Amphiphilic hyaluronic acid derivative with the bioreducible bond: Synthesis and its implication for intracellular drug delivery

Poly(oligo-D-arginine) With Internal Disulfide Linkages as a Cytoplasm-sensitive Carrier for siRNA Delivery

Context: Surface-modified pH-sensitive liposomal system may be useful for intracellular delivery of chemotherapeutics.Objective: Achieving site-specific targeting with over-expressed hyaluronic acid (HA) receptors along with using pH sensitive liposome carrier for intracellular drug delivery was the aim of this study.Materials and methods: Stealth HA-targeted pH-sensitive liposomes (SL-pH-HA) were developed and evaluated to achieve effective intracellular delivery of doxorubicin (DOX) vis–a-vis enhanced antitumor activity.Results: The in vitro release studies demonstrated that the release of DOX from SL-pH-HA was pH-dependent, i.e. faster at mildly acidic pH ∼5, compared to physiological pH ∼7.4. SLpH-HA was evaluated for their cytotoxicity potential on CD44 receptor expressing MCF-7 cells. The half maximal inhibitory concentration (IC50) of SL-pH-HA and SL-HA were about 1.9 and 2.5 μM, respectively, after 48 h of incubation. The quantitative uptake study revealed higher localization of targeted liposomes in the receptor positive cells, which was further confirmed by fluorescent microscopy. The antitumor efficacy of the DOX-loaded HA-targeted pH-sensitive liposomes was also verified in a tumor xenograft mouse model.Discussion: DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 receptor interaction. The major side-effect of conventional DOX formulation, i.e. cardiotoxicity was also estimated by measuring serum enzyme levels of LDH and CPK and found to be minimized with developed formulation. Overall, HA targeted pH-sensitive liposomes were significantly more potent than the non-targeted liposomes in cells expressing high levels of CD44.Conclusion: Results strongly implies the promise of such liposomal system as an intracellular drug delivery carrier developed for potential anticancer treatment.

Binding sites and endocytosis of heparin and polylysine are changed when the two molecules are given as a complex to chinese hamster ovary cells

Polyelectrolyte microcapsules were prepared by the layer-by-layer assembly of hyaluronic acid (HA) and a polycationic polymer, poly(allylamine) (PAH) or poly(lysine) (PLL). The influence of the polycationic partner on the morphology, stability, permeability properties, and enzymatic degradation of microcapsules was thoroughly analyzed. It was found that these properties could be tuned by shell cross-linking. Confocal microscopy studies of cellular uptake of the capsules showed that the polyelectrolyte shells remain stable outside the cells but readily break open once internalized by cells, suggesting their potential as carrier for intracellular drug delivery.

Investigation of the cellular uptake of E-Selectin-targeted immunoliposomes by activated human endothelial cells

Release after transmission: Arginine-rich, cell-penetrating peptides (CPPs) mediate cytoplasmic delivery of trimethoprim (TMP)-terbium complex conjugates and selective, intracellular labeling of E. coli dihydrofolate reductase (eDHFR) fusion proteins. A disulfide bond linking CPP and cargo is reduced following uptake. CPP conjugation can be used to deliver otherwise cell-impermeable, ligand-fluorophore conjugates.

