Non-modified Liposomes Research Articles

Use of leptin-conjugated phosphatidic acid liposomes with resveratrol and epigallocatechin gallate to protect dopaminergic neurons against apoptosis for Parkinson's disease therapy

Complex liposomes were assembled with 1,2-distearoyl-sn-glycero-3-phosphocholine, dihexadecyl phosphate (DHDP), cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (PA) to act as drug carriers for resveratrol (RES) and epigallocatechin gallate (EGCG). The liposomes were modified with leptin (Lep) on the surface to cross the blood-brain barrier (BBB) and to rescue degenerated dopaminergic neurons. The activity of RES and EGCG against neurotoxicity was investigated using an in vitro neurodegenerative model established by SH-SY5Y cells with an insult of 1-methyl-4-phenylpyridinium (MPP+). The results indicated that increasing the mole percentage of DHDP and PA increased the particle size and absolute zeta potential value, and improved the entrapment efficiency of RES and EGCG; however, this increase reduced the release rate of RES and EGCG and the grafting efficiency of Lep. The ability of Lep/RES-EGCG-PA-liposomes to cross the BBB was found to be higher than that of non-modified liposomes. Further, the addition of PA and Lep into liposomes enhanced cell viability and target efficiency. The immunofluorescence results demonstrated that the conjugation of Lep with liposomes enabled the docking of HBMECs and SH-SY5Y cells via Lep receptor, and enhanced their ability to permeate the BBB and cellular uptake. Immunofluorescence and western blot analysis also revealed that RES and EGCG encapsulated into liposomes could be a neural defensive strategy by reducing the apoptosis promotor protein Bcl-2 associated X protein and α-synuclein, and enhancement in the apoptosis inhibitor protein B cell lymphoma 2, tyrosine hydroxylase, and the dopamine transporter. Hence, Lep-PA-liposomes can be an excellent choice of potential delivery system for PD treatment.

PH-dependent reversibly activatable cell-penetrating peptides improve the antitumor effect of artemisinin-loaded liposomes

Artemisinin (ART) is well known as an antimalarial drug, and it can also be used to treat inflammation as well as cancer. Although many researchers have reported the antitumor activity of ART, most of these studies were investigated in vitro. In addition, ART is sparingly soluble in water, limiting its clinical relevance in drug development. Based on the data from our preliminary study, ART is not cytotoxic at low micromolar concentrations. Thus, we hypothesized that smart nanocarriers are beneficial for not only increasing the solubility of ART but also elevating the concentration of the drug at the target, thereby inducing the ideal antitumor effect. In this article, a reversibly activatable cell-penetrating peptide ((HE)10-G5-R6 or HE-R6) was introduced to modify artemisinin (ART)-loaded liposomes (ART-Lip-HE-R6) against tumors, and in vitro and in vivo performance were investigated. ART-Lip-HE-R6 exhibited sustained release under different pH conditions. The internalization and cytotoxicity of liposomes were enhanced at low pH, i.e., 6.5, after modification with HE-R6 versus nonmodified liposomes. Moreover, a longer retention time in tumors could be observed in the ART-Lip-HE-R6 group, followed by higher efficiency of tumor suppression. In conclusion, Lip-HE-R6 might be a promising delivery system for ART in cancer therapy.

Prolonged distribution of aerosolized PEGylated liposomes in the lungs of mice with bleomycin-induced pulmonary fibrosis

Objective Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic lung disease characterized by abnormal remodeling of the lung parenchyma with subsequent scarring of the alveolar structure. In this study, we examined the distribution characteristics of aerosolized polyethylene glycol (PEG)ylated liposomes in the lungs of mice with bleomycin-induced pulmonary fibrosis. Significance The present study details the utility of aerosolized PEGylated liposomes for improving intrapulmonary pharmacokinetics in fibrotic lungs. Methods Aerosolized PEGylated liposomes were administered to fibrotic mouse lungs using a MicroSprayer. Intrapulmonary pharmacokinetics was evaluated via in vivo imaging, measurement of liposome concentrations in bronchoalveolar lavage fluid (BALF) and alveolar macrophages (AMs), and observation of lung tissue sections. In addition, in vitro accumulation experiments using WI-38, A549, and RAW264.7 cells were performed. Results The decrease of the fluorescence intensity of the PEGylated liposomes was slower than that of the non-modified liposomes. Compared with the non-modified liposomes, the PEGylated liposomes were determined higher in BALF, whereas those in the AMs were lower. Both PEGylated and non-modified liposomes were widely dispersed in fibrotic regions in tissue sections. No difference in accumulation in WI-38 and A549 cells was noted between PEGylated and non-modified liposomes, whereas the PEGylated liposomes exhibited lower intracellular accumulation than non-modified liposomes in RAW264.7 cells. Conclusion Aerosolized drug delivery systems using PEGylated liposomes exhibited prolonged distribution in both healthy and fibrotic mouse lungs. PEGylated liposomes were determined to be efficient drug delivery systems for anti-fibrotic agents targeting lung fibroblasts and alveolar epithelial cells for optimizing the treatment of IPF.

