Remotely Controlling Drug Release by Light-Responsive Cholesteric Liquid Crystal Microcapsules Triggered by Molecular Motors.

Stimuli-responsive smart nanocarriers are an emerging class of materials applicable in fields including drug delivery and tissue engineering. Instead of constructing responsive polymer shells to control the release and delivery of drugs, in this work, we put forward a novel strategy to endow the internal drugs with light responsivity. The microcapsule consisted of molecular motor (MM)-doped cholesteric liquid crystals (CLCs) and drugs. The drug in gelatin-gum arabic microcapsules can protect the carried drugs for a long time with a low release speed totally resulting from drug diffusion. Under UV light, the MM isomerizes and the chirality changes, inducing the alteration of the superstructure of the CLCs. In this process, the cooperative molecular disturbance accelerates the diffusion of the drugs from the microcapsule core to the outside. As a result, thanks to the cooperative effect of liquid crystalline mesogens, molecular-scale geometric changes of motors could be amplified to the microscale disturbance of the self-organized superstructure of the CLCs, resulting in the acceleration of the drug release. This method is hoped to provide opportunities in the design and fabrication of novel functional drug delivery systems.

Synthesis and biological evaluation of cationic TopFluor cholesterol analogues.

Cholesterol is not only a major component of the cell membrane, but also plays an important role in a wide range of biological processes and pathologies. It is therefore crucial to develop appropriate tools for visualizing intracellular cholesterol transport. Here, we describe new cationic analogues of BODIPY-Cholesterol (TopFluor-Cholesterol, TF-Chol), which combine a positive charge on the sterol side chain and a BODIPY group connected via a C-4 linker. In contrast to TF-Chol, the new analogues TF-1 and TF-3 possessing acetyl groups on the A ring (C-3 position on steroid) internalized much faster and displayed slightly different levels of intracellular localization. Their applicability for cholesterol monitoring was indicated by the fact that they strongly label compartments with accumulated cholesterol in cells carrying a mutation of the Niemann-Pick disease-associated cholesterol transporter, NPC1.

Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents.

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.

Cholesteryl glucosides signal through the carbohydrate recognition domain of the macrophage inducible C-type lectin (mincle).

Cholesteryl α-d-glucosides (αGCs) are unique metabolic products of the cancer-causing human pathogen Helicobacter pylori. Via signalling through the Macrophage inducible C-type lectin (Mincle) and the induction of a pro-inflammatory response, they are thought to play a role in the development of gastric atrophy. Herein, we prepared the first library of steryl d-glucosides and determined that they preferentially signal through the carbohydrate recognition domain of human Mincle, rather than the amino acid consensus motif. Lipidated steryl d-glucosides exhibited enhanced Mincle agonist activity, with C18 cholesteryl 6-O-acyl-α-d-glucoside (2c) being the most potent activator of human monocytes. Despite exhibiting strong Mincle signalling, sito- (5b) and stigmasterol glycosides (6b) led to a poor inflammatory response in primary cells, suggesting that Mincle is a potential therapeutic target for preventing H. pylori-mediated inflammation and cancer.

Cholesterol: Bioactivities, Structural Modification, Mechanisms of Action, and Structure-Activity Relationships.

Cholesterol, steroid alcohol, was discovered by M.E. Chevreul in 1815. Cholesterol and its derivatives showed a large variety of biological properties such as anticancer activity, anticardiac activity, anti-inflammatory activity, antimicrobial activity, anti-psychotic activity, antioxidant activity, drug-loaded activity, etc. In this mini-review, the advances of structural modification of cholesterol from 2014 to 2020 are summarized. In addition, the bioactivities, mechanisms of action and structureactivity relationships of cholesterol and its related derivatives are also discussed.

Erlotinib entrapped in cholesterol-depleting cyclodextrin nanoparticles shows improved antitumoral efficacy in 3D spheroid tumors of the lung and the liver.

