Physical characterization and cellular uptake of propylene glycol liposomes in vitro

In order to facilitate the intracellular delivery of therapeutic agents, a new type of liposomes–propylene glycol liposomes (PGL) were prepared, and their cell translocation capability in vitro was examined. PGL was composed of hydrogenated egg yolk lecithin, cholesterol, Tween 80 and propylene glycol. With curcumin as a model drug, characterization of loaded PGL were measured including surface morphology, particle size, elasticity, encapsulation efficiency of curcumin and physical stability. Using curcumin-loaded conventional liposomes as the control, the cell uptake capacity of loaded PGL was evaluated by detection the concentration of curcumin in cytoplasm. Compared with conventional liposomes, PGL exhibited such advantages as high encapsulation efficiency (92.74% ± 3.44%), small particle size (182.4?±?89.2?nm), high deformability (Elasticity index?=?48.6) and high stability both at normal temperature (about 25°C) and low temperature at 4°C. From cell experiment in vitro, PGL exhibited the highest uptake of curcumin compared with that of conventional liposomes and free curcumin solution. Little toxic effect on cellular viability was observed by methyl tetrazolium assay. In conclusion, PGL might be developed as a promising intracellular delivery carrier for therapeutic agents.     

Physical characterization and cellular uptake of propylene glycol liposomes in vitro

In order to facilitate the intracellular delivery of therapeutic agents, a new type of liposomes-propylene glycol liposomes (PGL) were prepared, and their cell translocation capability in vitro was examined. PGL was composed of hydrogenated egg yolk lecithin, cholesterol, Tween 80 and propylene glycol. With curcumin as a model drug, characterization of loaded PGL were measured including surface morphology, particle size, elasticity, encapsulation efficiency of curcumin and physical stability. Using curcumin-loaded conventional liposomes as the control, the cell uptake capacity of loaded PGL was evaluated by detection the concentration of curcumin in cytoplasm. Compared with conventional liposomes, PGL exhibited such advantages as high encapsulation efficiency (92.74% ± 3.44%), small particle size (182.4?±?89.2?nm), high deformability (Elasticity index?=?48.6) and high stability both at normal temperature (about 25°C) and low temperature at 4°C. From cell experiment in vitro, PGL exhibited the highest uptake of curcumin compared with that of conventional liposomes and free curcumin solution. Little toxic effect on cellular viability was observed by methyl tetrazolium assay. In conclusion, PGL might be developed as a promising intracellular delivery carrier for therapeutic agents.     

Evaluation of in vitro stability of large unilamellar liposomes coated with a modified polysaccharide (O-palmitoylpullulan)

Aiming at encapsulation of a hydrosoluble drug, large unilamellar liposomes (LUV) of egg phosphatidylcholine (PC) were coated with a natural polysaccharide derivative, O-palmitoylpullulan (OPP), and its in vitro stability evaluated using fluorescent probes. This coating (in OPP/PC weight ratio of 3) improved significantly the in vitro stability of LUV by decreasing both the permeability and fluidity of the liposomal membrane.This paper was accepted for publication after the 1995 Conference of the European Society of Biomaterials, Oporto, Portugal, 10–13 September.     

Synthesis,characterization and in vitro studies of pegylated melphalan conjugates

Melphalan, a drug used for the treatment of breast, ovaries and a certain type of cancer in the bone marrow, was conjugated to linear methoxy poly (ethylene glycol) (M-PEG) of 2000 and 5000, Da. An ester linkage between polymer and drug was used in the coupling to yield a polymeric prodrug. Purified esters were characterized by Maldi-Tof and IR spectroscopy methods. The modification allowed overcoming the known melphalan aqueous solubility problem (0.1 µg/ml) leading us to obtain a polymer-drug bioconjugate more suitable for oral and parental administration. It was found that molecular weight of M-PEG is critical for the conjugates stability, aqueous solubility (80 times and 123 times higher aqueous solubility for M-PEG 2000 and M-PEG 5000, respectively), and hemolytic activity. The melphalan caused 100% hemolysis above the concentration 3.5 µg/ml in 1?h. whereas conjugate of M-PEG 2000 and M-PEG 5000 shows 81.3?±?0.5% and 48.8?±?1.5% hemolysis, respectively at 32 µg/ml after1?h. Further In vitro anticancer activity of melphalan and its conjugates was performed with breast cancer MCF-7 cell lines. It shows that LD50 concentration was higher 1.14 and 2 µm for M-PEG 2000 and M-PEG 5000, respectively in comparison to pure melphalan (0.74 µm). Above studies revealed improved pharmacokinetics properties upon conjugation.     

Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials

The interactions of nanoparticles with the soft surfaces of biological systems like cells play key roles in executing their biomedical functions and in toxicity. The discovery or design of new biomedical functions, or the prediction of the toxicological consequences of nanoparticles in vivo, first require knowledge of the interplay processes of the nanoparticles with the target cells. This article focusses on the cellular uptake, location and translocation, and any biological consequences, such as cytotoxicity, of the most widely studied and used nanoparticles, such as carbon-based nanoparticles, metallic nanoparticles, and quantum dots. The relevance of the size and shape, composition, charge, and surface chemistry of the nanoparticles in cells is considered. The intracellular uptake pathways of the nanoparticles and the cellular responses, with potential signaling pathways activated by nanoparticle interactions, are also discussed.     

Electroinjection of colloid particles and biopolymers into single unilamellar liposomes and cells for bioanalytical applications

A combined electroporation and pressure-driven microinjection method for efficient loading of biopolymers and colloidal particles into single-cell-sized unilamellar liposomes was developed. Single liposomes were positioned between a approximately 2-microm tip diameter solute-filled glass micropipet, equipped with a Pt electrode, and a 5-microm-diameter carbon fiber electrode. A transient, 1-10 ms, rectangular waveform dc voltage pulse (10-40 V/cm) was applied between the electrodes, thus focusing the electric field over the liposome. Dielectric membrane breakdown induced by the applied voltage pulse caused the micropipet tip to enter the liposome and a small volume (typically 50-500 x 10(-15) L) of fluorescein, YOYO-intercalated T7-phage DNA, 100-nm-diameter unilamellar liposomes, or fluorescent latex spheres could be injected into the intraliposomal compartment. We also demonstrate initiation of a chemical intercalation reaction between T2-phage DNA and YOYO-1 by dual injection into a single giant unilamellar liposome. The method was also successfully applied for loading of single cultured cells.     

Profiling of drugs for membrane activity using liposomes as an in vitro model system

The increasing size of chemical libraries being analyzed by high-throughput screening results in a growing number of active compounds that need to be assessed before moving forward in the drug development process. As a consequence, more rapid and highly sensitive strategies are required to accelerate the process of drug discovery without increasing the cost. Due to the fact that significant numbers of compounds from combinatorial libraries are hydrophobic in nature, approaches are needed to evaluate the potential for these compounds to interfere with the functions of biological membranes. The liposome system was used to detect agents that act as follows: (i) ionophores able to induce specific ion permeability, e.g., valinomycin for K⁺ and protonophoric uncouplers for H⁺; (ii) ion antiporters which exchange H⁺ for other ions, e.g., nigericin; (iii) agents that form low specificity ion channels in the membrane, e.g., gramicidin; and (iv) detergents and other membrane-disrupting agents. We propose using this liposome assay during the drug development process to identify compounds that have membrane activity and, as a consequence, produce a biological effect by altering the physico-chemical properties of the cell membrane rather than interacting with a protein target. Screening of a representative set of biologically-active compounds (198) indicated that the majority of systemic antimicrobial drugs, but not topical drugs, lack membrane activity in this model system.     

