Cholesteryl Chloroformate Research Articles

Cationic Lipid Derived from a Basic Amino Acid: Design and Synthesis

One of the major challenges in gene therapy is the efficient and safe introduction of nucleic acids into eukaryotic cells. This process requires overcoming various biological barriers and navigating complex pathways to reach target cells and achieve their biological function. To address this obstacle, numerous transfection methods have been developed, including physical techniques and the use of genetic vectors, both viral and non-viral. However, to date, no transfection method is 100% safe and efficient. Within the spectrum of non-viral genetic vectors, cationic liposomes formed by cationic lipids stand out for their ability to protect and deliver therapeutic NA. These liposomes offer greater biocompatibility and lower immunogenicity compared to viral vectors, although they still do not match the efficiency of viral delivery systems. Consequently, ongoing research focuses on synthesizing a wide variety of cationic lipids in the search for compounds that provide high transfection efficiency with minimal cytotoxicity. This study aimed to design and synthesize a novel cationic lipid (CholCadLys) derived from natural cellular molecules for transferring genetic material to eukaryotic cells. The lipid was synthesized using cholesteryl chloroformate for the hydrophobic region, cadaverine as a linker, and lysine for the polar region, connected by carbamate and amide bonds, respectively. Identification was confirmed through thin-layer chromatography, purification through preparative chromatography, and characterization via infrared spectroscopy and mass spectrometry. The synthesis yielded a 60% success rate, with stable nanoliposomes averaging 76 nm in diameter. Liposomes were formed using this CL and commercial neutral lipids, characterized by transmission electron microscopy and Nanoparticle Tracking Analysis. These liposomes, combined with plasmid DNA, formed lipoplexes used to transfect Hek-293 FT cells, achieving up to 40% transfection efficiency without cytotoxicity in the mixture of CholCadLys and CholCad. This novel CL demonstrates potential as an efficient, safe, and cost-effective gene transfer system, facilitating further development in gene therapy.

Self-assembled PHEA-based block copolymers for the synthesis of gold nanoparticles

Copolymer based on poly(2-hydroxyethyl acrylate) comprising cholesteryl and cholic acid and their participation in forming AuNPs.

The study on cholesteryl chloroformate liquid crystal for separation of isomers and determination of its surface properties

ABSTRACT The ability to separate isomer series and the surface properties of cholesteryl chloroformate (CC) liquid crystal were investigated by inverse gas chromatography (IGC). The retention diagrams of the selected solvents were plotted between 308.2 and 333.2 K using the IGC technique. The selectivity coefficients, α, of the structural isomers were determined for the CC liquid crystal in the infinite dilution. Besides, the dispersive energy, , the Gibbs specific energy, , the adsorption enthalpy, , the adsorption entropy, , of the selected solvents were also determined. The values were associated with the number of acidic and basic group to calculate the acidity constant () and the basicity constant (). When the values calculated in experimental studies were examined, it was seen that the adsorption takes place exothermically. According to and constants, CC surface was found to be basic character (.

The Inclusion of a Matrix Metalloproteinase-9 Responsive Sequence in Self-assembled Peptide-based Brain-Targeting Nanoparticles Improves the Efficiency of Nanoparticles Crossing the Blood-Brain Barrier at Elevated MMP-9 Levels.

This study investigated whether the inclusion of a matrix metalloproteinase-9 (MMP-9) responsive sequence in self-assembled peptide-based brain-targeting nanoparticles (NPs) would enhance the blood-brain barrier (BBB) penetration when MMP-9 levels are elevated both in the brain and blood circulation. Brain-targeting peptides were conjugated at the N-terminus to MMP-9-responsive peptides, and these were conjugated at the N-terminus to lipid moiety (cholesteryl chloroformate or palmitic acid). Two constructs did not have MMP-9-responsive peptides. NPs were characterised for size, charge, critical micelle concentration, toxicity, blood compatibility, neural cell uptake, release profiles, and invitro BBB permeability simulating normal or elevated MMP-9 levels. The inclusion of MMP-9-sensitive sequences did not improve the release of a model drug in the presence of active MMP-9 from NPs compared to distilled water. 19F NMR studies suggested the burial of MMP-9-sensitive sequences inside the NPs making them inaccessible to MMP-9. Only cholesterol-GGGCKAPETALC (responsive to MMP-9) NPs showed <5% haemolysis, <1pg/mL release of IL-1β at 500μg/mL from THP1 cells, with 70.75±5.78% of NPs crossing the BBB at 24h in presence of active MMP-9. In conclusion, brain-targeting NPs showed higher transport across the BBB model when MMP-9 levels were elevated and the brain-targeting ligand was responsive to MMP-9.

