Delivery of Antibody Mimics into Mammalian Cells via Anthrax Toxin Protective Antigen

Antibody mimics have significant scientific and therapeutic utility for the disruption of protein–protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N‐terminal domain of LF (LF_N). In contrast, a cell‐penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol. The refolding and binding of a transported tandem monobody to Bcr‐Abl (its protein target) in chronic myeloid leukemia cells were confirmed by co‐immunoprecipitation. We also observed inhibition of Bcr‐Abl kinase activity and induction of apoptosis caused by the monobody. In a separate case, we show disruption of key interactions in the MAPK signaling pathway after PA‐mediated delivery of an affibody binder that targets hRaf‐1. We show for the first time that PA can deliver bioactive antibody mimics to disrupt intracellular protein–protein interactions. This technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu.     

Cellular Internalization of Water‐Soluble Helical Aromatic Amide Foldamers

The intracellular transport of drugs and therapeutics represents one of the most exciting and challenging areas at the interface of chemistry, biology, and medicine. Most of the effort in this field so far has been devoted to the development of peptide‐based delivery systems that can translocate therapeutic agents into their intracellular targets. More recently, the use of bioinspired non‐natural foldamers has resulted in the successful delivery of cargo molecules, which possess a wide range of sizes and physicochemical properties across the cell membrane. We report herein the synthesis of aromatic amide foldamers and their biological evaluation as cell‐penetrating agents. By using a well‐established synthetic route, a series of fluorescein‐labeled cationic aryl amide conjugates has been constructed, and their cellular uptake into various human cell lines has been analyzed by flow cytometry and fluorescence microscopy. The assays revealed that longer oligomers achieve greater cellular translocation, with octamer Q8 proving to be a remarkable vehicle for all three cell lines. Biological studies have also indicated that these helices are biocompatible, thus showing promise in their application as cell‐penetrating agents and as vehicles to deliver biologically active molecules into cells.     

Live‐Cell MicroRNA Imaging through MnO2 Nanosheet‐Mediated DD‐A Hybridization Chain Reaction

Innovative techniques to visualize native microRNAs (miRNAs) in live cells can dramatically impact current research on the roles of miRNA in biology and medicine. Here, we report a novel approach for live‐cell miRNA imaging using a biodegradable MnO₂ nanosheet‐mediated DD‐A FRET hybridization chain reaction (HCR). The MnO₂ nanosheets can adsorb DNA hairpin probes and deliver them into live cells. After entering cells, the MnO₂ nanosheets are degraded by cellular GSH. Then, the target miR‐21 triggers cascaded assembly of the liberated hairpin probes into long dsDNA polymers, which brings each two FAMs (d onor) and one TAMRA (a cceptor) into close proximity to generate significantly enhanced DD‐A FRET signals, which was discovered and proven by our previous report. We think the developed approach can serve as an excellent intracellular miRNAs detection tool, which promises the potential for biological and disease studies.     

Passage of Trojan Peptoids into Plant Cells

Efficient drug delivery is essential for many therapeutic applications. In this context, Trojan peptoids have attracted attention as powerful tools to deliver bioactive molecules into living cells. Certain cell‐penetrating peptides, peptide mimetics, and peptoids have been shown to be endowed with a transport function and the structural features of this function have been characterized. However, most of the research has been done by using mammalian cell cultures as model organisms and the actual cellular mechanism of membrane passage has not been elucidated. Plant cells, which are encased in a cellulosic cell wall and differ in membrane composition, represent an alternative experimental system to address this issue, but so far, have attracted only little attention for both peptide‐ and peptoid‐based carrier systems. Moreover, efficient delivery of nonproteinaceous bioactive macromolecules into living plant cells could complement genetic engineering in biotechnological applications, such as metabolic engineering and molecular farming. In the present study, we investigated carrier peptoids with or without guanidinium side chains with regard to their uptake into plant cells, the cellular mechanism of uptake, and intracellular localization. We can show that in contrast to polyamine peptoids (polylysine‐like) fluorescently labeled polyguanidine peptoids (polyarginine‐like) enter rapidly into tobacco BY‐2 cells without affecting the viability of these cells. A quantitative comparison of this uptake with endocytosis of fluorescently labeled dextranes indicates that the main uptake of the guanidinium peptoids occurs between 30–60 min after the start of incubation and clearly precedes endocytosis. Dual visualization with the endosomal marker FM4‐64 shows that the intracellular guanidinium peptoid is distinct from endocytotic vesicles. Once the polyguanidine peptoids have entered the cell, they associate with actin filaments and microtubules. By pharmacological manipulation of the cytoskeleton we tested whether the association with the cytoskeleton is necessary for uptake, and observed that the actin inhibitor latrunculin B as well as the microtubule inhibitor oryzalin impaired uptake and intracellular spread of the guanidinium carrier to a certain extent. These findings are discussed with respect to the potential mechanisms of uptake and with respect to the potential of Trojan peptoids as tools for metabolic engineering in plant biotechnology.     

