Effect of covalent photoconjugation of affibodies to epidermal growth factor receptor (EGFR) on cellular quiescence.

Cellular quiescence is a reversible state of cell cycle arrest whereby cells are temporarily maintained in the nondividing phase. Inducing quiescence in cancer cells by targeting growth receptors is a treatment strategy to slow cell growth in certain aggressive tumors, which in turn increases the efficacy of treatments such as surgery or systemic chemotherapy. However, ligand interactions with cell receptors induce receptor-mediated endocytosis followed by proteolytic degradation, which limits the duration of cellular quiescence. Here, we report the effects of targeted covalent affibody photoconjugation to epidermal growth factor receptors (EGFR) on EGFR-positive MDA-MB-468 breast cancer cells. First, covalently conjugating affibodies to cells increased doubling time two-fold and reduced ERK activity by 30% as compared to cells treated with an FDA-approved anti-EGFR antibody Cetuximab, which binds to EGFR noncovalently. The distribution of cells in each phase of the cell cycle was determined, and cells conjugated with the affibody demonstrated an accumulation in the G1 phase, indicative of G1 cell cycle arrest. Finally, the proliferative capacity of the cells was determined by the incorporation of 5-ethynyl-2-deoxyuridine and Ki67 Elisa assay, which showed that the percentage of proliferative cells with photoconjugated affibody was half of that found for the untreated control.

Nongenetic Bioconjugation Strategies for Modifying Cell Membranes and Membrane Proteins: A Review.

The cell membrane possesses an extensive library of proteins, carbohydrates, and lipids that control a significant portion of inter- and intracellular functions, including signaling, proliferation, migration, and adhesion, among others. Augmenting the cell surface composition would open possibilities for advances in therapy, tissue engineering, and probing fundamental cell processes. While genetic engineering has proven effective for many in vitro applications, these techniques result in irreversible changes to cells and are difficult to apply in vivo. Another approach is to instead attach exogenous functional groups to the cell membrane without changing the genetic nature of the cell. This review focuses on more recent approaches of nongenetic methods of cell surface modification through metabolic pathways, anchorage by hydrophobic interactions, and chemical conjugation. Benefits and drawbacks of each approach are considered, followed by a discussion of potential applications for nongenetic cell surface modification and an outlook on the future of the field.

Enzymes Photo-Cross-Linked to Live Cell Receptors Retain Activity and EGFR Inhibition after Both Internalization and Recycling.

In this work, we show that a prodrug enzyme covalently photoconjugated to live cell receptors survives endosomal proteolysis and retains its catalytic activity over multiple days. Here, a fusion protein was designed with both an antiepidermal growth factor receptor (EGFR) affibody and the prodrug enzyme cytosine deaminase, which can convert prodrug 5-fluorocytosine to the anticancer drug 5-fluorouracil. A benzophenone group was added at a site-specific mutation within the affibody, and the fusion protein was selectively photoconjugated to EGFR receptors expressed on membranes of MDA-MB-468 breast cancer cells. The fusion protein was next labeled with two dyes for tracking uptake: AlexaFluor 488 and pH-sensitive pHAb. Flow cytometry showed that fusion proteins photo-cross-linked to EGFR first underwent receptor-mediated endocytosis within 12 h, followed by recycling back to the cell membrane within 24 h. These findings were also confirmed by confocal microscopy. The unique cross-linking of the affibody-enzyme fusion proteins was utilized for two anticancer treatments. First, the covalent linking of the protein to the EGFR led to inhibition of ERK signaling over a two-day period, whereas conventional antibody therapy only led to 6 h of inhibition. Second, when the affibody-CodA fusion proteins were photo-cross-linked to EGFR overexpressed on MDA-MB-468 breast cancer cells, prodrug conversion was found even 48 h postincubation without any apparent decrease in cell killing, while without photo-cross-linking no cell killing was observed 8 h postincubation. These studies show that affinity-mediated covalent conjugation of the affibody-enzymes to cell receptors allows for prolonged expression on membranes and retained enzymatic activity without genetic engineering.

Design and Application of Stimulus-Responsive Droplets and Bubbles Stabilized by Phospholipid Monolayers.

