Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

Encapsulation of Vincristine in Liposomes Reduces its Toxicity and Improves its Anti-Tumor Efficacy

Abstract

Vincristine is a potent therapeutic agent with activity against a variety of tumor types. It is a cell-cycle specific agent which has exhibited enhanced anti-tumor activity when delivered in liposomal form. Vincristine can be encapsulated into large unilamellar vesicles in response to a transmembrane pH gradient with trapping efficiencies approaching 100%. The extent of vincristine encapsulation, and the subsequent retention of the drug within the liposomes, both in vitro and in vivo, are strongly dependent on the lipid composition of the liposome and on the magnitude of the transmembrane pH gradient. Liposomal formulations of vincristine have been optimized for both liposome circulation longevity, drug retention characteristics and in vivo antitumor activity. When compared to free vincristine, these formulations significantly increase the levels of vincristine remaining in the plasma after i.v. administration. These formulations also significantly increase the delivery of vincristine to tumor sites. As a consequence of the improved accumulation of vincristine at tumor sites, liposomal formulations of vincristine exhibit dramatically improved efficacy against a variety of ascitic and solid murine and human tumors than does free vincristine. Liposomal vincristine is expected to be of wide utility in a variety of human malignancies.     

Synergistic breast cancer suppression efficacy of doxorubicin by combination with glycyrrhetinic acid as an angiogenesis inhibitor

BackgroundTherapeutic regimens of breast cancer treatment are increasingly inclined to adopt combination strategy based on the broad spectrum antitumor effect of doxorubicin (Dox). Currently, combination therapy comprises of conventional anti-cancer drugs and angiogenesis inhibitors have been corroborated as an effective approach in cancer treatment.PurposeWe explored the ability of a natural anti-angiogenic compound glycyrrhetinic acid (GA), derived from an edible-medicinal herb licorice, to enhance the breast cancer suppression effect of Dox.Study designThe drug ratio of GA and Dox with synergistic anticancer effect against MCF-7 cells was optimized by combination index (CI) value in vitro, followed by evaluation of the improved anticancer effects and reduced side-effects of this combination in vitro and in vivo.MethodsCell viability was measured by MTT assay. Analyses of mitochondrial membrane potential and cell apoptosis on MCF-7 cells were performed by JC-1 dye and Annexin V-FITC/PI assays. The cellular accumulation of Dox when combined with GA was evaluated. Levels of apoptosis-related proteins in MCF-7 cells were measured by Western blot analysis. Synergistic anti-angiogenic effects on HUVECs were evaluated. A breast cancer mouse model was established to investigate the anti-tumor effects in vivo.ResultsBased on the optimization by CI value, Dox and GA at 1:20 molar ratio was chosen as the optimal combination drug ratio that exhibited synergistic effect against MCF-7 breast cancer cells. In addition, the combination of GA and Dox exhibited significantly enhanced cytotoxicity, apoptosis, and loss of mitochondrial membrane potential via the upregulation of a mitochondrial-dependent apoptosis pathway against MCF-7 cells. Interestingly, the addition of GA increased the intracellular accumulation of Dox in MCF-7 cells. Moreover, VEGF-induced HUVECs proliferation, migration, and tube formation were strongly inhibited by Dox when used with GA via the significant down-regulation of VEGFR2-mediated pathway, indicating that the combination of Dox and GA could exhibit ideal synergistic anti-angiogenesis effect. Expectedly, the enhanced anti-tumor efficacy of Dox and reduced Dox-induced cardiotoxicity when used in combination with GA were evident in a mouse breast tumor model.ConclusionsThese findings support that the combination of Dox with GA is a novel and promising therapeutic strategy for the treatment of breast cancer.     