Advances in biomedical research have significantly enhanced our capability to identify drug targets for medical intervention. At same time, major technological breakthroughs such as combinatorial chemistry, computer-assisted drug design, robotic screening, and molecular cloning have made thousands of small compounds and macromolecules such as proteins and nucleic acids available for therapeutic consideration. Despite this progress, two major obstacles need to be overcome before we are able to move these products from basic research into clinical application. The first of these obstacles is the lack of specificity of drug compounds toward target cells. Although exposure of intended cellular targets to a drug results in a therapeutic effect, exposure of unintended nontarget cells often results in undesirable toxic effects. The other obstacle is the inability of most drugs, especially those that are hydrophilic and those of high molecular weight, to pass through the cell membrane and enter cells. The cell membrane imposes a significant physical barrier to the entrance of drug molecules into cells. The current strategy for overcoming the first obstacle is to develop drugs that are cell specific, or pro-drugs that remain inactive until they are inside the intended target cells. A common approach to resolve the second limitation is to employ receptor-mediated endocytosis, pinocytosis, or phagocytosis to enhance drug internalization. Despite being extensively studied and well-documented, these approaches have nevertheless had limited success and have met with serious obstacles, including the lack of proper receptors, insufficient endosomal release or avoidance of lysosomal degradation, and low delivery efficiency. At present, only a handful of delivery systems have the potential to overcome these obstacles and none are capable of high-efficiency delivery of hydrophilic molecules, especially macromolecules. Therefore, development of a highly effective carrier for intracellular drug delivery has been the goal of much research effort in both the pharmaceutical industry and academic institutions.KeywordsHuman Immunodeficiency Virus TypeProtein TransductionIntracellular DeliveryBasic Amino Acid ResidueNuclear Localization Signal SequenceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Administration of drugs is often associated with basic problems such as toxicity, instability and improper biodistribution. Encapsulation of the drug in a suitable carrier such as liposomes is one of the well recognized methods that protects the drug from the body milieu to improve stability, safety and targeting efficiency of the drug towards the target site. However, while encapsulation provides a favorable biodistribution, low toxicity and improved stability of drugs, delivery carriers should be capable of releasing encapsulated material at the appropriate site to exert their activity. Effective intracellular drug delivery is desired for many therapeutic agents those having specific molecular targets in the cytoplasm, nucleus, or other subcellular compartments of a cell such as mitochondria. Several nanocarriers that are designed to release drugs at desired sites have been developed so far. Among these, exhaustive research has established liposomes as an effective triggerable drug carrier. This mini review covers various aspects of triggering modalities examined to date such as temperature, pH, enzymes and light using liposomes. Keywords: Drug delivery, liposomes, pH sensitive, photosensitive, thermo sensitive.

Novel poly{(lactic acid)-co-[(glycolic acid)-alt-(L-glutamic acid)]}-g-monomethyl poly(ethylene glycol) (PLGGE) micelles were prepared and used as carriers for anti-tumor drug delivery. Three PEGylated PLGG copolymers (PLGGE2000, PLGGE1100 and PLGGE500) were characterized by XRD, TG and DSC. The critical micelle concentrations (CMCs) of the amphiphilic copolymers were 1.04, 0.55 and 0.13 μg/mL, respectively. The TEM, AFM and DLS measurements revealed that the micelles were homogeneous spherical nanoparticles with the diameters ranged from 50 to 150 nm when THF was used as solvent in the preparation of the micelles. Interestingly, extended cylindrical micelles were obtained using CHCl3 as solvent. The micelles could trap doxorubicin (DOX) in the core with the highest drug loading content up to 23.7%. The mean diameter of drug loaded micelles was much bigger than that of blank micelles. The in vitro drug release of the micelles was diffusion-controlled release within the first 36 h and initial burst release was not obvious. However, after 36 h, the release rate in pH 5.0 was faster than that in pH 7.4 due to the degradation. The PLGGE micelles were nontoxic to both NIH 3T3 fibroblasts and HepG2 cells. The in vitro cytotoxicity against HepG2 cells demonstrated that the drug loaded micelles exhibited high inhibition activity to cancer cells. CLSM observation of HepG2 cells showed that DOX released from the micelles could be delivered into cell cytoplasm and cell nuclei. PLGGE micelles are potential promising carriers for anti-tumor drug delivery.