Transferrin Modified Dioscin Loaded PEGylated Liposomes: Characterization and In Vitro Antitumor Effect.

In this study, a novel transferrin modified liposomal dioscin was prepared by the film dispersion method. The transferrin modified dioscin loaded liposomes (Tf-Lip/Dio) were near-spherical in morphology and had an average particle size of 140.07±1.33 nm, a narrow polydispersity index of <0.2 and a relatively stable zeta potential of -23.7±1.16 mV. The drug entrapment efficiency (EE) and drug loading (DL) of Tf-Lip/Dio were 88.94±1.02% and 4.16±0.05%, respectively. Tf-Lip/Dio exhibited a sustained release characterization of approximately 30% of the total dioscin content after 72 h at 37 °C. Tf-Lip/Dio showed higher cytotoxic efficacy after incubation for 24 h in both HeLa cells and HepG2 cells than in nonmodified liposomes. The enhanced antitumor activity of Tf-Lip/Dio might be due to the increased intracellular uptake, which was corroborated by laser scanning confocal microscopy and flow cytometry. Furthermore, hemolysis experiments preliminarily verified the safety of its intravenous injection. Overall, this study demonstrates Tf-Lip/Dio to be a favorable delivery vehicle for dioscin in future cancer therapy.

Delivery of folic acid-modified liposomal curcumin for targeted cervical carcinoma therapy.

Introduction: In this study, novel folic acid (FA)-modified curcumin (CUR) liposomes (LPs) were developed and evaluated for their antitumor activity in vitro and in vivo.Methods: Characterization of the LPs, including transmission electron microscopy, morphology, particle size, and zeta potential studies, was carried out. Drug entrapment efficiency, drug-loading capacity, and release properties in vitro were tested. The in vitro growth inhibition activity, cellular uptake efficiency, and cell apoptosis of FA-modified CUR LPs were also investigated by a cervical cancer HeLa cell model.Results: The optimized distearoyl-l-a-phosphatidylethanolamine (DSPE)-PEG2000-FA-LPs/CUR formed spherical vesicles of nanometer sizes and had particle sizes of 112.3±4.6 nm, polydispersity index of 0.19±0.03, and zeta potential of −15.3±1.4 mV. In addition, the EE% and DL% of (DSPE)-PEG2000-FA-LPs/CUR were 87.6% and 7.9%, respectively. Compared with the free drug, FA-modified CUR LPs had sustained-release properties in vitro. In vivo, a strong green fluorescence was observed in the cytoplasmic region after incubation of (DSPE)-PEG2000-FA-LPs/CUR for 2 hrs.Conclusion: (DSPE)-PEG2000-FA-LPs/CUR showed a superior antiproliferative effect on HeLa cells and had a better antitumor effect in vivo than the non-modified LPs. These results indicated that (DSPE)-PEG2000-FA-LPs/CUR was a promising candidate for antitumor drug delivery.

In vitro and in vivo evaluation of novel NGR-modified liposomes containing brucine.

