Dual-targeting for brain-specific liposomes drug delivery system: Synthesis and preliminary evaluation

The treatment of glioma has become a great challenge because of the existence of brain barrier (BB). In order to develop an efficient brain targeting drug delivery system to greatly improve the brain permeability of anti-cancer drugs, a novel brain-targeted glucose-vitamin C (Glu-Vc) derivative was designed and synthesized as liposome ligand for preparing liposome to effectively deliver paclitaxel (PTX). The liposome was prepared and its particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis and cytotoxicity were also characterized. What’s more, the cellular uptake of CFPE-labeled Glu-Vc-Lip on GLUT₁- and SVCT₂-overexpressed C6 cells was 4.79-, 1.95-, 4.00- and 1.53-fold higher than that of Lip, Glu-Lip, Vc-Lip and Glu?+?Vc-Lip. Also, the Glu-Vc modified liposomes showed superior targeting ability in vivo evaluation compared with naked paclitaxel, non-coated, singly-modified and co-modified by physical blending liposomes. The relative uptake efficiency was enhanced by 7.53 fold to that of naked paclitaxel, while the concentration efficiency was up to 7.89 times. What’s more, the Glu-Vc modified liposomes also displayed the maximum accumulation of DiD-loaded liposomes at tumor sites with the strongest fluorescence in the brain in vivo imaging. Our results suggest that chemical modification of liposomes with warheads of glucose and vitamin C represents a promising and efficient strategy for the development of brain-specific liposomes drug delivery system by utilizing the endogenous transportation mechanism of the warheads.     

Biotin and glucose dual-targeting,ligand-modified liposomes promote breast tumor-specific drug delivery

Breast cancer is the second leading cause of cancer-related deaths in women. Ligand-modified liposomes are used for breast tumor-specific drug delivery to improve the efficacy and reduce the side effects of chemotherapy; however, only a few liposomes with high targeting efficiency have been developed because the mono-targeting, ligand-modified liposomes are generally unable to deliver an adequate therapeutic dose. In this study, we designed biotin-glucose branched ligand-modified, dual-targeting liposomes (Bio-Glu-Lip) and evaluated their potential as a targeted chemotherapy delivery system in vitro and in vivo. When compared with the non-targeting liposome (Lip), Bio-Lip, and Glu-Lip, Bio-Glu-Lip had the highest cell uptake in 4T1 cells (3.00-fold, 1.60-fold, and 1.95-fold higher, respectively) and in MCF-7 cells (2.63-fold, 1.63-fold, and 1.85-fold higher, respectively). The subsequent cytotoxicity and in vivo assays further supported the dual-targeting liposome is a promising drug delivery carrier for the treatment of breast cancer.     

Development of a Cell-Based Bioassay for Phospholipase A2-Triggered Liposomal Drug Release

The feasibility of exploiting secretory phospholipase A₂ (sPLA₂) enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA₂-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA₂-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA₂ enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA₂-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG) 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA₂ releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA₂. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA₂-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.     

Sustained Liver Targeting and Improved Antiproliferative Effect of Doxorubicin Liposomes Modified with Galactosylated Lipid and PEG-Lipid

In this study, a cleavable PEG-lipid (methoxypolyethyleneglycol 2000-cholesteryl hemisuccinate, PEG₂₀₀₀-CHEMS) linked via ester bond and galactosylated lipid ((5-cholesten-3β-yl) 4-oxo-4-[2-(lactobionyl amido) ethylamido] butanoate, CHS-ED-LA) were used to modify doxorubicin (DOX) liposome. DOX was encapsulated into conventional liposomes (CL), galactosylated liposomes (modified with CHS-ED-LA, GalL), pegylated liposomes (modified with PEG₂₀₀₀-CHEMS, PEG-CL), and pegylated galactosylated liposomes (modified with CHS-ED-LA and PEG₂₀₀₀-CHEMS, PEG-GalL) using an ammonium sulfate gradient loading method and then intravenously injected to normal mice. Both PEG-GalL DOX and GalL DOX gave relatively high overall drug targeting efficiencies to liver ((T _e)_liver) and were mainly taken up by hepatocyte. However, PEG-GalL DOX showed unique “sustained targeting” characterized by slowed transfer of DOX to liver and reduced peak concentrations in the liver. The biodistribution and antitumor efficacy of various DOX preparations were studied in hepatocarcinoma 22 (H22) tumor-bearing mice. The inhibitory rate of PEG-GalL DOX to H22 tumors was up to 94%, significantly higher than that of PEG-CL DOX, GalL DOX, CL DOX, and free DOX, although the tumor distribution of DOX revealed no difference between PEG-GalL DOX and PEG-CL DOX. Meanwhile, the gradual increase in the liver DOX concentration due to the sustained uptake of PEG-GalL DOX formulations resulted in lower damage to liver. In conclusion, the present investigation indicated that double modification of liposomes with PEG₂₀₀₀-CHEMS, and CHS-ED-LA represents a potentially advantageous strategy in the therapy of liver cancers or other liver diseases.     