Enhancing the performance of PEG-b-PCL-based nanocarriers for curcumin through its conjugation with lipophilic biomolecules

Curcumin is a natural substance extracted from Curcuma longa Linn with beneficial pharmaceutical properties such as anticancer activity against several cellular lines. However, it presents poor bioavailability due to its low solubility in aqueous media and chemical instability. In this research, curcumin was encapsulated in polymer micelles obtained by the self-assembly of a biodegradable poly (ethylene glycol)-block-poly(ɛ-caprolactone) copolymer conjugated with cholesterol or oleic acid. A hydroxyl-terminated poly (ethylene glycol)-block-poly(ɛ-caprolactone) was reacted with cholesteryl chloroformate or oleyl chloride to obtain conjugated copolymers. The resulting polymeric materials were characterised through proton nuclear magnetic resonance, gel permeation chromatography and differential scanning calorimetry, and their critical aggregation concentration was measured through fluorescence spectroscopy. Poly (ethylene glycol)-block-poly(ɛ-caprolactone) conjugated with cholesterol and oleic acid posed an improved capacity of encapsulating curcumin, resulting in the loading capacities of 8.8% and 15.2%, respectively. Cell viability studies confirmed that curcumin loaded in polymer micelles maintained its anticancer activity against MCF-7 human breast cancer cells but presented low cytotoxicity against mouse fibroblasts. Hence, these formulations have good potential for applications in drug delivery systems for breast cancer treatment.

High Molecular Weight Poly(ethylenimine)-Based Water-Soluble Lipopolymer for Transfection of Cancer Cells.

Over the past decade, search for novel materials for nucleic acid delivery has prompted a special interest in polymeric nanoparticles (NPs). In this study, the biological applicability of a water-soluble cationic lipopolymer (WSLP) obtained by the modification of high molecular weight branched poly(ethylenimine) (PEI) with cholesteryl chloroformate is characterized and assessed for better cellular membrane permeability. To test the delivery efficiency of the produced lipopolymer, plasmid DNA (pDNA) encoding the enhanced green fluorescent protein and WSLP are mixed at different charge ratios. WSLP and WSLP/pDNA complexes are characterized by dynamic and static light scattering, particle charge detection, scanning electron microscopy, and transmission electron microscopy. The pDNA loading of WSLP is also verified by agarose gel electrophoresis. Cytotoxicity of PEI, WSLP, and of WSLP/pDNA is evaluated on human A549 and HeLa cells. A remarkable dependence of the toxicity on the dose, cholesterylation, and charge ratio is detected. Transfection is monitored by flow cytometry and by fluorescence microscopy. Importantly, cholesterylation decreases the toxicity of the polymer, while promoting high transfection efficiency in both cell lines. This work indicates a possible optimization mode of the high molecular weight PEI-based WSLP rendering it a promising candidate for gene delivery.

Combined Hydrophobization of Polyethylenimine with Cholesterol and Perfluorobutyrate Improves siRNA Delivery.

Polyethylenimine (PEI) is a promising delivery vector of nucleic acids, but cytotoxicity and only moderate transfection efficacy with small RNAs limit its applications. Here we hypothesized that hydrophobization of PEI by combined modification with perfluorinated moieties (F) and cholesterol (Ch) will help in addressing both the cytotoxicity and siRNA delivery efficacy. To test the hypothesis, we synthesized a series of copolymers (F-PEI-Ch) by modifying PEI by reaction with heptafluorobutyric anhydride and cholesteryl chloroformate. We investigated and compared the effect of the modifications on siRNA delivery in vitro and in vivo. We found that the F-PEI-Ch copolymers assembled into micellar structures and that the copolymer with the highest Ch content exhibited the best siRNA delivery performance, including lower cytotoxicity, enhanced cell uptake, improved endosomal escape, and the best siRNA silencing efficacy in vitro and in vivo when compared with control PEI, F-PEI, and PEI-Ch. Overall, hydrophobization of PEI with a combination of cholesterol and superhydrophobic perfluorinated moieties represents a promising approach to the design of siRNA delivery vectors with decreased toxicity and enhanced transfection efficacy.