Biomimetic colloidal particles are promising agents for biosensing, but current technologies fall far short of Nature's capabilities for sensing, assessing, and responding to stimuli. Phospholipid-containing cell membranes are capable of binding and responding to an enormous variety of biomolecules by virtue of membrane organization and the presence of receptor proteins. By tuning the composition and functionalization of simulated membranes, soft colloids such as droplets and bubbles can be designed to respond to various stimuli. Moreover, because lipid monolayers can surround almost any hydrophobic phase, the interior of the colloid can be selected to provide a sensitive readout, for example in the form of optical microscopy or acoustic detection. In this work, we review some advances made by our group and others in the formulation of lipid-coated particles with different internal phases such as fluorocarbons, hydrocarbons, or liquid crystals. In some cases, binding or displacement of stabilizing lipids gives rise to conformational changes or disruptions in local membrane geometry, which can be amplified by the interior phase. In other cases, multivalent analytes can promote aggregation or even membrane fusion, which can be utilized for optical or acoustic readout. By highlighting a few recent examples, we hope to show that lipid monolayers represent an extremely versatile biosensing platform that can react to and detect biomolecules by leveraging the unique capabilities of phospholipid membranes.

Click Nucleic Acid Mediated Loading of Prodrug Activating Enzymes in PEG-PLGA Nanoparticles for Combination Chemotherapy.

The simultaneous delivery of multiple therapeutics to a single site has shown promise for cancer targeting and treatment. However, because of the inherent differences in charge and size between drugs and biomolecules, new approaches are required for colocalization of unlike components in one delivery vehicle. In this work, we demonstrate that triblock copolymers containing click nucleic acids (CNAs) can be used to simultaneously load a prodrug enzyme (cytosine deaminase, CodA) and a chemotherapy drug (doxorubicin, DOX) in a single polymer nanoparticle. CNAs are synthetic analogs of DNA comprised of a thiolene backbone and nucleotide bases that can hybridize to complementary strands of DNA. In this study, CodA was appended with complementary DNA sequences and fluorescent dyes to allow its encapsulation in PEG-CNA-PLGA nanoparticles. The DNA-modified CodA was found to retain its enzyme activity for converting prodrug 5-fluorocytosine (5-FC) to active 5-fluorouracil (5-FU) using a modified fluorescent assay. The DNA-conjugated CodA was then loaded into the PEG-CNA-PLGA nanoparticles and tested for cell cytotoxicity in the presence of the 5-FC prodrug. To study the effect of coloading DOX and CodA within a single nanoparticle, cell toxicity assays were run to compare dually loaded nanoparticles with nanoparticles loaded only with either DOX or CodA. We show that the highest level of cell death occurred when both DOX and CodA were simultaneously entrapped and delivered to cells in the presence of 5-FC.

Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors.

A significant challenge for solid tumor treatment is ensuring that a sufficient concentration of therapeutic agent is delivered to the tumor site at doses that can be tolerated by the patient. Biomolecular targeting can bias accumulation in tumors by taking advantage of specific interactions with receptors overexpressed on cancerous cells. However, while antibody-based immunoconjugates show high binding to specific cells, their low dissociation constants ( KD) and large Stokes radii hinder their ability to penetrate deep into tumor tissue, leading to incomplete cell killing and tumor recurrence. To address this, we demonstrate the design and production of a photo-cross-linkable affibody that can form a covalent bond to epidermal growth factor receptor (EGFR) under near UV irradiation. Twelve cysteine mutations were created of an EGFR affibody and conjugated with maleimide-benzophenone. Of these only one exhibited photoconjugation to EGFR, as demonstrated by SDS-PAGE and Western blot. Next this modified affibody was shown to not only bind EGFR expressing cells but also show enhanced retention in a 3D tumor spheroid model, with minimal loss up to 24 h as compared to either unmodified EGFR-binding affibodies or nonbinding, photo-cross-linkable affibodies. Finally, in order to show utility of photo-cross-linking at clinically relevant wavelengths, upconverting nanoparticles (UCNPs) were synthesized that could convert 980 nm light to UV and blue light. In the presence of UCNPs, both direct photoconjugation to EGFR and enhanced retention in tumor spheroids could be obtained using near-infrared illumination. Thus, the photoactive affibodies developed here may be utilized as a platform technology for engineering new therapy conjugates that can penetrate deep into tumor tissue and be retained long enough for effective tumor therapy.

