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In this issue of ACS Nano, Chen et al. provide in vitro and in vivo evidence for monoclonal anti-poly(ethylene glycol) (anti-PEG) antibody-triggered, complement terminal complex-mediated damage to PEGylated liposomal doxorubicin, entailing the release of the encapsulated drug from the vesicles. These results reveal a new dimension of the potential damage of anti-PEG antibody-mediated complement activation on PEGylated nanomedicines in addition to previous observations on infusion hypersensitivity reactions and the accelerated blood clearance effect. The possibility of a destructive attack of the complement system on the liposome drug carrier may have safety implications in patients displaying high levels of preformed anti-PEG antibodies. In this Perspective, we summarize the experimental and clinical data highlighting the relationships among the above adverse immune phenomena and the options available for reducing the risk of immune damage caused by PEGylated nanomedicines.

Several liposome products have been approved for the treatment of cancer. In all of them, the active agents are encapsulated in the liposome water phase passively or by transmembrane ion gradients. An alternative approach in liposomal drug delivery consists of chemically modifying drugs to form lipophilic prodrugs with strong association to the liposomal bilayer. Based on this approach, we synthesized a mitomycin c-derived lipidic prodrug (MLP) which is entrapped in the bilayer of PEGylated liposomes (PL-MLP, Promitil®), and activated by thiolytic cleavage. PL-MLP is stable in plasma with thiolytic activation of MLP occurring exclusively in tissues and is more effective and less toxic than conventional chemotherapy in various tumor models. PL-MLP has completed phase I clinical development where it has shown a favorable safety profile and a 3-fold reduction in toxicity as compared to free mitomycin c. Clinical and pharmacokinetic studies in patients with advanced colo-rectal carcinoma have indicated a significant rate of disease stabilization (39%) in this chemo-refractory population and significant prolongation of median survival in patients attaining stable disease (13.9 months) versus progressive disease patients (6.35 months). The pharmacokinetics of MLP was typically stealth with long T½ (~1 day), slow clearance and small volume of distribution. Interestingly, a longer T½, and slower clearance were both correlated with disease stabilization and longer survival. This association of pharmacokinetic parameters with patient outcome suggests that arrest of tumor growth is related to the enhanced tumor localization of long-circulating liposomes and highlights the importance of personalized pharmacokinetic evaluation in the clinical use of nanomedicines. Another important area where PL-MLP may have an added value is in chemoradiotherapy, where it has shown a strong radiosensitizing effect in animal models based on a unique mechanism of enhanced prodrug activation and encouraging results in early human testing.

The effect of a lipidated prodrug of mitomycin C (MLP) on the membrane of a pegylated liposome formulation (PL-MLP), also known as Promitil, was characterized through high-sensitivity differential scanning calorimetry (DSC) and cryo-TEM. The thermodynamic analysis demonstrated that MLP led to the formation of heterogeneous domains in the membrane plane of PL-MLP. MLP concentrated in prodrug-rich domains, arranged in high-ordered crystal-like structures, as suggested by the sharp and high enthalpy endotherm in the first heating scanning. After thiolytic cleavage of mitomycin C from MLP by dithiothreitol (DTT) treatment, the crystal-like prodrug domain disappears and a homogeneous membrane with stronger lipid interactions and higher phase transition temperature compared with the blank (MLP-free) liposomes is observed by DSC. In parallel, the rod-like discoid liposomes and the "kissing liposomes" seen by cryo-TEM in the PL-MLP formulation disappear, and liposome mean size and polydispersity increase after DTT treatment. Both MLP and the residual postcleavage lipophilic moiety of the prodrug increased the rigidity of the liposome membrane as indicated by DSC. These results confirm that MLP is inserted in the PL-MLP liposome membrane via its lipophilic anchor, and its mitomycin C moiety located mainly at the region of the phospholipid glycerol backbone and polar headgroup. We hypothesize that π-π stacking between the planar aromatic rings of the mitomycin C moieties leads to the formation of prodrug-rich domains with highly ordered structure on the PL-MLP liposome membrane. This thermodynamically stable conformation may explain the high stability of the PL-MLP formulation. These results also provide us with an interesting example of the application of high sensitivity DSC in understanding the composition-structure-behavior dynamics of liposomal nanocarriers having a lipid-based drug as pharmaceutical ingredient.

Gammadelta T (γδ-T) cells are strong candidates for adoptive immunotherapy in oncology due to their cytotoxicity, ease of expansion, and favorable safety profile. The development of γδ-T cell therapies would benefit from non-invasive cell-tracking methods and increased targeting to tumor sites. Here we report the use of [89Zr]Zr(oxinate)4 to track Vγ9Vδ2 T cells in vivo by positron emission tomography (PET). In vitro, we showed that 89Zr-labeled Vγ9Vδ2 T cells retained their viability, proliferative capacity, and anti-cancer cytotoxicity with minimal DNA damage for amounts of 89Zr ≤20 mBq/cell. Using a mouse xenograft model of human breast cancer, 89Zr-labeled γδ-T cells were tracked by PET imaging over 1 week. To increase tumor antigen expression, the mice were pre-treated with PEGylated liposomal alendronate. Liposomal alendronate, but not placebo liposomes or non-liposomal alendronate, significantly increased the 89Zr signal in the tumors, suggesting increased homing of γδ-T cells to the tumors. γδ-T cell trafficking to tumors occurred within 48 hr of administration. The presence of γδ-T cells in tumors, liver, and spleen was confirmed by histology. Our results demonstrate the suitability of [89Zr]Zr(oxinate)4 as a cell-labeling agent for therapeutic T cells and the potential benefits of liposomal bisphosphonate treatment before γδ-T cell administration., Graphical Abstract, Man et al. demonstrate that [89Zr]Zr(oxinate)4 allows in vivo PET imaging of therapeutic γδ-T cells over 1 week. Furthermore, the trafficking of γδ-T cells to the tumor is increased by treatment with a liposomal aminobisphosphonate drug. This radiotracer is potentially translatable for clinical trials of therapeutic T cells.

Esben Christensen,1–3 Jonas R Henriksen,2,4 Jesper T Jørgensen,3 Yasmine Amitay,5 Hilary Shmeeda,5 Alberto A Gabizon,5 Andreas Kjær,3 Thomas L Andresen,1,2 Anders E Hansen1–3 1Department of Micro- and Nanotechnology, DTU Nanotech, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark; 2Center for Nanomedicine and Theranostics, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark; 3Cluster for Molecular Imaging, Department of Biomedical Sciences and Department of Clinical Physiology, Nuclear Medicine & PET, University of Copenhagen and Rigshospitalet, DK-2200 & DK-2100, Copenhagen, Denmark; 4Department of Chemistry, DTU Chemistry, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark; 5Shaare Zedek Medical Center and Hebrew University – School of Medicine, Jerusalem, Israel Background: Active, ligand-mediated, targeting of functionalized liposomes to folate receptors (FRs) overexpressed on cancer cells could potentially improve drug delivery and specificity. Studies on folate-targeting liposomes (FTLs) have, however, yielded varying results and generally fail to display a clear benefit of FR targeting.Method: Tumor accumulating potential of FTLs and NTLs were investigated in a FR overexpressing xenograft model by positron emission tomography/computed tomography imaging.Results: Tumors displayed significantly lower activity of FTLs than NTLs. Furthermore, FTLs displayed worse circulating properties and increased liver-accumulation than NTLs. Conclusion: This study underlines that long-circulating properties of liposomes must be achieved to take advantage of EPR-dependent tumor accumulation which may be lost by functionalization. FR-functionalization negatively affected both tumor accumulation and circulation properties. Keywords: liposomes, folate, cancer, imaging, PET, EPR

