Liposomes modified with double-branched biotin: A novel and effective way to promote breast cancer targeting

Although active targeting liposomes with cancer-specific ligands can bind and internalize into cancer cells, only a few high-efficiency liposomes have been developed so far because traditional single branched ligand modified liposomes generally failed to deliver adequate therapeutic payload. In this paper, we broke the traditional design concept and synthesized the double branched biotin modified cholesterol (Bio₂-Chol) for the first time. On this basis, different biotin density modified liposomes ((Bio-Chol)Lip, (Bio-Chol)₂Lip and (Bio₂-Chol)Lip) were successfully prepared and used as active targeting drug delivery systems for the treatment of breast cancer. The in vitro and in vivo breast cancer-targeting ability of these liposomes were systemically studied using paclitaxel (PTX) as the model drug. And the uptake mechanism of (Bio₂-Chol)Lip was investigated. The results showed that (Bio₂-Chol)Lip had the best breast cancer-targeting ability compared with naked paclitaxel, unmodified Lip, (Bio-Chol)Lip and (Bio-Chol)₂Lip. In particular, the relative uptake efficiency (RE) and concentration efficiency (CE) of (Bio₂-Chol)Lip were respectively enhanced by 5.61- and 5.06-fold compared to that of naked paclitaxel. Both distribution data and pharmacokinetic parameters suggested that the double branched biotin modified liposome ((Bio₂-Chol)Lip) is a very promising drug delivery carrier for breast cancer.     

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.     

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.     

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.     

Targeted Drug Delivery Systems Mediated by a Novel Peptide in Breast Cancer Therapy and Imaging

Targeted delivery of drugs to tumors represents a significant advance in cancer diagnosis and therapy. Therefore, development of novel tumor-specific ligands or pharmaceutical nanocarriers is highly desirable. In this study, we utilized phage display to identify a new targeting peptide, SP90, which specifically binds to breast cancer cells, and recognizes tumor tissues from breast cancer patients. We used confocal and electron microscopy to reveal that conjugation of SP90 with liposomes enables efficient delivery of drugs into cancer cells through endocytosis. Furthermore, in vivo fluorescent imaging demonstrated that SP90-conjugated quantum dots possess tumor-targeting properties. In tumor xenograft and orthotopic models, SP90-conjugated liposomal doxorubicin was found to improve the therapeutic index of the chemotherapeutic drug by selectively increasing its accumulation in tumors. We conclude that the targeting peptide SP90 has significant potential in improving the clinical benefits of chemotherapy in the treatment and the diagnosis of breast cancer.     

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.     

Pre-targeting and direct immunotargeting of liposomal drug carriers to ovarian carcinoma

Background

Epidermal growth factor receptor (EGFR) is overexpressed in many solid tumor types, such as ovarian carcinoma. Immunoliposome based drug targeting has shown promising results in drug delivery to the tumors. However, the ratio of tumor-to-normal tissue concentrations should be increased to minimize the adverse effects of cytostatic drugs.

Methodology/Principal Findings

We studied the EGFR-targeted doxorubicin immunoliposomes using pre-targeting and local intraperitoneal (i.p.) administration of the liposomes. This approach was used to increase drug delivery to tumors as compared to direct intravenous (i.v.) administration of liposomes. EGFR antibodies were attached on the surface of PEG coated liposomes using biotin-neutravidin binding. Receptor mediated cellular uptake and cytotoxic efficacy of EGFR-targeted liposomes were investigated in human ovarian adenocarcinoma (SKOV-3 and SKOV3.ip1) cells. In vivo distribution of the liposomes in mice was explored using direct and pre-targeting approaches and SPECT/CT imaging. Targeted liposomes showed efficient and specific receptor-mediated binding to ovarian carcinoma cells in vitro, but the difference in cytotoxicity between targeted and non-targeted liposomes remained small. The relatively low cytotoxic efficacy is probably due to insufficient doxorubicin release from the liposomes rather than lack of target binding. Tumor uptake of targeted liposomes in vivo was comparable to that of non-targeted liposomes after both direct and pre-targeting administration. For both EGFR-targeted and non-targeted liposomes, the i.p. administration increased liposome accumulation to the tumors compared to i.v. injections.