Cholesterol efflux from bovine sperm. I. Induction of the acrosome reaction with lysophosphatidylcholine after reducing sperm cholesterol

Methods were developed to quantitatively reduce the cholesterol (Chol)/phospholipid (PL) ratio of bovine sperm and to determine the effectiveness of this treatment in capacitating sperm. Washed sperm (2 × 10⁸) were incubated in 1.0 ml of modified Tyrode's solution (TS) containing unilamellar liposomes of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and [¹⁴C]-Chol (35:35:30 molar ratio, 300 nmol total PL). [³H]-triolein was included as a nonexchangable marker. After 90 min at 39°C, a 13% net exchange of [¹⁴C]-Chol from liposomes to sperm was observed (n = 4), and sperm motility was 80%. Sperm were then washed and 50 × 10⁶ sperm were incubated as before with PC/PE liposomes containing no Chol. After 90 min, sperm were separated from liposomes by centrifugation. Measurement of [¹⁴C]-Chol in the liposomes (supernatant) and parallel gas chromatographic analysis of extracted, saponified liposomes (n = 4) indicated that 30% of sperm Chol was removed by this procedure. Chol efflux decreased percent motile sperm by less than 10% but reduced sperm velocity by more than 50%. Sperm incubated with no liposomes (control), with liposomes containing Chol ( + Chol), and with Chol-free liposomes (—Chol) were washed and resuspended in TS with 0.2% BSA and 30 μg lysophosphatidylcholine (LPC)/mg bovine serum albumin (BSA). Percent sperm undergoing the acrosome reaction (AR) upon incubation with LPC-BSA was used as a measure of sperm capacitation. After 60 min of exposure to LPC-BSA at 39°C, the mean (± SE) percent motile sperm for control, +Chol, and —Chol treatments was 57.0 ± 4.9, 60.0 ± 4.7, and 57.0 ± 6.8, respectively. Corresponding values for percent AR were 14.0 ± 3.4, 20.3 ± 4.4, and 39.7 ± 1.2. These results suggest that loss of Chol from bovine sperm may be an early step in sperm capacitation in this species.     

Efficient Drug Delivery of Paclitaxel Glycoside: A Novel Solubility Gradient Encapsulation into Liposomes Coupled with Immunoliposomes Preparation

Although the encapsulation of paclitaxel into liposomes has been extensively studied, its significant hydrophobic and uncharged character has generated substantial difficulties concerning its efficient encapsulation into the inner water core of liposomes. We found that a more hydrophilic paclitaxel molecule, 7-glucosyloxyacetylpaclitaxel, retained tubulin polymerization stabilization activity. The hydrophilic nature of 7-glucosyloxyacetylpaclitaxel allowed its efficient encapsulation into the inner water core of liposomes, which was successfully accomplished using a remote loading method with a solubility gradient between 40% ethylene glycol and Cremophor EL/ethanol in PBS. Trastuzumab was then conjugated onto the surface of liposomes as immunoliposomes to selectively target human epidermal growth factor receptor-2 (HER2)-overexpressing cancer cells. In vitro cytotoxicity assays revealed that the immunoliposomes enhanced the toxicity of 7-glucosyloxyacetylpaclitaxel in HER2-overexpressing cancer cells and showed more rapid suppression of cell growth. The immunoliposomes strongly inhibited the tumor growth of HT-29 cells xenografted in nude mice. Notably, mice survived when treated with the immunoliposomes formulation, even when administered at a lethal dose of 7-glucosyloxyacetylpaclitaxel in vivo. This data successfully demonstrates immunoliposomes as a promising candidate for the efficient delivery of paclitaxel glycoside.     