Pegylated liposomal (PL) mitomycin C lipid-based prodrug (MLP) has recently entered clinical testing. We studied here the preclinical pharmacology of PL-MLP. The stability, pharmacokinetics, biodistribution, and other pharmacologic parameters of PL-MLP were examined. Thiolytic cleavage of MLP and release of active mitomycin C (MMC) were studied using dithiothreitol (DTT), and by incubation with tissue homogenates. MLP was incorporated in the bilayer at 10% molar ratio with nearly 100% entrapment efficiency, resulting in a formulation with high plasma stability. In vitro, DTT induced cleavage of MLP with predictable kinetics, generating MMC and enhancing pharmacological activity. A long circulation half-life of MLP (10–15 h) was observed in rodents and minipigs. Free MMC is either extremely low or undetectable in plasma. However, urine from PL-MLP injected rats revealed delayed but significant excretion of MMC indicating in vivo activation of MLP. Studies in mice injected with H3-cholesterol radiolabeled PL-MLP demonstrated relatively greater tissue levels of H3-cholesterol than MLP. MLP levels were highest in tumor and spleen, and very low or undetectable in liver and lung. Rapid cleavage of MLP in various tissues, particularly in liver, was shown in ex-vivo experiments of PL-MLP with tissue homogenates. PL-MLP was less toxic in vivo than equivalent doses of MMC. Therapeutic studies in C26 mouse tumor models demonstrated dose-dependent improved efficacy of PL-MLP over MMC. Thiolytic activation of PL-MLP occurs in tissues but not in plasma. Liposomal delivery of MLP confers a favorable pharmacological profile and greater therapeutic index than MMC.

Mitomycin C (MMC) has potent cytotoxicity but cumulative toxicity limits widespread use. In animals, pegylated liposomal mitomycin C lipid-based prodrug (PL-MLP) was well tolerated and more effective than free MMC. We evaluated PL-MLP in patients with advanced cancer. Twenty-seven patients were treated in escalating dose cohorts of 0.5–3.5 mg/kg (equivalent to 0.15–1.03 mg/kg MMC) every 4 weeks for up to 12 cycles, unless disease progression or unacceptable toxicity occurred. Pharmacokinetics were assessed during cycles 1 and 3. Per protocol maximum tolerated dose was not reached at 3.5 mg/kg. However, prolonged thrombocytopenia developed after repeated doses of 3 mg/kg or cumulative doses of 10–12 mg/kg. Dose-related grade 3 or higher adverse events included fatigue, anemia, and decreased platelets. Cmax and AUC0-∞ increased linearly over the dose range 0.5–2.0 mg/kg, and greater than linearly from 2.5 to 3.5 mg/kg; there were no significant differences in clearance of MLP between cycles 1 and 3. Median t1/2 was 23 h among dose cohorts, with no trend by dose or cycle. One patient had a partial response. Stable disease was observed in 10 patients across all dose levels. PL-MLP has a long circulation time, was well tolerated, and can be administered to heavily pretreated patients at a single dose of 3.0 mg/kg and cumulative dose of 10–12 mg/kg before development of prolonged thrombocytopenia; this is nearly threefold the equivalent dose of MMC tolerated historically. This formulation may be active in a variety of tumor types and is better tolerated than free MMC.

Anthracyclines are powerful anticancer agents and among the most important tools in the chemotherapy armamentarium of medical oncologists. They are approved for use in the treatment of a broad variety of solid and hematologic neoplasms. However, the usefulness of these agents, particularly doxorubicin, the most widely used anthracycline, is limited by considerable toxicity, especially damage to the cardiac muscle, which is cumulative and mostly irreversible, restricting extended use of this drug. In the last 30 years, extensive research with a variety of drug-delivery systems has attempted to overcome this limitation, with clinical success mostly confined to liposome formulations. Liposomal doxorubicin, and particularly pegylated liposomal doxorubicin, has shown significant pharmacologic advantages and an added clinical value over doxorubicin. Here, we review the mechanisms of action and toxicity of doxorubicin, and ways to reduce toxicity, with a focus on liposome-based drug-delivery systems.

Folate-targeted liposomes (FTL) were tested as drug delivery vehicles to PSMA-positive cancer cells. We used FL with co-entrapped mitomycin C lipophilic prodrug (MLP) and doxorubicin (DOX), and the LNCaP prostate cancer cell line which expresses PSMA but is negative for folate receptor. A major increase in cell drug levels was observed when LNCaP cells were incubated with FTL as compared to non-targeted liposomes (NTL). MLP was activated to mitomycin C, and intracellular and nuclear fluorescence of DOX was detected, indicating FTL processing and drug bioavailability. PMPA (2-(phosphonomethyl)-pentanedioic acid), a specific inhibitor of PSMA, blocked the uptake of FTL into LNCaP cells, but did not affect the uptake of FTL into PSMA-deficient and folate receptor-positive KB cells. The cytotoxic activity of drug-loaded FTL was found significantly enhanced when compared to NTL in LNCaP cells. FTL may provide a new tool for targeted therapy of cancers that over-express the PSMA receptor.

The development of stable and long-circulating liposomes provides protection of the drug cargo from degradation and increases tumor drug delivery, leading to the design of liposome formulations with great potential in cancer therapy. However, despite the sound pharmacologic basis, many liposomal as well as other nanoparticle-based drug formulations have failed to meet regulatory criteria for approval. The question that arises is whether we have missed key liposome-host interactions that can account for the gap between the major pharmacologic advantages in preclinical studies and the modest impact of the clinical effects in humans. We will discuss here the nanoparticle-immune system interactions that may undermine the antitumor effect of the nanodrug formulations and contribute to this gap. To overcome this challenge and increase clinical translation, new preclinical models need to be adopted along with comprehensive immunopharmacologic studies and strategies for patient selection in the clinical phase.

Background Zoledronic acid (Zol) is a potent inhibitor of farnesyl-pyrophosphate synthase with broad clinical use in the treatment of osteoporosis, and bone metastases. We have previously shown that encapsulation of Zol in liposomes targeted to the folate receptor (FR) greatly enhances its in vitro cytotoxicity. To examine whether targeted liposomal delivery of Zol could be a useful therapeutic approach, we investigated here the in vivo pharmacology of i.v. administered liposomal Zol (L-Zol) in murine models. Methods Zol was passively entrapped in the water phase of liposomes containing a small fraction of either dipalmitoyl-phosphatidylglycerol (DPPG) or a polyethylene-glycol (PEG)-conjugated phospholipid with or without insertion of a folate lipophilic conjugate. Radiolabeled formulations were used for pharmacokinetic (PK) and biodistribution studies. Toxicity was evaluated by clinical, hematological, biochemical, and histopathological parameters. Therapeutic studies comparing free Zol, nontargeted and folate targeted L-Zol were performed in FR-expressing human tumor models. Results Encapsulation of Zol in liposomes resulted in major PK changes including sustained high plasma levels and very slow clearance. DPPG-L-Zol was cleared faster than PEG-L-Zol. Grafting of folate lipophilic conjugates on liposomes further accelerated the clearance of Zol. L-Zol caused a major shift in drug tissue distribution when compared to free Zol, with a major increase (20 to 100-fold) in liver and spleen, a substantial increase (7 to 10-fold) in tumor, and a modest increase (2-fold) in bone. Liposomal formulations proved to be highly toxic, up to 50-fold more than free Zol. PEG-L-Zol was more toxic than DPPG-L-Zol. Toxicity was non-cumulative and appears to involve macrophage/monocyte activation and release of cytokines. Co-injection of L-Zol with a large dose of blank liposomes, or injection of a very low Zol-to-phospholipid ratio liposome formulation reduced toxicity by 2–4-fold suggesting that diluting macrophage exposure below a threshold Zol concentration is important to overcome toxicity. L-Zol failed to significantly enhance the therapeutic activity of Zol vis-a-vis free ZOL and doxorubicin. Folate-targeted L-Zol was marginally better than other treatment modalities in the KB tumor model but toxic deaths greatly affected the outcome. Conclusions Liposome delivery of Zol causes a major change in tissue drug distribution and an increase in tumor Zol levels. However, the severe in vivo toxicity of L-Zol seriously limits its dose and its utility for in vivo tumor cell targeting. This strategy is under evaluation using liposomes carrying less toxic bisphosphonates.

