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The intricate cooperation between cancer cells and nontumor stromal cells within melanoma microenvironment (MME) enables tumor progression and metastasis. We previously demonstrated that the interplay between tumor-associated macrophages (TAMs) and melanoma cells can be disrupted by using long-circulating liposomes (LCLs) encapsulating prednisolone phosphate (PLP) (LCL-PLP) that inhibited tumor angiogenesis coordinated by TAMs. In this study, our goal was to improve LCL specificity for protumor macrophages (M2-like (i.e., TAMs) macrophages) and to induce a more precise accumulation at tumor site by loading PLP into IL-13-conjugated liposomes (IL-13-LCL-PLP), since IL-13 receptor is overexpressed in this type of macrophages. The IL-13-LCL-PLP liposomal formulation was obtained by covalent attachment of thiolated IL-13 to maleimide-functionalized LCL-PLP. C57BL/6 mice bearing B16.F10  s.c melanoma tumors were used to investigate the antitumor action of LCL-PLP and IL-13-LCL-PLP. Our results showed that IL-13-LCL-PLP formulation remained stable in biological fluids after 24h and it was preferentially taken up by M2 polarized macrophages. IL-13-LCL-PLP induced strong tumor growth inhibition compared to nonfunctionalized LCL-PLP at the same dose, by altering TAMs-mediated angiogenesis and oxidative stress, limiting resistance to apoptosis and invasive features in MME. These findings suggest IL-13-LCL-PLP might become a promising delivery platform for chemotherapeutic agents in melanoma.

Anti-angiogenic therapies for melanoma have not yet been translated into meaningful clinical benefit for patients, due to the development of drug-induced resistance in cancer cells, mainly caused by hypoxia-inducible factor 1α (HIF-1α) overexpression and enhanced oxidative stress mediated by tumor-associated macrophages (TAMs). Our previous study demonstrated synergistic antitumor actions of simvastatin (SIM) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on an in vitro melanoma model via suppression of the aggressive phenotype of melanoma cells and inhibition of TAMs-mediated angiogenesis. Therefore, we took the advantage of long circulating liposomes (LCL) superior tumor targeting capacity to efficiently deliver SIM and DMXAA to B16.F10 melanoma in vivo, with the final aim of improving the outcome of the anti-angiogenic therapy. Thus, we assessed the effects of this novel combined tumor-targeted treatment on s.c. B16.F10 murine melanoma growth and on the production of critical markers involved in tumor development and progression. Our results showed that the combined liposomal therapy almost totally inhibited (> 90%) the growth of melanoma tumors, due to the enhancement of anti-angiogenic effects of LCL-DMXAA by LCL-SIM and simultaneous induction of a pro-apoptotic state of tumor cells in the tumor microenvironment (TME). These effects were accompanied by the partial re-education of TAMs towards an M1 phenotype and augmented by combined therapy-induced suppression of major invasion and metastasis promoters (HIF-1α, pAP-1 c-Jun, and MMPs). Thus, this novel therapy holds the potential to remodel the TME, by suppressing its most important malignant biological capabilities., (© 2021. The Author(s).)

Regardless of recent progress, melanoma is very difficult to treat, mainly due to the drug resistance modulated by tumor cells as well as by the tumor microenvironment (TME). Among the immune cells recruited at the tumor site, tumor associated macrophages (TAMs) are the most abundant, promoting important tumorigenic processes: angiogenesis, inflammation and invasiveness. Furthermore, it has been shown that TAMs are involved in mediating the drug resistance of melanoma cells. Thus, in the present study, we used liposomal formulation of prednisolone disodium phosphate (LCL-PLP) to inhibit the protumor function of TAMs with the aim to sensitize the melanoma cells to the cytotoxic drug doxorubicin (DOX) to which human melanoma has intrinsic resistance. Consequently, we evaluated the in vivo effects of the concomitant administration of LCL-PLP and liposomal formulation of DOX (LCL-DOX) on B16.F10 melanoma growth and on the production of key molecular markers for tumor development. Our results demonstrated that the concomitant administration of LCL-PLP and LCL-DOX induced a strong inhibition of tumor growth, primarily by inhibiting TAMs-mediated angiogenesis as well as the tumor production of MMP-2 and AP-1. Moreover, our data suggested that the combined therapy also affected TME as the number of infiltrated macrophages in melanoma microenvironment was reduced significantly.