In this study, a novel NGR (Asn-Gly-Arg) peptide-modified liposomal brucine was prepared by using spray-drying method. The surface morphology of the liposomes, encapsulation efficiency and particle size were investigated. The data showed that the addition of NGR did not produce any significant influence on brucine liposomes in terms of particle size or zeta potential. In addition, after 3 months of storage, no dramatic change such as visible aggregation, drug content changes or precipitation in the appearance of NGR-brucine liposomes occurred. The in vitro release results indicated that the release of brucine from NGR liposomes was similar to that of liposomes, demonstrating that the NGR modification did not affect brucine release. The in vitro drug-release kinetic model of NGR-brucine liposomes fitted well with the Weibull’s equation. In vivo, NGR-brucine liposomes could significantly extend the bioavailability of brucine; however, there was no significant difference observed in the pharmacokinetic parameters between liposomes and NGR liposomes after intravenous administration. Antitumor activity results showed that NGR-modified liposomes exhibited less toxicity and much higher efficacy in HepG2-bearing mice compared with non-modified liposomes. The enhanced antitumor activity might have occurred because brucine was specifically recognized by NGR receptor on the surface of tumor cells, which enhanced the intracellular uptake of drugs.

Lactose-based glycoconjugates with variable spacers for design of liver-targeted liposomes

Asialoglycoprotein receptors are highly abundant on the hepatocyte surface and have specific binding sites for blood serum glycoproteins. Such discovery resulted in development of liver-targeted drug delivery systems because modification of the liposomal surface by carbohydrate derivatives results in an increase of endocytosis, which facilitates selective uptake of such systems by hepatocytes. In this study we have synthesized novel lactose derivatives containing a palmitic hydrophobic domain. They were used for modification of the liposome surface. Transfection activity of modified liposomes was analyzed on the HepG2 cell line (hepatocytes) and showed an increase in the transfection efficiency as compared to the non-modified liposomes. At the same time transfection activities of modified and non-modified liposomes were similar in the case of a non-hepatocyte cell line (293T). The novel lactose-based glycoconjugates may be a promising tool for developing efficient vectors for delivery of nucleic acids to hepatocytes.

Sustained distribution of aerosolized PEGylated liposomes in epithelial lining fluids on alveolar surfaces.

The distribution characteristics of aerosolized PEGylated liposomes in alveolar epithelial lining fluid (ELF) were examined in rats, and the ensuing mechanisms were investigated in the in vitro uptake and protein adsorption experiments. Nonmodified or PEGylated liposomes (particle size 100nm) were aerosolized into rat lungs. PEGylated liposomes were distributed more sustainably in ELFs than nonmodified liposomes. Furthermore, the uptake of PEGylated liposomes by alveolar macrophages (AMs) was less than that of nonmodified liposomes. In further in vitro uptake experiments, nonmodified and PEGylated liposomes were opsonized with rat ELF components and then added to NR8383 cells as cultured rat AMs. The uptake of opsonized PEGylated liposomes by NR8383 cells was lower than that of opsonized nonmodified liposomes. Moreover, the protein absorption levels in opsonized PEGylated liposomes were lower than those in opsonized nonmodified liposomes. These findings suggest that sustained distributions of aerosolized PEGylated liposomes in ELFs reflect evasion of liposomal opsonization with surfactant proteins and consequent reductions in uptake by AMs. These data indicate the potential of PEGylated liposomes as aerosol-based drug delivery system that target ELF for the treatment of respiratory diseases.

Multi-functional Liposomes Enhancing Target and Antibacterial Immunity for Antimicrobial and Anti-Biofilm Against Methicillin-Resistant Staphylococcus aureus.

The aim of this study was to prepare wheat germ agglutinin (WGA)-modified liposomes encapsulating clarithromycin and to evaluate their in vitro and in vivo efficacy against Methicillin-resistant Staphylococcus aureus (MRSA). Physicochemical parameters, minimum inhibitory concentrations, in vitro killing kinetic, cellular uptake, biofilm formation inhibition and pre-formed biofilm destruction, biodistribution, in vivo antibacterial efficacy against MRSA, and phagocytosis into macrophages for liposomes loading clarithromycin were determined. The minimum inhibitory concentration and the time-kill curve for WGA-modified liposomal clarithromycin were better than those of free and nonmodified liposomal clarithromycin. Flow cytometry analysis displayed that liposomes could deliver more Coumarin 6, a fluorescent probe, into bacteria because of the conjugation of WGA. Besides, WGA-modified liposomal clarithromycin inhibited formation of S. aureus (ATCC 29213) and MRSA biofiom, and prompted the biofilm disassembly at lower concentrations below MIC. Effective accumulation of liposomes was displayed in the enterocoelia of the mice because of WGA. The number of MRSA colony-forming units in the kidney and spleen in mice treated with WGA-modified liposomal clarithromycin was significantly lower than that treated with free and nonmodified clarithromycin (p < 0.05). Intracellular localization of MRSA occurred in a significantly higher proportion of macrophage exposed to WGA-modified liposomes compared to those exposed to nonmodified liposomes. Liposome modified by WGA is a promising formulation for bacteria targeted delivery and immunity defensive system through macrophage improving uptake of bacteria, biodistribution, in vitro and in vivo antibacterial efficacy against MRSA.

