Novel liposomal glatiramer acetate: Preparation and immunomodulatory evaluation in murine model of multiple sclerosis.

The frequent administration rate required for Glatiramer acetate (GA), a first-line therapy for Multiple sclerosis (MS), poses patient compliance issues. Only a small portion of the subcutaneously administered GA is available for phagocytosis by macrophages, as most of it is hydrolyzed at its administration site or excreted renally. To unravel these hurdles, we have prepared liposomal formulations of GA through thin film-hydration method plus extrusion. The clinical and histopathological efficacy of GA-loaded liposomes were assessed in prophylactic and therapeutic manners on murine model of MS (experimental autoimmune encephalomyelitis (EAE)). The selected GA liposomal formulation showed favorable size (275 nm on average), high loading efficiency, and high macrophage localization. Moreover, administration of GA-liposomes in mice robustly suppressed the inflammatory responses and decreased the inflammatory and demyelinated lesion regions in CNS compared to the free GA with subsequent reduction of the EAE clinical score. Our study indicated that liposomal GA could be served as a reliable nanomedicine-based platform to hopefully curb MS-related aberrant autoreactive immune responses with higher efficacy, longer duration of action, fewer administration frequencies, and higher delivery rate to macrophages. This platform has the potential to be introduced as a vaccine for MS after clinical translation and merits further investigations.

Surface-modified cationic liposomes with a matrix metalloproteinase-degradable polyethylene glycol derivative improved doxorubicin delivery in murine colon cancer.

PEGylation is a commonly used approach to prolong the blood circulation time of cationic liposomes. However, PEGylation is associated with the "PEG dilemma", which hinders binding and uptake into tumor cells. The cleavable PEG products are a possible solution to this problem. In the current research, doxorubicin-loaded cationic liposomes (Dox-CLs) surface-conjugated with a matrix metalloproteinase-2 (MMP-2)-sensitive octapeptide linker-PEG derivative were prepared and compared to non-PEGylated and PEGylated CLs in terms of size, surface charge, drug encapsulation and release, uptake, in vivo pharmacokinetics, and anticancer efficacy. It was postulated that PEG deshielding in response to the overexpressed MMP-2 in the tumor microenvironment increases the interaction of protected CLs with cellular membranes and improves their uptake by tumor cells/vasculature. MMP2-responsive Dox-CLs had particle sizes of ∼115-140 nm, surface charges of ∼+25 mV, and encapsulation efficiencies of ∼85-95%. In vitro cytotoxicity assessments showed significantly enhanced uptake and cytotoxicity of PEG-cleavable CLs compared to their non-cleavable PEG-coated counterparts or Caelyx®. Also, the chick chorioallantoic membrane assay showed great antiangiogenesis ability of Dox-CLs leading to target and prevent tumor neovascularization. Besides, in vivo studies showed an effective therapeutic efficacy of PEG-cleavable Dox-CLs in murine colorectal cancer with negligible hematological and histopathological toxicity. Altogether, our results showed that MMP2-responsive Dox-CLs could be served as a promising approach to improve tumor drug delivery and uptake.

Targeting the tumor microenvironment by liposomal Epacadostat in combination with liposomal gp100 vaccine.

Indoleamine-2,3-dioxygenase (IDO1) pathway has vital role in cancer immune escape and its upregulation leads to immunosuppressive environment which is associated with poor prognosis and progression in various cancers like melanoma. Previously, we showed the antitumoral efficacy of nanoliposomal form of Epacadostat (Lip-EPA), as an IDO1 inhibitor. Herein, we used Lip-EPA as a combination approach with liposomal gp100 (Lip-gp100) anti-cancer vaccine in melanoma model. Here, we showed that B16F10 tumor express IDO1 so using Lip-EPA will enhance the efficacy of vaccine therapy. The biodistribution of ICG-labelled liposomal form of EPA showed the remarkable accumulation of drug at tumor site. In an in vivo study, Lip-EPA enhanced the antitumor efficacy of Lip-gp100 in which the IDO mRNA expression was decreased (~ fourfold) in tumor samples. Also, we identified a significant increase in the number of infiltrated T lymphocytes (p < 0.0001) with enhanced in interferon gamma (IFN-γ) production (p < 0.0001). Additionally, Lip-EPA + Lip-gp100 significantly modulated intratumoral regulatory T cells which altogether resulted in the highest delay in tumor growth (TGD = 56.54%) and increased life span (ILS > 47.36%) in treated mice. Our study demonstrated that novel combination of Lip-EPA and Lip-gp100 was effective treatment with capability of being used in further clinical studies.