Folate receptor targeted NIR cleavable liposomal delivery system augment penetration and therapeutic efficacy in breast cancer

BackgroundLiposomes are predominantly used sorts of nanocarriers for active a targeted delivery through surface functionalization using targeting ligand. The folate receptors are overexpressed in various cancers including breast cancer and because of its binding aptitude specifically to folate receptors, folic acid became the attractive ligand.MethodsIn this research, we have developed a folate and Poly-l-Lysine conjugate and coated this conjugate onto the liposomes. The prepared liposomes were characterized using DLS, FTIR, NMR, SEM, TEM, XRD, AFM, stability and drug release studies. Furthermore, in vitro studies were carried out on FR overexpressed breast cancer cell line.ResultsThe FA-LUT-ABC-Lip have diameter of 183 ± 3.17 nm with positive surface charge +33.65 ± 3 mV and the drug release studies confirm the NIR responsive payload cleavage. The coated formulation (in presence of NIR light) effectively reduced the IC₅₀ values and kills breast cancer cells through FR mediated internalization and accelerated drug release. Moreover, LUT Formulation shows anticancer effect due to significant inhibition of cell migration and proliferation by regulating VEGF expression and induced apoptosis through the caspase-3 up-regulation.ConclusionIt is evident from the in vitro studies that the formulation was found to be very effective and can be explored for triggered and targeted delivery of the substances through active targeting.General significanceCombining receptor mediated drug delivery with triggered release aid in more amounts of drug reaching the target site and achieving enhanced therapeutic activity.     

Enhanced Active Targeting via Cooperative Binding of Ligands on Liposomes to Target Receptors

To achieve effective active targeting in a drug delivery system, we previously developed dual-targeting (DT) liposomes decorated with both vascular endothelial growth factor receptor-1 (VEGFR-1)-targeted APRPG and CD13-targeted GNGRG peptide ligands for tumor neovessels, and observed the enhanced suppression of tumor growth in Colon26 NL-17 tumor-bearing mice by the treatment with the DT liposomes encapsulating doxorubicin. In this present study, we examined the binding characteristics of DT liposomes having a different couple of ligands, namely, APRPG and integrin α_vβ₃-targeted GRGDS peptides. These DT liposomes synergistically associated to stimulated human umbilical vein endothelial cells compared with single-targeting (ST) liposomes decorated with APRPG or GRGDS. The results of a surface plasmon resonance assay showed that ST liposomes modified with APRPG or GRGDS peptide selectively bound to immobilized VEGFR-1 or integrin α_vβ₃, respectively. DT liposomes showed a higher affinity for a mixture of VEGFR-1 and integrin α_vβ₃ compared with ST liposomes, suggesting the cooperative binding of these 2 kinds of ligand on the liposomal surface. In a biodistribution assay, the DT liposomes accumulated to a significantly greater extent in the tumors of Colon26 NL-17 tumor-bearing mice compared with other liposomes. Moreover, the intratumoral distribution of the liposomes examined by confocal microscopy suggested that the DT liposomes targeted not only angiogenic endothelial cells but also tumor cells due to GRGDS-decoration. These findings suggest that "dual-targeting" augmented the affinity of the liposomes for the target cells and would thus be useful for active-targeting drug delivery for cancer treatment.     

Microtubule-targeting anticancer drug eribulin induces drug efflux transporter P-glycoprotein

This study examined the effects of microtubule-targeting anticancer drugs (paclitaxel, cabazitaxel, and eribulin) on the expression of drug efflux transporter P-glycoprotein, which is encoded by MDR1. Paclitaxel and eribulin induced MDR1 promoter activity in a concentration-dependent manner, while cabazitaxel had little effect in human intestinal epithelial LS174T cells. Overexpression of the nuclear receptor pregnane X receptor (PXR) gene (NR1I2) enhanced paclitaxel- and eribulin-induced MDR1 activation, but expression of the nuclear receptor co-repressor silencing mediator for retinoid and thyroid receptors (SMRT) gene (NCOR2) repressed MDR1 activation. Eribulin increased the mRNA and protein expression of P-glycoprotein in LS174T cells. Cellular uptake of rhodamine 123 and calcein-acetoxymethyl ester (calcein-AM), P-glycoprotein substrates, decreased in paclitaxel- or eribulin-treated LS174T cells. Eribulin also increased MDR1 promoter activity in human breast cancer MCF7 cells. The results suggest that the microtubule-targeting anticancer drug eribulin can induce the drug efflux transporter P-glycoprotein via PXR in human intestinal and breast cancer cells and thus influence the efficacy of anticancer drugs.     