Currently, tumor-targeted nanocarriers self-assembled from amphiphilic polymer-drug conjugates are of great demand. The appeal of these carriers arises mainly through their excellent loading efficiency of homologous drug molecules with microenvironment-triggered drug release. Herein, doxorubicin (DOX) was constructed to a hyaluronic acid (HA) backbone through hydrazone and disulfide linkages to construct pH and reduction coresponsive prodrug conjugates (HA-ss-DOX). During formulation, the amphipathic HA-ss-DOX spontaneously assembled into distinct core/shell micelles in aqueous media and showed conspicuous physical DOX loading capabilities (29.1%, DOX/HA-ss-DOX) based on homologous compatibility. DOX/HA-ss-DOX micelles were shown to be stable in normal physiological environments, while accomplishing selective, rapid DOX release at acidic pH and/or highly reducing conditions. The efficacy of DOX/HA-ss-DOX micelles was tested on A549 human lung cancer cells, wherein flow cytometry and confocal microscopy analysis revealed their HA receptor-mediated endocytosis mechanism. In comparison, DOX-loaded redox-insensitive micelles (DOX/HA-DOX) still demonstrated pH-dependent drug release. However, a more rapid intracellular DOX release profile was achieved in DOX/HA-ss-DOX micelles because of their sensitivity to both acidic and reducing environments. Resultantly, DOX/HA-ss-DOX exhibited the strongest cytotoxicity and apoptosis-inducing ability among all tested groups when tested on an A549 cell line and xenograft model.

Antibody-drug conjugates (ADCs) are currently used for the targeted delivery of drugs to diseased cells, but intracellular drug delivery and therefore efficacy may be suboptimal because of the large size, slow internalization and ineffective intracellular trafficking of the antibody. Using a phage display method selecting internalizing phages only, we developed internalizing single domain antibodies (sdAbs) with high binding affinity to rat PDGFRβ, a receptor involved in different types of diseases. We demonstrate that these constructs have different characteristics with respect to internalization rates but all traffic to lysosomes. To compare their efficacy in targeted drug delivery, we conjugated the sdAbs to a cytotoxic drug. The conjugates showed improved cytotoxicity correlating to their internalization speed. The efficacy of the conjugates was inhibited in the presence of vacuolin-1, an inhibitor of lysosomal maturation, suggesting lysosomal trafficking is needed for efficient drug release. In conclusion, sdAb constructs with different internalization rates can be designed against the same target, and sdAbs with a high internalization rate induce more cell killing than sdAbs with a lower internalization rate in vitro. Even though the overall efficacy should also be tested in vivo, sdAbs are particularly interesting formats to be explored to obtain different internalization rates.

We have studied the effects of polysaccharide and protein crowding agents on the refolding of oxidized and reduced hen lysozyme in order to test the prediction that association constants of interacting macromolecules in living cells are greatly increased by macromolecular crowding relative to their values in dilute solutions. We demonstrate that whereas refolding of oxidized lysozyme is hardly affected by crowding, correct refolding of the reduced protein is essentially abolished due to aggregation at high concentrations of crowding agents. The results show that the protein folding catalyst protein disulfide isomerase is particularly effective in preventing lysozyme aggregation under crowded conditions, suggesting that crowding enhances its chaperone activity. Our findings suggest that the effects of macromolecular crowding could have major implications for our understanding of how protein folding occurs inside cells.

Programmed pH/reduction-responsive nanoparticles for efficient delivery of antitumor agents in vivo

Poly(ethylene glycol)-b-poly(lysine) copolymer bearing nitroaromatics for hypoxia-sensitive drug delivery

A novel drug carrier with dual triggerable release properties based on keratin graft poly(ethylene glycol) (keratin-g-PEG) copolymers is reported. Keratin-g-PEG copolymers with different graft densities are synthesized through thiol–ene click chemistry. Taking advantage of the amphiphilicity and the thiol groups of the graft copolymer, nanoparticles stabilized with PEG chains and keratin as the core, bearing glutathione (GSH) cleavable cross-links, are fabricated in aqueous solutions. The keratin-g-PEG copolymer nanoparticles can serve as excellent carriers for doxorubicin hydrochloride salt (DOX·HCl) with a highest loading capacity of 18.1% (w/w). The release of the loaded DOX is sensitive to the concentration of GSH, especially at a GSH concentration of cellular level. Trypsin can further trigger the release of the loaded DOX in the nanoparticles. In vitro cellular uptake experiments indicate that DOX released from the DOX-loaded keratin-g-PEG nanoparticles can be internalized into the cells efficiently, and the loaded DOX shows a faster release into the nuclei of the cells under higher GSH concentrations. The carriers have promising applications as drug carriers for intracellular drug delivery for cancer therapy.