We developed a pegylated liposome formulation of a dissociable salt of a nitrogen-containing bisphosphonate, alendronate (Ald), coencapsulated with the anthracycline, doxorubicin (Dox), a commonly used chemotherapeutic agent. Liposome-encapsulated ammonium Ald generates a gradient driving Dox into liposomes, forming a salt that holds both drugs in the liposome water phase. The resulting formulation (PLAD) allows for a high-loading efficiency of Dox, comparable to that of clinically approved pegylated liposomal doxorubicin sulfate (PLD) and is very stable in plasma stability assays. Cytotoxicity tests indicate greater potency for PLAD compared to PLD. This appears to be related to a synergistic effect of the coencapsulated Ald and Dox. PLAD and PLD differed in in vitro monocyte-induced IL-1β release (greater for PLAD) and complement activation (greater for PLD). A molar ratio Ald/Dox of ∼1:1 seems to provide an optimal compromise between loading efficiency of Dox, circulation time and in vivo toxicity of PLAD. In mice, the circulation half-life and tumor uptake of PLAD were comparable to PLD. In the M109R and 4T1 tumor models in immunocompetent mice, PLAD was superior to PLD in the growth inhibition of subcutaneous tumor implants. This new formulation appears to be a promising tool to exploit the antitumor effects of aminobisphosphonates in synergy with chemotherapy.

Mitomycin C (MMC) is a powerful anti-bacterial, anti-fungal and anti-tumor antibiotic, often active against multidrug resistant cells. Despite a broad spectrum of antitumor activity, MMC clinical use is relatively limited due to its fast clearance and dose-limiting toxicity. To exploit the potential antitumor activity of MMC and reduce its toxicity we have previously developed a formulation of pegylated liposomes with a lipophilic prodrug of MMC (PL-MLP), activated by endogenous reducing agents which are abundant in the tumor cell environment in the form of different thiols. PL-MLP has minimal in vitro cytotoxicity unless reducing agents are added to the cell culture to activate the prodrug. In the present study, we hypothesized that targeting PL-MLP via folate receptors will facilitate intracellular activation of prodrug and enhance cytotoxic activity without added reducing agents. We grafted a lipophilic folate conjugate (folate-PEG(5000)-DSPE) to formulate folate targeted liposomes (FT-PL-MLP) and examined in vitro cell uptake and cytotoxic activity in cancer cell lines with high folate receptors (HiFR). 3H-cholesterol-hexadecyl ether (3H-Chol)-radiolabeled liposomes were prepared to study liposome-cell binding in parallel to cellular uptake of prodrug MLP. 3H-Chol and MLP cell uptake levels were 4-fold and 9-fold greater in KB HiFR cells when FT-PL-MLP is compared to non-targeted PL-MLP liposomes. The cytotoxic activity of FT-PL-MLP liposomes was significantly increased up to ~5-fold compared with PL-MLP liposomes in all tested HiFR expressing cell lines. The enhanced uptake and intracytoplasmic liposome delivery was confirmed by confocal fluorescence studies with Rhodamine-labeled liposomes. In vivo, no significant differences in pharmacokinetics and biodistribution were observed when PL-MLP was compared to FT-PL-MLP by the intravenous route. However, when liposomes were directly injected into the peritoneal cavity of mice with malignant ascites of J6456 HiFR lymphoma cells, the tumor cell levels of MLP were significantly greater with the folate-targeted liposomes. Thus, folate targeting enhances liposome uptake by tumor cells enabling intracellular activation of prodrug in the absence of exogenous reducing agents, and leading to increased cytotoxicity. These results may be particularly relevant to the application of folate-targeted PL-MLP in intracavitary or intravesical treatment of cancer.

In recent years the importance of molecular and diagnostic imaging has increased dramatically in the treatment planning of many diseases and in particular in cancer therapy. Within nanomedicine there are particularly interesting possibilities for combining imaging and therapy. Engineered liposomes that selectively localize in tumor tissue can transport both drugs and imaging agents, which allows for a theranostic approach with great potential in personalized medicine. Radiolabeling of liposomes have for many years been used in preclinical studies for evaluating liposome in vivo performance and has been an important tool in the development of liposomal drugs. However, advanced imaging systems now provide new possibilities for non-invasive monitoring of liposome biodistribution in humans. Thus, advances in imaging and developments in liposome radiolabeling techniques allow us to enter a new arena where we start to consider how to use imaging for patient selection and treatment monitoring in connection to nanocarrier based medicines. Nanocarrier imaging agents could furthermore have interesting properties for disease diagnostics and staging. Here, we review the major advances in the development of radiolabeled liposomes for imaging as a tool in personalized medicine.

Background A mitomycin-C lipid-based prodrug (MLP) formulated in pegylated liposomes (PL-MLP) was previously reported to have significant antitumor activity and reduced toxicity in mouse tumor models (Clin Cancer Res 12:1913–20, 2006). MLP is activated by thiolysis releasing mitomycin-C (MMC) which rapidly dissociates from liposomes. The purpose of this study was to examine the plasma stability, pharmacokinetics, and antitumor activity of PL-MLP in mouse models of human gastroentero-pancreatic tumors. Methods MLP was incorporated with almost 100% efficiency in pegylated liposomes composed of hydrogenated phosphatidylcholine, with or without cholesterol (Chol). Mean vesicle size was 45–65 nm for liposome preparations downsized by homogenization, and 80–100 nm when downsized by extrusion, the latter displaying narrower polydispersity. MLP to phospholipid mole ratio was 5% (~20 μg MMC-equivalents/μmol). Therapeutic studies were carried out in the N87 gastric carcinoma (Ca), HCT15 colon Ca, and Panc-1 pancreatic Ca models implanted s.c. in CD1 nude mice. Treatment was administered i.v. in mice with established tumors. Results PL-MLP was very stable when incubated in plasma, and whole blood with a maximum of 5% release and activation to free MMC after 24 h. In the presence of a strong reducing agent (dithiotreitol), MLP was almost entirely activated to free MMC. Pharmacokinetic studies revealed major differences in plasma clearance between free MMC and PL-MLP. The longest half-lives were observed for extruded and Chol-containing preparations. Using a liposome radiolabel, it was found that the plasma levels of liposomes and prodrug were nearly superimposable confirming the absence of drug leakage in circulation. In vivo prodrug activation was significantly increased by co-injection of a large dose of a biocompatible reducing agent, N-acetylcysteine. PL-MLP was significantly more effective in delaying tumor growth and resulted in more tumor regressions than irinotecan in the N87 and HCT15 models, and than gemcitabine in the Panc-1 model. PL-MLP was ~3-fold less toxic than free MMC at MMC-equivalent doses, and displayed mild myelosuppression at therapeutic doses. Conclusions Delivery of MLP in pegylated liposomes is more effective than conventional chemotherapy in the treatment of gastroentero-pancreatic ectopic tumor models, and may represent an effective tool for treatment of these malignancies in the clinical setting with improved safety over free MMC. Reducing agents offer a tool for controlling in vivo prodrug release.