Cholesterol derivative-based liposomes for gemcitabine delivery: preparation,in vitro,and in vivo characterization

As an anti-tumor drug, gemcitabine (Gem) is commonly used for the treatment of non-small cell lung cancer and pancreatic cancer. However, there are several clinical drawbacks to using Gem, including its extremely short plasma half-life and side effects. To prolong its half-life and reduce its side effects, we synthesized a derivative of Gem using cholesterol (Chol). This derivative, called gemcitabine-cholesterol (Gem-Chol), was entrapped into liposomes by a thin-film dispersion method. The particle size of the Gem-Chol liposomes was 112.57?±?1.25?nm, the encapsulation efficiency was above 99%, and the drug loading efficiency was about 50%. In vitro studies revealed that the Gem-Chol liposomes showed delayed drug release and long-term stability at 4?°C for up to 2 months. In vivo studies also showed the superiority of the Gem-Chol liposomes, and compared with free Gem, the Gem-Chol liposomes had longer circulation time. Moreover, an anti-tumor study in H₂₂ and S180 tumor models showed that liposomal entrapment of Gem-Chol improved the anti-tumor effect of Gem. This study reports a potential formulation of Gem for clinical application.     

Silver/dendrimer nanocomposites as biomarkers: fabrication, characterization, in vitro toxicity, and intracellular detection

We have synthesized water-soluble, biocompatible, fluorescent, and stable silver/dendrimer nanocomposites that exhibit a potential for in vitro cell labeling. Amino-, hydroxyl-, and carboxyl-terminated ethylenediamine core generation 5 poly(amidoamine) dendrimers were utilized to prepare aqueous silver(I)-dendrimer complexes (with the molar ratio of 25 Ag+ per dendrimer) at the biologic pH of 7.4. Conversion of silver(I)-dendrimer complexes into dendrimer nanocomposites was achieved by irradiating the solutions with UV light to reduce the bound Ag+ cations to zerovalent Ag0 atoms, which were simultaneously trapped in the dendrimer network, resulting in the formation of {(Ag0)25-PAMAM_E5.NH2}, {(Ag0)25-PAMAM_E5.NGly}, and {(Ag0)25-PAMAM_E5.NSAH} dendrimer nanocomposites (DNC), respectively. The silver-DNCs were characterized by means of UV-vis, fluorescence spectroscopy, dynamic light-scattering, zeta potential measurements, high-resolution transmission electron microscopy, X-ray energy dispersive spectroscopy, and selected area electron diffraction. The cytotoxicity of dendrimers and related silver nanocomposites was evaluated using an XTT colorimetric assay of cellular viability. The cellular uptake of nanoparticles was examined by transmission electron and confocal microscopy. Results indicate that {(Ag0)25-PAMAM_E5.NH2}, {(Ag0-)25-PAMAM_E5.NGly}, and {(Ag0)25-PAMAM_E5.NSAH} form primarily single particles with diameters between 3 and 7 nm. The dendrimer nanocomposites are fluorescent, and their surface charge, cellular internalization, toxicity, and cell labeling capabilities are determined by the surface functionalities of dendrimer templates. The {(Ag0)25-PAMAM_E5.NH2} and {(Ag0)25-PAMAM_E5.NSAH} nanocomposites exhibit potential application as cell biomarkers.     