Conclusions/Significance

Intraperitoneal administration of liposomes may be a beneficial approach to treat the tumors in the abdominal cavity. The i.p. pre-targeting method warrants further studies as a potential approach in cancer therapy.     

Formulation of liposomes functionalized with Lotus lectin and effective in targeting highly proliferative cells

BackgroundLiposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism.MethodsWe used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy.ResultsWe confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells.ConclusionsLiposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity.General significanceDoxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.     

Examination of the specificity of tumor cell derived exosomes with tumor cells in vitro

Small endogenous vesicles called exosomes are beginning to be explored as drug delivery vehicles. The in vivo targets of exosomes are poorly understood; however, they are believed to be important in cell-to-cell communication and may play a prominent role in cancer metastasis. We aimed to elucidate whether cancer derived exosomes can be used as drug delivery vehicles that innately target tumors over normal tissue. Our in vitro results suggest that while there is some specificity towards cancer cells over “immortalized” cells, it is unclear if the difference is sufficient to achieve precise in vivo targeting. Additionally, we found that exosomes associate with their cellular targets to a significantly greater extent (> 10-fold) than liposomes of a similar size. Studies on the association of liposomes mimicking the unique lipid content of exosomes revealed that the lipid composition contributes significantly to cellular adherence/internalization. Cleavage of exosome surface proteins yielded exosomes exhibiting reduced association with their cellular targets, demonstrating the importance of proteins in binding/internalization. Furthermore, although acidic conditions are known to augment the metastatic potential of tumors, we found that cells cultured at low pH released exosomes with significantly less potential for cellular association than cells cultured at physiological pH.     

Peptide-Mediated Liposomal Doxorubicin Enhances Drug Delivery Efficiency and Therapeutic Efficacy in Animal Models

Lung cancer ranks among the most common malignancies, and is the leading cause of cancer-related mortality worldwide. Chemotherapy for lung cancer can be made more specific to tumor cells, and less toxic to normal tissues, through the use of ligand-mediated drug delivery systems. In this study, we investigated the targeting mechanism of the ligand-mediated drug delivery system using a peptide, SP5-2, which specifically binds to non-small cell lung cancer (NSCLC) cells. Conjugation of SP5-2 to liposomes enhanced the amount of drug delivered directly into NSCLC cells, through receptor-mediated endocytosis. Functional SP5-2 improved the therapeutic index of Lipo-Dox by enhancing therapeutic efficacy, reducing side effects, and increasing the survival rate of tumor-bearing mice in syngenic, metastatic and orthotopic animal models. Accumulation of SP5-2-conjugated liposomal doxorubicin (SP5-2-LD) in tumor tissues was 11.2-fold higher than that of free doxorubicin, and the area under the concentration-time curve (AUC_{0–72 hours}) was increased 159.2-fold. Furthermore, the experiment of bioavailability was assessed to confirm that SP5-2 elevates the uptake of the liposomal drugs by the tumor cells in vivo. In conclusion, the use of SP5-2-conjugated liposomes enhances pharmacokinetic properties, improves efficacy and safety profiles, and allows for controlled biodistribution and drug release.     

Quantitative Modeling of the High-Throughput Production and In Vivo Kinetics of (Drug-Encapsulating) Liposomes

In developing liposomes for in vivo use, it is important to design the liposomes to have optimal in vivo kinetics, and it is also necessary to identify optimal high-throughput production conditions for these liposomes. Previous work has not definitively established the general relationship between liposomes'' configuration and composition, and their in vivo kinetics. Also, no straightforward method exists to calculate optimal liposome high-throughput production conditions for specific liposome compositions. This work presents first-principles quantitative correlations describing liposomes'' in vivo drug leakage and vascular mass transfer kinetics. This work further presents a simple quantitative model relating specific liposome compositions to ideal high-throughput production parameters. The results have implications for the identification of promising liposome compositions via high-throughput screening methodologies, as well as the design and optimization of high-throughput reactors for liposome production.     