Doxorubicin-loaded liposomes surface engineered with the matrix metalloproteinase-2 cleavable polyethylene glycol conjugate for cancer therapy.

Background: Colorectal cancer is one of the prominent leading causes of fatality worldwide. Despite recent advancements within the field of cancer therapy, the cure rates and long-term survivals of patients suffering from colorectal cancer have changed little. The application of conventional chemotherapeutic agents like doxorubicin is limited by some drawbacks such as cardiotoxicity and hematotoxicity. Therefore, nanotechnology has been exploited as a promising solution to address these problems. In this study, we synthesized and compared the anticancer efficacy of doxorubicin-loaded liposomes that were surface engineered with the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-matrix metalloproteinase-2 (MMP-2) cleavable peptide-polyethylene glycol (PEG) conjugate. The peptide linker was used to cleave in response to the upregulated MMP-2 in the tumor microenvironment, thus exposing a positive charge via PEG-deshielding and enhancing liposomal uptake by tumor cells/vasculature. Liposomal formulations were characterized in terms of size, surface charge and morphology, drug loading, release properties, cell binding and uptake, and cytotoxicity. Results: The formulations had particle sizes of ~ 100-170 nm, narrow distribution (PDI ˂ 0.2), and various surface charges (- 10.2 mV to + 17.6 mV). MMP-2 overexpression was shown in several cancer cell lines (C26, 4T1, and B16F10) as compared to the normal NIH-3T3 fibroblast cells by gelatin zymography and qRT-PCR. In vitro results demonstrated enhanced antitumor efficacy of the PEG-cleavable cationic liposomes (CLs) as compared to the commercial Caelyx® (up to fivefold) and the chick chorioallantoic membrane assay showed their great antiangiogenesis potential to target and suppress tumor neovascularization. The pharmacokinetics and efficacy studies also indicated higher tumor accumulation and extended survival rates in C26 tumor-bearing mice treated with the MMP-2 cleavable CLs as compared to the non-cleavable CLs with no remarkable sign of toxicity in healthy tissues. Conclusion: Altogether, the MMP-2-cleavable CLs have great potency to improve tumor-targeted drug delivery and cellular/tumor-vasculature uptake which merits further investigation. Supplementary Information: The online version contains supplementary material available at 10.1186/s12645-023-00169-8.

Liposomal celecoxib combined with dendritic cell therapy enhances antitumor efficacy in melanoma.

Cancer vaccine efficacy is limited by the immunosuppressive nature of the tumor microenvironment created by inflammation, immune inhibitory factors, and regulatory T cells (Tregs). Inspired by the role of cyclooxygenase-2 (COX-2) in inflammation in the tumor site, we proposed that normalization of the tumor microenvironment by celecoxib as a COX-2 inhibitor might improve the efficacy of Dendritic Cell (DC) therapy in a melanoma model. In the present study, liposomal celecoxib (Lip-CLX) was combined with ex vivo generated DC vaccines pulsed with gp100 peptide (in liposomal and non-liposomal forms) for prophylactic and therapeutic evaluation in the B16F10 melanoma model. Tumor site analysis by flow cytometry demonstrated that intravenous administration of Lip-CLX at a dose of 1 mg/kg in four doses effectively normalized the tumor microenvironment by reducing Tregs and IL-10 production. Furthermore, in combination with DC vaccination (DC + Lip-peptide+Lip-CLX), it significantly increased tumor-infiltrating CD4+ and CD8+ T cells and secretion of IFN-γ. This combinatorial strategy produced an effective prophylactic and therapeutic antitumor response, which reduced tumor growth and prolonged the overall survival. In conclusion, our findings suggest that the liposomal celecoxib targets the inhibitory mechanisms of the tumor microenvironment and broadens the impact of DC therapy to improve the outcome of immunotherapy in solid tumors.

The comparison of biodistribution of glutathione PEGylated nanoliposomal doxorubicin formulations prepared by pre-insertion and post-insertion methods for brain delivery in normal mice.

