Acid-cleavable Linker Research Articles

A Simplified and Ultrafast Pipeline for Site-Specific Quantitative Chemical Proteomics.

Activity-based proteome profiling (ABPP) is a powerful chemoproteomic technology for global profiling of protein activity and modifications. The tandem orthogonal proteolysis-ABPP (TOP-ABPP) strategy utilizes a clickable enrichment tag with cleavable linkers to enable direct identification of probe-labeled residue sites within the target proteins. However, such a site-specific chemoproteomic workflow requires a long operation time and complex sample preparation procedures, limiting its wide applications. In the current study, we developed a simplified and ultrafast peptide enrichment and release TOP-ABPP ("superTOP-ABPP") pipeline for site-specific quantitative chemoproteomic analysis with special agarose resins that are functionalized with azide groups and acid-cleavable linkers. The azide groups allow enrichment of peptides that are labeled by the alkynyl probe through a one-step click reaction, which can be conveniently released by acid cleavage for subsequent LC-MS/MS analysis. In comparison with the traditional TOP-ABPP method, superTOP-ABPP cuts down the averaged sample preparation time from 25 to 9 h, and significantly improves the sensitivity and coverage of site-specific cysteinome profiling. The method can also be seamlessly integrated with reductive dimethylation to enable quantitative chemoproteomic analysis with a high accuracy. The simplified and ultrafast superTOP-ABPP will become a valuable tool for site-specific quantitative chemoproteomic studies.

Towards the use of an amino acid cleavable linker for solid-phase chemical synthesis of peptides and proteins.

The synthesis of proteins by solid-phase chemical ligation (SPCL) suffers from the paucity of linkers that can be cleaved under mild conditions. Here, we deployed a spontaneous nickel-assisted cleavage (SNAC) tag, known to undergo spontaneous cleavage in the presence of nickel(II), as a linker for C-to-N SPCL.

Prostate-Specific Membrane Antigen-Targeted Turn-on Probe for Imaging Cargo Release in Prostate Cancer Cells.

The tunable nature of phosphoramidate linkers enables broad applicability as pH-triggered controlled-release platforms, particularly in the context of antibody- and small-molecule-drug conjugates (ADCs and SMDCs), where there remains a need for new linker technology. Herein, we explored in-depth the release of turn-on fluorogenic payloads from a homoserinyl-based phosphoramidate acid-cleavable linker. Kinetics of payload release from the scaffold was observed in buffers representing the pH conditions of systemic circulation, early and late endosomes, and lysosomes. It was found that payload release takes place in two key consecutive steps: (1) P-N bond hydrolysis and (2) spacer immolation. These two steps were found to follow pseudo-first-order kinetics and had opposite dependencies on pH. P-N bond hydrolysis increased with decreasing pH, while spacer immolation was most rapid at physiological pH. Despite the contrasting release kinetics of these two steps, maximal payload release was observed at the mildly acidic pH (5.0-5.5), while minimal payload release occurred at physiological pH. We integrated this phosphoramidate-payload linker system into a PSMA-targeted fluorescent turn-on probe to study the intracellular trafficking and release of a fluorescent payload in PSMA-expressing prostate cancer cells. Results showed excellent turn-on and accumulation of the coumarin payload in the late endosomal and lysosomal compartments of these cells. The release properties of this linker mark it as an attractive alternative in the modular design of ADCs and SMDCs, which demand selective intracellular payload release triggered by the pH changes that accompany intracellular trafficking.

Imidazole-Mediated Dual Location Disassembly of Acid-Degradable Intracellular Drug Delivery Block Copolymer Nanoassemblies.