Polyacrylamide Hydrogels Produce Hydrogen Peroxide from Osmotic Swelling in Aqueous Media.

This work demonstrates that hydrogen peroxide (H2O2) is generated in weak polyacrylamide hydrogels due to mechanochemical reactions to osmotic swelling. Hydrogels are important tools and materials for many biomedical applications, particularly for growth of stem cells. However, swollen gels are under constant tension, which makes their individual chains susceptible to mechanochemical bond breakage. In this work, an assay was developed to measure the generation of H2O2 as a result of hydrogel swelling. Polyacrylamide hydrogels with both weak disulfide and strong PEG-diacrylate crosslinkers were synthesized and swelled. H2O2 generation increased in the presence of weaker crosslinkers, up to 30 µM H2O2, whereas stronger crosslinkers reduced this to 5 µM H2O2. H2O2 levels decreased when swelled in the presence of dextran to reduce osmotic stress or increased if the gels were conjugated to an acrylated surface. Finally, H2O2 continued to form for days after the gels had reached their equilibrium sizes, independently of dissolved oxygen. The results of this work impact those working in the 3D cell culture community and demonstrate that even well-characterized systems undergo mechanochemical processes in mild environments.

DNA-Assembled Core-Satellite Upconverting-Metal-Organic Framework Nanoparticle Superstructures for Efficient Photodynamic Therapy.

DNA-mediated assembly of core-satellite structures composed of Zr(IV)-based porphyrinic metal-organic framework (MOF) and NaYF4 ,Yb,Er upconverting nanoparticles (UCNPs) for photodynamic therapy (PDT) is reported. MOF NPs generate singlet oxygen (1 O2 ) upon photoirradiation with visible light without the need for additional small molecule, diffusional photosensitizers such as porphyrins. Using DNA as a templating agent, well-defined MOF-UCNP clusters are produced where UCNPs are spatially organized around a centrally located MOF NP. Under NIR irradiation, visible light emitted from the UCNPs is absorbed by the core MOF NP to produce 1 O2 at significantly greater amounts than what can be produced from simply mixing UCNPs and MOF NPs. The MOF-UCNP core-satellite superstructures also induce strong cell cytotoxicity against cancer cells, which are further enhanced by attaching epidermal growth factor receptor targeting affibodies to the PDT clusters, highlighting their promise as theranostic photodynamic agents.

DNA Hybridization-Mediated Liposome Fusion at the Aqueous Liquid Crystal Interface.

The prominence of receptor-mediated bilayer fusion in cellular biology motivates development of biomimetic strategies for studying fusogenic mechanisms. An approach is reported here for monitoring receptor-mediated fusion that exploits the unique physical and optical properties of liquid crystals (LC). PEG-functionalized lipids are used to create an interfacial environment capable of inhibiting spontaneous liposome fusion with an aqueous/LC interface. Then, DNA hybridization between oligonucleotides within bulk phase liposomes and a PEG-lipid monolayer at an aqueous/LC interface is exploited to induce receptor-mediated liposome fusion. These hybridization events induce strain within the liposome bilayer, promote lipid mixing with the LC interface, and consequently create an interfacial environment favoring re-orientation of the LC to a homeotropic (perpendicular) state. Furthermore, the bi-functionality of aptamers is exploited to modulate DNA hybridization-mediated liposome fusion by regulating the availability of the appropriate ligand (i.e., thrombin). Here, a LC-based approach for monitoring receptor (i.e., DNA hybridization)-mediated liposome fusion is demonstrated, liposome properties that dictate fusion dynamics are explored, and an example of how this approach may be used in a biosensing scheme is provided.

On-demand droplet fusion: a strategy for stimulus-responsive biosensing in solution.