Synthesis of cationic magnetite nanoparticles for intracellular protein delivery

Intracellular protein delivery shows great promise in the treatment of various diseases. However, therapeutic applications of this method are limited by its low delivery efficiency and poor targeting ability. As one of most important drug delivery cargoes, Fe₃O₄ nanoparticles (nFe₃O₄) have attracted much attention for both therapeutic and diagnostic applications, especially for targeting drug delivery. To use nFe₃O₄ for protein delivery, a simple but effective modification of nFe₃O₄ is critical to attach proteins on its surface. In this work, by designing and synthesizing cationic poly(2‐(dimethylamino)ethyl methacrylate) (PDMA)‐grafted nFe₃O₄ via in situ atom transfer radical polymerization (ATRP), we demonstrate a simple solution to improve interactions between nFe₃O₄ and proteins. With the grafted PDMA on the surface, nFe₃O₄ exhibits not only significant enhancement in dispersibility and stability in aqueous phase, but also an excellent capability to attach negative‐charged proteins. Moreover, with the assistance of external magnetic field, PDMA‐grafted nFe₃O₄ can be used as a targetable vector to deliver proteins into specific cells. This work provides a novel platform based on cationic magnetite nanoparticles that can deliver therapeutic proteins into specific sites for the treatment of various diseases. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40260.     

Spatio-Temporal Photoactivation of Cytotoxic Proteins

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.     

Esterified Trehalose Analogues Protect Mammalian Cells from Heat Shock

Trehalose is a disaccharide produced by many organisms to better enable them to survive environmental stresses, including heat, cold, desiccation, and reactive oxygen species. Mammalian cells do not naturally biosynthesize trehalose; however, when introduced into mammalian cells, trehalose provides protection from damage associated with freezing and drying. One of the major difficulties in using trehalose as a cellular protectant for mammalian cells is the delivery of this disaccharide into the intracellular environment; mammalian cell membranes are impermeable to the hydrophilic sugar trehalose. A panel of cell‐permeable trehalose analogues, in which the hydrophilic hydroxyl groups of trehalose are masked as esters, have been synthesized and the ability of these analogues to load trehalose into mammalian cells has been evaluated. Two of these analogues deliver millimolar concentrations of free trehalose into a variety of mammalian cells. Critically, Jurkat cells incubated with these analogues show improved survival after heat shock, relative to untreated Jurkat cells. The method reported herein thus paves the way for the use of esterified analogues of trehalose as a facile means to deliver high concentrations of trehalose into mammalian cells for use as a cellular protectant.     

Evaluation of a 7‐Methoxycoumarin‐3‐carboxylic Acid Ester Derivative as a Fluorescent,Cell‐Cleavable,Phosphonate Protecting Group

Cell‐cleavable protecting groups often enhance cellular delivery of species that are charged at physiological pH. Although several phosphonate protecting groups have achieved clinical success, it remains difficult to use these prodrugs in live cells to clarify biological mechanisms. Here, we present a strategy that uses a 7‐methoxycoumarin‐3‐carboxylic acid ester as a fluorescent protecting group. This strategy was applied to synthesis of an (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate (HMBPP) analogue to assess cellular uptake and human Vγ9Vδ2 T cell activation. The fluorescent ester displayed low cellular toxicity (IC₅₀>100 μm ) and strong T cell activation (EC₅₀=0.018 μm ) relative to the unprotected anion (EC₅₀=23 μm ). The coumarin‐derived analogue allowed no‐wash analysis of biological deprotection, which revealed rapid internalization of the prodrug. These results demonstrate that fluorescent groups can be applied both as functional drug delivery tools and useful biological probes of drug uptake.     

