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Liposomal nanoparticles are the most commonly used drug nano-delivery platforms. However, recent reports show that certain pegylated liposomal nanoparticles (PLNs) and polymeric nanoparticles have the potential to enhance tumor growth and inhibit antitumor immunity in murine cancer models. We sought herein to identify the mechanisms and determine whether PLN-associated immunosuppression and tumor growth can be reversed using alendronate, an immune modulatory drug. By conducting in vivo and ex vivo experiments with the immunocompetent TC-1 murine tumor model, we found that macrophages were the primary cells that internalized PLN in the tumor microenvironment and that PLN-induced tumor growth was dependent on macrophages. Treatment with PLN increased immunosuppression as evidenced by increased expression of arginase-1 in CD11b+Gr1+ cells, diminished M1 functionality in macrophages, and globally suppressed T-cell cytokine production. Encapsulating alendronate in PLN reversed these effects on myeloid cells and shifted the profile of multi-cytokine producing T-cells towards an IFNγ+ perforin+ response, suggesting increased cytotoxic functionality. Importantly, we also found that PLN-encapsulated alendronate (PLN-alen), but not free alendronate, abrogated PLN-induced tumor growth and increased progression-free survival. In summary, we have identified a novel mechanism of PLN-induced tumor growth through macrophage polarization and immunosuppression that can be targeted and inactivated to improve the anticancer efficacy of PLN-delivered drugs. Importantly, we also determined that PLN-alen not only reversed protumoral effects of the PLN carrier, but also had moderate antitumor activity. Our findings strongly support the inclusion of immune-responsive tumor models and in-depth immune functional studies in the preclinical drug development paradigm for cancer nanomedicines, and the further development of chemo-immunotherapy strategies to co-deliver alendronate and chemotherapy for the treatment of cancer.

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.

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.

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.

Quantum dots (QDs) are fluorescent semiconductor nanocrystals with superior optical properties compared to organic dyes currently undergoing rapid development for biological applications, particularly in fluorescence imaging. The folate receptor, overexpressed in a broad spectrum of malignant tumors, is an attractive target for selective delivery of imaging agents to tumor cells. This study examines nanoparticles containing QDs entrapped in a lipid shell, and post-loaded with a folate-lipid conjugate for targeting to mouse and human tumor cells expressing the folate receptor. Hydrophobic QDs were mixed with 1,2 dipalmitoyl-sn-glycero-3 phosphocholine and methoxy-polyethylene-glycol-distearoyl-phosphatidyl-ethanolamine (mPEG-DSPE) generating a nanoparticle referred to as lipodot, with a mean diameter size of approximately 100 nm. Folate-derivatized PEG-DSPE was post-loaded into the lipodots at 0.5% lipid molar concentration. Mouse J6456 lymphoma cells (J6456-FR) and human head and neck KB cancer cells (KB-FR), up-regulated for their folate receptors, were incubated with folate-targeted and non-targeted lipodots in vitro. Using fluorescence microscopy, it was found that only folate-targeted lipodots were taken up by tumor cells. Confocal depth scanning showed substantial internalization. Confirming the specificity of folate-targeted lipodots, binding and internalization were inhibited by free folate, and no uptake was found in a folate-receptor negative cell line. Selective binding and uptake of folate-targeted lipodots by J6456-FR cells was also observed in vivo after intra-peritoneal injection in mice bearing ascitic J6456-FR tumors based on FACS analysis and confocal imaging of harvested cells from the peritoneal cavity. Folate-targeted lipodots represent an attractive approach for tumor cell labeling both in vitro and in vivo.

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.