Acid-cleavable Linker Research Articles

A pH-responsive polymer/mesoporous silica nano-container linked through an acid cleavable linker for intracellular controlled release and tumor therapy in vivo

This paper proposes a pH-responsive polymer/mesoporous silica nano-container linked through an acid cleavable linker for intracellular controlled release and tumor therapy in vivo. In this system, the poly(acrylic acid) homopolymer (PAA), as a nanoscopic cap, is grafted onto the MSN through an acid cleavable linker (PAA-ACL-MSN). Doxorubicin (DOX), as a model drug, is used to assess the drug release behaviors and tumor therapy. At neutral pH, the linker is intact, resulting in blockage of pores and package of DOX. By the degradation of the linker at acidic pH, the grafted PAA is removed, which gives rise to uncapping and the subsequent pH-responsive controlled release of DOX. In vitro studies using a nasopharyngeal carcinoma cell line (HNE-1) prove that DOX loaded PAA-ACL-MSN (DOX@PAA-ACL-MSN) is endocytosed and demonstrates efficient operation at lysosomal pH, leading to significant cytotoxicity. As a preliminary tumor therapy in vivo, the progressive tumor development and inhibition following DOX@PAA-ACL-MSN treatment is monitored using bioluminescence imaging. By the examination of cell proliferation in tumor tissues and the comparison of body weight, it is revealed that the DOX@PAA-ACL-MSN is superior to free DOX in terms of therapeutic efficacy and side effects due to the enhanced permeability and retention effects and lower pH in tumor areas. We believe that this developed MSN based delivery system will provide a promising nanodevice for tumor therapy.

Two step mechanisms of tumor selective delivery of N-(2-hydroxypropyl)methacrylamide copolymer conjugated with pirarubicin via an acid-cleavable linkage

N-(2-Hydroxypropyl)methacrylamide copolymer containing hydrazide groups (PHPMA) conjugated with pirarubicin (THP) via a hydrazone bond (PHPMA-hyd-THP) is a drug conjugate that releases THP in the acidic milieu of a tumor. PHPMA-hyd-THP has an apparent Mw of 40,000 and a hydrodynamic diameter of 8.2±1.7nm but no apparent plasma protein binding. PHPMA-hyd-THP possesses two mechanisms of selectivity toward solid tumors and has potent antitumor action. The first one is drug accumulation in tumors that depends on the enhanced permeability and retention (EPR) effect, which results in a 4–20 times higher concentration of drug in the tumor than in normal tissues such as the heart, lung, and intestine. This accumulation in tumor tissue is in great contrast to that of conventional low-Mw THP. The second one is pH-dependent release of drug from PHPMA-hyd-THP: this conjugate released free THP more efficiently at a lower pH, which exists in tumors, and exerts cytotoxic activity. Free THP is known for its much faster uptake into tumor cells compared with doxorubicin. Thus, in our in vitro study, PHPMA-hyd-THP showed a higher cytotoxicity at the lower pH of tumor tissue than at the neutral pH of normal tissue. Furthermore, much more THP was liberated from the conjugate in acidic tumor tissue than in normal tissue. The EPR effect-dependent accumulation of PHPMA-hyd-THP and tumor-selective THP release in the tumor tissues led to highly tumor-selective drug accumulation, which continued for more than 72h, whereas the lowest free drug concentration was detected in normal tissues at 24h and no longer at a later time. In conclusion, we determined in our study here that the acid-cleavable PHPMA-hyd-THP conjugate had an excellent antitumor effect without appreciable adverse effects.

Acid-cleavable thiomaleamic acid linker for homogeneous antibody–drug conjugation

In this communication we describe a novel acid-cleavable linker strategy for antibody-drug conjugation. Functional disulfide bridging of the single interchain disulfide bond of a trastuzumab Fab fragment yields a homogeneous antibody-drug conjugate bearing a thiomaleamic acid linker. This linker is stable at physiological pH and temperature, but quantitatively cleaves at lysosomal pH to release the drug payload.