Cholesterol-conjugated PEGylated PAMAM as an efficient nanocarrier for plasmid encoding interleukin-12 immunogene delivery toward colon cancer cells.

IL-12 is a pleiotropic cytokine, which shows an ideal applicant for tumor immunotherapy, because of its features of creating an interconnection between innate (NK cells) and adaptive (cytotoxic T lymphocyte) immunity. IL-12 gene therapy is a useful technique to deliver an immune-modulatory gene directly into tumor site thereby limiting the adverse effects of systemic administration of IL-12 proteins. One of the most largely investigated non-viral gene carriers is polyamidoamine (PAMAM). In the current research, 5 and 3% of PAMAM primary amines were substituted to transmit the plasmid encoding IL-12 gene to cells by cholesteryl chloroformate and alkyl-PEG, respectively. The features of modified PAMAMs containing size and surface charge density, cytotoxicity, and transfection efficiency were investigated in colon cancer cells. in vitro experiment showed that this modified carrier with average size of about 160 nm and zeta potential of 30 mV was able to increase the level of IL-12 production up to two folds as compared to that of the unmodified PAMAM. Improvement of the polymer hydrophobic balance along with of the modulation of the surface positive charge could provide an efficient and safe non-viral IL-12 gene for colon cancer immunogene therapy.

Cholesterol-modified lignin: A new avenue for green nanoparticles, meltable materials, and drug delivery

Two fractions of kraft lignin of low and high molecular weight were reacted with cholesteryl chloroformate (Chol.Cl) to produce a modified lignin that demonstrated very high hydrophobicity. Surprisingly, both fractions displayed discernible melting points as opposed to the starting lignin. The suspension in water also gave rise to nanoparticles that displayed sizes in the range of 200−500 nm that were shown to satisfactorily load and release folic acid, a representative hydrophobic molecule, within the context of drug delivery.

Mapping out the Degree of Freedom of Hosted Enzymes in Confined Spatial Environments

Summary The integration of enzymes with solid materials is crucial for promoting their industrialization. Understanding the enzyme behavior upon association within a confined space, though of fundamental importance for biocomposite development, remains a persistent, unresolved challenge. Here, we present a comprehensive elucidation of the spatial environment’s impact on hosted enzymes’ degree of freedom and, consequently, their accompanying reactivity. Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy allows the direct detection of host-guest interactions at atomic resolution, while the tailorable synthesis of covalent organic frameworks (COFs) enables an evaluation of factors affecting such interactions. Specifically, lysozyme is found to be more constrained and less active along with increasing hydrophilicity of the COFs. These results support the establishment of a connection between the hydrophilicity of the spatial environment and the resulting biocomposites’ reactivity, enabling the prediction of the performance of unknown biocomposites. This study provides a unique insight into the mechanistic pathways underpinning biocatalysis.

Co-delivery of doxorubicin and TRAIL plasmid by modified PAMAM dendrimer in colon cancer cells, in vitro and in vivo evaluation

One strategy for cancer treatment is combination therapy using nanoparticles (NPs), which has resulted in enhanced anti-cancer effects and reduced cytotoxicity of therapeutic agents. Polyamidoamine dendrimer (PAMAM) has attracted considerable attention because of its potential applications ranging from drug delivery to molecular encapsulation and gene therapy. In this study, PAMAM G5 modified with cholesteryl chloroformate and alkyl-PEG was applied for co-delivery of doxorubicin (DOX) and plasmid encoding TRAIL into colon cancer cells, in vitro and in vivo. The results showed DOX was efficiently encapsulated in modified carrier (M-PAMAM) with loading level about 90%, and the resulting DOX-loaded M-PAMAM complexed with TRAIL plasmid showed much stronger antitumor effect than M-PAMAM containing DOX or TRAIL plasmid. On the other hand, the obtained results demonstrated that the treatment of mice bearing C26 colon carcinoma with this developed co-delivery system significantly decreased tumor growth rate. Thus, this modified PAMAM G5 can be considered as a potential carrier for co-delivery of drug and gene in cancer therapy.

