用于基因转染的阳离子脂质体研究进展

开发高效、低毒、具有靶向性的基因载体是基因治疗的关键。阳离子脂质体因具有低毒性与免疫原性、生物相容性好、易于制备等优点而受到广泛关注,具有良好的应用前景。近年来,人们通过使用各种辅助性成分研发新型材料,对脂质材料进行表面修饰以及制备工艺调整等方法改进阳离子脂质体的性能。从上述几方面综述了此类基因传递载体的研究进展。     

Chimeric Stimuli-Responsive Liposomes as Nanocarriers for the Delivery of the Anti-Glioma Agent TRAM-34

Nanocarriers are delivery platforms of drugs, peptides, nucleic acids and other therapeutic molecules that are indicated for severe human diseases. Gliomas are the most frequent type of brain tumor, with glioblastoma being the most common and malignant type. The current state of glioma treatment requires innovative approaches that will lead to efficient and safe therapies. Advanced nanosystems and stimuli-responsive materials are available and well-studied technologies that may contribute to this effort. The present study deals with the development of functional chimeric nanocarriers composed of a phospholipid and a diblock copolymer, for the incorporation, delivery and pH-responsive release of the antiglioma agent TRAM-34 inside glioblastoma cells. Nanocarrier analysis included light scattering, protein incubation and electron microscopy, and fluorescence anisotropy and thermal analysis techniques were also applied. Biological assays were carried out in order to evaluate the nanocarrier nanotoxicity in vitro and in vivo, as well as to evaluate antiglioma activity. The nanosystems were able to successfully manifest functional properties under pH conditions, and their biocompatibility and cellular internalization were also evident. The chimeric nanoplatforms presented herein have shown promise for biomedical applications so far and should be further studied in terms of their ability to deliver TRAM-34 and other therapeutic molecules to glioblastoma cells.     

Targeted Delivery of Drugs and Genes Using Polymer Nanocarriers for Cancer Therapy

Cancer is one of the primary causes of worldwide human deaths. Most cancer patients receive chemotherapy and radiotherapy, but these treatments are usually only partially efficacious and lead to a variety of serious side effects. Therefore, it is necessary to develop new therapeutic strategies. The emergence of nanotechnology has had a profound impact on general clinical treatment. The application of nanotechnology has facilitated the development of nano-drug delivery systems (NDDSs) that are highly tumor selective and allow for the slow release of active anticancer drugs. In recent years, vehicles such as liposomes, dendrimers and polymer nanomaterials have been considered promising carriers for tumor-specific drug delivery, reducing toxicity and improving biocompatibility. Among them, polymer nanoparticles (NPs) are one of the most innovative methods of non-invasive drug delivery. Here, we review the application of polymer NPs in drug delivery, gene therapy, and early diagnostics for cancer therapy.     

Efficient Delivery of Plasmid DNA Using Cholesterol-Based Cationic Lipids Containing Polyamines and Ether Linkages

Cationic liposomes are broadly used as non-viral vectors to deliver genetic materials that can be used to treat various diseases including cancer. To circumvent problems associated with cationic liposome-mediated delivery systems such as low transfection efficiency and serum-induced inhibition, cholesterol-based cationic lipids have been synthesized that resist the effects of serum. The introduction of an ether-type linkage and extension of the aminopropyl head group on the cholesterol backbone increased the transfection efficiency and DNA binding affinity compared to a carbamoyl-type linkage and a mono aminopropyl head group, respectively. Under optimal conditions, each liposome formulation showed higher transfection efficiency in AGS and Huh-7 cells than commercially available cationic liposomes, particularly in the presence of serum. The following molecular structures were found to have a positive effect on transfection properties: (i) extended aminopropyl head groups for a strong binding affinity to plasmid DNA; (ii) an ether linkage that favors electrostatic binding to plasmid DNA; and (iii) a cholesterol backbone for serum resistance.     