Dual‐Responsive Breakdown of Nanostructures with High Doxorubicin Payload for Apoptotic Anticancer Therapy

Self-assembled nanoaggregates co-encapsulating doxorubicin (DOX) and oligonucleotide are prepared for dual-responsive breakdown of the nanostructure with complete disappearance characteristics. Four-arm poly(ethylene glycol) is co-conjugated with DOX and anti-bcl-2 oligonucleotide with reducible linkers and acid-cleavable linkers, respectively. The conjugate is hydrophobically self-assembled into nanoaggregates in aqueous solution. Elemental scanning of the nanoaggregates reveals their core-shell structure with DOX and oligonucleotide located at the core and the shell, respectively. The tracking of size modulation suggests the complete disappearance of the particles under reducing conditions and the liberation of oligonucleotide at low pH, which is confirmed by dynamic light scattering and electron microscopy. The release of DOX and oligonucleotide is controlled by the pH and the reducing potential of the medium, and most of the drug and DNA are released in 24 h. The released fractions are analyzed by reversed-phase chromatography, which indicates facile cleavage of DOX and oligonucleotide from the carriers. The nanoaggregates with both DOX and oligonucleotide show the lowest IC(50) value when a cytotoxicity assay is performed against A549 cells. Apoptosis assay also confirms that cells treated with the nanoaggregates having both DOX and oligonucleotide show higher fluorescence intensity of antiapoptotic antibody than native DOX.

Before and after Endosomal Escape: Roles of Stimuli-Converting siRNA/Polymer Interactions in Determining Gene Silencing Efficiency

Silencing the expression of a target gene by RNA interference (RNAi) shows promise as a potentially revolutionizing strategy for manipulating biological (pathological) pathways at the translational level. However, the lack of reliable, efficient, versatile, and safe means for the delivery of small interfering RNA (siRNA) molecules, which are large in molecular weight, negatively charged, and subject to degradation, has impeded their use in basic research and therapy. Polyplexes of siRNA and polymers are the predominant mode of siRNA delivery, but innovative synthetic strategies are needed to further evolve them to generate the desired biological and therapeutic effects. This Account focuses on the design of polymeric vehicles for siRNA delivery based on an understanding of the molecular interactions between siRNA and cationic polymers. Ideal siRNA/polymer polyplexes should address an inherent design dilemma for successful gene silencing: (1) Cationic polymers must form tight complexes with siRNA via attractive electrostatic interactions during circulation and cellular internalization and (2) siRNA must dissociate from its cationic carrier in the cytoplasm before they are loaded into RNA-induced silencing complex (RISC) and initiate gene silencing. The physicochemical properties of polymers, which dictate their molecular affinity to siRNA, can be programmed to be altered by intracellular stimuli, such as acidic pH in the endosome and cytosolic reducers, subsequently inducing the siRNA/polymer polyplex to disassemble. Specific design goals include the reduction of the cationic density and the molecular weight, the loss of branched structure, and changes in the hydrophilicity/hydrophobicity of the polymeric siRNA carriers, via acid-responsive degradation and protonation processes within the endosome and glutathione (GSH)-mediated reduction in the cytoplasm, possibly in combination with gradual stimuli-independent hydrolysis. Acetals/ketals are acid-cleavable linkages that have been incorporated into polymeric materials for stimuli-responsive gene and drug delivery. Tailoring the ketalization ratio and the molecular weight of ketalized branched PEI (K-BPEI) offers molecular control of the intracellular trafficking of siRNA/polymer polyplexes and, therefore, the gene silencing efficiency. The ketalization of linear PEI (K-LPEI) enhances gene silencing in vitro and in vivo by improving siRNA complexation with the polymer during circulation and cellular internalization, supplementing proton buffering efficiency of the polymer in the endosome, and facilitating siRNA dissociation from the polymer in the cytoplasm, in a serum-resistant manner. Spermine polymerization via ketalization and esterification for multistep intracellular degradations provides an additional polymeric platform for improved siRNA delivery and highly biocompatible gene silencing. The chemistry presented in this Account will help lay the foundation for the development of innovative and strategic approaches that advance RNAi technology.

Isotope-coded chemical reporter and acid-cleavable affinity reagents for monitoring protein sulfenic acids

We have developed an approach that allows relative quantification of protein sulfenic acids using a pair of light and heavy isotope labled probes, DAz-2 and d(6)-DAz-2. In conjunction with a new complementary acid-cleavable linker, Yn-ACL, we demonstrate that tagged peptides are successfully labeled, enriched, and fully characterized by LC-MS/MS analysis. Overall, this method can be applied to map sites of cysteine oxidation and compare protein sulfenylation in normal and disease states.