Paclitaxel-Induced Apoptosis Is BAK-Dependent,but BAX and BIM-Independent in Breast Tumor

Paclitaxel (Taxol)-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death) plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim^−/− MEFs (mouse embryonic fibroblasts), the bim^−/− mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak ^−/− MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax^−/− MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance.     

Serum mannan binding protein inhibits mannosylated liposome-mediated transfection to macrophages

The effects of serum mannan binding proteins (MBP) in the transfection of plasmid DNA/Man–liposome complex via mannose receptor-mediated endocytosis was studied in vitro using cultured mouse peritoneal macrophages. Plasmid DNA encoding luciferase gene was complexed with cationic mannosylated liposomes (Man–liposomes), composed of cholesten-5-yloxy-N-(4-((1-imino-2-d-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and dioleoyl phosphatidylethanolamine (DOPE). The transfection efficiency, as well as the binding and uptake of the plasmid DNA/Man–liposome complex, was investigated with or without serum MBP. The in vitro transfection efficiency of the complex was significantly reduced on increasing the amount of serum MBP. In addition, the cellular association of the complex was also reduced. These results indicate that serum MBP specifically binds to the mannose moieties on the complex and suppresses its cellular uptake, resulting in inhibition of the gene transfection in macrophages. Such an interaction is an obstacle to mannose receptor-mediated in vivo gene transfer to mannose receptor-positive cells using mannosylated gene carriers.     

Antiproliferative Activity and VEGF Expression Reduction in MCF7 and PC-3 Cancer Cells by Paclitaxel and Imatinib Co-encapsulation in Folate-Targeted Liposomes

Co-encapsulation of anticancer drugs paclitaxel and imatinib in nanocarriers is a promising strategy to optimize cancer treatment. Aiming to combine the cytotoxic and antiangiogenic properties of the drugs, a liposome formulation targeted to folate receptor co-encapsulating paclitaxel and imatinib was designed in this work. An efficient method was optimized for the synthesis of the lipid anchor DSPE-PEG(2000)-folic acid (FA). The structure of the obtained product was confirmed by RMN, FT-IR, and ESI-MS techniques. A new analytical method was developed and validated for simultaneous quantification of the drugs by liquid chromatography. Liposomes, composed of phosphatidylcholine, cholesterol, and DSPE-mPEG(2000), were prepared by extrusion. Their surface was modified by post-insertion of DSPE-PEG(2000)-FA. Reaction yield for DSPE-PEG(2000)-FA synthesis was 87%. Liposomes had a mean diameter of 122.85 ± 1.48 nm and polydispersity index of 0.19 ± 0.01. Lyophilized formulations remained stable for 60 days in terms of size and drug loading. FA-targeted liposomes had a higher effect on MCF7 cell viability reduction (p < 0.05) when compared with non-targeted liposomes and free paclitaxel. On PC-3 cells, viability reduction was greater (p < 0.01) when cells were exposed to targeted vesicles co-encapsulating both drugs, compared with the non-targeted formulation. VEGF gene expression was reduced in MCF7 and PC-3 cells (p < 0.0001), with targeted vesicles exhibiting better performance than non-targeted liposomes. Our results demonstrate that multifunctional liposomes associating molecular targeting and multidrug co-encapsulation are an interesting strategy to achieve enhanced internalization and accumulation of drugs in targeted cells, combining multiple antitumor strategies.     

Characterization of a novel lipase and its specific foldase from Acinetobacter sp. XMZ-26

A novel operon containing a lipase gene (lip26) and its specific foldase gene (lif26) was discovered from Acinetobacter sp. XMZ-26 by creating and screening a gene library and then using genome walking. The amino acid sequence of Lip26 and Lif26 showed only 46.4% and 37.3% identity with the LipA and LipB (Lif) sequences from Acinetobacter sp. SY-01, respectively. The expressed recombinant Lip26 formed inactive inclusion bodies in Escherichia coli. However, the active Lip26 was refolded by the dilution refolding method with the assistance of purified recombinant Lif26, and the refolded Lip26 had a high specific activity. Lip26 hydrolyzed p-nitrophenyl (pNP) esters of fatty acids with C₂ to C₁₆ acyl chain lengths and had a substrate preference for pNP myristate. Maximal Lip26 activity was dependent on both the temperature (55 °C) and pH (9.0). In addition, Lip26 was capable of maintaining its activity in the presence of many detergents and organic solutions, and its activity was enhanced by the presence of Ca²⁺, Mn²⁺, and Ba²⁺. To directly obtain active Lip26, an E. coli strain was co-transformed with two expression plasmids containing the lip26 and lif26 genes. The co-expression of both proteins in vivo resulted in the expression of half of the recombinant Lip26 as a soluble protein with demonstrable lipase activity. A direct protein interaction between Lif26 and Lip26A was detected by both a pull-down assay and a yeast two-hybrid experiment.     