PAMAM Dendrimers Mediate siRNA Delivery to Target Hsp27 and Produce Potent Antiproliferative Effects on Prostate Cancer Cells

RNA interference (RNAi) holds great promise for the treatment of inherited and acquired diseases, provided that safe and efficient delivery systems are available. Herein we report that structurally flexible triethanolamine (TEA) core PAMAM dendrimers are able to deliver an Hsp27 siRNA effectively into prostate cancer (PC‐3) cells by forming stable nanoparticles with siRNA, protecting the siRNA nanoparticles from enzymatic degradation, and enhancing cellular uptake of siRNA. The Hsp27 siRNA resulted in potent and specific gene silencing of heat‐shock protein 27, an attractive therapeutic target in castrate‐resistant prostate cancer. Silencing of the hsp27 gene led to induction of caspase‐3/7‐dependent apoptosis and inhibition of PC‐3 cell growth in vitro. In addition, the siRNA–dendrimer complexes are non‐cytotoxic under the conditions used for siRNA delivery. Altogether, TEA core PAMAM dendrimer‐mediated siRNA delivery, in combination with RNAi that specifically targets Hsp27, may constitute a promising approach for combating castrate‐resistant prostate cancer, for which there is no efficacious treatment.     

A TAT-streptavidin fusion protein directs uptake of biotinylated cargo into mammalian cells

The HIV-1 TAT peptide has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For protein delivery, a variety of TAT-fusion proteins have been described which link the TAT coding sequence to the protein coding sequence of interest. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. Fluorescence activated cell sorting (FACS) analysis and confocal microscopy characterization showed that TAT-SA is internalized by Jurkat T-cells and NIH 3T3 cells alone and when complexed to phycoerythrin, whereas the native streptavidin is not. Additionally, biotinylated alkaline phosphatase is successfully internalized and retains its activity when complexed to TAT-SA and incubated with Jurkat T-cells. Confocal microscopy suggested, however, that internalized TAT-SA and TAT-SA complexes were largely compartmentalized in vesicular compartments, rather than freely diffusing in the cytoplasmic compartment. To effect cytoplasmic delivery, the endosomal releasing polymer, poly(propylacrylic acid) (PPAA), was biotinylated and complexed to TAT-SA. Endosomal release and cytoplasmic delivery of fluorescently labeled TAT-SA complexes with PPAA was shown by the diffuse distribution of fluorescent protein in the cytoplasm. Taken together, these results demonstrate that TAT-SA can be used to direct intracellular delivery of large biotinylated cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired.     

Fluorescent Visualization of Src by Using Dasatinib‐BODIPY

Many biological experiments are not compatible with the use of immunofluorescence, genetically encoded fluorescent tags, or FRET‐based reporters. Conjugation of existing kinase inhibitors to cell‐permeable fluorophores can provide a generalized approach to develop fluorescent probes of intracellular kinases. Here, we report the development of a small molecule probe of Src through conjugation of BODIPY to two well‐established dual Src‐Abl kinase inhibitors, dasatinib and saracatinib. We show that this approach is not successful for saracatinib but that dasatinib‐BODIPY largely retains the biological activity of its parent compound and can be used to monitor the presence of Src kinase in individual cells by flow cytometry. It can also be used to track the localization of Src by fixed and live‐cell fluorescence microscopy. This strategy could enable generation of additional kinase‐specific probes useful in systems not amenable to genetic manipulation or could be used together with fluorescent proteins to enable a multiplexed assay readout.     

Lipophilic 2′‐O‐Acetal Ester RNAs: Synthesis,Thermal Duplex Stability,Nuclease Resistance,Cellular Uptake,and siRNA Activity after Spontaneous Naked Delivery

The in vivo application of siRNA depends on its cellular uptake and intracellular release, and this is an unsatisfactorily resolved technical hurdle in medicinal applications. Promising concepts directed towards providing efficient cellular and intracellular delivery include lipophilic chemical modification of siRNA. Here we describe chemistry for the production of modified siRNAs designed to display improved transmembrane transport into human cells while preserving the potency of the RNAi‐based inhibitors. We report the synthesis and the biochemical and biophysical characteristics of 2′‐O‐phenylisobutyryloxymethyl (PiBuOM)‐modified siRNAs and their impact on biological activity. In the case of spontaneous cellular uptake of naked PiBuOM‐modified siRNA, we observed increased target suppression in human cells relative to unmodified or pivaloyloxymethyl (PivOM)‐modified siRNA. We provide evidence of improved spontaneous cellular uptake of naked PiBuOM‐modified siRNA and of substantial target suppression in human cells in serum‐containing medium.     

Complex aggregates formed with a hyperbranched polyglycerol derivative for drug delivery

An amphiphilic hyperbranched polyglycerol derivative (HPG‐C18) was synthesized by the anionic ring‐opening copolymerization with glycidol and 1,2‐epoxyloctadecane as the monomers. This hyperbranched polymer formed large complex aggregates as confirmed by dynamic light scattering and transmission electron microscopy tests. Because of its amphiphilic properties, HPG‐C18 was explored to load hydrophobic docetaxel, a clinical antitumor drug, and deliver it into breast cancer cell line (MCF‐7) cells. To investigate the application of the aggregates in drug delivery, blood compatibility was studied by hemolysis analysis, red blood cell observation, and thromboelastography assay. These results indicate that HPG‐C18 inhibited MCF‐7 proliferation effectively with good blood compatibility, and this suggested a potential application in tumor therapy. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 42895.     