In situ Delivery of Tumor Antigen– and Adjuvant-Loaded Liposomes Boosts Antigen-Specific T-Cell Responses by Human Dermal Dendritic Cells

Dendritic cells (DCs) have an important role in tumor control via the induction of tumor-specific T-cell responses and are therefore an ideal target for immunotherapy. The human skin is an attractive site for tumor vaccination as it contains various DC subsets. The simultaneous delivery of tumor antigen with an adjuvant is beneficial for cross-presentation and the induction of tumor-specific T-cell responses. We therefore developed liposomes that contain the melanoma-associated antigen glycoprotein 100280-288 peptide and Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) as adjuvant. These liposomes are efficiently taken up by monocyte-derived DCs, and antigen presentation to CD8(+) T cells was significantly higher with MPLA-modified liposomes as compared with non-modified liposomes or the co-administration of soluble MPLA. We used a human skin explant model to evaluate the efficiency of intradermal delivery of liposomes. Liposomes were efficiently taken up by CD1a(+) and especially CD14(+) dermal DCs. Induction of CD8(+) T-cell responses by emigrated dermal DCs was significantly higher when MPLA was incorporated into the liposomes as compared with non-modified liposomes or co-administration of soluble MPLA. Thus, the modification of antigen-carrying liposomes with TLR ligand MPLA significantly enhances tumor-specific T-cell responses by dermal DCs and is an attractive vaccination strategy in human skin.

Efficient delivery to human lung fibroblasts (WI-38) of pirfenidone incorporated into liposomes modified with truncated basic fibroblast growth factor and its inhibitory effect on collagen synthesis in idiopathic pulmonary fibrosis.

In the present in vitro study, we assessed the delivery of pirfenidone incorporated into liposomes modified with truncated basic fibroblast growth factor (tbFGF) to lung fibroblasts and investigated the anti-fibrotic effect of the drug. The tbFGF peptide, KRTGQYKLC, was used to modify the surface of liposomes (tbFGF-liposomes). We used the thin-layer evaporation method, followed by sonication, to prepare tbFGF-liposomes containing pirfenidone. The cellular accumulation of tbFGF-liposomes was 1.7-fold greater than that of non-modified liposomes in WI-38 cells used as a model of lung fibroblasts. Confocal laser scanning microscopy showed that tbFGF-liposomes were widely localized in WI-38 cells. The inhibitory effects of pirfenidone incorporated into tbFGF-liposomes on transforming growth factor-β1 (TGF-β1)-induced collagen synthesis in WI-38 cells were evaluated by measuring the level of intracellular hydroxyproline, a major component of the protein collagen. Pirfenidone incorporated into tbFGF-liposomes at concentrations of 10, 30, and 100 µM significantly decreased the TGF-β1-induced hydroxyproline content in WI-38 cells. The anti-fibrotic effect of pirfenidone incorporated into tbFGF-liposomes was enhanced compared with that of pirfenidone solution. These results indicate that tbFGF-liposomes are a useful drug delivery system of anti-fibrotic drugs to lung fibroblasts for the treatment of idiopathic pulmonary fibrosis.

Preparation and ocular pharmacokinetics of hyaluronan acid-modified mucoadhesive liposomes

The aim of this research was to formulate a liposomal preparation of DOX to be applied topically, and to investigate the in vitro and in vivo performance of the prepared liposomes. DOX liposomes were prepared by the solvent evaporation method, and then modified with bioadhesive material HA. Through MTT assay, we found that the safe concentration of liposomes delivered would hit 1 mg/mL. Cellular uptake studies showed that DOX liposomes coated with HA are much more targetable to cell nucleus. Their ocular pharmacokinetics in rabbits were investigated through the comparison with those obtained after dosing with non-modified liposomes and DOX solution. The in vitro transcorneal permeability of DOX in both kinds of liposomes was found to be slower than that of the solution because of sustained release. After in vivo instillation in rabbits, HA-modified liposomes had the longest retention time, following with naked liposomes. Significantly, the area under the curve of the aqueous humor concentration–time profiles of DOX liposomes was found to be 1.7-fold higher than that of DOX solution. The confocal experiment confirmed that HA-modified liposomes were able to maintain a higher DOX concentration and residence time than that of non-modified liposomes and free DOX. These results suggest that our liposomal preparation was of great help to improve the bioavailability of DOX.