Modulation of Multidrug Resistance in Cancer Cells by Liposome Encapsulated Doxorubicin

Abstract

A major problem in the chemotherapy of solid tumors and hematologic malignancies is the intrinsic as well as acquired cross resistance to multiple chemotherapeutic agents. Recently, this type of multidrug resistance has been related to a gene, MDR1, and its gene product, p-glycoprotein, which functions as the efflux pump, prevents accumulation of drugs and alters their cytotoxicity. Many drug-resistant human tumors express the MDR1 gene and MDR1 RNA levels are elevated in many cancers that have not responded to chemotherapy. The same persistent observation has been made in recurrent tumors who have responded initially to chemotherapy.

Doxorubicin is one of the most important anticancer agent having significant single agent activity in a variety of cancer types and is now the cornerstone of some widely used combination regimens. Despite the clinical effectiveness of the drug, doxorubicin resistance that arises in malignant cells following repeated courses of treatment is the major problem in the clinical management of neoplastic diseases. Recently, extensive studies have demonstrated that liposome encapsulated doxorubicin effectively modulates the multidrug resistance phenotype in cancer cells by altering the function of p-glycoprotein. This modulation of MDR phenotype by liposomes has been demonstrated in vitro in human breast cancer cells, ovarian cancer cells, human promyelocytic leukemia cells and in human colon cancer cells and in vivo in transgenic mice transfected with a functional MDR1 gene. It appears liposomes can play an effective role as a new modality of treatment for human cancers which have become refractory to chemotherapy. An exciting area of research which soon will emerge will exploit the different binding sites on p-glycoprotein by using combination of liposomes with other pharmacological modulators of MDR to impart maximal overcoming of multidrug resistance in cancer patients.     

Novel liposome-based formulations for prolonged delivery of proteins and vaccines

Abstract

Two strategies for increasing liposome stability in vivo are described in this review. The first strategy involves the encapsulation of liposomes within polymeric microcapsules of alginate-poly(L-lysine) that retained the liposomes inside but allowed the outward diffusion of proteins of 100 kDa or less, once they were released from the encapsulated liposomes. In vivo studies revealed that the microencapsulated liposome systems (MELs) extended the delivery of a model antigen, bovine serum albumin (BSA), for more that 80 days, resulting in the prolonged production of high levels of antigen-specific antibodies. The antibody levels were higher that those obtained with rats injected with BSA in complete Freund's adjuvant, or in liposomes. The unique construction of MELs enabled also the enzymatically-triggered pulsatile delivery of proteins from encapsulated liposomes, which was not possible before with liposomes.     

PEGylated liposomes of anastrozole for long-term treatment of breast cancer: in vitro and in vivo evaluation

The aim of present study was to develop conventional and PEGylated (long circulating), liposomes containing anastrozole (ANS) for effective treatment of breast cancer. ANS is a third-generation non-steroidal aromatase inhibitor of the triazole class used for the treatment of advanced and late-stage breast cancer in post-menopausal women. Under such disease conditions the median duration of therapy should be prolonged until tumor regression ends (>31 months). Liposomes were prepared by the thin film hydration method by using ANS and various lipids such as soyaphosphatidyl choline, cholesterol and methoxy polyethylene glycol distearoyl ethanolamine in different concentration ratios and evaluated for physical characteristics, in vitro drug release and stability. Optimized formulations of liposome were studied for in vitro cytotoxic activity against the BT-549 and MCF-7 cell lines and in vivo behavior in Wistar rats. Preformulation studies, both Fourier transform infrared study and differential scanning calorimetry analysis showed no interaction between the drug and the excipients used in the formulations. The optimized formulations AL-07 and AL-09 liposomes showed encapsulation efficiencies in the range 65.12?±?1.05% to 69.85?±?3.2% with desired mean particle size distribution of 101.1?±?5.9 and 120.2?±?2.8?nm and zeta potentials of ?43.7?±?4.7 and ?62.9?±?3.5 mV. All the optimized formulations followed Higuchi-matrix release kinetics and when plotted in accordance with the Korsemeyer–Peppas method, the n-value 0.5?n?in vitro cytotoxicity studies (p?(0–∞) values when compared to pure drug (p?相似文献   