Pulmonary Delivery of Liposomal Drugs

Abstract

This overview will discuss our studies of liposomes aerosols to treat diseases of the lung and will entail (i) formulation and characterization of liposome aerosols, including dry liposome powder aerosols, (ii) modulation of the pharmacokinetic profile of liposomal drugs delivered by aerosol or intratracheal instillation, (iii) liposome-alveolar macrophage interactions in vitro and in vivo, and (iv) safety of liposome aerosols in vivo in mice, sheep and healthy human volunteers. Water-soluble agents can be retained in liposomes during aerosolization with air-pressure nebulizers within certain limitations of liposome composition, size, and operating conditions. Dry powder liposome aerosols have been formulated and deliver water-soluble encapsulated substances efficiently. Pharmacokinetic profiles of liposomal drugs delivered via intratracheal instillation exhibit typical slow release plasma profiles indicating that the carrier is the rate-limiting barrier for release. Accordingly, pulmonary mean residence times are significantly prolonged and systemic concentrations remain low. Liposomes do not inhibit the phagocytic activity of alveolar macrophages in vitro and in vivo, have no apparent histopathologic effects on lung architecture even after chronic administration, and do not alter dynamic compliance, lung resistance, p_aO₂ and p_aCO₂ in awake, unanesthetized sheep and in healthy human volunteers. In conclusion, liposomes are a promising innocuous aerosol delivery system for drugs to achieve prolonged localized drug concentrations in the lung or intracellular drug targeting to alveolar macrophages.     

Multifunctional Nanoparticles Delivering Small Interfering RNA and Doxorubicin Overcome Drug Resistance in Cancer

Drug resistance is a major challenge to the effective treatment of cancer. We have developed two nanoparticle formulations, cationic liposome-polycation-DNA (LPD) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (MDR) tumors. In this study, we have provided four strategies to overcome drug resistance. First, we formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, we have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, we further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, we delivered a therapeutic siRNA to inhibit MDR transporter. We demonstrated the first evidence of c-Myc siRNA delivered by the LPD-II nanoparticles down-regulating MDR expression and increasing Dox uptake in vivo. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition. This study highlights a potential clinical use for the multifunctional nanoparticles with an effective delivery property and a function to overcome drug resistance in cancer. The activity and the toxicity of LPD- and LPD-II-mediated therapy are compared.     

Daunoxome® Treatment of Solid Tumors: Preclinical and Clinical Investigations

Abstract

Daunorubicin has been entrapped into small unilamellar vesicles (50-80 nm dia) composed of a 2:1 mole ratio of highly purified DSPC:cholesterol. In earlier studies, liposomes of this size and composition had been demonstrated to deliver their entrapped contents selectively to a wide range of solid tumors in vivo. Preclinical and initial clinical investigations of these daunorubicin liposomes (DaunoXome) are discussed. In one murine solid tumor model (P1798 lymphosarcoma), a ten-fold increased delivery of entrapped daunorubicin to tumor tissue was observed. Efficacy studies in the same model indicated improved tumor regression and extended life spans that correlated with the observed degree of enhanced tumor drug delivery. In a second tumor model (MA16C mammary adenocarcinoma), a ten-fold enhancement in efficacy again was demonstrated. In terms of median survival times and long term survival rate, DaunoXome dosed at 2 mg/kg (daunorubicin) demonstrated an efficacy comparable to free drug at 20 mg/kg. Clinical pharmacokinetics paralleled findings from animal studies. In humans, DaunoXome produced daunorubicin plasma AUC levels that were more than 35-fold greater than those reported for comparable doses of free drug at 80 mg/m². Response rates above 50% have been shown for treatment of Kaposi's sarcoma. A low incidence of side effects has been observed and HIV positive patients have been able to continue antiviral therapy during DaunoXome treatments. Cardiotoxicity has not manifested clinically even for patients receiving in excess of 1 gram/m² cumulative daunorubicin.     

Resveratrol enhances chemosensitivity of doxorubicin in multidrug-resistant human breast cancer cells via increased cellular influx of doxorubicin

Background

Multidrug resistance is a major problem in the treatment of breast cancer, and a number of studies have attempted to find an efficient strategy with which to overcome it. In this study, we investigate the synergistic anticancer effects of resveratrol (RSV) and doxorubicin (Dox) against human breast cancer cell lines.

Methods

The synergistic effects of RSV on chemosensitivity were examined in Dox-resistant breast cancer (MCF-7/adr) and MDA-MB-231 cells. In vivo experiments were performed using a nude mouse xenograft model to investigate the combined sensitization effect of RSV and Dox.

Results and conclusion

RSV markedly enhanced Dox-induced cytotoxicity in MCF-7/adr and MDA-MB-231 cells. Treatment with a combination of RSV and Dox significantly increased the cellular accumulation of Dox by down-regulating the expression levels of ATP-binding cassette (ABC) transporter genes, MDR1, and MRP1. Further in vivo experiments in the xenograft model revealed that treatment with a combination of RSV and Dox significantly inhibited tumor volume by 60%, relative to the control group.