Inhibition of Metastatic Tumor Growth and Metastasis via Targeting Metastatic Breast Cancer by Chlorotoxin-Modified Liposomes

A liposome system modified with chlorotoxin (ClTx), a scorpion venom peptide previously utilized for targeting brain tumors, was established. Its targeting efficiency and antimetastasis behavior against metastatic breast cancer highly expressed MMP-2, the receptor of ClTx, were investigated. 4T1, a metastatic breast cancer cell line derived from a murine breast tumor, was selected as the cell model. As results, the ClTx-modified liposomes displayed specific binding to 4T1 as determined by flow cytometry and confocal imaging. The cytotoxicity assay revealed that the ClTx modification increased the toxicity compared with nonmodified liposomes. In addition, the modified liposomes also exhibited high in vivo targeting efficiency in the BALB/c mice bearing 4T1 tumors. Importantly, this system inhibited the growth of metastatic tumor and prevented the incidence of lung metastasis in mice bearing 4T1 tumors with only low systemic toxicity. The data obtained from the in vitro and in vivo studies confirmed that the ClTx-modified liposomes increased the drug delivery to metastatic breast cancers. This study proved that the ClTx-modified liposomes had targeting ability to metastatic breast cancer in addition to brain cancer, and displayed an obvious antimetastasis effect. Generally, it may provide a promising strategy for metastatic breast cancer therapy.

Oral absorption enhancement of salmon calcitonin by using both N-trimethyl chitosan chloride and oligoarginines-modified liposomes as the carriers

In this study both N-trimethyl chitosan chloride (TMC) and oligoarginine (Arg8) were utilized to modify liposomes as the multifunctional carriers (TMC-Arg8-Lips) for enhancing the oral absorption of salmon calcitonin. Two permeation enhancers with positive charges were sequentially adsorbed on the liposomal surface with negative charges by electrostatic interaction. Instead of salmon calcitonin, fluorescein isothiocyanate dextran (FD4) was loaded in TMC-Arg8-Lips for Caco-2 cell permeation test in vitro and penetration examination in rat intestinal tract in vivo. The results showed that the apparent permeability coefficient (Papp) of TMC-Arg8-Lips containing FD4 were 7.0-, 4.4-, 1.8- and 1.4-folds higher than FD4 solution, FD4-TMC solution, non-modified liposomes (Non-Lips) and TMC modified liposomes (TMC-Lips), respectively. A strong fluorescence was observed by confocal laser scanning microscope (CLSM) at rat intestinal wall isolated in different times after the FD4 loaded carriers were intragastrically administrated. Furthermore, the images revealed that TMC-Arg8-Lips could penetrate deeply inside the mucosal membrane. The pharmacodynamic study indicated that TMC-Arg8-Lips containing calcitonin were more efficient in enhancing the absorption and prolonging the reduction of blood calcemia in rats. The area above the plasma calcium concentration–time curve (AAC) of TMC-Arg8-Lips containing calcitonin was increased by more than 16.6- and 1.6-fold when compared to Non-Lips and TMC-Lips, respectively.

Doxorubicin in TAT peptide-modified multifunctional immunoliposomes demonstrates increased activity against both drug-sensitive and drug-resistant ovarian cancer models