Some therapeutic liposomes and lipid excipient-based anticancer drugs are recognized by the immune system as foreign, leading to a variety of adverse immune phenomena. One of them is complement (C) activation, the cause, or major contributing factor to a hypersensitivity syndrome called C activation-related pseudoallergy (CARPA). CARPA represents a novel subcategory of acute (type I) hypersensitivity reactions (HSR), which is mostly mild, transient, and preventable by appropriate precautions. However, in an occasional patient, it can be severe or even lethal. Because a main manifestation of C activation is cardiopulmonary distress, CARPA may be a safety issue primarily in cardiac patients. Along with an overview of the various types of liposome-immune system interactions, this review updates the experimental and clinical information on CARPA to different therapeutic liposomes and lipid excipient-based (micellar) anticancer drugs, including PEGylated liposomal doxorubicin sulfate (PLD, Doxil®) and paclitaxel (Taxol®). The substantial individual variation of in vitro and in vivo findings reflects an extremely complex immune phenomenon involving multiple, redundant pathways of C activation, signal transduction in allergy-mediating cells and vasoactive mediator actions at the effector cell level. The latest advances in this field include the proposal of doxorubicin-induced shape changes and aggregation of liposomes in Doxil as possible contributing factors to CARPA caused by PLD, and the finding that Doxil-induced immune suppression prevents HSR to co-administered carboplatin, a significant benefit of Doxil in combination chemotherapy with carboplatin. The review evaluates the use of in vitro C assays and the porcine liposome-induced cardiopulmonary distress model for predicting CARPA. It is concluded that CARPA may become a frequent safety issue in the upcoming era of nanomedicines, necessitating its prevention at an early stage of nanomedicine R&D.

The folate receptor (FR) is overexpressed in a broad spectrum of malignant tumors and represents an attractive target for selective delivery of anti-cancer agents to FR-expressing tumors. Targeting liposomes to the FR has been proposed as a way to enhance the effects of liposome-based chemotherapy. Folate–polyethylene glycol–distearoyl–phosphatidyl–ethanolamine conjugate was inserted into pegylated liposomal doxorubicin (PLD). The therapeutic activity of folate-targeted (FT-PLD) and non-targeted (PLD) pegylated liposomal doxorubicin was tested in two human tumor models (KB, KB-V) and in one mouse ascitic tumor model (FR-expressing J6456) by the i.v. systemic route in all models, and by the i.p. intracavitary route in the ascitic tumor model only. Consistent with previous studies, PLD was clearly superior to free doxorubicin in all tumor models. When targeted and non-targeted liposome formulations were compared, FT-PLD was more effective than PLD in the KB and KB-V xenograft models, and in the J6456 intra-cavitary therapy model. The therapeutic effect was dose-dependent in the KB model and schedule-dependent in the J6456 intra-cavitary therapy model. In some experiments, toxic deaths aggravated by folate-depleted diet were a major confounding factor. In a non-FR expressing J6456 model, FT-PLD was as active as PLD indicating that its activity is not limited to FR-expressing tumors. Folate-targeting confers a significant albeit modest therapeutic improvement to PLD in FR-expressing tumor models, which appears particularly valuable in intracavitary therapy. The potential clinical added value of this approach has yet to be determined.

Background Receptor-directed targeting of ligand-bearing liposomes to tumor cells may enhance therapeutic efficacy by intracellular delivery of a concentrated payload of liposomal drug. The goal of this study was to assess whether Her2-targeted pegylated liposomal doxorubicin (PLD) retains its binding ability to Her2-expressing target cells through circulation in the blood and extravasation to tumor interstitial fluid. Methods PLD was grafted with a lipophilic conjugate of an anti-Her2 scFv antibody fragment at an approximate ratio of 7.5, 15, or 30 ligands per liposome. BALB/c mice were injected with J6456 lymphoma cells into the peritoneal cavity to generate malignant ascites used as a model for tumor interstitial fluid. When abdominal swelling developed, Her2-targeted (HT-) PLD and non-targeted PLD were injected into the mice i.v. at a dose of 15 mg/kg. The ascitic fluid was collected 48 h later, ascitic tumor cells were removed, and the doxorubicin levels in the cell-free ascitic fluid and plasma were determined. Binding of the cell-free ascitic fluid was tested in vitro against two Her2-expressing human tumor cell lines (N87, SKBR-3) and compared to the binding of shelf formulations (not passaged in vivo) of HT-PLD and PLD, by measuring the amount of cell-associated doxorubicin. Results Plasma and ascitic fluid levels of HT-PLD were only slightly below those of PLD indicating that, the Her2 ligand did not cause any significant change in the clearance rate of PLD. The in vitro binding of HT-PLD containing ascitic fluid to Her2-expressing cells was increased 10 to 20-fold above that of PLD-containing ascitic fluid, similarly to the 20-fold difference in binding between shelf Her2-PLD and PLD. The in vitro cytotoxicity of ascitic fluid containing HT-PLD tested against Her2-expressing tumor cells was far greater than that of PLD, and similar to that of the shelf formulations, indicating that the selective pharmacological activity of HT-PLD is preserved after in vivo passage. Optimal results were obtained with HT-PLD formulated with 15 ligands per liposome. Conclusions HT-PLD retains most of its original binding capacity to Her2-expressing cells after in vivo passage indicating that the ligand is stably maintained in vivo in association with the doxorubicin liposomal carrier, and confirming the validity of the post-formulation ligand grafting approach for liposome targeting. Targeting of PLD using a Her2 antibody fragment provides an important means of in vivo selective drug delivery to tumors expressing the Her2 receptor.

Ligand-receptor mediated targeting may affect differently the performance of supramolecular drug carriers depending on the nature of the nanocarrier. In this study, we compare the selectivity, safety and activity of doxorubicin (Dox) entrapped in liposomes versus Dox conjugated to polymeric nanocarriers in the presence or absence of a folic acid (FA)-targeting ligand to cancer cells that overexpress the folate receptor (FR). Two pullulan (Pull)-based conjugates of Dox were synthesized, (FA-PEG)-Pull-(Cyst-Dox) and (NH2-PEG)-Pull-(Cyst-Dox). The other delivery systems are Dox loaded PEGylated liposomes (PLD, Doxil®) and the FR-targeted version (PLD-FA) obtained by ligand post-insertion into the commercial formulation. Both receptor-targeted drug delivery systems (DDS) were shown to interact in vitro specifically with cells via the folate ligand. Treatment of FR-overexpressing human cervical carcinoma KB tumor-bearing mice with three-weekly injections resulted in slightly enhanced anticancer activity of PLD-FA compared to PLD and no activity for both pullulan-based conjugates. When the DDS were administered intravenously every other day, the folated-Pull conjugate and the non-folated-Pull conjugate displayed similar and low antitumor activity as free Dox. At this dosing regimen, the liposome-based formulations displayed enhanced antitumor activity with an advantage to the non-folated liposome. However, both liposomal formulations suffered from toxicity that was reversible following treatment discontinuation. Using a daily dosing schedule, with higher cumulative dose, the folated-Pull conjugate strongly inhibited tumor growth while free Dox was toxic at this regimen. For polymeric constructs, increasing dose intensity and cumulative dose strongly affects the therapeutic index and reveals a major therapeutic advantage for the FR-targeted formulation. All DDS were able to abrogate doxorubicin-induced cardiotoxicity. This study constitutes the first side-by-side comparison of two receptor-targeted ligand-bearing systems, polymer therapeutics versus nanoparticulate systems, evaluated in the same mouse tumor model at several dosing regimens.