Several obstacles limit the efficacy of brain tumour treatment, most notably the blood-brain barrier (BBB), which prevents the brain uptake of the majority of accessible medicines due to tight junctions. The presence of glutathione (GSH) receptors on the BBB surface has been demonstrated in numerous papers; consequently, products containing glutathione as a targeting ligand coupled with nanoliposomes are used to enhance drug delivery across the BBB. Here, the 5% pre-inserted PEG2000-GSH PEGylated liposomal doxorubicin was conducted according to 2B3-101 being tested in clinical trials. In addition, PEGylated nanoliposomal doxorubicin connected to the spacer-GSH targeting ligand (GSGGCE) and the PEG3400 was conducted using post-insertion method. Next, in vivo biodistribution of the produced formulations was tested on healthy mice to see if GSGGCE, as the targeted ligand, could cross the BBB compared to 5% pre-inserted PEG2000-GSH and Caelyx® . Compared to the pre-inserted formulation and Caelyx® , the post-inserted formulations' concentration was lower in the heart and higher in brain tissues, resulting in boosting the brain concentration of accumulated doxorubicin with fewer possible side effects, including cardiotoxicity. In comparison to the pre-insertion procedure, the post-insertion method is easier, faster, and more cost-effective. Moreover, employing PEG3400 and the post-insertion approach in the PEG3400-GSGGCE liposomal formulations was found to be effective in crossing the BBB.

A novel regulatory facet for hypertriglyceridemia: The role of microRNAs in the regulation of triglyceride-rich lipoprotein biosynthesis.

Atherosclerotic cardiovascular disease (ASCVD) is one of the major leading global causes of death. Genetic and epidemiological studies strongly support the causal association between triacylglycerol-rich lipoproteins (TAGRL) and atherogenesis, even in statin-treated patients. Recent genetic evidence has clarified that variants in several key genes implicated in TAGRL metabolism are strongly linked to the increased ASCVD risk. There are several triacylglycerol-lowering agents; however, new therapeutic options are in development, among which are miRNA-based therapeutic approaches. MicroRNAs (miRNAs) are small non-coding RNAs (18-25 nucleotides) that negatively modulate gene expression through translational repression or degradation of target mRNAs, thereby reducing the levels of functional genes. MiRNAs play a crucial role in the development of hypertriglyceridemia as several miRNAs are dysregulated in both synthesis and clearance of TAGRL particles. MiRNA-based therapies in ASCVD have not yet been applied in human trials but are attractive. This review provides a concise overview of current interventions for hypertriglyceridemia and the development of novel miRNA and siRNA-based drugs. We summarize the miRNAs involved in the regulation of key genes in the TAGRLs synthesis pathway, which has gained attention as a novel target for therapeutic applications in CVD.

Nanophytosomes of hesperidin and of hesperetin: Preparation, characterization, and in vivo evaluation.

Hesperidin and hesperetin are two important plant flavanones abundantly found in citrus fruit. They have discovered many biological activities to treat diseases, including cancer, diabetes, and Alzheimer's disease. Despite their various benefits, they have poor solubility, which reduces their bioavailability and absorption. In this study, nanophytosomes have been utilized to improve their payload's solubility and bioavailability. In the current study, hesperetin or hesperidin was complexed with Phospholipon 90G with a 2:1 or 3:1 molar ratio, respectively. The formation of associations between active compounds and phospholipid were confirmed by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and nuclear magnetic resonance (NMR) techniques. Dynamic light scattering data show that the prepared associations in the presence of body fluids can make nanoparticles in the range of 200-250 nm. In addition, oral administration demonstrated that Cmax of hesperidin and hesperetin was increased (up to four times) after complexation with the lipid. It is concluded that phospholipid association may be used as a suitable and straightforward strategy to improve therapeutic activities of hesperidin and hesperetin by increasing their solubility and bioavailability.

Redox-sensitive doxorubicin liposome: a formulation approach for targeted tumor therapy.

In this study redox-sensitive (RS) liposomes manufactured using 10,10'-diselanediylbis decanoic acid (DDA), an organoselenium RS compound, to enhance the therapeutic performance of doxorubicin (Dox). The DDA structure was confirmed by 1H NMR and LC-MS/MS. Various liposomal formulations (33 formulations) were prepared using DOPE, Egg PC, and DOPC with Tm Ë‚ 0 and DDA. Some formulations had mPEG2000-DSPE and cholesterol. After extrusion, the external phase was exchanged with sodium bicarbonate to create a pH gradient. Then, Dox was remotely loaded into liposomes. The optimum formulations indicated a burst release of 30% in the presence of 0.1% hydrogen peroxide at pH 6.5, thanks to the redox-sensitive role of DDA moieties; conversely, Caelyx (PEGylated liposomal Dox) showed negligible release at this condition. RS liposomes consisting of DOPE/Egg PC/DDA at 37.5 /60/2.5% molar ratio, efficiently inhibited C26 tumors among other formulations. The release of Dox from RS liposomes in the TME through the DDA link fracture triggered by ROS or glutathione is seemingly the prerequisite for the formulations to exert their therapeutic action. These findings suggest the potential application of such intelligent formulations in the treatment of various malignancies where the TME redox feature could be exploited to achieve an improved therapeutic response.