In Vitro and in Vivo Evaluation of Lactoferrin-Conjugated Liposomes as a Novel Carrier to Improve the Brain Delivery

In this study, lactoferrin-conjugated PEGylated liposomes (PL), a potential drug carrier for brain delivery, was loaded with radioisotope complex, ^99mTc labeled N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine (^99mTc-BMEDA) for in vitro and in vivo evaluations. The hydrophilicity of liposomes was enhanced by PEGylation which was not an ideal brain delivery system for crossing the blood brain barrier (BBB). With the modification of a brain-targeting ligand, lactoferrin (Lf), the PEGylated liposome (PL) might become a potential brain delivery vehicle. In order to test the hypothesis in vitro and in vivo, ^99mTc-BMEDA was loaded into the liposomes as a reporter with or without Lf-conjugation. The mouse brain endothelia cell line, bEnd.3 cells, was cultured to investigate the potential uptake of liposomes in vitro. The in vivo uptake by the mouse brain of the liposomes was detected by tissue biodistribution study. The results indicated that Lf-conjugated PEGylated liposome showed more than three times better uptake efficiency in vitro and two-fold higher of brain uptake in vivo than PEGlyated liposome. With the success of loading the potential Single Photon Emission Tomography (SPECT) imaging probe, ^99mTc-BMEDA, Lf-PL might serve as a promising brain delivery system for loading diagnostics or therapeutics of various brain disorders.     

Mathematical Model for Evaluation of Tumor Response in Targeted Radionuclide Therapy with 211At Using Implanted Mouse Tumor

Recent introduction of alpha-emitting radionuclides in targeted radionuclide therapy has stimulated the development of new radiopharmaceuticals. Preclinical evaluation using an animal experiment with an implanted tumor model is frequently used to examine the efficiency of the treatment method and to predict the treatment response before clinical trials. Here, we propose a mathematical model for evaluation of the tumor response in an implanted tumor model and apply it to the data obtained from the previous experiment of ²¹¹At treatment in a thyroid cancer mouse model. The proposed model is based on the set of differential equations, describing the kinetics of radiopharmaceuticals, the tumor growth, and the treatment response. First, the tumor growth rate was estimated from the control data without injection of ²¹¹At. The kinetic behavior of the injected radionuclide was used to estimate the radiation dose profile to the target tumor, which can suppress the tumor growth in a dose-dependent manner. An additional two factors, including the time delay for the reduction of tumor volume and the impaired recovery of tumor regrowth after the treatment, were needed to simulate the temporal changes of tumor size after treatment. Finally, the parameters obtained from the simulated tumor growth curve were able to predict the tumor response in other experimental settings. The model can provide valuable information for planning the administration dose of radiopharmaceuticals in clinical trials, especially to determine the starting dose at which efficacy can be expected with a sufficient safety margin.     

Charge Conversion Polymer–Liposome Complexes to Overcome the Limitations of Cationic Liposomes in Mitochondrial-Targeting Drug Delivery

Mitochondrial-targeting therapy is considered an important strategy for cancer treatment. (3-Carboxypropyl) triphenyl phosphonium (CTPP) is one of the candidate molecules that can drive drugs or nanomedicines to target mitochondria via electrostatic interactions. However, the mitochondrial-targeting effectiveness of CTPP is low. Therefore, pH-sensitive polymer–liposome complexes with charge-conversion copolymers and CTPP-containing cationic liposomes were designed for efficiently delivering an anti-cancer agent, ceramide, into cancer cellular mitochondria. The charge-conversion copolymers, methoxypoly(ethylene glycol)-block-poly(methacrylic acid-g-histidine), were anionic and helped in absorbing and shielding the positive charges of cationic liposomes at pH 7.4. In contrast, charge-conversion copolymers became neutral in order to depart from cationic liposomes and induced endosomal escape for releasing cationic liposomes into cytosol at acidic endosomes. The experimental results reveal that these pH-sensitive polymer–liposome complexes could rapidly escape from MCF-7 cell endosomes and target MCF-7 mitochondria within 3 h, thereby leading to the generation of reactive oxygen species and cell apoptosis. These findings provide a promising solution for cationic liposomes in cancer mitochondrial-targeting drug delivery.     