Background: Emerging treatment options for colon cancer are needed to overcome the limitations regarding the side effects of current chemotherapeutics and drug resistance. The goal of this study was to assess the antitumor actions of PEGylated long-circulating liposomes (LCL) co-delivering curcumin (CURC) and doxorubicin (DOX) on murine colon carcinoma cells (C26)., Methods: The cytotoxicity of CURC and DOX, administered alone or in combination, either in free or LCL form, was evaluated with regard to antiproliferative effects on C26 cells and to protumor processes that might be affected., Results: Our results indicated that PEGylated LCL-CURC-DOX exerted strong antiproliferative effects on C26 cells, slightly exceeding those induced by free CURC-DOX, but higher than either agent administered alone in their free form. These effects of LCL-CURC-DOX were due to the inhibition of the production of angiogenic/inflammatory proteins in a NF-κB-dependent manner, but were independent of ROS production or AP-1 c-Jun activation. Notable, the anti-angiogenic actions of LCL-CURC-DOX appeared to be much stronger than those induced by the co-administration of CURC and DOX in their free form, on C26 colon cancer cells., Conclusion: LCL-CURC-DOX demonstrated enhanced cytotoxicity on C26 murine colon cancer cells by inhibiting the production of the majority of factors involved in tumor-associated angiogenesis and inflammation and is now being evaluated in vivo regarding its efficacy towards tumor growth in colon cancer., (Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Quality by design principles (QbD) were used to assist the formulation of prednisolone-loaded long-circulating liposomes (LCL-PLP) in order to gain a more comprehensive understanding of the preparation process. This approach enables us to improve the final product quality in terms of liposomal drug concentration, encapsulation efficiency and size, and to minimize preparation variability. A 19-run D-optimal experimental design was used to study the impact of the highest risk factors on PLP liposomal concentration (Y 1 - μg/ml), encapsulation efficiency (Y 2 -%) and size (Y 3 -nm). Out of six investigated factors, four of them were identified as critical parameters affecting the studied responses. PLP molar concentration and the molar ratio of DPPC to MPEG-2000-DSPE had a positive impact on both Y 1 and Y 2 , while the rotation speed at the formation of the lipid film had a negative impact. Y 3 was highly influenced by prednisolone molar concentration and extrusion temperature. The accuracy and robustness of the model was further on confirmed. The developed model was used to optimize the formulation of LCL-PLP for efficient accumulation of the drug to tumor tissue. The cytotoxicity of the optimized LCL-PLP on C26 murine colon carcinoma cells was assessed. LCL-PLP exerted significant anti-angiogenic and anti-inflammatory effects on M2 macrophages, affecting indirectly the C26 colon carcinoma cell proliferation and development.

Statins possess antitumor actions at doses 100- to 500-fold higher than those needed to lower cholesterol levels. Thus, the antitumor efficacy of statins could be improved greatly by using tumor-targeted delivery systems. Therefore the present work aims to investigate the antitumor activity of long-circulating liposome-encapsulated simvastatin (LCL-SIM) versus free SIM in B16.F10 murine melanoma-bearing mice. Our results showed that LCL-SIM inhibits strongly the B16.F10 melanoma growth (by 85%) whereas free SIM was ineffective. Moreover, the antitumor activity of LCL-SIM depends on the presence of functional tumor-associated macrophages (TAM) in tumor tissue and is mainly based on the reduction of the TAM-mediated oxidative stress as well as of the production of the hypoxia-inducible factor 1 α (HIF-1 α) in tumors. In conclusion, our findings suggest that the antitumor activity of LCL-SIM on B16.F10 melanoma growth is a result of the tumor-targeting property of the liposome formulation and is tightly dependent on the presence of TAM in tumor tissue., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