Erlotinib (ERL), a tyrosine kinase inhibitor approved for therapeutic use in non-small cell lung cancer is further researched for eventual liver cancer treatment. However, conventional ERL has important bioavailability problems resulting from oral administration, poor solubility and gastrointestinal degradation into inactive metabolites. Alternative administration routes and nanoparticulate drug delivery systems are studied to prevent or reduce these drawbacks. In this study, ERL-loaded CD nanosphere and nanocapsule formulations capable of cholesterol depletion in resistant cancer cells were evaluated for ERL delivery. Drug loading and release profile depended largely on the surface charge of nanoparticles. Antiproliferative activity data obtained from 2D and 3D cell culture models demonstrated that polycationic ßCD nanocapsules were the most effective formulation for ERL delivery to lung and liver cancer cells. 3D tumour tumoral penetration studies further revealed that nanocapsule formulations penetrated deeper into the tumour through the multilayered cells. Furthermore, all formulations were able to extract membrane cholesterol from lung and liver cancer cell lines, indicating the induction of apoptosis and overcoming drug resistance. In conclusion, given their tumoral penetration and cell membrane cholesterol depletion abilities, amphiphilic CD nanocapsules emerge as promising alternatives to improve the safety and efficiency of ERL treatment of both liver and lung tumours.

Synthesis of some steroidal mitocans of nanomolar cytotoxicity acting by apoptosis.

Several steroids (abiraterone, prednisone, testosterone, cholesterol) and the BCL-2 inhibitor bexarotene were used as starting materials to synthesize iperazinyl-spacered rhodamine B conjugates. The conjugates were screened for their cytotoxicity in SRB assays against several human tumor cell lines and found to be active in a low µM to nM range. The conjugate derived from testosterone held an EC50 = 59 nM against MCF-7 tumor cells and acted mainly by necrosis. The prednisone conjugate, however, was less cytotoxic but acted mainly by apoptosis and held a moderate selectivity against MCF-7 tumor cells.

Design, preparation and evaluation of different branched biotin modified liposomes for targeting breast cancer.

A series of liposome ligands (Bio-Chol, Bio-Bio-Chol, tri-Bio-Chol and tetra-Bio-Chol) modified by different branched biotins that can recognize the SMVT receptors over-expressed in breast cancer cells were synthesized. And four liposomes (Bio-Lip, Bio-Bio-Lip, tri-Bio-Lip and tetra-Bio-Lip) modified by above mentioned ligands as well as the unmodified liposome (Lip) were prepared to study the targeting ability for breast cancer. The cytotoxicity study and apoptosis assay of paclitaxel-loaded liposomes showed that tri-Bio-Lip had the strongest anti-proliferative effect on breast cancer cells. The cellular uptake studies on mice breast cancer cells (4T1) and human breast cancer cells (MCF-7) indicated tri-Bio-Lip possessed the strongest internalization ability, which was 5.21 times of Lip, 2.60 times of Bio-Lip, 1.67 times of Bio-Bio-Lip and 1.17 times of tetra-Bio-Lip, respectively. Moreover, the 4T1 tumor-bearing BALB/c mice were used to evaluate the in vivo targeting ability. The data showed the enrichment of liposomes at tumor sites were tri-Bio-Lip > tetra-Bio-Lip > Bio-Bio-Lip > Bio-Lip > Lip, which were consistent with the results of in vitro targeting studies. In conclusion, increasing the density of targeting molecules on the surface of liposomes can effectively enhance the breast cancer targeting ability, and the branching structure and spatial distance of biotin residues may also have an important influence on the affinity to SMVT receptors. Therefore, tri-Bio-Lip could be a promising drug delivery system for targeting breast cancer.

The morphology, self-assembly, and host-guest properties of cellulose nanocrystals surface grafted with cholesterol.

Cellulose nanocrystals (CNC) were prepared using acid hydrolysis of cellulose fiber. The CNC modified topo-chemically by grafting of bulky cholesterol moieties which changed subsequent morphology, thermal behavior, lyotropic crystalline properties, and host-guest release behavior. Bond formation between the cellulose nanocrystals surfaces and cholesterol was confirmed by FT-IR and solid-state NMR. The product indicated strong hydrophobic characteristics with an ordered chiral nematic self-assembly. This novel biomaterials were exploited through uptake of folic acid as part of a preliminary host-guest system. The guest molecule released as a function of physiologically relevant pHs was examined.

Side-chain deuterated cholesterol as a molecular probe to determine membrane order and cholesterol partitioning.