PrPC Aptamer Conjugated–Gold Nanoparticles for Targeted Delivery of Doxorubicin to Colorectal Cancer Cells

Anticancer drugs, such as fluorouracil (5-FU), oxaliplatin, and doxorubicin (Dox) are commonly used to treat colorectal cancer (CRC); however, owing to their low response rate and adverse effects, the development of efficient drug delivery systems (DDSs) is required. The cellular prion protein PrP^C, which is a cell surface glycoprotein, has been demonstrated to be overexpressed in CRC, however, there has been no research on the development of PrP^C-targeting DDSs for targeted drug delivery to CRC. In this study, PrP^C aptamer (Apt)-conjugated gold nanoparticles (AuNPs) were synthesized for targeted delivery of Dox to CRC. Thiol-terminated PrP^C-Apt was conjugated to AuNPs, followed by hybridization of its complementary DNA for drug loading. Finally, Dox was loaded onto the AuNPs to synthesize PrP^C-Apt-functionalized doxorubicin-oligomer-AuNPs (PrP^C-Apt DOA). The PrP^C-Apt DOA were spherical nanoparticles with an average diameter of 20 nm. Treatment of CRC cells with PrP^C-Apt DOA induced reactive oxygen species generation by decreasing catalase and superoxide dismutase activities. In addition, treatment with PrP^C-Apt DOA inhibited mitochondrial functions by decreasing the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, complex 4 activity, and oxygen consumption rates. Compared to free Dox, PrP^C-Apt DOA decreased proliferation and increased apoptosis of CRC cells to a greater degree. In this study, we demonstrated that PrP^C-Apt DOA targeting could effectively deliver Dox to CRC cells. PrP^C-Apt DOA can be used as a treatment for CRC, and have the potential to replace existing anticancer drugs, such as 5-FU, oxaliplatin, and Dox.     

Comparative Preclinical Evaluation of Peptide-Based Chelators for the Labeling of DARPin G3 with 99mTc for Radionuclide Imaging of HER2 Expression in Cancer

Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [^99mTc]Tc-(HE)₃-G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)₃), provided clear imaging of HER2 expressing breast cancer 2–4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E₃C), Gly-Gly-Gly-Cys (G₃C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser)₃-linker (designated as G3-(G₃S)₃C) would further improve the contrast of imaging using ^99mTc-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9–5 nM. Biodistribution of [^99mTc]Tc-G3-G₃C, [^99mTc]Tc-G3-(G₃S)₃C, and [^99mTc]Tc-G3-E₃C in mice was compared with the biodistribution of [^99mTc]Tc-(HE)₃-G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [^99mTc]Tc-(HE)₃-G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.     

3D Printed Composite Scaffolds Incorporating Ruthenium Complex–Loaded Liposomes as a Delivery System to Prevent the Proliferation of MG‐63 Cells

Owing to the propensity of osteosarcoma to recur and metastasize, the administration of a topical treatment to enhance the therapeutic effect of conventional therapy is necessary. To develop a locally released drug delivery system (DDS) to inhibit the growth and recurrence of osteosarcoma, hydrophobically modified silica nanoparticles (m‐SiO₂)/poly(ε‐caprolactone) (PCL) porous scaffolds are fabricated by 3D printing emulsion inks. Ruthenium‐loaded PEGylated liposomes (RL) are then incorporated into the scaffolds to obtain the Ruthenium‐loaded PEGylated liposome scaffold (RLS) composite. The emulsion inks and the density, porosity, morphology, and mechanical properties of the scaffolds are characterized. The results indicate that the composite DDS has a relatively uniform porous structure with good mechanical properties. Drug is released from RLS in a relatively sustained manner over 48 h, which demonstrates the potential of RLS as a drug carrier. In addition, the MG‐63 cell viability and apoptosis rate are evaluated by MTT assays. The cell experiments reveal that RLS triggers mitochondrial dysfunction resulting in MG‐63 cell apoptosis. All the results indicate that RLS provides a promising approach for improving the treatment of osteosarcoma.     