Preparation and pharmacokinetics in rabbits of breviscapine unilamellar vesicles

The purpose of the study was to prepare the unilamellar liposomal vesicles of breviscapine (Breviscapine-LUVs) and investigate the pharmacokinetics of Breviscapine-LUVs in rabbits. Breviscapine-LUVs were prepared by the film dispersion method and treated further by extrusion. Its size distribution and zeta potential were determined by photon correlation spectroscopy. The encapsulation efficiency (EE) and cumulative release of Breviscapine-LUVs were assayed by the dialysis method. The crossover design (two periods) was used in six rabbits, which were administered Breviscapine-LUVs and reference preparation. Results showed that the particle size of Breviscapine-LUVs was 50.8 nm, and the polydispersity index was 0.287. The zata potential was -24 mV ± 9 mV(n = 3), and the EE% was 81.1 ± 1.1% (n = 3). The cumulative release of vesicles in 0.9% NaCl was 17.2 ± 0.78%, 26.1 ± 0.68%, and 29.9 ± 0.81% in 2, 8, and 24 h, respectively. The mean concentration-time curves of breviscapine liposomes and reference preparation were both fitted to a two-compartment model with the main pharmacokinetic parameters as follows: t_1/2β of Breviscapine-LUVs and reference preparation were (42.5 ± 28.6) min and (6.01 ± 4.64) min, respectively; CL_(s) were (15.3 ± 9.03) mL × min^-1 and (84.6 ± 40.6) mL × min^-1, respectively; AUC_0-300 were (1267 ± 1083) μg × min × mL^-1 and (196 ± 107) μg × min × mL^-1, respectively. Compared with the reference preparation, breviscapine liposomes had a much higher concentration in plasma and contained characteristic of sustained-release, which ameliorated the pharmacokinetic properties of scutellarin.     

Using acoustic cavitation to enhance chemotherapy of DOX liposomes: experiment in vitro and in vivo

Experiments in vitro and in vivo were designed to investigate tumor growth inhibition of chemotherapeutics-loaded liposomes enhanced by acoustic cavitation. Doxorubicin-loaded liposomes (DOX liposomes) were used in experiments to investigate acoustic cavitation mediated effects on cell viability and chemotherapeutic function. The influence of lingering sensitive period after acoustic cavitation on tumor inhibition was also investigated. Animal experiment was carried out to verify the practicability of this technique in vivo. From experiment results, blank phospholipid-based microbubbles (PBM) combined with ultrasound (US) at intensity below 0.3 W/cm(2) could produce acoustic cavitation which maintained cell viability at high level. Compared with DOX solution, DOX liposomes combined with acoustic cavitation exerted effective tumor inhibition in vitro and in vivo. The lingering sensitive period after acoustic cavitation could also enhance the susceptibility of tumor to chemotherapeutic drugs. DOX liposomes could also exert certain tumor inhibition under preliminary acoustic cavitation. Acoustic cavitation could enhance the absorption efficiency of DOX liposomes, which could be used to reduce DOX adverse effect on normal organs in clinical chemotherapy.     

Inhalable liposomes of Glycyrrhiza glabra extract for use in tuberculosis: formulation,in vitro characterization,in vivo lung deposition,and in vivo pharmacodynamic studies

Objective: The current study involves the development of liposomal dry powder for inhalation (LDPI) containing licorice extract (LE) for use in tuberculosis.

Significance: The current epidemiology of tuberculosis along with the increasing emergence of resistant forms of tuberculosis necessitates the need for developing alternative efficacious medicines for treatment. Licorice is a medicinal herb with reported activity against Mycobacterium tuberculosis.

Methods: Liposomes with LE were prepared by thin film hydration technique and freeze dried to obtain LDPI. The comprehensive in vitro and in vivo characterization of the LDPI formulation was carried out.

Results: The particle size of liposomes was around 210?nm with drug entrapment of almost 75%. Transmission electron microscopy revealed spherical shape of liposome vesicles. The flow properties of the LDPI were within acceptable limits. Anderson Cascade Impactor studies showed the mean median aerodynamic diameter, geometric standard deviation and fine particle fraction of the LDPI to be 4.29?µm, 1.23, and 54.68%, respectively. In vivo lung deposition studies of LDPI in mice showed that almost 46% of the drug administered reaches the lungs and 16% of administered drug is retained in the lungs after 24?hours of administration. The in vivo pharmacodynamic evaluation of the LDPI showed significant reduction in bacterial counts in lungs as well as spleen of TB-infected mice.

Conclusions: LE LDPI thus has a promising potential to be explored as an effective anti-tubercular medicine or as an adjunct to existing anti-tubercular drugs.     