A novel strategy is reported for biochemically controlled fusion of oil-in-water (O/W) droplets as an in-solution sensor for biological targets. Inspired by the SNARE complex in cells, the emulsions were stabilized by a combination of phospholipids, phospholipid-poly(ethylene glycol) conjugates, and cholesterol-anchored oligonucleotides. Prior to oligonucleotide binding, the droplets were stable in aqueous media, but hybridization of the oligonucleotides in a zipperlike fashion was shown to initiate droplet fusion. Using image analysis of content mixing of dye-loaded droplets, fusion specificity was studied and optimized as a function of interfacial chemistry. Changing the orientation of the anchored oligonucleotides, using long-chain phospholipids (C18 and C22), and binding a complementary oligonucleotide slowed or even halted fusion completely. Based on these studies, a sensor for the biomarker thrombin was designed using competitive binding of aptamer strands, with droplet fusion increasing as a function of thrombin addition in accordance with a simple binding model, with sensitivity down to 100 nM and with results in as little as 15 min. Future efforts will focus on utilizing this mechanism of content mixing to facilitate highly sensitive detection via modalities such as magnetoresistance or chemiluminescence.

Imparting the unique properties of DNA into complex material architectures and functions.

While the remarkable chemical and biological properties of DNA have been known for decades, these properties have only been imparted into materials with unprecedented function much more recently. The inimitable ability of DNA to form programmable, complex assemblies through stable, specific, and reversible molecular recognition has allowed the creation of new materials through DNA's ability to control a material's architecture and properties. In this review we discuss recent progress in how DNA has brought unmatched function to materials, focusing specifically on new advances in delivery agents, devices, and sensors.

Controlling Cell Organization in 3D Coculture Spheroids Using DNA Interactions.

The role of stromal and immune cells in transforming the tumor microenvironment is a key consideration in understanding tumor cell behavior and anticancer drug development. To better model these systems in vitro, 3D coculture tumor spheroids have been engineered using a variety of techniques including centrifugation to microwells, hanging drop, low adhesion cultures, and culture of cells in a microfluidic platform. Aside from using bioprinting, however, it has remained more challenging to direct the spatial organization of heterotypic cells in standalone 3D spheroids. To address this, we present an in vitro 3D coculture tumor model where we modulated the interactions between cancer cells and fibroblasts through DNA hybridization. When native heterotypic cells are simply mixed, the cell aggregates typically show cell sorting behavior to form phase separated structures composed of single cell types. In this work, we demonstrate that when MDA-MB-468 breast cancer and NIH/3T3 fibroblasts are directed to associate via complementary DNA, a uniform distribution of the two cell types within a single spheroid was observed. In contrast, in the absence of specific DNA interactions between the cancer cells and fibroblasts, individual clusters of the NIH/3T3 cells formed in each spheroid due to cell sorting. To better understand the effect of heterotypic cell organization on either cell-cell contacts or matrix protein production, the spheroids were further stained with anti-E-cadherin and antifibronectin antibodies. While the amounts of E-cadherin appeared to be similar between the spheroids, a significantly higher amount of fibronectin secretion was observed in the coculture spheroids with uniform mixing of two cell types. This result showed that different heterotypic cell distributions within 3D architecture can influence the ECM protein production that can again alter the properties of the tumor or tumor microenvironment. The present study thus describes the use of DNA templating to direct the organization of cells in coculture spheroids, which can provide mechanistic biological insight into how heterotypic distribution in tumor spheroids can influence tumor progression, metastasis, and drug resistance.

Enzyme Prodrug Therapy with Photo-Cross-Linkable Anti-EGFR Affibodies Conjugated to Upconverting Nanoparticles.