Multidrug resistance (MDR) is a hallmark of cancer cells and a crucial factor in chemotherapy failure, cancer reappearance, and patient deterioration. We have previously described the physicochemical characteristics and the in vitro anticancer properties of a multifunctional doxorubicin-loaded liposomal formulation. Lipodox®, a commercially available PEGylated liposomal doxorubicin, was made multifunctional by surface-decorating with a cell-penetrating peptide, TATp, conjugated to PEG1000-PE, to enhance liposomal cell uptake. A pH-sensitive polymer, PEG2000-Hz-PE, with a pH-sensitive hydrazone (Hz) bond to shield the peptide in the body and expose it only at the acidic tumor cell surface, was used as well. In addition, an anti-nucleosome monoclonal antibody 2C5 attached to a long-chain polymer to target nucleosomes overexpressed on the tumor cell surface was also present. Here, we report the in vitro cell uptake and cytotoxicity of the modified multifunctional immunoliposomes as well as the in vivo studies on tumor xenografts developed subcutaneously in nude mice with MDR and drug-sensitive human ovarian cancer cells (SKOV-3). Our results show the ability of multifunctional immunoliposomes to overcome MDR by enhancing cytotoxicity in drug-resistant cells, compared with non-modified liposomes. Furthermore, in comparison with the non-modified liposomes, upon intravenous injection of these multifunctional immunoliposomes into mice with tumor xenografts, a significant reduction in tumor growth and enhanced therapeutic efficacy of the drug in both drug-resistant and drug-sensitive mice was obtained. The use of “smart” multifunctional delivery systems may provide the basis for an effective strategy to develop, improve, and overcome MDR cancers in the future.

PSA-responsive and PSMA-mediated multifunctional liposomes for targeted therapy of prostate cancer

In the hormone-refractory stage of prostate cancer (PC), the expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) often remains highly active. Accumulating studies have demonstrated that these two proteins are attractive targets for specific delivery of functional molecules to advanced PC, not merely as potential sensitive markers for PC detection. In this study, we constructed a dual-modified liposome that incorporated PSA-responsive and PSMA-mediated liposomes and potentially offers double selectivity for PC. The folate moiety binds quickly to PSMA-positive tumors, and the PSA-responsive moiety is cleaved by PSA that was enriched in tumor tissues. The activated liposomes (folate and cell-penetrating peptides dual-modifications) are subsequently taken up by the tumor cells via polyarginine's penetrating effects and receptor-mediated endocytosis. To corroborate these assumptions, a series of experiments were conducted, including PSA-responsive peptide hydrolysis kinetics, cellular uptake, internalization mechanism and escape from endosomes in PC-3 and/or 22Rv1 cells, biodistribution and antitumor activity of siRNA-loaded liposomes after systemic administration, gene silencing and cell apoptosis in vitro and in vivo. The results reveal that multivalent interactions play a key role in enhancing PC cell recognition and uptake while reducing nonspecific uptake. The dual-modified liposomes carrying small interfering RNA (siRNA) have significant advantages over the control liposomes, including single-modified (folate, CPP, PSA-responsive only) and non-modified liposomes. The dual-modified liposomes elevated cellular uptake, downregulated expression of polo-like kinase 1 (PLK-1) and augmented cell apoptosis in prostate tumor cells. The entry of the dual-modified liposomes into 22Rv1 cells occurred via multiple endocytic pathways, including clathrin-mediated endocytosis and macropinocytosis, followed by an effective endosomal escape of the entrapped siRNA into the cytoplasm. In vivo studies conducted on a 22Rv1 xenograft murine model demonstrated that the dual-modified liposomes demonstrated the maximized accumulation, retention and knockdown of PLK-1 in tumor cells, as well as the strongest inhibition of tumor growth and induction of tumor cell apoptosis. In terms of targeting capacity and therapeutic potency, the combination of a PSA-responsive and PSMA-mediated liposome presents a promising platform for therapy and diagnosis of PSMA/PSA-positive PC.

Pluronic L61 as a long-circulating modifier for enhanced liposomal delivery of cancer drugs

Pluronic copolymer L61 was found to possess a stealth function for liposomal drug delivery, comparing favorably to PEG. The liposomes modified with 0.1% L61 (w/w) were long-circulating, and preferentially accumulated in the tumor site. The paclitaxel-loaded L61-liposomes with a single injection dose could successfully arrest the tumor growth, lasting for five days, and displayed better treatment outcomes compared to the non-modified liposomes and even the PEGylated liposomes.