Acid-degradable (or acid-cleavable) polymeric nanoassemblies have witnessed significant progress in anti-cancer drug delivery. However, conventional nanoassemblies designed with acid-cleavable linkages at a single location have several challenges, such as, sluggish degradation, undesired aggregation of degraded products, and difficulty in controlled and on-demand drug release. Herein, a strategy that enables the synthesis of acid-cleavable nanoassemblies labeled with acetaldehyde acetal groups in both hydrophobic cores and at core/corona interfaces, exhibiting synergistic response to acidic pH at dual locations and thus inducing rapid drug release is reported. The systematic analyses suggest that the acid-catalyzed degradation and disassembly are further enhanced by decreasing copolymer concentration (i.e., increasing proton/acetal mole ratio). Moreover, incorporation of acid-ionizable imidazole pendants in the hydrophobic cores improve the encapsulation of doxorubicin, the anticancer drug, through π-π interactions and enhance the acid-catalyzed hydrolysis of acetal linkages situated in the dual locations. Furthermore, the presence of the imidazole pendants induce the occurrence of core-crosslinking that compensates the kinetics of acetal hydrolysis and drug release. These results, combined with in vitro cell toxicity and cellular uptake, suggest the versatility of the dual location acid-degradation strategy in the design and development of effective intracellular drug delivery nanocarriers.

Overcoming Challenges in the Development of Anticancer ADCs

Overcoming Challenges in the Development of Anticancer ADCs

Novel Silyl Ether-Based Acid-Cleavable Antibody-MMAE Conjugates with Appropriate Stability and Efficacy.

Antibody-drug conjugate (ADC) is a novel efficient drug delivery system that has been successfully used in clinical practice, and it has become a research hotspot in the anti-tumor drug field. Acid-cleavable linkers were first used in clinical ADCs, but their structural variety (e.g., hydrazone and carbonate) is still limited, and their stability is usually insufficient. Designing novel acid-cleavable linkers for the conjugation of the popular cytotoxin monomethyl auristatin E (MMAE) has always been a significant topic. In this paper, we generate a novel, silyl ether-based acid-cleavable antibody-MMAE conjugate, which skillfully achieves efficient combination of amino-conjugated MMAE with the acid-triggered silyl ether group by introducing p-hydroxybenzyl alcohol (PHB). The stability, acid-dependence cleavage, effective mechanism, efficacy and safety of the resulting ADC were systematically studied; the results show that it exhibits a significant improvement in stability, while maintaining appropriate efficacy and controlled therapeutic toxicity. This strategy is expected to expand a new type of acid-cleavable linkers for the development of ADCs with highly potent payloads.

Folate-PEG/Hyd-curcumin/C18-g-PSI micelles for site specific delivery of curcumin to colon cancer cells via Wnt/β-catenin signaling pathway

Curcumin shows a potential anticancer activity, as it is involved in signaling pathway suppressing β-catenin response transcription. In this study, the effects of curcumin released from the biocompatible and biodegradable polyaspartamide based micelles on colon cancer treatment via the Wnt/β-catenin signaling pathway are investigated. Hydrophobic octadecylamine (C18) and hydrophilic O-(2-aminoethyl) polyethylene glycol (PEG) were grafted on a polysuccinimide (PSI) backbone for micelle formation. Folic acid (FA) was employed to facilitate the targeting activity to colon cancer cells and curcumin was conjugated via acid cleavable linkage, hydrazone (Hyd) to provide the pH sensitive drug release. Two types of micellar structures, Folate-PEG/Hyd-Curcumin/C18-g-PSI (FA-Cur) and PEG/Hyd-Curcumin/C18-g-PSI (NFA-Cur), were synthesized and their chemical structure was identified by 1H NMR spectroscopy. The cytotoxicity carried out by MTT assay informed that the cell viability of FA-Cur treated SW480 was much lower than that of NFA-Cur treated one at the concentration > 0.25 μg mL−1. Western blot assay showed that FA-Cur inhibited the target genes, cyclin D1 and c-myc, more strongly than NFA-Cur at the concentration > 0.5 μg mL−1. From these results, FA-Cur micelles are expected to be a promising candidate for colon anti-cancer via inhibiting Wnt/β-catenin pathway.