A novel aptamer developed for breast cancer cell internalization

Breast cancer affects one in eight women in the United States, with a mortality rate that is second only to lung cancer. Although chemotherapy is widely used in breast cancer treatment, its side effects remain a challenge. One way to address this problem is through drug delivery by the internalization of cell-type-specific probes. Although nucleic acid aptamers are excellent probes for molecular recognition, only a few studies have demonstrated that aptamers can be internalized into living cells. Therefore, herein we report the development of a cancer-cell-specific DNA aptamer probe, KMF2-1a. By using the cell-SELEX method, this aptamer was selected against breast cancer cell line MCF-10AT1. Our results show that KMF2-1a is internalized efficiently and specifically to the endosome of target breast cancer cells. These results indicate that KMF2-1a is a promising agent for cell-type-specific intracellular delivery with both diagnostic and therapeutic implications.     

Clickable Polyamine Derivatives as Chemical Probes for the Polyamine Transport System

Polyamines are essential for cell growth and differentiation, but their trafficking by the polyamine transport system is not fully understood. Herein, the synthesis of several azido‐derivatized polyamines for easy conjugation by click chemistry is described. Attachment of a 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) dye gave fluorescent polyamine probes, which were tested in cell culture. The linear probe series showed superior cellular uptake compared with that of probes in which the dye was attached to a branch on one of the central amines. Interestingly, the linear probes accumulated rapidly in cancer cells (MCF‐7), but not in nontumorigenic cells (MCF‐10A). The fluorescent polyamine probes are therefore applicable to the study of polyamine trafficking, whereas the azido polyamines may be further utilized to transport cargo into cancer cells by exploiting the polyamine transport system.     

PEGylated polylysine derived copolymers with reduction‐responsive side chains for anticancer drug delivery

Reduction‐responsive drug delivery systems have recently gained intense attention in intracellular delivery of anticancer drugs. In this study, we developed a PEGylated polypeptide, poly(ethylene glycol)‐block‐poly(?‐propargyloxycarbonyl‐l ‐lysine) (PEG₁₁₃‐b‐PPAL), as a novel clickable substrate for conjugation of reduction‐responsive side chains for antineoplastic drug delivery. PEG₁₁₃‐b‐PPAL was synthesized through ring‐opening polymerization of alkyne‐containing N‐carboxyanhydride monomers. A designed disulfide‐containing side chain was introduced onto the PEGylated polypeptide by click reaction. The obtained copolymer PEG₁₁₃‐b‐P(Lys‐DSA) formed micelles by self‐assembly, which exhibited reduction‐responsive behavior under the stimulus of 10 mmol L^–1 glutathione (GSH) in water. A small molecule intermediate, compound 2 , was used as a model to investigate the thiol reduction mechanism of PEG₁₁₃‐b‐P(Lys‐DSA) copolymers. The anticancer drug doxorubicin (DOX) was then loaded into the micelles with a drug loading content of 6.73 wt% and a loading efficiency of 40.3%. Both the blank and the drug‐loaded micelles (DOX‐loaded polylysine derived polymeric micelles (LMs/DOX)) adopted a spherical morphology, with average diameters of 48.0 ± 13.1 and 63.8 ± 20.0 nm, respectively. The in vitro drug release results indicated that DOX could be released faster from the micelles by the trigger of GSH in phosphate buffered saline. Confocal laser scanning microscopy and flow cytometer analysis further proved the intracellular delivery of DOX by LMs/DOX and their GSH‐sensitive release behavior. A 3‐(4,5‐dimethyl‐thiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay showed that the polymers exhibited negligible cytotoxicity towards normal L929 cells or cancer MCF‐7 cells with a treated concentration up to 1.0 mg mL^–1. In conclusion, our synthesized biocompatible and biodegradable PEGylated polypeptides hold great promise for intracellular antineoplastic drug delivery. © 2019 Society of Chemical Industry     

