be used to probe the kinetics of nanoparticle internalization and the mechanisms by which the nanoparticles are taken up by cells. This information is fundamental for engineering more effective nanoparticle delivery systems. The SHIP assay is a simple and a high-throughput technique that could have wide application in therapeutic delivery research.
Next generation, sequentially assembled ultrathin films: Beyond electrostatics
Publisher: Royal Society of Chemistry (RSC)
Date: 2007
DOI: 10.1039/B610778H
Abstract: Over the last 15 years, the layer-by-layer (LbL) assembly technology has proven to be a versatile method for surface modification. This approach is likely to find widespread application because of its simplicity and versatility however, the conventional use of highly charged materials with limited responsive behaviour presents some key limitations. In this tutorial review, the formation of multilayer thin films prepared through non-electrostatic interactions is reviewed. We discuss the assembly of films via a number of different methodologies, with particular emphasis on those that provide enhanced orientational control, stimuli-responsive behaviour, and improved film stability.
Quantifying Cellular Internalization with a Fluorescent Click Sensor
Publisher: American Chemical Society (ACS)
Date: 20-04-2018
DOI: 10.1021/ACSSENSORS.8B00219
Abstract: The ability to determine the amount of material endocytosed by a cell is important for our understanding of cell biology and in the design of effective carriers for drug delivery. To quantify internalization by fluorescence, the signal from material remaining on the cell surface must be differentiated from endocytosed material. Sensors for internalization offer advantages over traditional methods for achieving this as they exhibit improved sensitivity, allow for multiple fluorescent markers to be used simultaneously, and are amenable to high-throughput analysis. We have developed a small fluorescent internalization sensor, similar in size to a standard fluorescent dye, that can be conjugated to proteins and uses the rapid and highly specific bio-orthogonal reaction between a tetrazine and a trans-cyclooctene group to switch off the surface signal. The sensor can be attached to a variety of materials using simple chemistry and is compatible with flow cytometry and fluorescence microscopy, making it a useful tool to study the uptake of material into cells.
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