Abstract 4641: Improved chemotherapeutic delivery to brain metastases with targeted nanoparticles in preclinical breast cancer brain metastasis models

Abstract Brain metastases present an increasing challenge to long-term survival for HER2-amplified breast cancer patients. While HER2 targeted therapies effectively control systemic metastases, they are largely ineffective against brain metastases due to their inability to cross the blood-brain barrier. We have recently demonstrated that brain uptake of targeted nanoparticles can be increased by adding an acid-cleavable linkage between transferrin (Tf) and the nanoparticle core [1]. Our lab has also developed a method for assembling targeted nanoparticles by complexing diol-containing polymer-drug conjugates and boronic acid-containing targeting agents [2]. Here, we report the first efficacy results of targeted nanoparticles formed via this assembly strategy in a preclinical model of breast cancer brain metastasis. Female Rag2-/-;Il2rg-/- mice were intracranially injected with human HER2-amplified BT-474 breast cancer cells, and formation of brain tumors was monitored by MRI. Mice were randomized into four groups of six mice per group (targeted nanoparticle, non-targeted nanoparticle, free drug, and saline). Targeted and non-targeted nanoparticles were prepared by functionalizing nanoparticle cores consisting of a mucic acid polymer conjugate of camptothecin (CPT) with either Tf-PEG or methoxy-PEG, respectively, using boronic acid-diol chemistry. The different formulations were administered intravenously once per week for four weeks at a dose of 4 mg/kg (CPT basis), and tumor volume was measured weekly by MRI. Targeted nanoparticles significantly reduced tumor growth in mice bearing intracranially implanted tumors compared to saline, free drug, and non-targeted nanoparticle controls. However, free drug and non-targeted nanoparticles also reduced tumor growth compared to saline, albeit to a lesser extent. Taken together, the results are highly suggestive that there isn't a completely intact blood-brain or blood-tumor barrier, and furthermore that artificial transport pathways may be introduced following intracranial implantation. To overcome limitations of the intracranial injection model, female Rag2-/-;Il2rg-/- mice were intracardiacly or intravenously (tail vein) injected with human HER2-amplified BT-474 breast cancer cells, and formation of metastatic brain tumors was monitored by MRI. Unlike brain tumors from intracranially injected cells, brain tumors formed from intravenous cell injections were not affected by systemic doses of either lapatinib or trastuzumab. Current investigations involve collection of efficacy data for the different formulations in the intracardiac and intravenous models that may provide further insight into the blood-brain barrier integrity and nanoparticle delivery. [1] Clark, AJ, Davis, ME, PNAS, 112 (2015) 12486-12491. [2] Han, H, Davis, ME, Mol. Pharmaceutics, 10 (2013) 2558-2567. Citation Format: Emily A. Wyatt, Mark E. Davis. Improved chemotherapeutic delivery to brain metastases with targeted nanoparticles in preclinical breast cancer brain metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4641.

Stimuli-disassembling gold nanoclusters for diagnosis of early stage oral cancer by optical coherence tomography

A key design consideration in developing contrast agents is obtaining distinct, multiple signal changes in diseased tissue. Plasmonic gold nanoparticles (Au NPs) have been developed as contrast agents due to their strong surface plasmon resonance (SPR). This study aims to demonstrate that stimuli-responsive plasmonic Au nanoclusters (Au NCs) can be used as a contrast agent for optical coherence tomography (OCT) in detecting early-stage cancer. Au NPs were clustered via acid-cleavable linkers to synthesize Au NCs that disassemble under mildly acidic conditions into individual Au NPs, simultaneously diminishing SPR effect (quantified by scattering intensity) and increasing Brownian motion (quantified by Doppler variance). The acid-triggered morphological and accompanying optico-physical property changes of the acid-disassembling Au NCs were confirmed by TEM, DLS, UV/Vis, and OCT. Stimuli-responsive Au NCs were applied in a hamster check pouch model carrying early-stage squamous carcinoma tissue. The tissue was visualized by OCT imaging, which showed reduced scattering intensity and increased Doppler variance in the dysplastic tissue. This study demonstrates the promise of diagnosing early-stage cancer using molecularly programmable, inorganic nanomaterial-based contrast agents that are capable of generating multiple, stimuli-triggered diagnostic signals in early-stage cancer.

Hyperbranched Polyglycerol Loaded with (Zinc-)Porphyrins: Photosensitizer Release Under Reductive and Acidic Conditions for Improved Photodynamic Therapy.