Preparation, characterization, and biodistribution of glutathione PEGylated nanoliposomal doxorubicin for brain drug delivery with a post-insertion approach.

Objectives: Brain cancer treatments have mainly failed due to their inability to cross the blood-brain barrier. Several studies have confirmed the presence of glutathione (GSH) receptors on BBB's surface, as a result, products like 2B3-101, which contain over 5% pre-inserted GSH PEGylated liposomal doxorubicin, are being tested in clinical trials. Here we conducted the PEGylated nanoliposomal doxorubicin particles that are covalently attached to the glutathione using the post-insertion technique. Compared with the pre-insertion approach, the post-insertion method is notably simpler, faster, and more cost-effective, making it ideal for large-scale pharmaceutical manufacturing. Materials and Methods: The ligands of the DSPE PEG(2000) Maleimide-GSH were introduced in the amounts of 25, 50, 100, 200, and 400 on the available Caelyx. Following physicochemical evaluations, animal experiments such as biodistribution, fluorescence microscopy, and pharmacokinetics were done. Results: In comparison with Caelyx, the 200L and 400L treatment arms were the most promising formulations. We showed that nanocarriers containing 40 times fewer GSH micelles than 2B3-101 significantly increased blood-brain barrier penetrance. Due to the expressed GSH receptors on tissues as an endogenous antioxidant, doxorubicin will likely concentrate in the liver, spleen, heart, and lung in comparison with Caelyx, according to other tissue analyses. Conclusion: The post-insertion technique was found a successful approach with more pharmaceutical aspects for large-scale production. Moreover, further investigations are highly recommended to determine the efficacy of 5% post-inserted GSH targeted nanoliposomes versus 2B3-101 as a similar formulation with a different preparation method.

Nanocarriers Call the Last Shot in the Treatment of Brain Cancers.

Our brain is protected by physio-biological barriers. The blood-brain barrier (BBB) main mechanism of protection relates to the abundance of tight junctions (TJs) and efflux pumps. Although BBB is crucial for healthy brain protection against toxins, it also leads to failure in a devastating disease like brain cancer. Recently, nanocarriers have been shown to pass through the BBB and improve patients' survival rates, thus becoming promising treatment strategies. Among nanocarriers, inorganic nanocarriers, solid lipid nanoparticles, liposomes, polymers, micelles, and dendrimers have reached clinical trials after delivering promising results in preclinical investigations. The size of these nanocarriers is between 10 and 1000 nm and is modified by surface attachment of proteins, peptides, antibodies, or surfactants. Multiple research groups have reported transcellular entrance as the main mechanism allowing for these nanocarriers to cross BBB. Transport proteins and transcellular lipophilic pathways exist in BBB for small and lipophilic molecules. Nanocarriers cannot enter via the paracellular route, which is limited to water-soluble agents due to the TJs and their small pore size. There are currently several nanocarriers in clinical trials for the treatment of brain cancer. This article reviews challenges as well as fitting attributes of nanocarriers for brain tumor treatment in preclinical and clinical studies.

Recent advancements in nanoparticle-mediated approaches for restoration of multiple sclerosis.

Multiple Sclerosis (MS) is an autoimmune disease with complicated immunopathology which necessitates considering multifactorial aspects for its management. Nano-sized pharmaceutical carriers named nanoparticles (NPs) can support impressive management of disease not only in early detection and prognosis level but also in a therapeutic manner. The most prominent initiator of MS is the domination of cellular immunity to humoral immunity and increment of inflammatory cytokines. The administration of several platforms of NPs for MS management holds great promise so far. The efforts for MS management through in vitro and in vivo (experimental animal models) evaluations, pave a new way to a highly efficient therapeutic means and aiding its translation to the clinic in the near future.

An insight into the role of liposomal therapeutics in the reversion of multiple sclerosis.