In this work, we demonstrate that a photo-cross-linkable conjugate of upconverting nanoparticles and cytosine deaminase can catalyze prodrug conversion specifically at tumor sites in vivo. Non-covalent association of proteins and peptides with cellular surfaces leads to receptor-mediated endocytosis and catabolic degradation. Recently, we showed that covalent attachment of proteins such as affibodies to cell receptors yields extended expression on cell surfaces with preservation of protein function. To adapt this technology for in vivo applications, conjugates were prepared from upconverting nanoparticles and fusion proteins of affibody and cytosine deaminase enzyme (UC-ACD). The affibody allows covalent photo-cross-linking to epidermal growth factor receptors (EGFRs) overexpressed on Caco-2 human colorectal cancer cells under near-infrared (NIR) light. Once bound, the cytosine deaminase portion of the fusion protein converts the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU). NIR covalent photoconjugation of UC-ACD to Caco-2 cells showed 4-fold higher retention than observed with cells that were not irradiated in vitro. Next, athymic mice expressing Caco-2 tumors showed 5-fold greater UC-ACD accumulation in the tumors than either conjugates without the CD enzyme or UC-ACDs in the absence of NIR excitation. With oral administration of 5-FC prodrug, tumors with photoconjugated UC-ACD yielded 2-fold slower growth than control groups, and median mouse survival increased from 28 days to 35 days. These experiments demonstrate that enzyme-decorated nanoparticles can remain viable after a single covalent photoconjugation in vivo, which can in turn localize prodrug conversion to tumor sites for multiple weeks.

Silica-coated gold nanorods with hydrophobic modification show both enhanced two-photon fluorescence and ultrasound drug release.

Hydrophobically-modified silica-coated gold nanorods are presented here as multifunctional theranostic agents. A single modification both increases two-photon fluorescence and promotes cavitation-based acoustic signal for imaging. A two-fold greater release of small molecule drugs was observed under ultrasound-mediated conditions as compared to passive release without ultrasound.

Facile One-Pot Synthesis of Polymer-Phospholipid Composite Microbubbles with Enhanced Drug Loading Capacity for Ultrasound-Triggered Therapy.

This paper reports the one-pot synthesis of perfluorocarbon microbubbles with crosslinked shells of poly(acrylic acid) and phospholipid that boast excellent ultrasound contrast enhancement, enhanced loading capacity, and the ability to retain or release their contents through variation in the level of ultrasound exposure.

Generation of 3D cellular spheroids using DNA modified cell receptors and programmable DNA interactions.

3D culture is known to provide more faithful tissue models than 2D culture, and thus it is a valuable tool for in vitro evaluation of biological models. However, many cell lines are unable to form desired 3D spheroids by traditional methods because the naturally occurring cell-cell adhesion is too weak. Here, we present a method to produce 3D cell spheroids by using DNA-mediated assembly. We first demonstrate an Affinity Mediated Photoconjugation Approach (AMCP) to covalently modify cell receptors with affibody-streptavidin fusion proteins, where the affibody chemically crosslinks to cell expressed EGFR and the streptavidin is used to attach DNA strands. The DNA conjugated cells were then mixed with complementary DNA 'linker strands' to impart cell-cell interactions. When incubated in wells coated with non-adhesive polymers, cells formed dense spherical aggregates larger than 500 microns in diameter. Each of these studies was carried out using human breast cancer cells (MBA-MB-468), aneuploid human keratinocytes (HaCaT), and human colon cancer cells (Caco-2). Without either DNA on the cells or in solution as linkers, no cell spheroids were observed. After 96 h of incubation, the cultured DNA assembled spheroids were found to be mechanically stable enough to be handled easily for further analysis and confocal imaging. The findings suggest that the proposed DNA assembly method can be considered as an attractive strategy for assembling cells into stable spheroids.

Mechanochemistry Activated Covalent Conjugation Reactions in Soft Hydrogels Induced by Interfacial Failure.

This work reports the development of a mechanochemistry activated covalent conjugation (MACC) reaction that shows areas of interfacial failure in soft hydrogels. Hydrogels are prone to delamination from rigid substrates due to the competition between swelling and adhesion, which can lead to bonding failure in a mechanism similar to crack propagation in harder materials. In this work, reductive amination was shown to occur when a ketone-bearing fluorescein derivative was bonded to an amine-functionalized hydrogel, as both of these moieties were found to be necessary for covalent conjugation into the gel network. For thin, circular polyacrylamide hydrogels, wrinkle patterns and regions of subsequent delamination at the edge of the gel were found to be selectively tagged by the dye. This reaction was then used to explore the effect of gel properties on patterns of interfacial failure. As cross-linker loading increased, the propagation of the delamination front and the area fraction of delamination were both found to increase, as shown by fluorescence images of gels. Increasing the thickness of the gel increased the fraction of delaminated area but did not change its propagation toward the center of the gel. This MACC reaction shows how mechanochemical reactions can be used for fluorescence tagging without incorporating mechanophores into the polymer gel matrix.