An adaptable approach toward cleavable nanoparticle carrier systems for photodynamic therapy (PDT) is presented, comprising a biocompatible carrier loaded with multiple photosensitizer (PS) molecules related to the clinically employed PS Temoporfin, two linkers cleavable under different triggers and glyco-targeting with mannose. A synthetic pathway to stimuli responsive hyperbranched polyglycerol (hPG) porphyrin conjugates via the copper(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC) or the strain-promoted alkyne-azide cycloaddition (SPAAC) has been developed. The PS 10,15,20-tris(3-hydroxyphenyl)-5-(2,3,4,5,6-pentafluorophenyl)porphyrin was functionalized with disulfide containing cystamine and acid-labile benzacetal linkers. Conjugates with reductively and pH labile linkers were thus obtained. Cleavage of the active PS agents from the polymer carrier is shown in several different release studies. The uptake of the conjugates into the cells is demonstrated via confocal laser scanning microscopy (CLSM) and flow cytometry. Finally, the antitumor and antibacterial phototoxicity of selected conjugates has been assessed in four different tumor cell lines and in cultures of the bacterium Staphylococcus aureus. The conjugates exhibited phototoxicity in several tumor cell lines in which conjugates with reductively cleavable linkers were more efficient compared to conjugates with acid-cleavable linkers. For S. aureus, strong phototoxicity was observed for a combination of the reductively cleavable and the pH labile linker and likewise for the cleavable conjugate with mannose targeting groups. The results thus suggest that the conjugates have potential for antitumor as well as antibacterial PDT.

Backbone-hydrazone-containing biodegradable copolymeric micelles for anticancer drug delivery

Well-defined biodegradable, pH-sensitive amphiphilic block polymers, poly(ethylene glycol)-Hyd-poly(lactic acid) (mPEG-Hyd-PLA) which have acid-cleavable linkages in their backbones, were synthesized via ring-opening polymerization initiated from hydrazone-containing macroinitiators. Introducing a hydrazone bond onto the backbone of an amphiphilic copolymer will find a broad-spectrum encapsulation of hydrophobic drugs. Dynamic light scattering (DLS) and transmission electron microscopy showed that the diblock copolymers self-assembled into stable micelles with average diameters of 100 nm. The mean diameters and size distribution of the hydrazone-containing micelles changed obviously in mildly acidic pH (multiple peaks from 1 to 202 nm appeared under a pH 4.0 condition) than in neutral, while there were no changes in the case of non-sensitive ones. Doxorubicin (DOX) and paclitaxel (PTX) were loaded with drug loading content ranging from 2.4 to 3.5 %, respectively. Interestingly, the anticancer drugs released from mPEG-Hyd-PLA micelles could also be promoted by the increased acidity. An in vitro cytotoxicity study showed that the DOX-loaded mPEG-Hyd-PLA micelles have significantly enhanced cytotoxicity against HepG2 cells compared with the non-sensitive poly(ethylene glycol)-block-poly(lactic acid) (mPEG-PLA) micelles. Confocal microscopy observation indicated that more DOX were delivered into the nuclei of cells following 6 or 12 h incubation with DOX-loaded mPEG-Hyd-PLA micelles. In vivo studies on H22-bearing Swiss mice demonstrated the superior anticancer activity of DOX-loaded mPEG-Hyd-PLA micelles over free DOX and DOX-loaded mPEG-PLA micelles. These hydrazone-containing pH-responsive degradable micelles provide a useful strategy for antitumor drug delivery.

Self-Assembling Doxorubicin Prodrug Forming Nanoparticles and Effectively Reversing Drug Resistance In Vitro and In Vivo.