Separation of Carrier-Free 90Y from 90Sr by SLM Technique Using D2EHPA in N-Dodecane as Carrier

Solvent extraction studies on Sr²⁺ and Y³⁺ are carried out from varying concentrations (0.01–6.0 M) of nitric acid using di-(2-ethylhexyl)phosphoric acid (D2EHPA) as extractant. Extraction of yttrium is observed to be higher than that of strontium at all the acidities and is found to increase substantially with decreasing concentration of nitric acid. Practically negligible extraction (D < 10^?3) of Sr²⁺ is observed from feed solutions containing nitric acid in the range of 1.0 to 4.0 M. These solvent extraction data are used to optimize the transport of ⁹⁰Sr and ⁹⁰Y across the supported liquid membrane (SLM) individually as well as from their mixture (due to insitue growth) under different experimental conditions. Selective separation of ⁹⁰Y (>90%) from ⁹⁰Sr is obtained in 6 h, when the concentration of nitric acid in feed is kept at 1.0 M and that of receiving phase is maintained at 4.0 M. 20% D2EHPA in n-dodecane is found to be the optimum carrier concentration for the efficient transport of ⁹⁰Y in SLM mode. Under these conditions transport of strontium is found to be negligible. Radiochemical purity of the product ⁹⁰Y is checked by following its decay as well as by extraction paper chromatography. The contamination of ⁹⁰Sr in ⁹⁰Y product is found to be < 0.001%. Based on the experimental results, a single stage SLM system for the generation of carrier-free ⁹⁰Y from ⁹⁰Sr source is described. The system is amenable for automation and scale up.     

Highly Selective and Efficient Membrane Transport of Copper as Cu(SCN)2− 4 Ion Using K+-Dicyclohexyl-18-crown-6 as Carrier

Abstract

Potassium-Dicyclohexyl-18-Crown-6 Complex Was Used As A Highly Efficient Carrier For The Uphill Transport Of Copper As Cu(SCN)^2? ₄ Complex Ion Through A Chloroform Bulk Liquid Membrane. By Using Histidine As A Metal Ion Acceptor In The Receiving Phase At The Optimum Ph Of 7.4, The Amount Of Copper Transported Across The Liquid Membrane After 2 Hours Was 90.2 ± 1.0%. The Selectivity And Efficiency Of Copper Transport From Aqueous Solutions Containing Equimolar Mixtures Of Ag⁺, Cd²⁺, Zn²⁺, Ni²⁺, Fe²⁺, Co²⁺, Pb²⁺, Mn²⁺, Fe³⁺, Bi³⁺, And Cr³⁺ Ions Were Investigated. In The Presence Of Pyrophosphate As A Suitable Masking Agent In The Source Phase, The Interfering Effects Of Co²⁺, Zn²⁺, Pb²⁺, And Cd²⁺ Ions Were Completely Eliminated.     

Synthesis of Biodegradable Diblock Copolymers of Glycolide and Poly(oxyethylene) Using a Montmorillonite Clay as Catalyst

Biodegradable diblock copolymers were prepared from glycolide and poly(oxyethylene) of M_n=600 (POE 600), 1500 (POE 1500) and 2000 (POE 2000). The copolymerization of glycolide and POE was induced in heterogeneous phase by “Maghnite-H⁺” under suitable conditions. Maghnite-H⁺ is a montmorillonite sheet silicate clay exchanged with protons. Various techniques, including ¹H NMR, ¹³C NMR, IR, DSC and Ubbelohde viscometer were used to elucidate structural characteristics and thermal properties of the resulting copolymers. The effect of the [glycolide]/[POE] molar ratio on the rate of copolymerization and on intrinsic viscosity of the resulting copolymers was studied. Data showed that the rate of copolymerization and intrinsic viscosity of copolymers increase with increasing [glycolide]/[POE] molar ratio.     