Purpose: A lipid-based prodrug of mitomycin C [MMC; 2,3-(distearoyloxy)propane-1-dithio-4′-benzyloxycarbonyl-MMC] was designed for liposome formulation. The purpose of this study was to examine the in vitro cytotoxicity, pharmacokinetics, in vivo toxicity, and in vivo antitumor activity of this new lipid-based prodrug formulated in polyethylene glycol–coated (pegylated) liposomes. Experimental Design: MMC was released from the MMC lipid–based prodrug (MLP) by thiolytic-induced cleavage with a variety of thiol-containing reducing agents. MLP was incorporated with nearly 100% efficiency in cholesterol-free pegylated liposomes with hydrogenated phosphatidylcholine as the main component and a mean vesicle size of ∼90 nm. This formulation was used for in vitro and in vivo tests in rodents. Results: In vitro, the cytotoxic activity of pegylated liposomal MLP (PL-MLP) was drastically reduced compared with free MMC. However, in the presence of reducing agents, such as cysteine or N-acetyl-cysteine, its activity increased to nearly comparable levels to those of free MMC. Intravenous administration of PL-MLP in rats resulted in a slow clearance indicating stable prodrug retention in liposomes and long circulation time kinetics, with a pharmacokinetic profile substantially different from that of free MMC. In vivo, PL-MLP was ∼3-fold less toxic than free MMC. The therapeutic index and absolute antitumor efficacy of PL-MLP were superior to that of free MMC in the three tumor models tested. In addition, PL-MLP was significantly more active than a formulation of doxorubicin in pegylated liposomes (DOXIL) in the M109R tumor model, a mouse tumor cell line with a multidrug-resistant phenotype. Conclusions: Delivery of MLP in pegylated liposomes is a potential approach for effective treatment of multidrug-resistant tumors while significantly buffering the toxicity of MMC.

Coating of liposomes with polyethylene-glycol (PEG) by incorporation in the liposome bilayer of PEG-derivatized lipids results in inhibition of liposome uptake by the reticulo-endothelial system and significant prolongation of liposome residence time in the blood stream. Parallel developments in drug loading technology have improved the efficiency and stability of drug entrapment in liposomes, particularly with regard to cationic amphiphiles such as anthracyclines. An example of this new generation of liposomes is a formulation of pegylated liposomal doxorubicin known as Doxil or Caelyx, whose clinical pharmacokinetic profile is characterized by slow plasma clearance and small volume of distribution. A hallmark of these long-circulating liposomal drug carriers is their enhanced accumulation in tumors. The mechanism underlying this passive targeting effect is the phenomenon known as enhanced permeability and retention (EPR) which has been described in a broad variety of experimental tumor types. Further to the passive targeting effect, the liposome drug delivery platform offers the possibility of grafting tumor-specific ligands on the liposome membrane for active targeting to tumor cells, and potentially intracellular drug delivery. The pros and cons of the liposome platform in cancer targeting are discussed vis-à-vis nontargeted drugs, using as an example a liposome drug delivery system targeted to the folate receptor.

Targeting of liposomes with phospholipid-anchored folate conjugates is an attractive approach to deliver chemotherapeutic agents to folate receptor (FR) expressing tumors. The use of polyethylene glycol (PEG)-coated liposomes with folate attached to the outer end of a small fraction of phospholipid-anchored PEG molecules appears to be the most appropriate way to combine long-circulating properties critical for liposome deposition in tumors and binding of liposomes to FR on tumor cells. Although a number of important formulation parameters remain to be optimized, there are indications, at least in one ascitic tumor model, that folate targeting shifts intra-tumor distribution of liposomes to the cellular compartment. In vitro, folate targeting enhances the cytotoxicity of liposomal drugs against FR-expressing tumor cells. In vivo, the therapeutic data are still fragmentary and appear to be formulation- and tumor model-dependent. Further studies are required to determine whether folate targeting can confer a clear advantage in efficacy and/or toxicity to liposomal drugs.

Liposomes have raised considerable interest as drug carriers in cancer chemotherapy [1] given their ability to alter the biodistribution and control the release rate of encapsulated drugs. Their ve...

Liposomes have tremendous potential as drug carriers in the treatment of cancer. However, despite enhanced tumor drug delivery and decreased toxicity, patient survival rates have not improved significantly compared to corresponding free drug treatments. Importantly, we found that a liposomal nanoparticle currently used as a drug carrier in cancer patients enhanced tumor growth in an immune competent murine model of cancer. This was associated with increased tumor angiogenesis and suppression of antitumor immune responses as indicated by decreased cytokine production by tumor macrophages and cytotoxic T cells, diminished tumor infiltration of tumor-specific T cells, and decreased number of dendritic cells in tumor draining lymph nodes. These results suggest that carrier-induced immunosuppression and angiogenesis have the potential to reduce the antitumor effects of drugs loaded within. These findings may have significant implications for the current use and future development of anticancer nanoparticles and further investigations are urgently needed. From the Clinical Editor This study discusses important implications of nanoliposome-based drug delivery systems in cancer therapy, and demonstrates that nanoliposomes may have immunosuppressive and angiogenetic properties, directly counterbalancing their anti-cancer activity, which may also have important clinical implications related to more widespread applications of such systems.

BACKGROUND Stealth liposomes hold promise as a mode of delivering cytotoxic agents selectively to tumors in cancer patients. The objective of this study was to determine whether stealth liposomal doxorubicin accumulates selectively in bone metastases based on clinical material obtained from two patients with breast carcinoma. METHODS Tumor tissue was obtained from two women (ages 33 years and 41 years, respectively) with metastatic breast carcinoma who responded to treatment with stealth liposomal doxorubicin and later underwent a surgical fixation procedure to treat a pathologic fracture of the femur. Drug levels in the tumor and adjacent muscle were examined by high performance liquid chromatography analysis in both patients and by fluorescence microscopy in one of the patients. RESULTS Bone tumor fragments obtained during surgery performed 6 days after the administration of the 12th course of stealth liposomal doxorubicin in 1 patient and 12 days after the administration of the 16th course of stealth liposomal doxorubicin in the second patient had a 10-fold greater concentration of liposomal doxorubicin than tumor free muscle. Doxorubicin fluorescence and specific nuclear staining showed good colocalization, thus confirming the presence of the liposome-delivered drug in the nuclei of tumor cells. CONCLUSIONS Using skeletal muscle as a comparator, stealth liposomal doxorubicin accumulates selectively in metastatic breast carcinoma cells within bone. Cancer 1999;86:72–8. © 1999 American Cancer Society.