PEG-PLA nanoparticles decorated with small-molecule PSMA ligand for targeted delivery of galbanic acid and docetaxel to prostate cancer cells

Prostate cancer (PCa) is one of the most prevalent non-drug delivery system cutaneous malignancies. Undoubtedly, introducing novel treatment options to achieve higher therapeutic index will be worthwhile. In this study, we report for the first time, a novel targeted self-assembled based on PEG-PLA nanoparticles (PEG-PLA NPs) containing galbanic acid (GBA) and docetaxel, which was targeted using ((S)-2-(3-((S)-5-amino-1-carboxypentyl) ureido) pentanedioic acid (ACUPA), a small molecule inhibitor targeting prostate-specific membrane antigen (PSMA), in prostate cancer cell line. The prepared NPs were characterized by different analytical methods. The MTT assay was used to compare the anti-proliferation of drugs-loaded PEG-PLA NPs and ACUPA-PEG-PLA against LNCaP (PSMA⁺) and PC3 (PSMA⁻) cells. PEG-PLA NPs with an average size of 130–140 nm had an enhanced release of GBA and docetaxel at pH 5.5 compared with pH 7.5. Spectrofluorometric analysis suggested that ACUPA-modified PEG-PLA could effectively enhance the drug uptake in PSMA⁺ prostate cancer cells. Cytotoxicity studies showed that the targeted NPs loaded with different concentrations of GBA and fixed concentration of docetaxel (4 nM) have shown higher toxicity (IC₅₀ 30 ± 3 µM) than both free GBA (80 ± 4.5 µM) and nontargeted NPs (IC₅₀ 40 ± 4.6 µM) in LNCaP cells. Collectively, these findings suggest that ACUPA-conjugated PEG-PLA nanosystem containing GBA and docetaxel is a viable delivery carrier for various cancer-targeting PSMA that suffer from short circulation half-life and limited therapeutic efficacy.     

CD44+ fibroblasts increases breast cancer cell survival and drug resistance via IGF2BP3‐CD44‐IGF2 signalling

CD44, a cell adhesion protein, involves in various process in cancer such as cell survival and metastasis. Most researches on CD44 in cancer focus on cancer cells. Recently, it is found that CD44 expression is high in fibroblasts of tumour microenvironment. However, its role in communication between fibroblasts and breast cancer cells is seldom known. In this study, CD44‐positive (CD44⁺Fbs) and CD44‐negative carcinoma‐associated fibroblasts (CD44^?Fbs) were isolated and cocultured with breast cancer cells for analysis of cell survival and drug resistance. We found that CD44⁺Fbs promoted breast cancer cell survival and paclitaxel resistance and inhibited paclitaxel‐induced apoptosis. Our further research for the molecular mechanism showed that IGF2BP3 bound to CD44 mRNA and enhanced CD44 expression, which increased IGF2 levels of fibroblasts and then stimulated breast cancer cell proliferation and drug resistance. IGF2 was found to activate Hedgehog signal pathway in breast cancer cells. In conclusion, the results illustrated that in CD44⁺Fbs, binding of IGF2BP3 and CD44 promotes IGF2 expression and then accelerates breast cancer cell proliferation, survival and induced chemotherapy resistance likely by activating Hedgehog signal pathways.     

A Liposomal Drug Platform Overrides Peptide Ligand Targeting to a Cancer Biomarker,Irrespective of Ligand Affinity or Density

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for α_vβ₆-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to α_vβ₆. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.     