Brucine-loaded liposomes composed of HSPC and DPPC at different ratios: in vitro and in vivo evaluation

Objective: The objective of this study is to test the hypothesis that the phase transition temperature (T_m), the main property of liposomes, can be easily controlled by changing the molar ratio of hydrogenated soy phosphatidylcholine (HSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphacholine (DPPC) after drug encapsulation.

Materials and methods: Brucine, an antitumor alkaloid, was encapsulated into the liposomes with different HSPC/DPPC compositions. The T_ms of the brucine-loaded liposomes (BLs) were determined by differential scanning calorimetry (DSC). Then the physicochemical properties and pharmacokinetics of the BLs with different HSPC/DPPC compositions were investigated and compared.

Results: The results of DSC revealed that HSPC and DPPC can combine into one phase. The findings of molecular modeling study suggested that HSPC interacts with DPPC via electrostatic interaction. The molar ratio of HSPC/DPPC influenced the sizes of BLs but had little effect on the entrapment efficiency (EE). The stability of BLs was improved with the increase of the HSPC ratios, especially with the presence of plasma. Following i.v. administration, it was found that AUC values of BLs in vivo were directly related to the HSPC/DPPC ratios of BLs, namely the T_ms of BLs.

Discussion: The behavior of liposomes, especially in vivo pharmacokinetic behavior, can be controlled by the modification of T_m.

Conclusion: The characterization of BLs in vitro and in vivo had demonstrated that the T_m could be flexibly modified for liposomes composed of both HSPC and DPPC. Using HSPC/DPPC composition may be an efficient strategy to control the T_m, thus control the in vivo pharmacokinetic behavio, of BLs.     

In vitro safety toxicology data for evaluation of gold nanoparticles-chronic cytotoxicity, genotoxicity and uptake

Safety and toxic effects of nanoparticles are still largely unexplored due to the multiple aspects that influence their behaviour toward biological systems. Here, we focus the attention on 12 nm spherical gold nanoparticle coated or not with hyaluronic acid compared to its precursor counterpart salt. Results ranging from the effects of a 10-days exposure in an in vitro model with BALB/c 3T3 fibroblast cells show how 12 nm spherical gold nanoparticles are internalized from 3T3 cells by endo-lysosomal pathway by an indirect measurement technique; and how gold nanoparticles, though not being a severe cytotoxicant, induce DNA damage probably through an indirect mechanism due to oxidative stress. While coating them with hyaluronic acid reduces gold nanoparticles cytotoxicity and slows their cell internalization. These results will be of great interest to medicine, since they indicate that gold nanoparticles (with or without coating) are suitable for therapeutic applications due to their tunable cell uptake and low toxicity.     

Preparation,characterization and in vitro anticancer activity of paclitaxel conjugated magnetic nanoparticles

In this study, magnetic nanoparticles (MNPs) coated with L-aspartic acid (F-Asp NPs) were synthesized through a co-precipitation method and conjugated with paclitaxel (PTX) (F-Asp-PTX NPs) by esterification reaction between the carboxylic acid end groups on MNPs surface and the hydroxyl groups of the PTX and studied its cytotoxic effect in vitro. The successful conjugating of PTX onto the nanoparticles (NPs) was confirmed by X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), fourier transform infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM) and transmission electron microscopy (TEM) techniques. The results showed that the average size was 46.11?±?7.8 (mean?±?SD (n?=?25)) nm. The cytotoxicity of void of PTX and F-Asp-PTX NPs were compared to each other by MTT assay of the treated MCF-7 cell line. The F-Asp-PTX NPs showed pH-dependent drug release behavior. These studies specify that F-Asp-PTX NPs have a very remarkable anticancer effect, for breast cancer cell line.     