Purpose. The aim of this study was to investigate the stability of doxorubicin encapsulated in polyethyleneglycol-coated liposomes (Doxil™) under UV-A light. Methods. High performance liquid chromatography and a fluorimetric method were used to quantify doxorubicin in bulk solution and doxorubicin in Doxil formulation. Results. The photodegradation of Doxil was significantly lower in comparison to the photodegradation of the free drug and showed no concentration dependency at the measured concentration range of 5−50 μg/ml. During and after UV-A irradiation, there was no leakage of the drug from liposomes to the medium. After induced leakage of doxorubicin from the liposomes by the ionophore nigericin, the degradation kinetics of Doxil were identical to that of free doxorubicin. Conclusions. High intraliposomal doxorubicin concentration and intraliposomal acidic pH are the two critical factors that protect DXR in Doxil from UV-A degradation.

The pharmacokinetics of liposome-encapsulated drugs are controlled by the interplay of two variables: the rate of plasma clearance of the liposome carrier, and the stability of the liposome-drug association in the blood stream. The pharmacokinetic properties of the liposomal drug, the vesicle size of the liposome carrier and the vascular permeability of individual tissues will determine the extravasation and biodistribution profile. The pharmacokinetics of polyethylene-glycol-(PEG)-liposomal doxorubicin are characterized by an extremely long circulating half-life, slow plasma clearance and reduced volume of distribution compared to free doxorubicin. These carrier systems show an improved extravasation profile with enhanced localization in tumors and superior therapeutic efficacy in comparison to doxorubicin in free form. These properties are the result of an optimized liposome composition and of a special drug-loading method which produces stable and long-circulating carriers. In clinical studies, doxorubicin encapsulated in PEG-coated liposomes shows a unique pharmacokinetic-toxicity profile and promising antitumor activity.

Inhibition of the rapid uptake of liposomes by the reticulo-endothelial system (RES) and reduction of the rate of drug leakage have resulted in long-circulating liposomal drug systems with valuable pharmacologic properties. Particularly, the coating of liposomes with polyethylene-glycol (PEG) confers optimal protection to the vesicles from RES-mediated clearance, while bilayer rigidification using high Tm phospholipids reduces the rate of leakage of liposome contents. These carrier systems display an improved extravasation profile with enhanced localization in tumors and possibly in other tissues, such as skin. An anti-cancer drug, doxorubicin, encapsulated in small-sized (< 100nm diameter), PEG-coated liposomes with a rigid bilayer shows a unique pharmacokinetic pattern, characterized by extremely long half-life, slow clearance, and small volume of distribution. Liposome longevity in circulation correlates positively with high drug levels in the extracellular tumor fluid and with enhanced therapeutic efficacy in a variety of tumor models regardless of the site of tumor growth. Examples of biodistribution studies will be presented for several murine tumors and human tumor xenografts inoculated by various routes, including a brain-implanted tumor. Liposome localization in tumors appears to be the result of an enhanced rate of extravasation through the abnormally permeable microvasculature of tumors coupled with an impaired lymphatic drainage. These results stress the potential of these long-circulating liposomal systems to manipulate the pharmacokinetics of anticancer drugs and enhance drug delivery to tumors. This therapeutic approach has been validated in AIDS-related Kaposi's sarcoma and is now undergoing extensive clinical testing in solid tumors.

Long-circulating (stealth) liposomes coated with polyethylene glycol (PEG), which show reduced uptake by the reticuloendothelial system (RES) and enhanced accumulation in tumours, were used for conjugation to monoclonal antibodies (MAbs) as a drug-targeting device. A MAb (N-12A5) directed against erbB-2 oncoprotein, a functional surface antigen, was used. Amplification and overexpression of the erbB-2 gene product, being unique to malignancy, confer onto this antibody-mediated therapy high tumour specificity. In vitro binding of [3H]cholesteryl ether ([3H]Chol ether) labelled anti-erbB-2 conjugated liposomes to N-87 cells (erbB-2-positive human gastric carcinoma) was compared with the binding of non-targeted liposomes and indicated a 16-fold increase in binding for the targeted liposomes. No difference in binding to OV1063 cells (erbB-2-negative human ovary carcinoma) was observed. These results indicate highly selective binding of antibody-targeted liposomes to erbB-2-overexpressing cells. Despite increased cell binding, doxorubicin (DOX) loaded in anti-erbB-2-conjugated liposomes did not cause increased in vitro cytotoxicity against N-87 cells, suggesting lack of liposome internalisation. In vivo, the critical factor needed to decrease the non-specific RES uptake and prolong the circulation time of antibody-conjugated liposomes is a low protein to phospholipid ratio ( < 60 micrograms mumol-1). Using these optimised liposome preparations loaded with DOX and by monitoring the drug levels and the [3H]Chol ether label, biodistribution studies in nude mice bearing subcutaneous implants of N-87 tumours were carried out. No significant differences in liver and spleen uptake between antibody-conjugated and plain liposomes were observed. Nevertheless, there was no enhancement of tumour liposome levels over plain liposomes. Both liposome preparations considerably enhanced DOX concentration in the tumour compared with free drug administration. Therapeutic experiments with N-87 tumour-bearing nude mice indicated that anti-tumour activity of targeted and non-targeted liposomes was similar, although both preparations had an increased therapeutic efficacy compared with the free drug. These studies suggest that efficacy is dependent on drug delivery to the tumour and that the rate-limiting factor of liposome accumulation in tumours is the liposome extravasation process, irrespective of liposome affinity or targeting to tumour cells.

The effect of liposome composition on drug delivery to tumors and therapeutic efficacy of liposome-encapsulated anthracyclines was investigated in two murine tumor models: an ascitic tumor (J6456 lymphoma) and a solid carcinoma (M-109). Longevity in circulation correlated positively with high drug levels in the extracellular (ascitic) tumor fluid and with delayed peak tumor levels. Using polyethylene-glycol(PEG)-coated liposomes, liposome stability (drug retention) was found to be an important determinant of therapeutic efficacy, as indicated by the superior survival conferred by high Tm phosphatidylcholines (hydrogenated, dipalmitoyl) over low Tm (egg phosphatidyl-choline). Replacing PEG with another negatively-charged surface headgroup (phosphatidyl-glycerol, phosphatidyl-inositol) resulted in relatively shorter longevity in circulation of the liposome-associated drug, but no detectable differences in anti-tumor efficacy. When neither the surface charged headgroup nor the PEG coating are present, the resulting drug formulation was significantly less effective than PEG and phosphatidylinositol-based formulations in both tumor models. In conclusion, longevity in circulation, as obtained with PEG coating, tends to improve the therapeutic efficacy of liposome-encapsulated anthracyclines. The current therapeutic models were however unable to detect differences between the therapeutic activity of PEG and other liposome formulations with relatively small differences in circulation longevity.

This short paper describes the problems, and their solutions, of formulating liposomal dosage forms for clinical use, as exemplified in tumor therapy by liposomal doxorubicin. We will demonstrate that the development of an efficacious drug delivery system requires fitting the delivery system to the micro-anatomy of the tumor. Many tumors have gaps in their capillarics which permit particles smaller than 120 nm to slowly extravasate into the tumor. Therefore small liposomes having a prolonged circulation time may be suitable drug carriers for tumor therapy. Recently a liposomal doxorubicin formulation (DOX-SL™) was recommended for approval by the US FDA, with indications of treating Kaposi's sarcoma. These liposomes seem to be efficacious in other types of tumors as well. The unique features of this formulation include: control of the biodistribution and pharmacokinetics of the intact liposomes through liposome lipid composition, especially the role of steric stabilization of the liposomes which e...