INTRODUCTION: Multiple Sclerosis (MS), as an autoimmune disease, has complicated immunopathology, which makes its management relevant to various factors. Novel pharmaceutical vehicles, especially liposomes, can support efficacious handling of this disease both in early detection and prognosis and also in a therapeutic manner. The most well-known triggers of MS onset are the predominance of cellular to humoral immunity and enhancement of inflammatory cytokines level. The installation of liposomes as nanoparticles to control this disease holds great promise up to now. AREAS COVERED: Various types of liposomes with different properties and purposes have been formulated and targeted immune cells with their surface manipulations. They may be encapsulated with anti-inflammatory, MS-related therapeutics, or immunodominant myelin-specific peptides for attaining a higher therapeutic efficacy of the drugs or tolerance induction. Cationic liposomes are also highly applicable for gene delivery of the anti-inflammatory cytokines or silencing the inflammatory cytokines. Liposomes have also been used as biotools for comprehending MS pathomechanisms or as diagnostic agents. EXPERT OPINION: The efforts to manage MS through nanomedicine, especially liposomal therapeutics, pave a new avenue to a high-throughput medication of this autoimmune disease and their translation to the clinic in the future for overcoming the challenges that MS patients confront.

The impact of nanocarriers in the induction of antigen-specific immunotolerance in autoimmune diseases.

Immunotolerance induction in an antigen-specific manner is the long-term goal of immunotherapy to treat autoimmune diseases. Nanocarriers (NCs) can be designed as a new generation of delivery systems to modulate the immune responses through targeted delivery of antigens and immunomodulators to antigen presenting cells (APCs). In this manuscript, several formulation factors in the preparation of NCs which affect their uptake using APCs and generation of tolerance have been reviewed. The physicochemical properties and composition of NCs have been shown to play essential roles in achieving the desired immunological outcome. Also, targeting of dendritic cells and macrophages as APCs and direct targeting of the autoreactive lymphocytes have been presented as two main ways for induction of antigen-specific tolerance by these tolerogenic nanocarriers (tNCs). These particles herald a promising approach to treat or even prevent unwanted immune reactions in humans specifically.

Comparison of two routes of administration of a cationic liposome formulation for a prophylactic DC vaccination in a murine melanoma model.

Dendritic cell (DC) vaccination can be achieved via straight loading of vaccine into DCs ex vivo or administration to DCs in vivo. However, there is no certain consensus on which approach is preferable, and each strategy has its advantages and disadvantages, which affect the efficacy and safety of vaccines. It will also be more complicated when a vaccine delivery system is included. In this study, the efficacy of ex vivo pulsed DC-based vaccine compared with in vivo subcutaneous administration of a cationic liposomes (CLs) formulation containing gp100 antigen (gp100-CLs) was evaluated in a murine melanoma model. In combination with an anti-PD-1 antibody, the ex vivo approach of gp100-CLs yielded a significant (P < 0.01) increase in the number of antigen-specific tumors infiltrated lymphocytes (TILs) with a significant upregulation of IFN-γ (P < 0.0001) and PD-1 (P < 0.0001) expression level. They also dampened the function of immunosuppressive regulatory T cells (Tregs) via significant downregulation of IL-10 and TGF-ß (P < 0.0001) expression level compared to in vivo approach in the tumor microenvironment (TME). Furthermore, prophylactic immunization with gp100-CLs pulsed DCs ex vivo delayed tumor growth and induced the survival benefit over in vivo immunization. Collectively, the ex vivo DC-based vaccination pulsed with gp100 encapsulated in liposomes synergizes with anti-PD-1 antibody and represents a preferable approach against melanoma.

Development of a stable and high loaded liposomal formulation of lapatinib with enhanced therapeutic effects for breast cancer in combination with Caelyx®: In vitro and in vivo evaluations.

Lapatinib, a dual tyrosine kinase inhibitor, has poor water solubility, which results in poor and incomplete absorption from the gastrointestinal tract. To overcome this obstacle, we designed a stable and high-loaded liposomal formulation encapsulating lapatinib and examined its therapeutic efficacy in vitro and in vivo on TUBO and 4T1 cell lines. We also assessed the impact of liposomal lapatinib on the extent of the tumor and spleen-infiltrating lymphocytes and the autophagy and apoptosis gene expression within the tumor site. Our results showed that liposomal lapatinib inhibits cell proliferation and significantly induces autophagy and apoptosis compared to control groups. Moreover, when it used in combination with liposomal doxorubicin, it extended the time to end from 22.4 ± 3.5 in the control group to 40 days in the TUBO cell line and from 29.2 ± 1.7 to 38.6 ± 2.2 days in 4T1 triple-negative breast cancer cell line, which reveals its promising effects on the survival of tumor-bearing mice. Our results indicated the need for further evaluations to understand liposomal lapatinib's potential effects on autophagy, apoptosis, and particularly on immune system cells.

pH-Sensitive PEGylated Liposomal Silybin: Synthesis, In Vitro and In Vivo Anti-Tumor Evaluation.