Positively and negatively charged liposomes as carriers for transdermal delivery of sumatriptan: in vitro characterization

Background: The influence of liposome composition, lamellarity, preparation method, and charge on the encapsulation efficiency, size, polydispersity, and surface charge of sumatriptan liposomes was studied. For this purpose, we studied multilamellar, unilamellar, and frozen and thawed liposomes. Positively or negatively charged liposomes were obtained using both phosphatidylcholine and cholesterol, in combination with stearylamine or dicetylphosphate. Liposomal formulations were characterized by confocal laser scanning microscopy and optical microscopy for vesicle formation, morphology, and lamellarity by dynamic laser light scattering for size distribution and polydispersity, and electrophoretic mobility for zeta potential determination. To obtain more information about the sumatriptan encapsulation, dynamic dialysis technique was employed. The sumatriptan amount was quantified by high-performance liquid chromatography. Results: Overall obtained results showed that liposomes may be interesting carriers for sumatriptan succinate. Statistical analysis evidenced that the preparation method does not affect the evaluated characterization parameters. However, the presence of charge inducer agents modified these characteristics. Highest loading efficiency of sumatriptan was exhibited for positively charged liposomes containing 6.58:10.34:3.73 mmolar ratio for phosphatidylcholine : cholesterol : stearylamine. The mean size was affected by the charge inducer, being smaller in positively charged liposomes. Logically, surface charge of liposomes varied as a function of the employed charged agent. Also, interesting results were obtained when vesicles were loaded with sumatriptan, showing a statistical significance between all pairs, comparing the formulations with and without drug. Conclusion: Results obtained revealed that the presence of sumatriptan into the vesicles has a different behavior in negatively and positively charged liposomes.     

Zinc oxide nanoparticles: a review of their biological synthesis,antimicrobial activity,uptake, translocation and biotransformation in plants

Over the past decade, incorporation of nanomaterials into agricultural practices like nanofertilizers and nanopesticides has gained a lot of attention. Progress and application of fertilizers in nanoforms are one of the effective options for considerable improvement of the agricultural yield worldwide. Zinc oxide nanoparticles (ZnO NPs) are considered as a biosafe material for biological species. Earlier studies have shown the potential of ZnO NPs in stimulation of seed germination and plant growth as well as disease suppression and plant protection by its antimicrobial activity. However, both positive and negative effects of ZnO NPs on plant growth and metabolism at various developmental periods have been documented. Uptake, translocation and accumulation of ZnO NPs by plants depend upon the features of NPs as well as the anatomy of the host plant. This review summarizes the applications of ZnO NPs as nanofertilizer in crop production and also attempts to examine and record the possible mechanism of antimicrobial activity of ZnO NPs. Biological synthesis of ZnO NPs and their uptake, translocation and biotransformation in plants via various routes have also been examined.     

Presentation matters: Identity of gold nanocluster capping agent governs intracellular uptake and cell metabolism

Au nanoclusters （AuNCs） hold tremendous potential to be employed in a wide variety of biological applications. Despite the rapid development in the field of NCs synthesis, a comprehensive understanding of how cells interact with this class of ultra-small nanoparticles （〈2 nm） having defined sizes and surface chemistry, remains poorly understood. In this study, we show that the choice of the surface ligand used to protect AuNCs can significantly perturb cellular uptake and intracellular redox signaling. A panel of monodisperse, atomically precise AuNCs with different core Au atom number （i.e., Auls, Au18 and Au25） protected with either mercaptopropionic acid （MPA） or glutathione （GSH） capping agent were synthesized and their effects on the generation of intracellular reactive oxygen species （ROS）, cytotoxicity and genotoxicity of the NCs were assessed. Both mitochondrial superoxide anion （O2^-） and cytoplasmic ROS were found to be higher in cells exposed to MPA but not GSH capped AuNCs. The unregulated state of intracellular ROS is correlated to the amount of internalized AuNCs. Interestingly, MPA-AuNCs induction of ROS level did not lead to any detrimental cellular effects such as cell death or DNA damage. Instead, it was observed that the increase in redox status corresponded to higher cellular metabolism and proliferative capacity. Our study illustrates that surface chemistry of AuNCs plays a pivotal role in affecting the biological outcomes and the new insights gained will be useful to form the basis of defining specific design rules to enable rational engineering of ultra-small complex nanostructures for biological applications.