The pharmacokinetics and biodistribution of liposome-encapsulated drugs are controlled by the interplay of two variables: the rate of plasma clearance of the liposome carrier, and the stability of the liposome-drug association in circulation. Inhibition of the rapid uptake of liposomes by the reticuloendothelial system and reduction of the rate of drug leakage have resulted in long-circulating liposomal drug systems with valuable pharmacologic properties. These carrier systems show an improved extravasation profile with enhanced localization in tumors and possibly in other tissues, such as skin. An anticancer drug, doxorubicin, encapsulated in polyethyleneglycol-coated, long-circulating liposomes, shows a unique pharmacokinetic/toxicity pattern and promising antitumor activity in initial clinical studies.

In this chapter, we will address the therapeutic potential of targeting liposomes and polymers to the folate receptor for delivery of anticancer drugs and review the experimental data in this field. By exploiting the pharmacologic attributes of liposomes and polymers together with their cellular internalization by folate receptor-mediated endocytosis, these systems should allow for improved selectivity and control of drug delivery to folate receptor expressing cancers. Particularly, extension of circulation time and enhanced deposition in tumors coupled with reduced toxicity of liposome- and polymer-associated drugs are important assets of the nanomedicine platform when compared to free drugs or low-molecular weight conjugates. In addition, the high drug:ligand ratio of liposomes and some polymers enable the delivery of a high drug payload per ligand–receptor interaction. However these systems have also some in vivo limitations that will be discussed. It remains unclear whether folate-targeted nanomedicines can provide a significant added value in clinical applications vis-a-vis folate-targeted drug conjugates.

We report here the design, development and performance of a novel formulation of liposome- encapsulated glucocorticoids (GCs). A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate. We demonstrate fabrication principles that derive from the physicochemical properties of the GC and the liposomal lipids, which play a crucial role in GC release rate and kinetics. These principles allow fabrication of formulations that exhibit either a fast, second-order (t(1/2) ~1 h), or a slow, zero-order release rate (t(1/2) ~ 50 h) kinetics. A high therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies.

Doxorubicin (DOX) has been encapsulated with high efficiency in the water phase of small-sized lipid vesicles. Plasma-induced drug leakage from these vesicles is minimal when hydrogenated phosphatidylcholine is present as the main component. A prolonged circulation time of liposome-encapsulated DOX is observed in animal models when a small fraction of polyethyleneglycol-derivatized phospholipid (PEG) is present in the liposome bilayer. Using these PEG-coated liposomes, we found that the concentration of DOX in tumor implants of the mouse M-109 carcinoma is significantly enhanced by liposome delivery. The antitumor activity of liposome-encapsulated DOX in a lung metastases model of the M-109 carcinoma is superior to that of free DOX. The minimal lethal dose of DOX to tumor-free mice was substantially increased by encapsulation in PEG-coated liposomes, indicating that toxicity is reduced. We also found that the vesicant of DOX after intradermal injection is prevented by liposome encapsulation. These...

The pharmacokinetics of doxorubicin (DOX) encapsulated in liposomes containing polyethylene glycol-derivatized distearoylphosphatidylethanolamine (PEG/DSPE) were investigated in rodents and dogs. The plasma levels of DOX obtained with PEG/DSPE-containing liposomes were consistently higher than those without PEG/DSPE or when PEG/DSPE was replaced with hydrogenated phosphatidylinositol (HPI). Despite the inclusion of PEG/DSPE in liposomes, there was a significant drop in the plasma levels of DOX when the main phospholipid component, hydrogenated phosphatidylcholine, was replaced with lipids of lower phase transition temperature (dipalmitoylphosphatidylcholine, egg phosphatidylcholine), indicating that phase transition temperature affects the pharmacokinetics of liposome-encapsulated DOX. In beagle dogs, clearance was significantly slower for DOX encapsulated in PEG/DSPE-containing liposomes than in HPI-containing liposomes, with distribution half-lives of 29 and 13 hr, respectively. In both instances, almost 100% of the drug measured in plasma was liposome-associated. The apparent volume of distribution was only slightly above the estimated plasma volume of the dogs, indicating that drug leakage from circulating liposomes is insignificant and that the distribution of liposomal drug is limited mostly to the intravascular compartment in healthy animals.

There is significant inter-patient variability in the pharmacokinetics of pegylated liposomal doxorubicin (PLD). Identification of factors affecting the pharmacokinetics of PLD would enable personalization of therapy. We previously reported that age, gender, body composition, and monocytes affect the clearance of other liposomal agents. Therefore, we evaluated how these factors affect the pharmacokinetics of PLD.Pharmacokinetic studies of PLD were performed as part of phase I and II studies in 70 patients with solid tumors or Kaposi's sarcoma. The effects of monocyte count, age, gender, and body composition on PLD clearance were examined.There was a 15.3-fold variability in PLD clearance. Body surface area-based dosing did not significantly reduce the variability in PLD clearance. The mean ± SD clearance for patients60 years old and ≥60 years old were 54.6 ± 28.5 and 23.3 ± 10.8 mL/h/m(2), respectively (P 0.0001), and for female and male patients were 23.7 ± 18.8 and 55.6 ± 26.8 mL/h/m(2), respectively (P 0.0001). A reduction in pre-cycle monocyte count was associated with a greater reduction in PLD clearance.Age, gender, and monocyte counts appear to correlate with PLD clearance. Further investigation of the association between these factors, PLD pharmacokinetics, and clinical outcomes (efficacy and toxicity) is warranted. These effects on the pharmacokinetics of PLD may be an approach for personalizing PLD therapy and may affect other pegylated liposomes and nanoparticle agents.

Introduction Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, is a potent inhibitor of farnesyl-pyrophosphate synthase with poor in vitro cytotoxic activity as a result of its limited diffusion into tumor cells. The purpose of this study was to investigate whether liposomes targeted to the folate receptor (FR) can effectively deliver ZOL to tumor cells and enhance its in vitro cytotoxicity. Methods ZOL was entrapped in the water phase of liposomes of various compositions with or without a lipophilic folate ligand. Stability and blood levels after i.v. injection were checked. The in vitro cytotoxic activity and cell uptake of liposomal ZOL (L-ZOL) were examined on various human and mouse cell lines. Results All formulations were highly stable and resulted in high blood levels in contrast to free ZOL which was rapidly cleared from plasma. Non-targeted L-ZOL was devoid of any in vitro activity at concentrations up to 200 µM. In contrast, potent cytotoxic activity of folate-targeted L-ZOL (FTL-ZOL) was observed, with optimal activity, reaching the sub-micromolar range, for dipalmitoyl-phosphatidylglycerol (DPPG)-containing liposomes and relatively lower activity for pegylated (PEG) formulations. IC50 values of FTL-ZOL on FR-expressing tumor cells were > 100-fold lower than those of free ZOL. Compared to doxorubicin, the cytotoxicity of DPPG-FTL-ZOL was equivalent in drug-sensitive cell lines, and greatly superior in drug-resistant cell lines. When tested on the non-FR upregulated cell lines, the cytotoxicity of FTL-ZOL was lower but still superior to that of L-ZOL. The uptake of ZOL by FR-expressing tumor cells was enhanced ∼ 25-fold with DPPG-FTL-ZOL, and only ∼ 4-fold with PEG-FTL-ZOL. Conclusions FR targeting of ZOL using liposomes is an effective means to exploit the tumor cell growth inhibitory properties of ZOL. DPPG-FTL-ZOL is significantly more efficient at intracellular delivery of ZOL than PEG-FTL-ZOL in FR-expressing tumor cells.