Cholesterol recognition system by molecular imprinting on self-assembled monolayer

In this study, the molecular imprinting technique and self-assembled monolayer technique were combined to increase the recognition ability of cholesterol. A self-assembled monolayer with 4-mercaptophenol and benzenethiol was formed on a gold plate, and the reaction of cholesteryl chloroformate with the phenol group in the 4-mercaptophenol was followed. This layer was then coated with poly(methyl methacrylate) (PMMA). In order to expose the cholesterol buried under the coating, the partial upper part of the coated PMMA was then removed by stroking the coated plate in acetone solvent. The molecular imprinted site was prepared by hydrolysis of the carbonate bond and extraction of the hydrolyzed cholesterol. This gold plate was used as a working electrode to test the recognition ability for cholesterol. The result showed that the plate obtained good recognition ability for cholesterol compared with cholic acid. The ratio of 4-mercaptophenol to benzenethiol was a very important factor in the ability to recognize cholesterol. Regulating the thickness of the coated PMMA was also one of the important factors to increase the ability to recognize cholesterol.

Modified PAMAM vehicles for effective TRAIL gene delivery to colon adenocarcinoma: in vitro and in vivo evaluation

TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) gene therapy is considered as one of the promising approaches for cancer treatment. Polyamidoamine (PAMAM) is one of the most extensively applied polymeric vector in gene delivery. In the current study, PAMAM (G4 and G5) dendrimers were modified with alkyl-carboxylate chain, PEG and cholesteryl chloroformate in order to enhance transfection efficiency through overcoming extracellular and intracellular barriers while reducing PAMAM cytotoxicity. Gene delivery efficiency of synthetized vectors was evaluated by both GFP (green fluorescent protein) reporter gene and TRAIL plasmid in colon cancer cells, in vitro and in vivo. The obtained results demonstrated that PAMAM G4-alkyl-PEG (3%)-Chol (5%)-TRAIL complexes at C/P ratio 4 could significantly increase cell death (29.45%) in comparison with unmodified PAMAM vector (15.5%). Moreover, in vivo study in C26 tumor-bearing BALB/c mice suggested that the prepared non-toxic safe vector could inhibit the tumor growth. This study represented the potent vehicle based on cholesterol-grafted PAMAM dendrimers with alkyl-PEG modification for efficient gene delivery in vitro and in vivo.

Preparation and properties evaluation of a novel pH-sensitive liposomes based on imidazole-modified cholesterol derivatives

As a new kind of drug carries, pH-sensitive liposomes have been widely studied in tumor therapy for their advantages of target ability and sustained-release. Here, we synthesized a pH-sensitive material, N-(3-Aminopropyl)imidazole-cholesterol (IM-Chol) and prepared a novel pH-sensitive liposomes using IM-Chol and phosphatidylcholine. IM-Chol was synthesized through amidation reaction between the amino group of N-(3-Aminopropyl)imidazole and acyl chloride group of cholesteryl chloroformate in a weak base solution. Optimal conditions to prepare liposomes were obtained by the orthogonal experiment with the higher encapsulation efficiency as the evaluation indicator. The properties of liposomes, such as particle size, zeta potential, morphology, encapsulation efficiency, drug release behavior and in vitro cell toxicity were evaluated by transmission electron microscopy (TEM), dynamic light scattering (DLS) and MTT assay respectively. The results showed that the average particle size of IM-Chol liposomes was 141nm (PDI 0.323). Liposomes can assemble into uniform spheres at pH 7.4, but under the condition of pH 5.0, the spherical structure of IM-Chol liposomes was broken, exhibiting pH-sensitive property. In vitro drug releasing studies demonstrated the controlled-release behavior of the curcumin (CUR) in the IM-Chol liposomes. The cumulative release of CUR reached to 72.5% in the first 24h at pH 5.0, faster than that at pH 7.4, which confirmed that the drug carrier displayed pH-sensitive release behaviors. In addition, the MTT assay was employed to test the cytotoxicity of IM-Chol liposomes and CUR IM-Chol liposomes. All cell viabilities were greater than 80% after incubating for 24h, even up to the highest dose of 500mg/L, indicating that IM-Chol liposomes had good biocompatibility. The tumor inhibitory results towards EC109 cells of free CUR and CUR-loaded IM-Chol liposomes indicated that IM-Chol liposomes indeed enhanced the cell killing effect of CUR. These results showed that the novel IM-Chol liposomes prepared in this paper had pH-sensitive property and were expected to play a huge potential in tumor treatment.