Deficiency of Thyroid Hormone Reduces Voltage-Gated Na+ Currents as Well as Expression of Na+/K+-ATPase in the Mouse Hippocampus

Mice lacking functional thyroid follicular cells, Pax8^−/− mice, die early postnatally, making them suitable models for extreme hypothyroidism. We have previously obtained evidence in postnatal rat neurons, that a down-regulation of Na⁺-current density could explain the reduced excitability of the nervous system in hypothyroidism. If such a mechanism underlies the development of coma and death in severe hypothyroidism, Pax8^−/− mice should show deficits in the expression of Na⁺ currents and potentially also in the expression of Na⁺/K⁺-ATPases, which are necessary to maintain low intracellular Na⁺ levels. We thus compared Na⁺ current densities in postnatal mice using the patch-clamp technique in the whole-cell configuration as well as the expression of three alpha and two beta-subunits of the Na⁺/K⁺-ATPase in wild type versus Pax8^−/− mice. Whereas the Na⁺ current density in hippocampal neurons from wild type mice was upregulated within the first postnatal week, the Na⁺ current density remained at a very low level in hippocampal neurons from Pax8^−/− mice. Pax8^−/− mice also showed significantly decreased protein expression levels of the catalytic α1 and α3 subunits of the Na⁺/K⁺-ATPase as well as decreased levels of the β2 isoform, with no changes in the α2 and β1 subunits.     

Comparative study on iodide and bromide isotopic exchange reactions by application of radioactive tracer technique

In the present investigation, a short lived radioactive tracer isotopes ¹³¹I and ⁸²Br were used to study the kinetics of isotopic exchange reactions. For bromide isotopic exchange reaction, the calculated values of specific reaction rate (min⁻¹), and amount of bromide ions exchanged (mmol) were obtained higher than that for iodide isotopic exchange reaction under identical experimental conditions. For both the exchange reactions it was observed that when the concentration of ionic solution was increased twice, the␣percentage of ions exchanged increases sharply at nearly same specific reaction rate, indicating that concentration greatly influences the exchange reactions.     

Randomness in Ziegler-Natta olefin copolymerizations as determined by C-NMR spectroscopy—the influence of chain heterogeneity

The chain heterogeneity of a polyethylene resin can be assessed by simple dissolution techniques followed by compositional analyses. This heterogeneity influences common measurements of the randomness of the polymerization process. Specifically, the values for the product of reactivity ratios and the relative monomer dispersities will suggest a less random polymerization process than is actually occurring on a molecular scale.For the system studied, compositional analysis of the parent resins demonstrates the blockiness (non-random) character commonly reported for heterogeneous Ziegler-Natta polymerizations. Compositional analyses of the soluble and insoluble fractions show at least two chain populations with significantly different ethylene/1-hexene molar ratios, each showing a near random incorporation of comonomer. These data are consistent with at least two catalyst sites, each with different comonomer incorporation efficiency.     

The Mode of SN38 Derivatives Interacting with Nicked DNA Mimics Biological Targeting of Topo I Poisons

The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered for advancement to the preclinical development stage. To gain better insights into the molecular mechanism with the biological target, here, we conducted an investigation into their interactions with model nicked DNA (1) using different techniques. In this work, we observed the complexity of the mechanism of action of the compounds 2 and 3, in addition to their decomposition products: compound 4 and SN38. Using DOSY experiments, evidence of the formation of strongly bonded molecular complexes of SN38 derivatives with DNA duplexes was provided. The molecular modeling based on cross-peaks from the NOESY spectrum also allowed us to assign the geometry of a molecular complex of DNA with compound 2. Confirmation of the alkylation reaction of both compounds was obtained using MALDI–MS. Additionally, in the case of 3, alkylation was confirmed in the recording of cross-peaks in the ¹H/¹³C HSQC spectrum of ¹³C-enriched compound 3. In this work, we showed that the studied compounds—parent compounds 2 and 3, and their potential metabolite 4 and SN38—interact inside the nick of 1, either forming the molecular complex or alkylating the DNA nitrogen bases. In order to confirm the influence of the studied compounds on the topoisomerase I relaxation activity of supercoiled DNA, the test was performed based upon the measurement of the fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The presented results confirmed that studied SN38 derivatives effectively block DNA relaxation mediated by Topo I, which means that they stop the machinery of Topo I activity.     