A New Mixed-Backbone Oligonucleotide against Glucosylceramide Synthase Sensitizes Multidrug-Resistant Tumors to Apoptosis

Enhanced ceramide glycosylation catalyzed by glucosylceramide synthase (GCS) limits therapeutic efficiencies of antineoplastic agents including doxorubicin in drug-resistant cancer cells. Aimed to determine the role of GCS in tumor response to chemotherapy, a new mixed-backbone oligonucleotide (MBO-asGCS) with higher stability and efficiency has been generated to silence human GCS gene. MBO-asGCS was taken up efficiently in both drug-sensitive and drug-resistant cells, but it selectively suppressed GCS overexpression, and sensitized drug-resistant cells. MBO-asGCS increased doxorubicin sensitivity by 83-fold in human NCI/ADR-RES, and 43-fold in murine EMT6/AR1 breast cancer cells, respectively. In tumor-bearing mice, MBO-asGCS treatment dramatically inhibited the growth of multidrug-resistant NCI/ADR-RE tumors, decreasing tumor volume to 37%, as compared with scrambled control. Furthermore, MBO-asGCS sensitized multidrug-resistant tumors to chemotherapy, increasing doxorubicin efficiency greater than 2-fold. The sensitization effects of MBO-asGCS relied on the decreases of gene expression and enzyme activity of GCS, and on the increases of C₁₈-ceramide and of caspase-executed apoptosis. MBO-asGCS was accumulation in tumor xenografts was greater in other tissues, excepting liver and kidneys; but MBO-asGCS did not exert significant toxic effects on liver and kidneys. This study, for the first time in vivo, has demonstrated that GCS is a promising therapeutic target for cancer drug resistance, and MBO-asGCS has the potential to be developed as an antineoplastic agent.     

Targeting of Liposomes Within Cardiovascular System

Abstract

Our studies on the targeting of liposomes and liposome-associated pharmaceuticals within the cardiovascular system are reviewed. The delivery of diagnostic and therapeutic agents in plain liposomes, immunoliposomes, long-circulating liposomes and long-circulating immunoliposomes into the sites of vascular injuries and myocardial infarction is discussed. In vitro, ex vivo, and in vivo experiments present a general view on the advantages and limitations of using liposome-mediated targeting. Liposomes capable of targeting pathological areas of the blood vessel wall both, in vitro and ex vivo are described, as well as liposome able to be internalized by normal endothelial cells. Liposome-mediated drug targeting to compromised myocardium is reviewed with a primary impact on liposomes with anti-cardiac myosin antibodies. Targeted visualization of myocardial infarction with diagnostic liposomes is discussed. Efficient accumulation of long-circulating immunoliposomes in the infarct zone is demonstrated, and a relative importance of different variables, such as liposome size, targetability, and prolonged circulation time, for target accumulation is analyzed. The use of immunoliposomes for targeted sealing of hypoxia-caused damages in plasmic membranes of cardiocytes is considered as a new approach in the therapeutic use of liposomes.     

Codelivery of Chemotherapeutics via Crosslinked Multilamellar Liposomal Vesicles to Overcome Multidrug Resistance in Tumor

Multidrug resistance (MDR) is a significant challenge to effective cancer chemotherapy treatment. However, the development of a drug delivery system that allows for the sustained release of combined drugs with improved vesicle stability could overcome MDR in cancer cells. To achieve this, we have demonstrated codelivery of doxorubicin (Dox) and paclitaxel (PTX) via a crosslinked multilamellar vesicle (cMLV). This combinatorial delivery system achieves enhanced drug accumulation and retention, in turn resulting in improved cytotoxicity against tumor cells, including drug-resistant cells. Moreover, this delivery approach significantly overcomes MDR by reducing the expression of P-glycoprotein (P-gp) in cancer cells, thus improving antitumor activity in vivo. Thus, by enhancing drug delivery to tumors and lowering the apoptotic threshold of individual drugs, this combinatorial delivery system represents a potentially promising multimodal therapeutic strategy to overcome MDR in cancer therapy.     