General significance

These results suggest that treatment with a combination of RSV and Dox would be a helpful strategy for increasing the efficacy of Dox by promoting an intracellular accumulation of Dox and decreasing multi-drug resistance in human breast cancer cells.     

In vivo longitudinal imaging of RNA interference‐induced endocrine therapy resistance in breast cancer

Endocrine therapy resistance in breast cancer is a major obstacle in the treatment of patients with estrogen receptor‐positive (ER+) tumors. Herein, we demonstrate the feasibility of longitudinal, noninvasive and semiquantitative in vivo molecular imaging of resistance to three endocrine therapies by using an inducible fluorescence‐labeled short hairpin RNA (shRNA) system in orthotopic mice xenograft tumors. We employed a dual fluorescent doxycycline (Dox)‐regulated lentiviral inducer system to transfect ER+ MCF7L breast cancer cells, with green fluorescent protein (GFP) expression as a marker of transfection and red fluorescent protein (RFP) expression as a surrogate marker of Dox‐induced tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockdown. Xenografted MCF7L tumor‐bearing nude mice were randomized to therapies comprising estrogen deprivation, tamoxifen or an ER degrader (fulvestrant) and an estrogen‐treated control group. Longitudinal imaging was performed by a home‐built multispectral imaging system based on a cooled image intensified charge coupled device camera. The GFP signal, which corresponds to number of viable tumor cells, exhibited excellent correlation to caliper‐measured tumor size (P << .05). RFP expression was substantially higher in mice exhibiting therapy resistance and strongly and significantly (P < 1e‐7) correlated with the tumor size progression for the mice with shRNA‐induced PTEN knockdown. PTEN loss was strongly correlated with resistance to estrogen deprivation, tamoxifen and fulvestrant therapies.      

Liposomes: From the Bench to the Bed

Abstract

Our recent in vivo studies have investigated the surface adsorption property of various circulating liposomes to blood proteins, and have related this property to liposome clearance behavior. In particular, we have investigated liposomes composed of different charged or neutral lipids, fatty acyl chain length and saturation, and cholesterol content. From these studies an apparent inverse relationship between the amount of blood protein that associates with large unilamellar vesicles and the circulation half-lives of the liposomes is observed, indicating that protein-mediated liposome clearance mechanisms are dominant. Furthermore, by comparing the protein profiles of rapidly cleared liposomes with liposomes exhibiting enhanced circulation times, key blood proteins have been identified and implicated in the clearance process.     

Folate-Targeted Liposomes for Drug Delivery

Abstract

The folate receptor has been identified as a marker for ovarian carcinomas and is also up-regulated in many other types of cancer. Folate-conjugation has been successfully applied in the tumor cell-selective targeting of liposomes. A long polyethyleneglycol (PEG) spacer between the targeting ligand (i.e. folic acid) and the liposome surface is required for receptor recognition. Ligand binding is compatible with the PEG-coating of the liposomes needed for prolonged systemic circulation. Folate-targeted liposomes have been shown to enhance the in vitro cytotoxicity of liposome-entrapped doxorubicin and antisense oligodeoxynucleotides to receptor-bearing tumor cells. Folate, as a targeting ligand, offers unique advantages over immunoliposomes, i.e., easy liposomal incorporation, low cost, high receptor affinity and tumor specificity, extended stability, and potential lack of immunogenicity.     

Polymer-Grafted Liposomes: Physical Basis for the “Stealth” Property

Abstract

Polymer-bearing lipids have recently been incorporated into liposomes that are used in in vivo drug delivery. This strategy has improved the liposome's ability to avoid the reticuloendothelial system and has thereby increased its circulation time in the bloodstream. In order to understand the physical basis for this, so called, Stealth® effect, we have begun a series of studies that characterize the surface structure, interactive properties and in vivo performance of the polymer-bearing, Stealth lipids. For a 1900 g/mol polyethylene glycol (PEG) moiety, we have used x-ray diffraction and micropipet manipulation methods to show that, (i) the polymer chains extend ～50Å out from the lipid bilayer surface; (ii) this surface polymer exerts a significant long range mutual repulsion between adjacent bilayers that prevents bilayer-bilayer adhesion. Furthermore, the measured polymer extension and repulsive pressure are well modelled by polymer scaling laws. These results imply that the interaction of macromolecules and cellular surfaces with the Stealth liposome is probably limited to a distance of ～50Å from the liposome surface. We conclude that the origin of the Stealth effect lies in a steric stabilization mechanism. By using fluorescence video microscopy to observe implanted tumor tissue, we have also shown that fluorescent Stealth liposomes extravasate through the leaky vessel walls of tumors. This method allows us to characterize, in real time, the accumulation of liposomes and release of drug at an implanted tumor site.     

