Delivery of doxorubicin loaded P18 conjugated-poly(2-ethyl-oxazoline)-DOPE nanoliposomes for targeted therapy of breast cancer.

Breast cancer, a heterogeneous disease, has the highest incidence rate and is a major cause of death in females worldwide. Drug delivery by using nanotechnology has shown great promise for improving cancer treatment. Nanoliposomes are known to have enhanced accumulation ability in tumors due to prolonged systemic circulation. Peptide 18 (P18), a tumor homing peptide targeting keratin-1 (KRT-1), was previously shown to have high binding affinity towards breast cancer cells. In this study, we investigate the ability of P18 conjugated PEtOx-DOPE nanoliposomes (P18-PEtOx-DOPE) for the targeted delivery of doxorubicin to AU565 breast cancer model. Toxicology studies of PEtOx-DOPE nanoliposomes performed on normal breast epithelial cells (MCF10A), showed minimal toxicity. Doxorubicin delivery by P18-PEtOx-DOPE to AU565 cells induces cytotoxicity in a dose and time dependent manner causing mitotic arrest in G2/M phase at 24 h. Anti-cancer activity of P18-PEtOx-DOPE-DOX nanoliposomes on AU565 cells was detected by Annexin V/PI apoptosis assay. In terms of in vivo antitumor efficacy, P18-PEtOx-DOPE-DOX nanoliposomes administration to AU565 CD-1 nu/nu mice model showed significant decrease in tumor volume suggesting that DOX delivered by these nanoliposomes elicited a strong antitumor response comparable to the free delivery of doxorubicin. Overall, our results offered preclinical proof for the use of P18-PEtOx-DOPE-DOX nanoliposomes in KRT-1+ breast cancer therapy.

Effectiveness and safety of 1L PEG-ASC preparation for colonoscopy in patients with inflammatory bowel diseases.

INTRODUCTION: The effectiveness of bowel cleansing is a key element for high-quality colonoscopy. Recently, a 1 L polyethylene glycol plus ascorbate (PEG-ASC) solution has been introduced, but effectiveness and safety of this preparation have not been assessed in IBD patients. This study aims to evaluate effectiveness and safety of 1 L PEG-ASC solution in patients with IBD compared to controls. METHODS: We retrospectively analysed prospectively collected data on a cohort of 411 patients performing a colonoscopy after preparation with 1 L PEG-ASC, consecutively enrolled in 5 Italian centres. RESULTS: Overall, 185/411 (45%) were patients with IBD and 226/411 (55%) served as controls. A significantly higher cleansing success was achieved in IBD patients (92.9% vs 85.4%, p = 0.02). The multiple regression model showed that presence of IBD (OR=2.514, 95%CI=1.165-5.426; P = 0.019), lower age (OR=0.981, 95%CI=0.967-0.996; P = 0.014), split preparation (OR=2.430, 95%CI=1.076-5.492; P = 0.033), absence of diabetes (OR=2.848, 95%CI=1.228-6.605; P = 0.015), and of chronic constipation (OR=3.350, 95%CI=1.429-7.852; P = 0.005), were independently associated with cleansing success. The number of treatment-emergent adverse events (TEAEs) (51 vs 62%, p = 0.821), and of patients with TEAEs (22.2% vs 21.2%, p = 0.821), were similar in IBD patients and in controls, respectively. CONCLUSIONS: Results from this study support the effectiveness and safety of 1 L PEG-ASC solution in IBD patients, which may improve the definition of endoscopic outcomes both in Crohn's disease and ulcerative colitis.

Effects of Dietary Supplementation with EPA-enriched Phosphatidylcholine and Phosphatidylethanolamine on Glycerophospholipid Profile in Cerebral Cortex of SAMP8 Mice fed with High-fat Diet.