Doxorubicin (DOX) is a widely used chemotherapeutic drug to treat a range of cancers. However, its unfavorable effects, particularly the cardiotoxicity and the induction of multidrug resistance (MDR), significantly limit its clinical applications. Herein, a novel doxorubicin prodrug, PEG2K -DOX, is synthesized by conjugating a deprotonated doxorubicin molecule with the polyethylene glycol (PEG, MW: 2K) chain via pH-responsive hydrazone bond, and its potential as a better alternative than doxorubicin is evaluated. The data show that the amphiphilic PEG2K -DOX can self-assemble into stable nanoparticles with a high and fixed doxorubicin loading content (≈20 wt%), a favorable size of 91.5 nm with a narrow polydispersity (PDI = 0.14), good stability, and pH-dependent release behavior due to the acid-cleavable linkage between PEG and doxorubicin. Although doxorubicin hardly accumulates in MDR cells, PEG2K -DOX nanoparticles significantly increase the cellular uptake and cell-killing activity of doxorubicin in two MDR cancer cell lines MCF-7/ADR and KBv200, with the IC50 values dropped to 1.130% and 42.467% of doxorubicin, respectively. More impressively, PEG2K -DOX nanoparticles exhibit significantly improved plasma pharmacokinetics, increased in vivo therapeutic efficacy against MDR xenograft tumors, and better in vivo safety compared with doxorubicin. PEG2K -DOX nanoparticles hold the promise to become a better alternative than doxorubicin for cancer treatment, especially for MDR tumors.

Pluronic mimicking fluorescent carbon nanoparticles conjugated with doxorubicin via acid-cleavable linkage for tumor-targeted drug delivery and bioimaging

Fluorescent carbon nanoparticles based target drug delivery with bio-imaging systems offer tremendous scope for future. We described Pluronic mimicking fluorescent nanoparticles (Plu-FNPs) surface decorated doxorubicin (DOX) via acid-cleavable hydrazone linkages with tumor target folic acid (FA) [Plu-FNPs-FA-DOX]. The acid labile of hydrazine linkage to DOX can easily break off by controlled pH inducing release of conjugated DOX at site of over-expressed folate receptors (FR). This nanoparticle system can deliver DOX to the target FR and trace the delivery pathway of cancer cells. The approaching platform demonstrates the selectivity and sensitivity of molecular targets, thereby able to maximize the therapeutic efficiency.

Electrochemical immunosensor for detection of prostate specific antigen based on an acid cleavable linker into MSN-based controlled release system

A mesoporous silica nanoparticle (MSN)-based controlled release system with acid cleavable linkage was developed to fabricate an electrochemical immunosensor for the quantitative detection of the prostate-specific antigen (PSA). 3,9-Bis(3-aminopropyl)-2,4,8,10-tetraoxaspiro[5.5]undecane functionalized mesoporous silica nanoparticles (MSN-Acetal) were used to immobilize the electron mediator thionine (Th). The encapsulation of Th molecules was achieved by capping the pores of MSN-Acetal with carboxylic acid modified Au nanoparticles (defined as MSN-Th-Au). Under the acidic conditions, the capped Au nanoparticles were removed from MSN-Th-Au through the hydrolysis of the acid-labile acetal linker, resulting in the release of encapsulated Th. In this work, the pH-responsive cargo release system was firstly used as the label of secondary anti-PSA for developing an electrochemical immunosensor, and amination Fe3O4 was used as the sensing matrix for immobilizing primary anti-PSA on magnetic carbon electrode surfaces. The specific recognition of PSA resulted in the attachment of MSN-Th-Au-secondary anti-PSA (MSN-Th-Au-Ab2) onto the electrode surfaces. Subsequently, the released Th was detected by differential pulse voltammetry under the acidic conditions. The developed cargo release system provided an innovative and reliable method for the detection of PSA because the response signal was correlated with the concentration of PSA. Under the optimal conditions, the electrochemical immunosensor exhibited a wide linear range of 0.001–5.0ng/mL with a low detection limit of 0.31pg/mL. Moreover, the developed immunosensor showed superior reproducibility and long-term stability, which has promising applications in bioassay and biosensing.