P-糖蛋白在多药耐药的K562细胞转运苯妥英纳与卡马西平中的作用

研究证实,多药转运体与难治性癫痫耐药机制密切相关,P-糖蛋白在其中起重要作用．主要研究P-糖蛋白拮抗剂维拉帕米对P-糖蛋白过表达的K562细胞耐药性及细胞内苯妥英纳与卡马西平浓度的影响．首先建立了P-糖蛋白高表达的K562/Dox(阿霉素诱导)耐药细胞株,比较耐药细胞株和P-糖蛋白表达阴性的K562细胞株对苯妥英纳和卡马西平的耐药性,并观察给予维拉帕米后,耐药细胞内抗癫痫药物的浓度变化．结果发现,苯妥英纳和卡马西平对K562/Dox细胞株的半数抑制浓度(IC₅₀)明显高于K562细胞株,加入维拉帕米后,苯妥英纳和卡马西平对K562/Dox 细胞的IC₅₀明显下降,逆转倍数分别为2.5和1.5．进一步研究发现,K562/Dox细胞内苯妥英纳和卡马西平的浓度均显著少于其药敏K562细胞,仅分别为正常K562细胞的23.6%和32.2%．当加入维拉帕米后,K562/Dox细胞内抗癫痫药物浓度明显升高(P < 0.05)．由此证明,高表达的P-糖蛋白参与了细胞的药物转运,在难治性癫痫的耐药机制中扮演重要角色．     

The Effect of Shear on the Desorption of Liposomes Adsorbed to Bacterial Biofilms

Abstract

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,β[N-(N¹,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immoblized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome–biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

番茄连作青枯病不同发病时期的非根际土壤细菌群落变化特征

[目的]研究连作条件下番茄青枯病不同发病时期的非根际土壤微生物群落差异,明确土壤微生物对青枯病发病时期的响应机制。[方法]本研究对16S rRNA V4-V5区进行实时荧光定量PCR和高通量测序,综合分析了连续种植第1、3、5和7季的发病高峰期和发病末期的番茄非根际土壤的病原菌数量、细菌群落多样性、群落组成、标志细菌类群和群落构建机制。[结果]发病高峰期的青枯菌数量(1.28×10⁷ copies/g)要高于发病末期(1.77×10⁶ copies/g)。随着连作时间的增加,发病时期对细菌群落多样性的影响逐渐增加。第3季和第5季不同发病时期之间的细菌群落alpha和beta多样性存在显著差异。LEfSe分析发现,番茄青枯病发病高峰期和发病末期的非根际土壤样品有其各自不同的标志细菌类群。此外,随连作时间的延长协助青枯菌致病的细菌逐渐累积。发病高峰期是微杆菌属(Microbacterium)和亚硝化螺菌属(Nitrosospira)协助青枯菌致病,而在发病末期,则由鞘脂菌属(Sphingobium)、norank_f...     

Seed-shedding in heather (Calluna vulgaris (L.) Hull

Abstract

Background: Accumulating evidence, mainly from small-scale experiments, suggests that species diversity helps stabilise ecological communities; however, this relationship needs testing at larger scales in a wider range of natural communities.

Aims: In experimentally burnt ericaceous shrub stands, we aimed to determine whether more diverse stands had more stable vegetation cover.

Methods: Using two prescribed fire management experiments – one on heathland and one in forest – at scales of 100–700 m², we compared pre-disturbance vegetation characteristics (measures of diversity, traits and composition) with resistance (degree of perturbation on disturbance), resilience (here defined as post-disturbance recovery rate) and stability (the inverse of temporal variability).

Results: Responses to disturbance were usually best explained by a measure of dominance: Simpson's index. High dominance was associated with high resilience, but low resistance and low stability. Within the forest, the shrub community had lower dominance and higher stability than it did on heathland. Dominance and diversity were strongly linked as opposite poles of a principal axis of vegetation variation; therefore, more diverse vegetation was more stable.