The destruction of lipid homeostasis is associated with nervous system diseases such as Alzheimer's disease (AD). It has been reported that dietary EPA-enriched phosphatidylcholine (EPA-PC) and phosphatidylethanolamine (EPA-PE) could improve brain function. However, it was unclear that whether EPA-PC and EPA-PE intervention could change the lipid composition of cerebral cortex in AD mice. All the senescence-accelerated mouse-prone 8 (SAMP8) mice were fed with a high-fat diet for 8 weeks. After another 8 weeks of intervention with EPA-PC and EPA-PE (1%, w/w), the cerebral cortex lipid levels were determined by lipidomics. Results demonstrated that dietary supplementation with EPA-PE and EPA PC for 8 weeks significantly increased the amount of choline plasmalogen (pPC) and Lyso phosphatidylethanolamine (LPE) in the cerebral cortex of SAMP8 mice fed with high fat diet. Meanwhile, administration with EPA-PE and EPA-PC could significantly decrease the level of docosapentaenoic acid (DPA)-containing phosphatidylserine (PS) as well as increase the levels of arachidonic acid (AA)-containing phosphatidylethanolamine and PS in cerebral cortex. EPA-PE and EPA-PC could restore the lipid homeostasis of dementia mice to a certain degree, which might provide a potential novel therapy strategy and direction of dietary intervention in patients with cognitive impairment.

Evaluation of antitumor efficacy of folate-poly(2-ethyl-2-oxazoline)-distearoyl phosphatidyl ethanolamine based liposome.

This study aims to explore and evaluate the antitumor efficacy of doxorubicin (DOX)-loaded liposomes containing the novel tri-block polymer folate-poly (2-ethyl-2-oxazoline)-distearoyl phosphatidyl ethanolamine (F-PEOz-DSPE), compared with folate-polyethylene glycol-distearoyl phosphatidyl ethanolamine (F-PEG-DSPE) to offer an alternative for PEG decorated carriers. PEOz, a pH-sensitive polymer, exhibits similar solubility and segmental flexibility to PEG. In our previous study, PEOz was employed to an F-PEOz-DSPE which was segmentally similar to F-PEG-DSPE and exhibited selective targeting and pH-sensitivity in tumor cells. In this work, DOX-loaded liposomes containing F-PEOz-DSPE (F-PEOz liposome) or F-PEG-DSPE (F-PEG liposome) were prepared. In vivo/vitro antitumor efficacy and biodistribution were compared between the two liposomes. F-PEOz liposome showed higher in vitro antitumor activity and significantly stronger inhibition of tumor growth in HeLa tumor-bearing nude mice (tumor inhibition rate, 81.20 vs 52.99% with the treatment of 9 mg/kg DOX-loaded F-PEOz liposome/F-PEG liposome) and much less toxicity than free DOX. In vivo fluorescence imaging experiment confirmed that F-PEOz liposome accumulated much more than F-PEG liposome in tumor. Based on the above, F-PEOz liposome may be a promising carrier in tumor chemotherapy to achieve better therapeutic efficacy.

Vascular-targeted micelles as a specific MRI contrast agent for molecular imaging of fibrin clots and cancer cells.

Molecular medical imaging is intended to increase the accuracy of diagnosis, particularly in cardiovascular and cancer-related diseases, where early detection could significantly increase the treatment success rate. In this study, we present mixed micelles formed from four building blocks as a magnetic resonance imaging targeted contrast agent for the detection of atheroma and cancer cells. The building blocks are a gadolinium-loaded DOTA ring responsible for contrast enhancement, a fibrin-specific CREKA pentapeptide responsible for targeting, a fluorescent dye and DSPE-PEG2000. The micelles were fully characterized in terms of their size, zeta potential, stability, relaxivity and toxicity. Target binding assays performed on fibrin clots were quantified by fluorescence and image signal intensities and proved the binding power. An additional internalization assay showed that the micelles were also designed to specifically enter into cancer cells. Overall, these multimodal mixed micelles represent a potential formulation for MRI molecular imaging of atheroma and cancer cells.