Synthesis and Anti‐herpetic Activity of Phosphoramidate ProTides

Among the many prodrug approaches aimed at delivering nucleoside monophosphates into cells, the phosphoramidate ProTide approach is one that has shown success, which has made it possible for some of the phosphoramidates to enter into clinical trials. Herein, we report the synthesis and antiviral activity of a series of phosphoramidate ProTides designed to bypass the thymidine kinase (TK) dependence of the parent nucleoside analogues. Phosphoramidate derivatives of (E)‐5‐(2‐bromovinyl)‐2′‐deoxyuridine (BVDU) that contain L ‐alanine or pivaloyloxymethyl iminodiacetate (IDA‐POM) exhibit anti‐HSV‐1 and anti‐VZV activity in cell cultures, but they largely lost antiviral potency against TK‐deficient virus strains. Among deazapurine nucleosides and their phosphoramidate derivatives, the 7‐deazaadenine containing nucleosides and their phosphoramidate triester derivatives showed weak antiviral activity against VZV. Apparently, intracellular nucleotide delivery with these phosphoramidates is partly successful. However, none of the compound prodrugs showed superior activity to their parent drugs.     

Antibiotic Algae by Chemical Surface Engineering

Chemical cell‐surface engineering is a tool for modifying and altering cellular functions. Herein, we report the introduction of an antibiotic phenotype to the green alga Chlamydomonas reinhardtii by chemically modifying its cell surface. Flow cytometry and confocal microscopy studies demonstrated that a hybrid of the antibiotic vancomycin and a 4‐hydroxyproline oligomer binds reversibly to the cell wall without affecting the viability or motility of the cells. The modified cells were used to inhibit bacterial growth of Gram‐positive Bacillus subtilis cultures. Delivery of the antibiotic from the microalgae to the bacterial cells was verified by microscopy. Our studies provide compelling evidence that 1) chemical surface engineering constitutes a useful tool for the introduction of new, previously unknown functionality, and 2) living microalgae can serve as new platforms for drug delivery.     

Synergistic effect and drug‐resistance relief of paclitaxel and cisplatin caused by Co‐delivery using polymeric micelles

Combination therapy of paclitaxel (PTX) and cisplatin has been used to treat several cancers in clinic practice, but often causes serious systemic toxicity. Co‐delivery of PTX and cisplatin by means of polymeric micelles can reduce the systemic toxicity, but often needs two carrier polymers because of the solubility difference between them. Therefore, a strategy is developed to co‐deliver both PTX and cisplatin with only one carrier polymer by encapsulating PTX in the core of a polymeric micelle and cross‐linking the micelle with cisplatin. The PTX and Pt contents in the micellar formulation M(PTX/Pt) were 10 and 14 wt %, respectively. In vitro cytotoxicity of M(PTX/Pt) was evaluated via 3‐(4,5‐dimethylthiazol‐2‐yl)?2,5‐diphenyltetrazolium bromide assay in comparison with PTX and its micelle M(PTX), cisplatin and its micelle M(Pt), and PTX/cisplatin combination towards human hepatocarcinoma (SMMC‐7721) cells and chemoresistant SMMC‐7721(SMMC‐7721R) cells. The M(PTX/Pt) exhibited a high synergistic effect in the inhibition of cell growth and proliferation of both SMMC‐7721 and SMMC‐7721R cells and showed reasonable drug‐resistance relief. The synergistic effect and resistance relief were further supported or explained by intracellular uptake measurement of dye‐labeled micelles and by the confocal laser scanning microscopy observation of SMMC‐7721 and SMMC‐7721R cells treated with various formulations. Therefore, M(PTX/Pt) micelles were expected to find potential application in cancer chemotherapy. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41440.     

pH‐Cleavable Nucleoside Lipids: A New Paradigm for Controlling the Stability of Lipid‐Based Delivery Systems

Lipid‐based delivery systems are an established technology with considerable clinical acceptance and several applications in human. Herein, we report the design, synthesis and evaluation of novel orthoester nucleoside lipids (ONLs) for the modulation of liposome stability. The ONLs contain head groups with 3′‐orthoester nucleoside derivatives featuring positive or negative charges. The insertion of the orthoester function in the NL structures allows the formation of pH‐sensitive liposomes. ONL‐based liposomes can be hydrolyzed to provide nontoxic products, including nucleoside derivatives and hexadecanol. To allow the release to be tunable at different hydrolysis rates, the charge of the polar head structure is modulated, and the head group can be released at a biologically relevant pH. Crucially, when ONLs are mixed with natural phosphocholine lipids (PC), the resultant liposome evolves toward the formation of a hexadecanol/PC lamellar system. Biological evaluation shows that stable nucleic acid lipid particles (SNALPs) formulated with ONLs and siRNAs can effectively enter into tumor cells and release their nucleic acid payload in response to an intracellular acidic environment. This results in a much higher antitumor activity than conventional SNALPs. The ability to use pH‐cleavable nucleolipids to control the stability of lipid‐based delivery systems represents a promising approach for the intracellular delivery of drug cargos.