Investigating the use of conducting oligomers and redox molecules in CdS-MoFeP biohybrids.

In this work we report the effect of incorporating conducting oligophenylenes and a cobaltocene-based redox mediator on photodriven electron transfer between thioglycolic acid (TGA) capped CdS nanorods (NR) and the native nitrogenase MoFe protein (MoFeP) by following the reduction of H+ to H2. First, we demonstrate that the addition of benzidine-a conductive diphenylene- to TGA-CdS and MoFeP increased catalytic activity by up to 3-fold as compared to CdS-MoFeP alone. In addition, in comparing the use of oligophenylenes composed of one (p-phenylenediamine), two (benzidine) or three (4,4''-diamino-p-terphenyl)phenylene groups, the largest gain in H2 was observed with the addition of benzidine and the lowest with phenylenediamine. As a comparison to the conductive oligophenylenes, a cobaltocene-based redox mediator was also tested with the TGA-CdS NRs and MoFeP. However, adding either cobaltocene diacid or diamine caused negligible gains in H2 production and at higher concentrations, caused a significant decrease. Agarose gel electrophoresis revealed little to no detectable interaction between benzidine and TGA-CdS but strong binding between cobaltocene and TGA-CdS. These results suggest that the tight binding of the cobaltocene mediator to CdS may hinder electron transfer between CdS and MoFe and cause the mediator to undergo continuous reduction/oxidation events at the surface of CdS.

Hydrophobically Modified Silica-Coated Gold Nanorods for Generating Nonlinear Photoacoustic Signals.

In this work, we report that gold nanorods coated with hydrophobically-modified mesoporous silica shells not only enhance photoacoustic (PA) signal over unmodified mesoporous silica coated gold nanorods, but that the relationship between PA amplitude and input laser fluence is strongly nonlinear. Mesoporous silica shells of ~14 nm thickness and with ~3 nm pores were grown on gold nanorods showing near infrared absorption. The silica was rendered hydrophobic with addition of dodecyltrichlorosilane, then re-suspended in aqueous media with a lipid monolayer. Analysis of the PA signal revealed not only an enhancement of PA signal compared to mesoporous silica coated gold nanorods at lower laser fluences, but also a nonlinear relationship between PA signal and laser fluence. We attribute each effect to the entrapment of solvent vapor in the mesopores: the vapor has both a larger expansion coefficient and thermal resistance than silica that enhances conversion to acoustic energy, and the hydrophobic porous surface is able to promote phase transition at the surface, leading to a nonlinear PA response even at fluences as low as 5 mJ cm-2. At 21 mJ cm-2, the highest laser fluence tested, the PA enhancement was >12-fold over mesoporous silica coated gold nanorods.

Hydrogel Coatings on Container Surfaces Reduce Protein Aggregation Caused by Mechanical Stress and Cavitation.

This work reports the ability of hydrogel coatings to protect therapeutic proteins from cavitation-induced aggregation caused by mechanical stress. Here, we show that polyacrylamide hydrogel coatings on container surfaces suppress mechanical shock-induced cavitation and the associated aggregation of intravenous immunoglobulin (IVIg). First, crosslinked polyacrylamide hydrogels were grown on the surfaces of borosilicate glass vials. Treatment with ultrasound showed that these hydrogel surfaces suppressed cavitation events to levels below those found for unfunctionalized borosilicate glass. Next, IVIg solutions were loaded into these vials and subjected to tumbling, horizontal shaking, and drop testing. Aggregation was quantified by bisANS fluorescence staining and particle counting by flow imaging microscopy (FIM). In all cases, the presence of polyacrylamide hydrogels on the vial surfaces reduced the amount of IVIg aggregation and the number of particulates. In addition, the polyacrylamide appeared to have a protective effect that prevented additional aggregates from forming at extended tumbling times. Finally, drop test studies showed that the polyacrylamide coatings suppressed detectable cavitation. This work reveals how even a simple hydrogel vial coating can have a profound effect on stabilizing protein therapeutics.