Synergistic combination cancer therapy based in lectin-targeted pH-responsive mesoporous silica nanoparticles

Event Abstract Back to Event Synergistic combination cancer therapy based in lectin-targeted pH-responsive mesoporous silica nanoparticles Marina Martínez-Carmona1, 2, 3, 4, Montserrat Colilla1, 2, 3, 4, Daniel Lozano1, 2, 3 and María Vallet-Regí1, 2, 3, 4 1 Universidad Complutense de Madrid, Química Inorgánica y Bioiniorgánica, Facultad de Farmacia, Spain 2 Instituto de Investigación Sanitaria Hospital 12 de Octubre i+12, Spain 3 Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain 4 CEI Campus Moncloa, UCM-UPM, Spain Introduction: The main challenge in the treatment of cancer therapy is the development of nanocarriers able to selectively transport and release cytotoxic drugs once the target has been reached[1]. Mesoporous silica nanoparticles (MSNs) have emerged as promising candidates to develop targeted stimuli-responsive drug delivery devices for the treatment of solid tumors[2]-[5]. Some specific combinations of cytotoxic agents exhibiting synergistic effects results in the increase of the tumor cell kill capability and the decrease of side effects[6]. Herein we propose the development of a targeted-pH-responsive mesoporous core@shell silica@polymer nanosystem entrapping the antitumor drug doxorubicin (Dox) and incorporating the lectin Concanavalin A (Con-A) in its outermost surface (Fig. 1). The use of Con-A aims at playing a dual role by acting as targeting ligand and therapeutic agent, thus improving the antitumor ability of the nanosystem. Materials and Methods: Pure silica MSNs were synthetized following the well-known modified Stöber method. Then MSNs were grafter with 3,9-Bis(3-aminopropyl)2,4,8-10-tetraoxaspiro[5,5] undecane (ATU), an acid cleavable linker[7], and loaded with Dox by soaking in a concentrated aqueous drug solution. The capping of the pores was accomplished using a polyacrylic acid polymer (PAA). Con-A was attached to the PAA shell via an EDC/NHS crosslinking method. The materials were characterized by TG, FTIR, 13C solid state NMR, N2 adsorption, SEM, HR-TEM, zeta-potential and DLS. The pH-responsive drug delivery behavior of the nanosystem was tested in vial. Finally, the in vitro behavior of the nanosystems regarding cell internalization and killing capability was evaluated in healthy (MC3T3-E1) and tumor (HOS) cell cultures. Results and Discussion: The physical-chemical, structural and textural characterization of the materials allowed confirming the successful incorporation of the different molecules to the spherical 2D-hexagonal MCM-41 type MSNs after each synthesis step. The amounts of PAA and Con-A grafted to MSNs were ca. 7% and 10% in weight, respectively, which accounts for the suitability of the functionalization methods here reported. The amount of Dox loaded was ca. 7%. The drug delivery behavior of the full nanosystem evaluated in vial showed that nearly 4-fold higher release was obtained in acidic media than in neutral pH, which demonstrates the pH-responsive drug release ability of the system (Fig. 2A). This finding proves the suitability of the nanosystem for smart Dox delivery system capable of releasing the drug in the acidic environment of the endosomes/lysosomes once it has been internalized by the cell. The in vitro tests indicated that the presence of Con-A as targeting ligand leads to higher nanosystem internalization degree in HOS than in MC3T3-E1 cells. Moreover, the cancer cell killing capability of Dox was synergistically enhanced by the presence of Con-A (Fig. 2B), as derived from cell viability assays. Conclusion: We have developed a novel pH-responsive drug delivery nanosystem incorporating Con-A and Dox. The combination of these two elements into the same nanosystem synergistically enhances its antitumor capacity in a selective fashion. These findings open promising expectations for the further in vivo evaluation of the nanosystems. Authors acknowledge Ministerio de Economía y Competitividad (MINECO), Secretaría de Estado de Investigación, Desarrollo e Innovación (SEIDI) supporting through projects MAT201235556 and CSO201011384E (Agening Network of Excellence). Marina MartínezCarmona acknowledges Moncloa Campus of International Excellence (UCM/UPM-ISCIII) for a PICATA predoctoral fellowship. Daniel Lozano acknowledges MINECO/SEIDI for a Postdoctoral Juan de la Cierva grant

Polymerizable phosphoramidites with an acid-cleavable linker for eco-friendly synthetic oligodeoxynucleotide purification

Purification at the hundred milligram scale is as convenient as at the sub-milligram scale. Long sequences are easily isolated from complex mixtures. The waste to product ratio is reduced by hundreds of times over known methods.

Synthesis and evaluations of an acid-cleavable, fluorescently labeled nucleotide as a reversible terminator for DNA sequencing.