Intravascular optical imaging of high-risk plaques in vivo by targeting macrophage mannose receptors.

Macrophages mediate atheroma expansion and disruption, and denote high-risk arterial plaques. Therefore, they are substantially gaining importance as a diagnostic imaging target for the detection of rupture-prone plaques. Here, we developed an injectable near-infrared fluorescence (NIRF) probe by chemically conjugating thiolated glycol chitosan with cholesteryl chloroformate, NIRF dye (cyanine 5.5 or 7), and maleimide-polyethylene glycol-mannose as mannose receptor binding ligands to specifically target a subset of macrophages abundant in high-risk plaques. This probe showed high affinity to mannose receptors, low toxicity, and allowed the direct visualization of plaque macrophages in murine carotid atheroma. After the scale-up of the MMR-NIRF probe, the administration of the probe facilitated in vivo intravascular imaging of plaque inflammation in coronary-sized vessels of atheromatous rabbits using a custom-built dual-modal optical coherence tomography (OCT)-NIRF catheter-based imaging system. This novel imaging approach represents a potential imaging strategy enabling the identification of high-risk plaques in vivo and holds promise for future clinical implications.

Functionalized Polymers from α‐Hydroxyalkylated N‐Vinylcaprolactam

Hydroxyalkylation of N‐vinylcaprolactam (NVCL) in α‐position via ring‐opening reaction of propylene oxide and ε‐caprolactone, respectively, yields in precursors for multifunctional NVCL derivatives. Homo‐ and copolymers of hydroxyfunctionalized NVCL derivatives, synthesized by free radical mechanism, are further investigated regarding their thermoresponsive behavior. Esterification of hydroxypropylated NVCL derivative with methacrylic anhydride is carried out yielding a versatile bifunctional cross‐linker. Networks are obtained either via free radical polymerization of the cross‐linker or anionic polymerization of only the methacrylic function and subsequent polymer analogous cross‐linking of the vinylic side groups. The rheological behavior during and after curing is investigated by oscillatory rheology. Furthermore, N‐vinylcaprolactam anion is used as an initiator for the anionic ring‐opening polymerization of ε‐caprolactone. Copolymerization of poly(ε‐caprolactone) macromonomer with NVCL yields graft copolymers. A polymerizable thermotropic liquid crystalline (LC) derivative is prepared by coupling cholesteryl chloroformate to NVCL. The thermal behavior of LC derivative is investigated by differential scanning calorimetry and polarized light microscopy. image

Incorporation of fluorophore–cholesterol conjugates into liposomal and mycobacterial membranes

Fluorescently-labeled steroids that emit intense blue light in nonpolar solvent (λem (CH2Cl2)≈440nm, ΦF=0.70) were prepared by treating cholesteryl chloroformate with 4-amino-1,8-naphthalimides. The lipid portion of the conjugates embeds into liposomal membrane bilayers in minutes, leaving the fluorophore exposed to the external aqueous environment. This causes a 40-nm red-shift in λem and significant quenching. DFT optimizations predict the conjugates to be about 30Å long when fully extended, but rotation about the linker group can bring the compounds into an ‘L’-shape. Such a conformation would allow the cholesteryl anchor to remain parallel to the acyl chains of a membrane while the fluorescent group resides in the interfacial region, instead of extending beyond it. When incubated with Mycobacterium smegmatis mc2 155, a bacterial species known to use natural cholesterol, the labeled steroids support growth and can be found localized in the membrane fraction of the cells using HPLC. These findings demonstrate stable integration of fluorescent cholesterols into bacterial membranes in vivo, indicating that these compounds may be useful for evaluating cholesterol uptake in prokaryotic organisms.