Acid cleavable surface enhanced raman tagging for protein detection

Dye conjugation is a common strategy improving the surface enhanced Raman detection sensitivity of biomolecules. Reported is a proof-of-concept study of a novel surface enhanced Raman spectroscopic tagging strategy termed as acid-cleavable SERS tag (ACST) method. Using Rhodamine B as the starting material, we prepared the first ACST prototype that consisted of, from the distal end, a SERS tag moiety (STM), an acid-cleavable linker, and a protein reactive moiety. Complete acid cleavage of the ACST tags was achieved at a very mild condition that is 1.5% trifluoroacetic acid (TFA) aqueous solution at room temperature. SERS detection of this ACST tagged protein was demonstrated using bovine serum albumin (BSA) as the model protein. While the SERS spectrum of intact ACST-BSA was entirely dominated by the fluorescent signal of STM, quality SERS spectra can be readily obtained with the acid cleaved ACST-BSA conjugates. Separation of the acid cleaved STM from protein further enhances the SERS sensitivity. Current SERS detection sensitivity, achieved with the acid cleaved ACST-BSA conjugate is ∼5 nM in terms of the BSA concentration and ∼1.5 nM in ACST content. The dynamic range of the cleaved ACST-BSA conjugate spans four orders of magnitudes from ∼10 nM to ∼100 μM in protein concentrations. Further improvement in the SERS sensitivity can be achieved with resonance Raman acquisition. This cleavable tagging strategy may also be used for elimination of protein interference in fluorescence based biomolecule detection.

Development of a novel polyethylene glycol-corticosteroid-conjugate with an acid-cleavable linker

Background: Corticosteroids like dexamethasone are often used in the treatment of inflammatory diseases. Despite efficacy, their use is limited by severe side-effects. Targeted drug-delivery to the site of inflammation would be advantageous for the patients. Macromolecules can be used for this approach, because they accumulate at sites of inflammation due to the enhanced permeability and retention effect.Purpose: Our aim was to develop a polymer-corticosteroid-conjugate for the treatment of inflammatory diseases. The authors covalently linked a derivative of dexamethasone to the macromolecule polyethylene glycol (PEG), using an acid-cleavable linker to achieve lysosomal drug-release.Methods: The corticosteroid-PEG-conjugate was synthesized and characterized by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry. Cleavage experiments were performed to study the nature of products after incubation at acidic pH, and the efficacy of the conjugate was tested in two model cell lines.Results: Acid hydrolysis of the novel corticosteroid-PEG-conjugate resulted in two new derivatives of dexamethasone. The conjugate was effective in both model cell lines showing lysosomal release and efficacy of the cleavage products.Discussion and conclusion: The authors new corticosteroid-PEG-conjugate shows glucocorticoid activity and should be developed further to treat inflammatory diseases with reduced side-effects while retaining drug efficacy.

Shell cross-linked and hepatocyte-targeting nanoparticles containing doxorubicin via acid-cleavable linkage

Hepatocyte-targeting and shell cross-linked nanoparticles with lactose moiety on the surface and doxorubicin (DOX) in the core were prepared from lactose-PEG-DOX conjugate. The process consists of the synthesis of a novel α-hydrazine- ω-propargyl poly(ethylene glycol) (PEG) with a double bond in the PEG backbone, followed by the bonding of a lactose molecule containing an azide group to the ω-end of PEG via “click” chemistry, and finally, the conjugation of DOX to the α-end of PEG via an acid-labile, hydrazone linkage. The resultant conjugate can be self-assembled into nanoparticles. Thiolated tri(ethylene glycol) was introduced into the shell of nanoparticles as a cross-linking agent. The release of DOX is more rapid from lactose-PEG-DOX at pH 5.0 than at pH 7.4. Fluorescent microscope studies suggest that the lactose-DOX nanoparticles are internalized by hepatoma cells through a lactose receptor–mediated mechanism, whereas the lactose-free nanoparticles are not endocytosed as rapidly as lactose-DOX nanoparticles. MTT assay also shows that lactose-DOX nanoparticles have a stronger inhibition against hepatoma cells than DOX nanoparticles and pure DOX. From the Clinical Editor In this basic science study, a highly efficient targeted doxorubicin delivery method to hepatocytes is presented.