Selective Targeting of Non-nuclear Estrogen Receptors with PaPE-1 as a New Treatment Strategy for Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial and severe neurodegenerative disorder characterized by progressive memory decline, the presence of Aß plaques and tau tangles, brain atrophy, and neuronal loss. Available therapies provide moderate symptomatic relief but do not alter disease progression. This study demonstrated that PaPE-1, which has been designed to selectively activate non-nuclear estrogen receptors (ERs), has anti-AD capacity, as evidenced in a cellular model of the disease. In this model, the treatment of mouse neocortical neurons with Aß (5 and 10 µM) induced apoptosis (loss of mitochondrial membrane potential, activation of caspase-3, induction of apoptosis-related genes and proteins) accompanied by increases in levels of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) as well as reduced cell viability. Following 24 h of exposure, PaPE-1 inhibited Aß-evoked effects, as shown by reduced parameters of neurotoxicity, oxidative stress, and apoptosis. Because PaPE-1 downregulated Aß-induced Fas/FAS expression but upregulated that of Aß-induced FasL, the role of PaPE-1 in controlling the external apoptotic pathway is controversial. However, PaPE-1 normalized Aß-induced loss of mitochondrial membrane potential and restored the BAX/BCL2 ratio, suggesting that the anti-AD capacity of PaPE-1 particularly relies on inhibition of the mitochondrial apoptotic pathway. These data provide new evidence for an anti-AD strategy that utilizes the selective targeting of non-nuclear ERs with PaPE-1.

The exogenous natural phospholipids, EPA-PC and EPA-PE, contribute to ameliorate inflammation and promote macrophage polarization.

Dietary intake of sea cucumber phospholipids, a rich source of eicosapentaenoic acid in the form of phospholipids (EPA-PLs), has been shown to improve obesity and related disorders. However, whether dietary eicosapentaenoic acid in the form of phosphatidylcholine and phosphatidylethanolamine (EPA-PC and EPA-PE, respectively) shows anti-inflammatory efficacy and its underlying mechanism has scarcely been investigated to date. Thus, the purpose of this study was to determine if EPA-PC and EPA-PE improve chronic inflammation and alter the interaction between macrophages and adipocytes. We found that EPA-PC and EPA-PE reduced the elevated levels of serum TNF-α, IL-6 and MCP1 and attenuated macrophage infiltration in the liver and iWAT of an HFSD-induced inflammatory model. Importantly, EPA-PC and EPA-PE promoted macrophage polarization in white adipose tissue. Furthermore, this effect on macrophage polarization was also observed in a 3T3L1 and Raw 264.7 Transwell co-culture system, which suggests that EPA-PC and EPA-PE attenuate chronic inflammation by promoting the M2-dominant polarization of macrophages in vitro. Our experiments in vitro illustrated that EPA-PC and EPA-PE attenuated the phosphorylation of p65 NFκB in Raw264.7 macrophages and increased PPARγ expression in 3T3-L1 adipocytes during the co-culture, which may contribute to the improvement in adipose inflammation. Thus, dietary eicosapentaenoic acid in the form of phosphatidylcholine and phosphatidylethanolamine may be a therapeutic strategy for chronic inflammation in obese adipose tissue.

Muramyl Tripeptide-Phosphatidyl Ethanolamine Encapsulated in Liposomes (L-MTP-PE) in the Treatment of Osteosarcoma.

The recruitment of autologous macrophages to attack osteosarcoma represents a novel immunotherapy approach to the treatment of osteosarcoma. Muramyl tripeptide-phosphatidyl ethanolamine encapsulated in liposomes (L-MTP-PE) was derived as a compound with the ability to stimulate macrophages to destroy autologous osteosarcoma tumor cells. Preclinical studies including studies in dogs with spontaneously arising osteosarcoma showed the ability of L-MTP-PE to control microscopic metastatic disease in osteosarcoma. A pivotal clinical trial led to the approval of L-MTP-PE for the treatment of newly diagnosed osteosarcoma in over 40 countries.

Fluorescence imaging-guided multifunctional liposomes for tumor-specific phototherapy for laryngeal carcinoma.

Reliable diagnosis and efficient targeted therapy are important and may lead to the effective treatment of laryngeal carcinoma. Multifunctional nano-theranostic agents demonstrate great potential in tumor theranostic applications. Thus, herein, we report novel targeting multifunctional theranostic nanoparticles, internalized RGD (iRGD)-modified indocyanine green (ICG) encapsulated liposomes (iLIPICG), for imaging-guided photothermal therapy (PTT) and photodynamic therapy (PDT) for the treatment of laryngeal carcinoma. The iRGD-PEG-DSPE lipid endowed iLIPICG with high affinity for tumor vascular targeting, tumor-penetration and tumor cell targeting. The in vivo results showed that iLIPICG exhibited excellent blood circulation and tumor accumulation. iLIPICG could be spatially and temporally controlled, simultaneously producing hyperthermia and reactive oxygen species as well as a fluorescence-guided effect through ICG to ablate laryngeal carcinoma cells under irradiation from an 808 nm laser. iLIPICG generated synergistic photodynamic-photothermal cytotoxicity against Hep-2 cells, resulting in the efficient ablation of laryngeal carcinoma. Thus, the iLIPICG system provides a promising strategy to improve the precision imaging and effective phototherapy for the treatment of laryngeal carcinoma.