Cholesterol is an essential and ubiquitous component in mammalian cell membranes. However, its distributions and interactions with phospholipids are often elusive, partly because chemical modifications for preparing cholesterol probes often cause significant perturbations in its membrane behavior. To overcome these problems, a 2H-labeled probe (24-d-cholesterol), which perfectly retained the original membrane properties, was synthesized by a stereoselective introduction of 2H into the side chain of cholesterol. A deuterium label at the side-chain more sensitively reflects membrane fluidity than the conventional labeling at the 3 position of a sterol core (3-d-cholesterol), thus providing 24-d-cholesterol with desirable properties to report membrane ordering. Solid state 2H NMR of 24-d-cholesterol with sphingomyelins (SM) and unsaturated phosphatidylcholine in the bilayer membranes clearly revealed the partitioning ratio of cholesterol in the raft-like liquid ordered (Lo) phase and the liquid disordered phase based on cholesterol interactions with surrounding lipids in each phase. This probe turned out to be superior to the widely used 3-d-cholesterol; e.g., 24-d-cholesterol clearly revealed a 10 mol% difference in the Lo distribution ratios of cholesterol between palmitoyl-SM and stearoyl-SM. The comprehensive use of 24-d-cholesterol in solid state 2H NMR will disclose the cholesterol-lipid interactions, distribution ratio of cholesterol, and membrane ordering in model bilayers as well as more complicated biological membranes.

Design of Peptidomimetic Functionalized Cholesterol Based Lipid Nanoparticles for Efficient Delivery of Therapeutic Nucleic Acids.

Lipid nanoparticles (LNP) are the most potent carriers for the delivery of nucleic acid-based therapeutics. The first FDA approved a short interfering RNA (siRNA) drug that uses a cationic LNP system for the delivery of siRNA against human transthyretin (hTTR). However, preparation of such LNP involves tedious multi-step synthesis with relatively low yields. In the present study, we synthesized cationic peptidomimetic functionalized cholesterol (denote Chorn) in straightforward chemical approaches with high yield. When formulated with helper lipids, Chorn LNPs complexed with siRNA to form nanoparticles with an average diameter of 150 nm to 200 nm. Chorn LNP mediated transfection of a green fluorescence protein (GFP) expressing plasmid resulted in 60% GFP positive cells. Moreover, Chorn LNP delivered siRNA against polo-like kinase 1 (Plk1), a disease related gene in cancer cells and efficiently suppressed the expression of the gene, resulting in significant morphological changes in the cell nuclei. Our data suggested that cholesterol based cationic LNP, prepared through a robust chemical strategy, may provide a promising siRNA delivery system.

Efficacious Doxorubicin Delivery Using Glutathione-Responsive Hollow Non-phospholipid Vesicles Bearing Lipoyl Cholesterols.

In this study, we developed redox-sensitive vesicles using synthesised lipoyl cholesterol derivatives, a non-ionic surfactant and an optimum level of free cholesterol. Interestingly, concentration-dependent self-assembly was observed by scanning electron microscopy, wherein vesicles manifested as hollow spherical (at 0.15 mm) and triangular (0.50 mm). The redoxresponsive characteristics of the vesicles was probed in the presence of dithiothreitol; they underwent a clear increase in size as observed by dynamic light scattering measurements. These vesicles could easily encapsulate an anticancer drug, doxorubicin, and were observed to be stable in the presence of serum. They showed substantial release of the drug in response to biologically relevant stimulus, that is, glutathione. A toxicity assessment on HeLa and HepG2 cancer cells demonstrated activities of the drug-loaded vesicles comparable to that of free drug, whereas significantly enhanced toxicity and apoptotic induction were observed against drug-resistant HeLa cells, which was determined by studying the cellular internalisation of doxorubicin.

Halting hyaluronidase activity with hyaluronan-based nanohydrogels: development of versatile injectable formulations.

Hyaluronan (HA) is among the most used biopolymers for viscosupplementation and dermocosmetics. However, the current injectable HA-based formulations present relevant limitations: I) unmodified HA is quickly degraded by endogenous hyaluronidases (HAase), resulting in short lasting properties; II) cross-linked HA, although shows enhanced stability against HAase, often contains toxic chemical cross-linkers. As such, herein, we present biocompatible self-assembled hyaluronan-cholesterol nanohydrogels (HA-CH NHs) able to bind to HAase and inhibit the enzyme activity in vitro, more efficiently than currently marketed HA-based cross-linked formulations (e.g. Jonexa™). HA-CH NHs inhibit HAase through a mixed mechanism, by which NHs bind to HAase with an affinity constant 7-fold higher than that of native HA. Similar NHs, based on gellan-CH, evidenced no binding to HAase, neither inhibition of the enzyme activity, suggesting this effect might be due to the specific binding of HA-CH to the active site of the enzyme. Therefore, HA-CH NHs were engineered into injectable hybrid HA mixtures or physical hydrogels, able to halt the enzymatic degradation of HA.