Highly enhanced phototoxicity of chlorin e6-conjugated poly(β-cyclodextrin) with gas forming capacity at an acidic tumor extracellular pH and its in vitro evaluation

In this study, we report a novel polysaccharidic drug conjugate consisting of poly(β-cyclodextrin) [poly(β-CD)] with gas-forming carbonate linkages resulting from the chemical coupling of the hydroxyl groups of poly(β-CD) and cholesteryl chloroformate (CC) and a photosensitizing drug (chlorin e6: Ce6). This drug conjugate was self-assembled in aqueous solution leading to the production of nanoparticles containing the poly(β-CD) on the hydrophilic outer shell and CC and Ce6 in the hydrophobic inner core. Cleavage (i.e. the detachment of CC moieties) of the carbonate linkage at a slightly acidic pH (~pH 6.5) produced carbon dioxide bubbles. More specifically, the nanoparticles (with autoquenched Ce6 molecules in their core at pH 7.4) were destabilized at pH 6.5, thereby dequenching the Ce6 molecules. These experimental results demonstrate that under light illumination the nanoparticles increased singlet oxygen generation at pH 6.5 compared to pH 7.4 and exhibited a higher phototoxicity for KB tumor cells at pH 6.5 compared to pH 7.4. This approach represents an effective photodynamic therapy for acidic tumors. Copyright © 2015 John Wiley & Sons, Ltd.

Development of high drug-loading nanomicelles targeting steroids to the brain

The objective of this research was to develop and evaluate high drug-loading ligand-modified nanomicelles to deliver a steroidal compound to the brain. YC1 (5α-cholestane-24-methylene-3β, 5α, 6β, 19-tetraol), with poor solubility and limited access to the brain, for the first time, has been proved to be an effective neuroprotective steroid by our previous studies. Based on the principle of ‘like dissolves like’, cholesterol, which shares the same steroidal parent nucleus with YC1, was selected to react with sodium alginate, producing amphiphilic sodium alginate– cholesterol derivatives (SACDs). To increase the grafting ratio and drug loading, cholesterol was converted to cholesteryl chloroformate, for the first time, before reacting with sodium alginate. Further, lactoferrin was conjugated on SACDs to provide lactoferrin-SACDs (Lf-SACD), which was established by immune electron microscopy (IEM) and self-assembled into brain-targeting nanomicelles. These nanomicelles were negatively charged and spherical in nature, with an average size of <200 nm. The YC1 drug loading was increased due to the cholesteryl inner cores of the nanomicelles, and the higher the grafting ratio was, the lower the critical micelle concentration (CMC) value of SACD, and the higher drug loading. The in vitro drug release, studied by bulk-equilibrium dialysis in 20 mL of 6% hydroxypropyl-β-cyclodextrin solution at 37°C, indicated a prolonged release profile. The YC1 concentration in mouse brain delivered by lactoferrin-modified nanomicelles was higher than in those delivered by non-modified nanomicelles and YC1 solution. The unique brain-targeting nanomicelle system may provide a promising carrier to deliver hydrophobic drugs across the blood–brain barrier for the treatment of brain diseases.

Improved thermooxidation and sedimentation stability of covalently-coated carbonyl iron particles with cholesteryl groups and their influence on magnetorheology

Sedimentation of particles in magnetorheological suspensions represents a crucial problem that concerns their efficient long-term application in practice. Prepared carbonyl iron (CI) microparticles coated with a low density substance, cholesteryl chloroformate, via a two-step reaction and immersed in silicone oil, exhibit three positive aspects: (1) the CI particle modification increased the compatibility between the particles and the silicone oil resulting in improved long-term stability (reduction in sedimentation); (2) the coating provided the particles with enhanced thermal stability in the oxygen atmosphere; and (3) rheological measurements proved a promising magnetorheological performance at different particle weight fractions.