We have investigated the in vitro cytotoxicity of free doxorubicin (DOX) and liposome-entrapped DOX (L-DOX) against a human ovarian carcinoma cell line (OV-1063) using a colorimetric assay. DOX was encapsulated in the inner water phase of liposomes by an ammonium sulfate-generated proton gradient. Liposomes varied in phospholipid composition but were of a similar size. It was found that the cytotoxic activity of L-DOX is substantially decreased when liposomes containing phospholipids of high phase-transition temperature (Tm) are used. The type of negatively charged headgroup did not have any significant influence on the cytotoxicity observed. Experiments using resin beads that bind free and protein-bound DOX, but do not interact with L-DOX, indicated that the cytotoxic effect is mediated by the release of drug from the liposomes into the extracellular medium; no evidence was found for direct cellular uptake of liposome-encapsulated drug. The use of the ionophore nigericin to induce the release of DOX from high-Tm liposomes increased cytotoxicity to a level comparable to free DOX, suggesting that 'remote release' techniques may substantially improve the efficiency of liposome-mediated drug delivery and allow for the full exploitation of the favorable pharmacokinetic properties of specific high-Tm formulations.

A liposome-associated doxorubicin formulation that has been utilized in two phase I clinical trials at two separate medical centers is described. This work deals with formulation optimization, process development, quality control assays on raw materials, characterization assays for these liposomes, and their short-term stability. One of the main goals of this work is to provide a model for the production of liposomal formulations of sufficient quantity and quality to be used in clinical trials.

The effect of negative surface charge and hydrophilic groups on liposome clearance from blood was investigated in mice using liposome-entrapped 67gallium-deferoxamine as a label. The presence of negatively-charged lipids may retard or accelerate liposome clearance. Physicochemical features contributing to optimal retardation of liposome clearance include a hydrophilic carbohydrate moiety and a sterically hindered negatively-charged group. The relevance of the negative charge steric effect is suggested by the finding that phosphatidylinositol phosphate (PIP) and trisialoganglioside (GT1) are less effective than phosphatidylinositol (PI) and monosialoganglioside (GM1), respectively, in retarding liposome clearance. The need for negative charge in addition to the carbohydrate group for optimal effect on retardation of clearance is indicated by the observation that asialoganglioside (AGM1) is less effective than GM1 in this respect. The negative charge effect is observed with liposome bilayers having both low and high temperature phase-transitions. Increasing the molar fraction of negatively-charged lipid (hydrogenated PI derived from soya) from 23 to 41% resulted in a dramatic acceleration of liposome clearance. The clearance-accelerating effect of the high negative charge was specifically directed to the liver with selective reduction of spleen uptake. Increasing liposome size also had an accelerating effect on clearance but in this case it was accompanied by a non-specific concomitant increase of both liver and spleen uptake.

Pharmacokinetic and imaging studies in 19 patients receiving liposome-entrapped adriamycin (L-ADM) were carried out within the framework of a Phase I clinical trial (Gabizon et al., 1989a). The formulation of L-ADM tested consisted of 0.2 microM-extruded multilamellar vesicles composed of egg phosphatidylcholine, egg-derived phosphatidyl-glycerol (PG), cholesterol, and ADM intercalated in the fluid lipid bilayer. Plasma clearance of total drug extracted from the plasma after L-ADM infusion followed a biexponential curve with a pattern similar to that reported for free ADM. The plasma concentration of drug circulating in liposome-associated from was also measured in a subgroup of seven patients. Liposome-associated drug was found to be rapidly cleared from plasma. Its ratio to non-liposome-associated drug appeared to correlate with liver reserve, with highest ratios in patients with normal liver function. Liposome clearance, as measured by the plasma concentration of PG in three patients was slower than the clearance of liposome-associated ADM, suggesting that liposomes lose part of their drug payload during circulation. To learn about the liposome organ distribution, imaging studies were carried out with 111Indium-deferoxamine labelled liposomes of the same composition. Liposomes were cleared predominantly by liver and spleen and to a lesser extent by bone marrow in seven out of nine patients. In two patients with active hepatitis and severe liver dysfunction, there was minimal liver uptake and increased spleen and bone marrow uptake. Except for one hepatoma patient, intrahepatic and extrahepatic tumours were not imaged by liposomes, suggesting that liposome uptake is restricted to cells of the reticulo-endothelial system (RES).(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 6 Figure 7 Figure 8 Figure 9 Figure 10

Therapeutic applications of intravenously injected liposomes have been limited by their rapid clearance from the bloodstream and their uptake by the macrophage cells of the liver and spleen (RES). Recently, however, liposomes which substantially evade the rapid uptake by the RES have been introduced. Since these liposomes exhibit dramatically different pharmacokinetics and biodistribution, new therapeutic opportunities have appeared. These include enhanced efficacy of antineoplastic agents against tumors, sites of inflammation, and targeting ligand-coupled liposomes to extravascular targets. Despite extensive experimental work, the mechanism underlying the ability of liposomes to avoid the rapid uptake by the RES is still not fully understood. Our approach is an alternative to seeking the answers in complex differential interactions of liposomes with various components of blood. We believe that the effect can be easily explained, at least in qualitative terms, by the fundamental principles of colloid stability. In this communication, we propose that steric stabilization of liposomes is responsible for their prolonged circulation times. We propose that stabilization results from local surface concentration of highly hydrated groups that sterically inhibit both electrostatic and hydrophobic interactions of a variety of blood components at the liposome surface.

The ability of low frequency ultrasound (LFUS) to trigger the release of drugs from nano sterically stabilized liposomes (nSSL) in vitro, without affecting the drugs' chemical integrity or biological potency, has been previously shown. Herein, the ability of LFUS to (a) trigger the release of cisplatin from nSSL in vivo, and (b) affect the therapeutic efficacy by locally releasing the drug, was studied. For this, nSSL loaded with the anti-cancer chemotherapeutic agent cisplatin were injected intraperitoneally (i.p.) to mice bearing well-developed J6456 murine lymphoma tumors in their peritoneal cavity. Then, LFUS was applied externally to the abdominal wall for 120 s, and drug release was quantified. Nearly 70% of the liposomal cisplatin was released in tumors exposed to LFUS, compared to

Pegylated liposomal doxorubicin (PLD) is a liposomal formulation with a distinct pharmacokinetic profile characterized by an extended circulation time and a reduced volume of distribution. Biodistribution animal studies indicate preferential accumulation of PLD into various implanted mouse-human tumors, with an enhancement of liposomal drug tumor levels compared with free drugs. The extended circulation time of pegylated liposomes and their ability to extravasate through the leaky vasculature of tumors results in the enhanced delivery of liposomal drug and/or radiotracers to the tumor site in patients with cancer. In malignant effusions, Kaposi sarcoma skin lesions, and a variety of solid tumors there is evidence of selective tumor uptake detected by various methods. Pegylated liposomal doxorubicin has been approved for clinical use in a variety of neoplastic conditions because of its antitumor efficacy and unique safety profile with an impressive reduction of cardiac toxicity in comparison with conventional doxorubicin.