Combined Multimodal Optical Imaging and Targeted Gene Silencing Using Stimuli-Transforming Nanotheragnostics

Combined diagnosis and therapy for cancer has been of great interest in medicine. Small interference RNA (siRNA)-encapsulating polyplexes were covalently coated with small gold nanoparticles (Au NPs) via acid-cleavable linkages in order to explore the possibility of achieving combined stimuli-responsive multimodal optical imaging and stimuli-enhanced gene silencing. In a mildly acidic tumor environment, Au NPs are dissociated from the siRNA-carrying polyplexes, generating various optical signal changes such as diminished scattering intensity, increased variance of Doppler frequency, and blue-shifted UV absorbance (stimuli-responsive imaging). Simultaneously, Au NP dissociation exposes the siRNA-carrying polyplex with elevated surface charge and results in enhanced cellular uptake and transfection (stimuli-enhanced therapy). In this study, the feasibility of achieving combined diagnosis and therapy for cancer (theragnostics) is demonstrated by (1) microscopic and spectrophotometric confirmation of acid-transformation of the nanoparticles, (2) reduced scattering intensity and increased variance of Doppler frequency in an acidic pH upon the nanoparticle's transformation, and (3) simultaneous optical signal changes and gene silencing in vitro under a tumor pH-mimicking condition. This novel type of stimuli-responsive nanotheragnostics will provide a new paradigm for pinpointed, multimodal, and combined imaging and therapy for cancer.

Dendrimeric Alkylated Polyethylenimine Nano-carriers with Acid-Cleavable Outer Cationic Shells Mediate Improved Transfection Efficiency Without Increasing Toxicity

Improved polycation-based non-viral DNA vectors were sought by preparing dendrimers with polyethylenimine cores surrounded by various shells incorporating structural features intended to facilitate steps in transfection mechanisms. Dendrimeric vectors were designed with (a) an outer oligocation shell, intended to facilitate DNA release inside cells, (b) a hydrophobic C-16 alkyl shell, and (c) a polycationic core, the latter two intended to combine lipid-depletion and osmotic burst endosome escape mechanisms, respectively, and were (d) attached through an a acid-cleavable linker reported to hydrolyze at endosomal pH values. Vectors and DNA complexes were characterized by dynamic and static light scattering. Flow cytometry was used to quantitate transfection activity and cytotoxicity in CHO-K1 cells. About 5-fold increased transfection activity was obtained for a vector constructed with an outer shell of oligocations attached through acid-cleavable linkers, relative to a control dendrimer with an acid-stable linker. The most effective oligocation component of outer shells tested was spermine. Neither modification was associated with increased cytotoxicity. This vector design did not permit an evaluation of the benefit of combining endosome release mechanisms. Using acid-cleavable linkers to attach an outer shell of short, highly-charged oligocations to a PEI-based dendrimeric vector substantially increased transfection efficiency without increasing cytotoxicity.

Synthesis, Characterization, Antitumor Activity of Pluronic Mimicking Copolymer Micelles Conjugated with Doxorubicin via Acid-Cleavable Linkage

Pluronic mimicking poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer having multiple hydroxyl groups in the PPO middle segment (core-functionalized Pluronic: CF-PLU) was synthesized for conjugation of doxorubicin (DOX). DOX was conjugated on the multiple hydroxyl groups of CF-PLU via an acid-labile hydrazone linkage (CF-PLU-DOX). In aqueous solution, CF-PLU-DOX copolymers self-assembled to form a core/shell-type micelle structure consisting of a hydrophobic DOX-conjugated PPO core and a hydrophilic PEO shell layer. The conjugated DOX from CF-PLU-DOX micelles was released out more rapidly at pH 5 than pH 7.4, indicating that the hydrazone linkage was cleaved under acidic condition. CF-PLU-DOX micelles exhibited greatly enhanced cytotoxicity for MCF-7 human breast cancer cells compared to naked DOX, while CF-PLU copolymer itself showed extremely low cytotoxicity. Flow cytometry analysis revealed that the extent of cellular uptake for CF-PLU-DOX micelles was greater than free DOX. Confocal image analysis also showed that CF-PLU-DOX micelles had a quite different intracellular distribution profile from free DOX. CF-PLU-DOX micelles were mainly distributed in the cytoplasm, endosomal/lysosomal vesicles, and nucleus, while free DOX was localized mainly within the nucleus, suggesting that CF-PLU-DOX micellar formulation might be advantageously used for overcoming the multidrug resistance (MDR) effect, which gradually develops in many tumor cells during repeated drug administration.

Proteomics Evaluation of Chemically Cleavable Activity-based Probes

Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system.