Increased apoptosis in cancer cells in vitro and in vivo by ceramides in transferrin-modified liposomes

Lysosomes are a promising therapeutic target for induction apoptosis in cancer cells due to lysosomal membrane permeabilization (LMP) leading to leakage of hydrolytic enzymes, especially the cathepsins, into the cytoplasm. We hypothesized that with the modification of the ceramide-loaded liposomes with transferrin (Tf), we would achieve both tumor targeting and increased delivery of lysosome-destabilizing agents, such as ceramides to lysosomes, to initiate LMP-induced apoptosis. We prepared Tf-modified (TL) and plain (PL) liposomes loaded with short (C6) or long (C16) N-acyl chain ceramides. Uptake, intracellular localization of liposomes, stability of the lysosomal membrane and release of cathepsin D were investigated on Hela cells by fluorescence microscopy and flow cytometry. Apoptosis was evaluated by binding of fluorescently-labeled Annexin V. Antitumor and pro-apoptotic effects of C6Cer-loaded Tf-liposomes were demonstrated in vivo in an A2780-ovarian carcinoma xenograft mouse model. TL were internalized specifically via the TfR-dependent endocytic pathway and localized within the endosome-lysosomal compartment. Ceramide-loaded Tf-liposomes significantly increased apoptosis compared with ceramide-free and ceramide-loaded non-modified liposomes. The treatment of cancer cells with TL led to increased LMP and cytoplasmic relocation of the intralysosomal cathepsin D. A strong antitumor and pro-apoptotic effect of C6Cer-loaded TL was also demonstrated in vivo in an A2780-ovarian carcinoma xenograft mouse model. The lysosomal accumulation of ceramides delivered by Tf-liposomes initiates the permeabilization of the lysosomal membranes required for the release of lysosomal cathepsins into the cytoplasm and initiation of the cancer cell apoptosis both in vitro and in vivo.

Chlorotoxin-modified stealth liposomes encapsulating levodopa for the targeting delivery against the Parkinson’s disease in the MPTP-induced mice model

Chlorotoxin (ClTx), originally isolated from scorpion venom of Leiurus quinquestriatus, is a 36-amino acid peptide and specifically binds to the brain gliomas and proliferating vascular endothelial cells. In this paper, it was first used to establish the ClTx-modified stealth liposomes (ClTx-LS) encapsulating levodopa (LD) for the targeting drug delivery against the Parkinson’s disease (PD). After the DSPE-PEG-ClTx was synthesized and identified, the ClTx-LS system was prepared and characterized. Its targeting capability was studied in vitro and in vivo, and finally its anti-PD activity was evaluated, with non-modified liposomes (LS) as control. It was demonstrated through flow cytometry and confocal microscopy that ClTx modification highly facilitated the uptake of LS by brain microvascular endothelial cells in vitro. After intraperitoneal injection to mice, the active targeting system loaded with LD significantly increased the distribution of dopamine and dihydroxyphenyl acetic acid, the metabolites of LD, in the substantia nigra and striata. In the methyl-phenyl-tetrahydropyridine (MPTP)-induced PD mice model, LD-loaded ClTx-LS significantly attenuated the serious behavioral disorders and diminished the MPTP-induced loss of tyrosine hydroxylase-positive dopaminergic neurons. In conclusion, ClTx-modified LS might serve as a targeting delivery system to transport more drugs into the brain for a better PD therapy.

Wheat Germ Agglutinin Modified Liposomes for the Photodynamic Inactivation of Bacteria†

Photodynamic inactivation (PDI) of bacteria is a promising approach for combating the increasing emergence of antibiotic resistance in pathogenic bacteria. To further improve the PDI efficiency on bacteria, a bacteria-targeting liposomal formulation was investigated. A generation II photosensitizer (temoporfin) was incorporated into liposomes, followed by conjugation with a specific lectin (wheat germ agglutinin, WGA) on the liposomal surface. WGA was successfully coupled to temoporfin-loaded liposomes using an activated phospholipid containing N-hydroxylsuccinimide residue. Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were selected to evaluate the WGA modified liposomes in terms of bacteria targeted delivery and in vitro PDI test. Fluorescence microscopy revealed that temoporfin was delivered to both kinds of bacteria, while flow cytometry demonstrated that WGA- modified liposomes delivered more temoporfin to bacteria compared to nonmodified liposomes. Consequently, the WGA- modified liposomes eradicated all MRSA and significantly enhanced the PDI of P. aeruginosa. In conclusion, the WGA- modified liposomes are a promising formulation for bacteria targeted delivery of temoporfin and for improving the PDI efficiency of temoporfin on both Gram-positive and Gram-negative bacterial cells.