Function of Graphene Oxide as the “Nanoquencher” for Hg2+ Detection Using an Exonuclease I-Assisted Biosensor

Graphene oxide is well known for its excellent fluorescence quenching ability. In this study, positively charged graphene oxide (pGO25000) was developed as a fluorescence quencher that is water-soluble and synthesized by grafting polyetherimide onto graphene oxide nanosheets by a carbodiimide reaction. Compared to graphene oxide, the fluorescence quenching ability of pGO25000 is significantly improved by the increase in the affinity between pGO25000 and the DNA strand, which is introduced by the additional electrostatic interaction. The FAM-labeled single-stranded DNA probe can be almost completely quenched at concentrations of pGO25000 as low as 0.1 μg/mL. A simple and novel FAM-labeled single-stranded DNA sensor was designed for Hg²⁺ detection to take advantage of exonuclease I-triggered single-stranded DNA hydrolysis, and pGO25000 acted as a fluorescence quencher. The FAM-labeled single-stranded DNA probe is present as a hairpin structure by the formation of T–Hg²⁺–T when Hg²⁺ is present, and no fluorescence is observed. It is digested by exonuclease I without Hg²⁺, and fluorescence is recovered. The fluorescence intensity of the proposed biosensor was positively correlated with the Hg²⁺ concentration in the range of 0–250 nM (R² = 0.9955), with a seasonable limit of detection (3σ) cal. 3.93 nM. It was successfully applied to real samples of pond water for Hg²⁺ detection, obtaining a recovery rate from 99.6% to 101.1%.     

Investigation of the Structure-Directing Effect of Surfactants on the Distribution of Silicate Species Using <Superscript>29</Superscript>Si-NMR Spectroscopy

Silicate anions were studied using ²⁹Si NMR to investigate the structure-directing effect of a surfactant (sodium dodecylsulfate-SDS) on the distribution of silicate species present in alkaline silicate solutions. The distribution of the anions in the presence of polyelectrolytes was also investigated by ²⁹Si NMR through the use of different bases including tetramethyl ammonium hydroxide (TMAOH) and sodium hydroxide (NaOH). Both SDS and the polyelectrolytic solutions were shown to stabilize low molecular weight silicate species and thus inhibit polymerization.

Experimental and modeling analysis of the formation of cuprous intermediate species formed during the copper deposition on a rotating disk electrode

Two comprehensive kinetic models reported in the literature for the copper deposition in sulfate media are modeled and compared with experimental data (linear sweep voltammetry) in order to disclose the role and phase of the cuprous species as intermediates. Differences in the phase of these species are considered for each model, i.e. Cu(I)_ads and Cu⁺. Modeling considers the formation of copper in two mono-electronic steps and account for mass transport by diffusion and convection in a RDE. Reasonable fits were obtained for both models at different experimental conditions (e.g. Cu²⁺ bulk concentration, rotation rate). It was found that this behavior is possible due to the low concentrations of cuprous species and their rapid consumption to form metallic copper, i.e. not rate-controlling step. Further insights of the kinetic and mass transport contributions of this system were obtained for both models by computing some variables that cannot be experimentally measured (i.e. surface concentrations). The first reaction in the mechanism was found to be the rate-determining step. A set of optimum kinetic parameters and constants obtained from the analysis of the models are also reported in this study.