The drug delivery systems improve the efficacy of chemotherapeutics through enhanced targeting and controlled release however, biological barriers of tumor microenvironment greatly impede the penetration of nanomedicine within the tumor. We report herein the fabrication of a PEG-detachable silybin (SLB) pH-sensitive liposome decorated with TAT-peptide. For this, Acyl hydrazide-activated PEG2000 was prepared and linked with ketone-derivatized DPPE via an acid-labile hydrazone bond to form mPEG2000-HZ-DPPE. TAT peptide was conjugated with a shorter -PEG1000-DSPE spacer and post-inserted into PEGylated liposome (DPPC: mPEG2000-DSPE: Chol). To prepare nanoliposomes (around 100 nm), first, a novel method was used to prepare SLB-Soya PC (SLB-SPC) complex, then this complex was incorporated into nanoliposomes. The pH-sensitivity and shielding effect of long PEG chain on TAT peptide was investigated using DiI liposome and FACS analysis. Pre-treatment to the lowered pH enhanced cellular association of TAT-modified pH-sensitive liposome due to the cleavage of hydrazone bond and TAT exposure. Besides, TAT-modified pH-sensitive liposomes significantly reduced cell viability compared to the plain liposome. In vivo results were very promising with pH-sensitive liposome by detaching PEG moieties upon exposure to the acidic tumor microenvironment, enhancing cellular uptake, retarding tumor growth, and prolonging the survival of 4T1 breast tumor-bearing BALB/c mice. TAT modification of pH-sensitive liposome improved cancer cell association and cytotoxicity and demonstrated potential intracellular delivery upon exposure to acidic pH. However, in in vivo studies, TAT as a targeting ligand significantly decreased the therapeutic efficacy of the formulation attributed to an inefficient tumor accumulation and higher release rate in the circulation. The results of this study indicated that pH-sensitive liposome containing SLB, which was prepared with a novel method with a significant SLB loading efficiency, is very effective in the treatment of 4T1 breast tumor-bearing BALB/c mice and merits further investigation.

Development of a novel formulation method to prepare liposomal Epacadostat.

BACKGROUND: One of the important metabolic pathways in cancer progression is tryptophan catabolism by the indoleamin-2,3-dioxygenase (IDO) enzyme, which suppresses the immune system and induces tolerance. Inhibition of IDO1 is an important therapeutic goal for immunotherapy in many cancers such as metastatic melanoma. Epacadostat (EPA) is a very strong inhibitor of IDO1, and its clinical studies are being performed in a higher clinical phase than other inhibitors. In this study, we have developed a new liposomal EPA formulation to reduce the dose, side effects, and treatment costs. METHODS: Liposomes containing EPA were formulated using a novel remote loading method. Their morphology, particle size, surface charge, total phospholipid content, and drug loading were evaluated. Validation method studies to assay of EPA were carried out according to ICHQ2B guidelines. For in-vivo study, B16F10 melanoma bearing C57BL/6 mice were treated with the free or liposomal forms of EPA, and then monitored for tumor size and survival rate. RESULTS: A validated method for EPA determination in liposomal form using UV-visible spectrophotometry was developed which was a precise, accurate and robust method. The particle size, zeta potential, and encapsulation efficacy of liposomes was 128.1 ± 1.1 nm, -16.5 ± 1 mV, and 64.9 ± 3.5, respectively. The half maximal inhibitory concentration (IC50) of liposomal EPA was 64 ng/ml that was lower than free EPA (128 ng/ml). In-vivo results also showed that tumor growth was slower in mice receiving liposomal EPA than in the group receiving free EPA. CONCLUSION: A new method was developed to load EPA into liposomes. Moreover, the use of the nanoliposomal EPA showed more efficacy than EPA in inhibiting the tumor growth in melanoma model. Therefore, it might be used in further clinical studies as a good candidate for immunotherapy alone or in combination with other treatments.