Conjugation of bile esters to cellulose by olefin cross-metathesis: A strategy for accessing complex polysaccharide structures.

Bile salts tend to form micelles in aqueous media and can thereby contribute to drug solubilization; they also exhibit crystallization inhibition properties that can stabilize supersaturated drug solutions. Herein, we explore conjugation of bile salts with polysaccharides to create new, amphiphilic polysaccharide derivatives with intriguing properties, portending broad utility in various applications. We introduce efficient conjugation of cholesterol (as a model steroid), lithocholic acid, and deoxycholic acid by mild, modular olefin cross-metathesis reactions. These small molecules were first modified with an acrylate group from the A-ring hydroxyl, then reacted with cellulose derivatives bearing olefin-terminated metathesis "handles". Successful conjugation of bile acids has demonstrated chemoselective cross-metathesis with complex, polyfunctional structures, and large multi-ring systems. It also enabled an efficient, general pathway for polysaccharide-bile salt conjugates, which promise synergy for applications such as amorphous solid dispersion (ASD).

Differentiation of Intracellular Hyaluronidase Isoform by Degradable Nanoassembly Coupled with RNA-Binding Fluorescence Amplification.

Hyaluronidase has two cruical isoforms, hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2), which are essential for cellular hyaluronic acid (HA) catabolism to generate different-sized oligosaccharide fragments for performing different physiological functions. In particular, Hyal-1 is the major tumor-derived hyaluronidase. Thus, specific detection of one hyaluronidase isoform, especially Hyal-1, in live cells is of scientific significance but remains challenging. Herein, by use of differentiated tolerance capability of an amphiphilic HA-based nanoassembly to Hyal-1 and Hyal-2, we rationally design a Hyal-1 specific nanosensor, consisting of cholesterylamine-modified HA nanoassembly (CHA) and RNA-binding fluorophores (RBF). The RBF molecules were entrapped in CHA to switch off their fluorescence via aggregation caused quenching. However, CHA can be disassembled by Hyal-1 to release RBF, resulting in fluorescence activation. Moreover, the fluorescence of the released RBF is further enhanced by cytoplasm RNA. Owing to this cascade signal amplification, this nanosensor RBF@CHA displays a significant change of signal-to-background-noise ratio (120-fold) toward 16 µg/mL Hyal-1 in cellular lysates. In contrast, it is resistant to Hyal-2. By virtue of its selective and sensitive characteristics under a complicated matrix, RBF@CHA had been successfully applied for specifically visualizing Hyal-1 over Hyal-2 inside live cells for the first time, detecting a low level of intracellular Hyal-1 and distinguishing normal and cancer cells with different expressions of Hyal-1. This approach would be useful to better understand biological functions and related diseases of intracellular Hyal-1.

Chemical synthesis of marine saponins.

Covering: 1989-2017 Saponins are characteristic metabolites of starfish and sea cucumbers, and occasionally are also found in sponges, soft coral, and small fish. These steroid or triterpenoid glycosides often show remarkable biological and pharmacological activities, such as antifungal, antifouling, shark repellent, antitumor and anti-inflammatory activities. Over one thousand marine saponins have been characterized; the majority of them can be categorized into three major structural types, i.e., asterosaponins, polyhydroxysteroid glycosides, and holostane glycosides. Thus far, only 12 marine saponins have been synthesized; those representing the major types were successfully synthesized recently. The syntheses involve preparation of the aglycones from the terrestrial steroid or triterpene materials, installation of the carbohydrate units, and manipulation of the protecting groups. Herein, we provide a comprehensive review on these syntheses.

Cooperation of Amphiphilicity and Smectic Order in Regulating the Self-Assembly of Cholesterol-Functionalized Brush-Like Block Copolymers.

Nanoparticle morphology significantly affects the application of nanometer-scale materials. Understanding nanoparticle formation mechanisms and directing morphological control in nanoparticle self-assembly processes have received wide attention. Herein, a series of brush-like amphiphilic liquid crystalline block copolymers, PChEMA m- b-POEGMA n, containing cholesteryl mesogens with different hydrophobic/hydrophilic block ratios were designed and synthesized. The self-assembly behaviors of the resulting PChEMA m- b-POEGMA n block copolymers in different solvents (tetrahydrofuran/H2O, 1,4-dioxane/H2O, and N, N-dimethylformamide) were investigated in detail. Desirable micellar aggregates with well-organized architectures, including short cylindrical micelles, nanofibers, fringed platelets, and ellipsoidal vesicles with smectic micellar cores, were observed in 1,4-dioxane/H2O with an increasing hydrophobic block ratio. Although both amphiphilicity and smectic order governed the self-assembly, these two factors were differently balanced in the different solvents. This unique supramolecular system provides a new strategy for the design of advanced functional nanomaterials with tunable morphologies.