Liposomes containing alkylated methotrexate analogues for phospholipase A2 mediated tumor targeted drug delivery

Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO III and HT-29 human colon cancer cells. Both analogues contained a C₁₆-alkyl chain attached to the γ-carboxylic acid and one of the analogues had an additional benzyl group attached to the α-carboxylic acid. The cytotoxicity of the γ-alkylated compound towards KATO III (IC₅₀ = 55 nM) and HT-29 (IC₅₀ = 400 nM) cell lines, was unaffected by the alkylation, whereas the additional benzyl group on the α-carboxyl group made the compound nontoxic. The γ-derivative with promising cytotoxicity was incorporated into liposomes that were designed to be particularly susceptible to a liposome degrading enzyme, secretory phospholipase A₂ (sPLA₂), which is found in high concentrations in tumors of several different cancer types. Liposome incorporation was investigated by differential scanning calorimetry (DSC), and sPLA₂ hydrolysis was examined by fluorescence spectroscopy and high performance liquid chromatography (HPLC). The results showed that the methotrexate (MTX)-analogue could be incorporated into liposomes that were degradable by sPLA₂. However, the in vitro cytotoxicity of the MTX-liposomes against KATO III and HT-29 cancer cells was found to be independent of sPLA₂ hydrolysis, indicating that the alkylated MTX-analogue was available for cancer cell uptake even in the absence of liposome hydrolysis. Using a DSC based method for assessing the anchoring stability of alkylated compounds in liposomes, it was demonstrated that the MTX-analogue partitioned into the water phase and thereby became available for cell uptake. It was concluded that liposomes containing alkylated MTX-analogues show promise as a drug delivery system, although the MTX-analogue needs to be more tightly anchored to the liposomal carrier. Also, the developed DSC-assay for studying the anchoring stability of alkylated drugs will be a useful tool in the development of liposomal drug delivery systems.     

Comparative biochemical characterization and in silico analysis of novel lipases Lip11 and Lip12 with Lip2 from Yarrowia lipolytica

Novel lipases lip11 and lip12 from Yarrowia lipolytica MSR80 were cloned and expressed in E. coli HB101 pEZZ18 system along with lip2. These enzymes were constitutively expressed as extracellular proteins with IgG tag. The enzymes were purified by affinity chromatography and analyzed by SDS-PAGE with specific activity of 314, 352 and 198?U/mg for Lip2, Lip11 and Lip12, respectively on olive oil. Biochemical characterization showed that all were active over broad range of pH 4.0?C9.0 and temperature 20?C80?°C with optima at pH 7 and 40?°C. All the three lipases were thermostable up to 80?°C with varying t_1/2. Activity on various substrates revealed that they were most active on oils?>?triacylglycerides?>?p-np-esters. Relatively Lip2 and Lip11 showed specificity for mid to long chain fatty acids, while Lip12 was mid chain specific. GC analysis of triolein hydrolysis by these lipases revealed that Lip2 and Lip11 are regioselective, while Lip12 is not. Effect of metal ions showed that Lip2 and Lip12 were activated by Ca²⁺ whereas Lip11 by Mg²⁺. All were thiol activated and inhibited by PMSF and N-bromosuccinimide. All were activated by non polar solvents and inhibited by polar solvents. Detailed sequence analysis and structural predictions revealed Lip11 and Lip12 shared 61 and 62?% homology with Lip2 (3O0D) and three dimensional superimposition revealed Lip2 was closer to Lip11 than to Lip12 as was observed during biochemical characterization. Finally, thermostability and substrate specificity has been explained on the basis of detailed amino acid analysis.     

Temperature-sensitive liposomes for co-delivery of tamoxifen and imatinib for synergistic breast cancer treatment

Co-delivery of chemotherapeutic agents using nanocarriers is a promising strategy for enhancing therapeutic efficacy of anticancer agents. The aim of this work was to develop tamoxifen and imatinib dual drug loaded temperature-sensitive liposomes to treat breast cancer. Liposomes were prepared using 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), monopalmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC), and different surface active agents. The liposomes were characterized for the average particle size, zeta potential, transition temperature, and drug release below and above liposomal transition temperature. The temperature-sensitive liposomes co-encapsulated with tamoxifen and imatinib were investigated for their synergistic activity against MCF-7 and MDA-MB-231 breast cancer cells. The liposomal nanoparticles showed a transition temperature of 39.4?°C and >70% encapsulation efficiency for tamoxifen and imatinib. The temperature-responsive liposomes showed more than 80% drug released within 30?min above transition temperature. Dual drug loaded liposomes showed synergistic growth inhibition against MCF-7 and MDA-MB-231 breast cancer cells. Co-delivery of tamoxifen and imatinib using temperature-sensitive liposomes can be developed as a potential targeting strategy against breast cancer.