Development of a Novel Chemical Probe for the Selective Enrichment of Phosphorylated Serine‐ and Threonine‐Containing Peptides

Gaining insight into phosphoproteomes is of the utmost importance for understanding regulation processes such as signal transduction and cellular differentiation. While the identification of phosphotyrosine-containing amino acid sequences in peptides and proteins is now becoming possible, mainly because of the availability of high-affinity antibodies, no general and robust methodology allowing the selective enrichment and analysis of serine- and threonine-phosphorylated proteins and peptides is presently available. The method presented here involves chemical modification of phosphorylated serine or threonine residues and their subsequent derivatization with the aid of a multifunctional probe molecule. The designed probe contains four parts: a reactive group that is used to bind specifically to the modified phosphopeptide, an optional part in which heavy isotopes can be incorporated, an acid-labile linker, and an affinity tag for the selective enrichment of modified phosphopeptides from complex mixtures. The acid-cleavable linker allows full recovery from the affinity-purified material and removal of the affinity tag prior to MS analysis. The preparation of a representative probe molecule containing a biotin affinity tag and its applicability in phosphoproteome analysis is shown in a number of well-defined model systems of increasing degrees of complexity. Amounts of phosphopeptide as low as 1 nmol can be modified and enriched from a mixture of peptides. During the development of the beta-elimination/nucleophilic addition protocol, special attention was paid to the different experimental parameters that might affect the chemical-modification steps carried out on phosphorylated residues.

Doxorubicin-conjugated biodegradable polymeric micelles having acid-cleavable linkages

Doxorubicin was chemically conjugated to the terminal end of a di-block copolymer composed of poly( l-lactic acid) (PLLA) and methoxy-poly(ethylene glycol) (mPEG) via two acid-cleavable linkages. A hydrazone bond and a cis-acotinyl bond were formed between doxorubicin and the terminal group of PLLA segment in the block copolymer. Doxorubicin-conjugated PLLA–mPEG di-block copolymers self-assembled to form micelles in aqueous solution. The doxorubicin-conjugated micelles were about 89.1 nm in diameter and their critical micelle concentration was 1.3 μg/ml. These values were comparable with those of unconjugated micelles. In an acidic condition, the conjugated doxorubicin in the hydrazone linkage was readily cleaved, releasing doxorubicin in an intact structure. Doxorubicin-conjugated PLLA–mPEG micelles were more potent in cell cytotoxicity than free doxorubicin, suggesting that they were more easily taken up within cells with concomitant rapid release of cleaved doxorubicin into the cytoplasm from acidic endosomes.

Zyn-Linked colchicines: Controlled-release lipophilic prodrugs with enhanced antitumor efficacy

Conjugation of antimitotic colchicine derivatives with proprietary lipophilic molecules (Zyn-Linkers™) via acid cleavable linkages (hydrazone or imine) produced prodrugs with enhanced antitumor activity. The pharmacodynamic properties (half-lives) of the pH-sensitive linkages were determined around values which might be expected in intracellular, especially lysosomal, environments. In buffered solutions at pH 4.1, 5.6, and 7.2, conjugates released 50% of the drug at rates from 0.1 h at pH 4.1 to over 3 months at neutral pH. In a tumor cell cytotoxicity assay, the slowest releasing molecules were up to 100-fold less active in vitro than the unlinked drug, which demonstrated that the linkages were stable and that a true prodrug had been generated. Binding of conjugate to cells, release of active drug and action on an intracellular target (tubulin) were demonstrated by the ability of the conjugates to block cells in the G2/M phase of the cell cycle 20 h after a 10 min exposure and was consistent with the rate of drug released. In vivo, a single injection of the Zyn-Linked conjugated colchicines enhanced the survival time of the mice to a greater extent than single doses of the unlinked colchicine analogues in a murine tumor model. Activity of the slowest releasing conjugate (ZYN 162) was shown to be comparable to a single LD 10 dose of doxorubicin. These data suggest that the Zyn-Linker conjugates described containing acid cleavable bonds form a body depot of prodrug which release an anti-mitotic agent and can significantly inhibit tumor growth.

An acid-cleavable linker stable at neutral pH that releases doxorubicin at lysosomal pH

A Kemp's triacid amide of doxorubicin (DOX), esterified at a second carboxyl, releases DOX at lysosomal pH at a reasonable rate but is extremely stable at neutral pH.