Novel steroid derivatives: synthesis, antileishmanial activity, mechanism of action, and in silico physicochemical and pharmacokinetics studies.

The search for new drugs for the treatment of leishmaniasis is an important strategy for improving the current therapeutic arsenal for the disease. There are several limitations to the available drugs including high toxicity, low efficacy, prolonged parenteral administration, and high costs. Steroids are a diverse group of compounds with various applications in pharmacology. However, the antileishmanial activity of this class of molecules has not yet been explored. Therefore, in the present study, we investigated the antileishmanial activity and cytotoxicity of novel steroids against murine macrophages with a focus on the derivatives of cholesterol (CD), cholic acid (CA), and deoxycholic acid (DA). Furthermore, the mechanism of action of the best compound was assessed, and in silico studies to evaluate the physicochemical and pharmacokinetic properties were also conducted. Among the sixteen derivatives, schiffbase2, CD2 and deoxycholic acid derivatives (DOCADs) were effective against promastigotes of Leishmania species. Despite their low toxicity to macrophages, the majority of DOCADs were active against intracellular amastigotes of L. amazonensis, and DOCAD5 exhibited the best biological effect against these parasitic stages (IC50 = 15.34 µM). Neither the CA derivatives (CAD) nor DA alone inhibited the intracellular parasites. Thus, the absence of hydroxyl in the C-7 position of the steroid nucleus, as well as the modification of the acid group in DOCADs were considered important for antileishmanial activity. The treatment of L. amazonensis promastigote forms with DOCAD5 induced biochemical changes such as depolarization of the mitochondrial membrane potential, increased ROS production and cell cycle arrest. No alterations in parasite plasma membrane integrity were observed. In silico physicochemical and pharmacokinetic studies suggest that DOCAD5 could be a good candidate for an oral drug. The data demonstrate the potential antileishmanial effect of certain steroid derivatives and encourage new in vivo studies.

Cholesterol-Based Compounds: Recent Advances in Synthesis and Applications.

This review reports on the latest developments (since 2014) in the chemistry of cholesterol and its applications in different research fields. These applications range from drug delivery or bioimaging applications to cholesterol-based liquid crystals and gelators. A brief overview of the most recent synthetic procedures to obtain new cholesterol derivatives is also provided, as well as the latest anticancer, antimicrobial, and antioxidant new cholesterol-based derivatives. This review discusses not only the synthetic details of the preparation of new cholesterol derivatives or conjugates, but also gives a short summary concerning the specific application of such compounds.

Byproduct-Free Intact Modification of Insulin by Cholesterol End-Modified Poly(ethylene glycol) for in Vivo Protein Delivery.

Insulin is a key peptide hormone used for the treatment of both type I and type II diabetes. To maximize the effect of the treatment of these diseases, the addition of poly(ethylene glycol) (PEGylation) methods for the insulin are widely developed. Here, to make these PEGylation methods the simplest, we report the byproduct-free intact modification of insulin by cholesterol end-modified poly(ethylene glycol) with urethane, propyl, and methoxy groups (that is, Chol-U-Pr-mPEG). The noncovalent PEGylation by the Chol-U-Pr-mPEG has been achieved by the simple mixing of insulin with the Chol-U-Pr-mPEG in aqueous solution, followed by freeze-drying. The formation of the Chol-U-Pr-mPEG/insulin complex has proceeded without byproducts, such as N-hydroxysuccinimide, formed by the conventional covalent PEGylation using an active ester group. The byproduct-free PEGylation has preserved insulin conformation as well as primary structure. The intact PEGylation has protected insulin from hydrolysis by protease. The resulting insulin modified by the Chol-U-Pr-mPEG has sustainably suppressed the level of blood glucose, as compared to naked insulin, in mice. Consequently, the Chol-U-Pr-mPEG/insulin complex formation offers the byproduct-free intact PEGylation of insulin for in vivo protein delivery.