This study evaluates whether the inhibitory effects of prednisolone phosphate (PLP) encapsulated in long-circulating liposomes (LCL-PLP) on tumor growth and tumor angiogenesis described previously can be generalized to other types of glucocorticoids (GC) encapsulated in LCL (LCL-GC). Four types of synthetic GC, i.e. budesonide disodium phosphate (BUP), dexamethasone disodium phosphate (DXP), methylprednisolone disodium phosphate (MPLP), and PLP, were selected based on the difference in their potency to activate the human glucocorticoid receptor. The effects of all LCL-GC on the production of angiogenic/inflammatory factors in vivo in the B16.F10 murine melanoma model as well as on the viability and proliferation of tumor cells and endothelial cells in vitro were investigated. Our results show that all four selected LCL-GC formulations inhibit tumor growth, albeit to different degrees. The differences in antitumor activity of LCL-GC correlate with their efficacy to suppress tumor angiogenesis and inflammation. The strongest antitumor effect is achieved by LCL-encapsulated BUP (LCL-BUP), due to the highest potency of BUP versus the other three GC types. The in vitro results presented herein suggest that LCL-BUP has strong cytotoxic effects on B16.F10 melanoma cells and the anti-proliferative effects of all LCL-GC towards angiogenic endothelial cells play a role in their antitumor activity.

Prednisolone disodium phosphate (PLP) encapsulated in long-circulating liposomes (LCL) (LCL-PLP) inhibited tumor growth by 80-90% after a single dose of 20 mg/kg, whereas PLP in the free form was completely ineffective at the same single dose. To generalize our findings with LCL-PLP, the antitumor activity and side effects of LCL containing synthetic glucocorticoids (LCL-GC) other than PLP were investigated. In addition to PLP, budesonide disodium phosphate, dexamethasone disodium phosphate, and methylprednisolone disodium phosphate were selected based on the difference in their potency to activate the human glucocorticoid receptor. The present study shows that the tumor localization of each GC is governed by the transport capacity of the LCL composed of dipalmitoylphosphatidylcholine, cholesterol, and polyethylene glycol 2000-distearoylphosphatidylethanolamine in a molar ratio of 1.85:1.0:0.15. The antitumor potency of the LCL-GC strongly depends on the potency of the type of GC encapsulated. LCL-encapsulated budesonide disodium phosphate (LCL-BUP) had the highest antitumor activity which is likely due to the much higher potency of BUP encapsulated in LCL versus the other three GC types. The high potency of LCL-BUP confers the risk for occurrence of strong side effects. However, at the dose of 3 mg/kg, LCL-BUP was highly efficacious without the occurrence of adverse effects.

Glucocorticoids can inhibit solid tumor growth via downregulation of tumor-associated inflammation/angiogenesis. In this minireview we describe the possible mechanisms of glucocorticoid action in tumor growth inhibition. We also present an overview of the current status of tumor-targeted glucocorticoid delivery. It appears that long-circulating liposomes are the only targeting system currently being explored for this purpose.

Prednisolone phosphate (PLP) encapsulated in long-circulating liposomes can inhibit tumor growth after intravenous administration (i.v.). These antitumor effects of liposomal PLP are the result of the tumor-targeting property of the liposome formulation. The mechanism by which liposomal PLP inhibits tumor growth is unclear. We investigated the effects of liposome-encapsulated PLP versus free PLP on angiogenic protein production in tumor tissue in vivo and on viability and proliferation of tumor and endothelial cells in vitro. In vivo, liposomal PLP had a stronger reducing effect on pro-angiogenic protein levels than free PLP, whereas levels of anti-angiogenic proteins were hardly affected. Cell viability was only slightly affected with either treatment. Liposomal PLP had strong anti-proliferative effects on human umbilical vein endothelial cells, whereas free PLP had hardly any effect. Taken together, the present study points to a strong inhibitory effect of liposomal PLP on tumor angiogenesis by reduction of the intratumoral production of the majority of pro-angiogenic factors studied and direct inhibition of endothelial cell proliferation, which is the result of high prolonged levels of prednisolone in the tumor by liposomal delivery.

Glucorticoids are potent drugs that have a multitude of pharmacological actions both at genomic and non-genomic levels. Many of the diseases in which glucorticoids are routinely administered are featured by angiogenesis and enhanced capillary permeability, permitting targeted delivery using long-circulating drug delivery systems. By encapsulation of glucorticoids in long-circulating liposomes, drug levels at the site of the pathology are markedly higher, increasing and prolonging therapeutic efficacy in models of rheumatoid arthritis, multiple sclerosis, and cancer.