An acid-cleavable linker based on a dimethylketal moiety was synthesized and used to connect a nucleotide with a fluorophore to produce a 3'-OH unblocked nucleotide analogue as an excellent reversible terminator for DNA sequencing by synthesis.

Polymeric micellar nanoplatforms for Fenton reaction as a new class of antibacterial agents

Reactive oxygen species (ROS) produced by host phagocytes exert antibacterial action against a variety of pathogens and ROS-induced oxidative stress is the governing mechanism for the antibacterial activity of major bactericidal antibiotics. In particular, hydroxyl radical is a strong and nonselective oxidant which can damage biomolecules such as DNA, proteins and lipids. Ferrous ion is known to convert mild oxidant hydrogen peroxide (H2O2) into highly reactive and toxic hydroxyl radicals, referred to as Fenton reaction. Herein, we report a new class of antibacterial agents based on Fenton reaction-performing nanostructures, composed of H2O2-generating polymer (PCAE) and iron-containing ferrocene. Amphiphilic PCAE was designed to incorporate H2O2-generating cinnamaldehyde through acid-cleavable linkages and self-assemble to form thermodynamically stable micelles which could encapsulate ferrocene in their hydrophobic core. All the experiments in vitro display that ferrocene-loaded PCAE micelles produce hydroxyl radicals to kill Escherichia coli and Pseudomonas aeruginosa through membrane damages. Intraperitoneally injected ferrocene-loaded PCAE micelles significantly reduced the lung damages and therefore increased the survival rate of mice infected with drug resistant P. aeruginosa. Given their potent antibacterial activity, ferrocene-loaded PCAE micelles hold great potential as a new class of ROS-manipulating antibacterial agents.

Increased brain uptake of targeted nanoparticles by adding an acid-cleavable linkage between transferrin and the nanoparticle core

Most therapeutic agents are excluded from entering the central nervous system by the blood-brain barrier (BBB). Receptor mediated transcytosis (RMT) is a common mechanism used by proteins, including transferrin (Tf), to traverse the BBB. Here, we prepared Tf-containing, 80-nm gold nanoparticles with an acid-cleavable linkage between the Tf and the nanoparticle core to facilitate nanoparticle RMT across the BBB. These nanoparticles are designed to bind to Tf receptors (TfRs) with high avidity on the blood side of the BBB, but separate from their multidentate Tf-TfR interactions upon acidification during the transcytosis process to allow release of the nanoparticle into the brain. These targeted nanoparticles show increased ability to cross an in vitro model of the BBB and, most important, enter the brain parenchyma of mice in greater amounts in vivo after systemic administration compared with similar high-avidity nanoparticles containing noncleavable Tf. In addition, we investigated this design with nanoparticles containing high-affinity antibodies (Abs) to TfR. With the Abs, the addition of the acid-cleavable linkage provided no improvement to in vivo brain uptake for Ab-containing nanoparticles, and overall brain uptake was decreased for all Ab-containing nanoparticles compared with Tf-containing ones. These results are consistent with recent reports of high-affinity anti-TfR Abs trafficking to the lysosome within BBB endothelium. In contrast, high-avidity, Tf-containing nanoparticles with the acid-cleavable linkage avoid major endothelium retention by shedding surface Tf during their transcytosis.

Design and Synthesis of New Acid Cleavable Linkers for DNA Sequencing by Synthesis

A new kind of acid sensitive tetrahydrofuranyl (THF) linker was synthesized and then reacted with 5-(6)-carboxytetramethylrhodaminesuccinimidyl ester (5(6)-TAMRA, SE), followed by di(N-succinimidyl) carbonate (DSC) and modified 2′-deoxyuridine triphosphate (dUTP); the final product, as a reversible terminator for DNA sequencing by synthesis (DNA SBS), was given obtained and confirmed by 1H-NMR, 31P-NMR, and HRMS with purity of up to 99%. The synthesized dye-labeled terminator incorporated into DNA strand successfully, and the fluorophore was cleaved completely under acidic conditions. The preliminary results encourage us to explore more acid-sensitive linkers for DNA SBS to increase the cleavage efficiency under weakly acidic conditions.