Conclusions: Ericaceous shrub cover is known to support important ecosystem services, such as soil protection, carbon sequestration, flood prevention and livestock grazing. Our results suggest that high shrub diversity would support stable provision of these services. Land managers who prioritise this aim in British heathlands and pinewoods should consider a wider range of management approaches than those of typical current practice.     

Effect of Macrophage Elimination Using Liposome-Encapsulated Dichloromethylene Diphosphonate on Tissue Distribution of Liposomes

Abstract

Effect of macrophage elimination using liposomal dichloromethylene diphosphonate (C1₂MDP)1 on tissue distribution of different types of liposomes was examined in mice. Intravenously administration into mice with CI2MDP encapsulated in liposomes composed of phosphatidylcholine, cholesterol and phosphatidylserine exhibits a temporary blockade of liver and spleen function for liposome uptake. At a low dose of 90 (ig/mouse, the liposome uptake by the liver was significantly decreased. Such decrease was accompanied by an increase in liposome accumulation in either spleen or blood depending on liposome composition and size. Direct correlation between the administration dose of liposomal CI2MDP and the liposome circulation time in blood was also obtained even for liposomes with an average diameter of more than 500 nm. These results indicate that temporary elimination of macrophages of the liver and spleen using liposomal CI2MDP may prove to be useful to enhance the drug delivery efficiency of liposomes.     

Potential for improvement in clinical decision-making: tumor imaging with in-111 labeled liposomes results of a phase ii-iii study

Abstract

Liposome scanning using In- 111 labeled VS102 liposomes (VesCan^R) has previously been shown to image a wide variety of common human tumors, probably related to tumor neovascular capIIIary fenestrations and binding of liposomes to tumor cells. We further tested In-III VS102 liposomes in a Phase II trial (27 patients) and a Phase III trial (38 patients). The sensitivity for detecting tumors in primary sites was 82% and in metastatic sites was 65% at the recommended lipid dose of 100 mg. There was 1 false positive scan (specificity 98%). Tumors which have been imaged include carcinomas of the breast, lung, head-neck, prostate, colon, ovary, cervix, thyroid, kidney, testes, melanoma, sarcoma and lymphoma. Sites imaged have included soft tissue, breast, mediastinum, bone, lung, lymph node, liver and pelvis. We also describe five patients in whom a In-111 liposome scan was performed in addition to standard tests, and in whom therapy plans were changed by use of liposome scan results. In two instances, no therapy would have been given without In-III liposome scan, but chemotherapy or radiotherapy were used based on liposome scan results and confirmatory tests. In one patient, surgery would have been used in the absence of In-III liposome scans, versus radiotherapy with In-III liposome scan results. In two other patients, palliative radiotherapy or chemotherapy would have been given without In-111 liposome scan. One of the patients would have required further therapy and the other needed curative surgery after liposome scan evaluation. These results suggest In-111 liposome scans may be useful to complement standard diagnostic tests in cancer patient management.     

Targeted liposomes in fungi: Modifying the therapeutic index of amphotericin b by its incorporation into negatively charged liposomes

Abstract

The mechanism of action of liposome—incorporated amphotericin B (ABLC) is not well understood. Most studies to date have dealt with the role of liposome size, lipid composition, and fluidity on ABLC toxicity and activity. However, little is known about the behavior of ABLC once injected into the circulation. This review describes the behavior of ABLC, both as a particle and in a lipophilic system, following intravenous administration in an attempt to better understand the mechanisms involved in ABLC's enhanced therapeutic index. Our data suggest that once ABLC is injected into the circulation, two major processes probably occur: (1) exchange with lipoproteins: both the liposomal lipid and the liposome—associated AmpB are actively exchanged with lipoproteins; and (2) phagocytosis: liposomes are taken up by phagocytes that potentially carry the internalized ABLC to infection sites. As a result of these interactions, ABLC is more effectively delivered to fungal cells where ABLC is released from the phagocyte and AmpB is liberated from the lipid by fungal lipases. Furthermore, ABLC predominantly distributes into high—density lipoproteins (HDL) following incubation in human serum for 1 hour at 37°C. This HDL—associated ABLC is less toxic to renal cells than either AmpB or LDL—associated AmpB because of the low level of expression of HDL receptors on renal cells. These findings demonstrate that the interaction of ABLC with plasma lipoproteins and blood phagocytes may be responsible for ABLC's enhanced therapeutic index.