A simple method for producing a technetium-99m-labeled liposome which is stable In Vivo

A new method for labeling preformed liposomes with technetium-99m (^99mTc) has been developed which is simple to perform and stable in vivo. Previous ^99mTc-liposome labels have had variable labeling efficiencies and stability. This method consistently achieves high labeling efficiencies (> 90%) with excellent stability. A commercially available radiopharmaceutical kit—hexamethylpropyleneamine oxime (HM-PAO)—is reconstituted with ^99mTcO^−₄ and then incubated with preformed liposomes that encapsulate glutathione. The incubation takes only 30 min at room temperature. Liposomes that co-encapsulate other proteins such as hemoglobin or albumin, in addition to glutathione, also label with high efficiency. Both in vitro and in vivo studies indicate good stability of this label. Rabbit images show significant spleen and liver uptake at 2 and 20 h after liposome infusion without visualization of thyroid, stomach or bladder activity.This labeling method can be used to study the biodistribution of a wide variety of liposome preparations that are being tested as novel drug delivery systems. This method of labeling liposomes with ^99mTc may also have applications in diagnostic imaging.     

Liposome-mediated delivery of deoxyribonucleic acid to cells: enhanced efficiency of delivery related to lipid composition and incubation conditions

Delivery of liposome-encapsulated simian virus 40 (SV40) DNA to African green monkey Related to been used as a probe to study liposome--cell interactions and to determine conditions which favor the intracellular delivery of liposome contents to cells. The efficiency of DNA delivery by various liposome preparations (monitored by infectivity assays) was found to be dependent both on the magnitude of vesicle binding to cells and on the resistance of liposomes to cell-induced leakage of contents. Acidic phospholipids were much more effective in both binding and delivery, and phosphatidylserine (PS) was the best in both aspects. The inclusion of 50 mol % cholesterol in liposomes reduces the cell-induced leakage of vesicle contents (2--5-fold) and substantially enhances the delivery of DNA to cells (2--10-fold). Following incubation of cells with negatively charged liposomes containing SV40 DNA, infectivity can be enhanced greatly by brief exposure of the cells to glycerol solutions. In contrast, only slight enhancement by glycerol was observed for SV40 DNA encapsulated in neutral or positively charged liposomes. The results of competition experiments between empty phosphatidylcholine liposomes and DNA-containing PS liposomes also suggest possible differences in the interaction of neutral and negatively charged liposome preparations with cells. Morphological studies indicate that the glycerol treatment stimulates membrane ruffling and vacuolization and suggest that the enhanced uptake of liposomes occurs by an endocytosis-like process. Results obtained with metabolic inhibitors are also consistent with the interpretation that the enhancement of liposome delivery in glycerol-treated cells occurs via an energy-dependent endocytotic pathway. Pretreatment of cells with chloroquine, a drug which alters lysosomal activity, further enhanced infectivity in glycerol-treated cells (4-fold). This observation suggests the involvement of a lysosomal processing step at some point in the expression of liposome-encapsulated DNA and, more importantly, illustrates the possibility of altering cellular mechanism to engineer more efficient delivery by liposomes. Under optimal conditions determined in this study, the efficiency of liposome-mediated SV40 DNA delivery was increased more than 1000-fold over that obtained by simply incubating cells with liposomes. It is also demonstrated that these conditions enhance delivery of other molecules, besides DNA, which are encapsulated in liposomes.     

In situ activation of murine macrophages by liposomes containing lymphokines

Murine alveolar and peritoneal macrophages harvested after injection of lymphokines encapsulated within multilamellar phospholipid vesicles (liposomes) were tumoricidal in vitro. The state and degree of activation depended on the route of liposome administration. Activation of peritoneal macrophages was achieved by intraperitoneal injection of liposomes and alveolar macrophages were activated by injecting liposomes intravenously but not intraperitoneally. The in vivo rendering of macrophages with tumoricidal properties might be useful toward destruction of tumor cells in vivo.