Naturally available hypericin undergoes electron transfer for type I photodynamic and photothermal synergistic therapy.

Naturally available compounds with bioactivity are potential candidates for cancer treatment. In this paper, we isolated hypericin (HC) from Hypericum sinense L. and investigated its antitumor activity both in vitro and in vivo. The nanoparticles (NPs) of HC were prepared by a nanoprecipitation process with 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG-2000). With light irradiation, HC NPs not only undergo efficient electron transfer to generate the superoxide radical (O2-˙) and the hydroxyl radical (OH˙) as well as energy transfer producing singlet oxygen (1O2) for photodynamic therapy (PDT), but also non-radiative decay to produce heat for photothermal therapy (PTT) with a photothermal conversion efficiency of 29.3%. This synergistic therapy, therefore, largely boosts the phototherapy efficacy of HC NPs on human cervical cancer cells (HeLa), guaranteeing a low half maximal inhibitory concentration (IC50) of only 5.6 µg mL-1. Furthermore, in vivo studies suggest that HC NPs are capable of inhibiting tumor proliferation after laser irradiation, and the main organs remain healthy, including the heart, kidneys, liver, lungs and spleen. Our results indicate that HC NPs derived from nature with excellent phototherapy efficacies are biocompatible candidates for type I PDT/PTT synergistic cancer therapy.

Cholic acid-based mixed micelles as siRNA delivery agents for gene therapy.

Gene therapy is a promising tool for the treatment of various cancers but is hindered by the physico-chemical properties of siRNA and needs a suitable vector for the delivery of siRNA to the target tissue. Bile acid-based block copolymers offers certain advantages for the loading and delivery of siRNA since they can efficiently complex siRNA and bile acids are biocompatible endogenous molecules. In this study, we demonstrate the use of lipids as co-surfactants for the preparation of mixed micelles to improve the siRNA delivery of cholic acid-based block copolymers. Poly(allyl glycidyl ether) (PAGE) and poly(ethylene glycol) (PEG) were polymerized on the surface of cholic acid to afford a star-shaped block copolymer with four arms (CA-PAGE-b-PEG)4. The allyl groups of PAGE were functionalized to bear primary or tertiary amines and folic acid was grafted onto the PEG chain end to increase cell uptake. (CA-PAGE-b-PEG)4 functionalized with either primary or tertiary amines show high siRNA complexation with close to 100% complexation at N/P ratio of 8. Uniform aggregates with diameters between 181 and 188 nm were obtained. DOPE, DSPE-PEG2k, and DSPE-PEG5k lipids were added as co-surfactants to help stabilize the nanoparticles in the cell culture media. Mixed micelles had high siRNA loading with close to 100% functionalization at N/P ratio of 16 and diameters ranging from 153 to 221 nm. The presence of lipids in the mixed micelles improved cell uptake with a concomitant siRNA transfection in HeLa and HeLa-GFP model cells, respectively.

In Vitro and in Vivo Behavior of Liposomes Decorated with PEGs with Different Chemical Features.

The colloidal stability, in vitro toxicity, cell association, and in vivo pharmacokinetic behavior of liposomes decorated with monomethoxy-poly(ethylene glycol)-lipids (mPEG-lipids) with different chemical features were comparatively investigated. Structural differences of the mPEG-lipids used in the study included: (a) surface-anchoring moiety [1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), cholesterol (Chol), and cholane (Chln)]; (b) mPEG molecular weight (2 kDa mPEG45 and 5 kDa mPEG114); and (c) mPEG shape (linear and branched PEG). In vitro results demonstrated that branched (mPEG114)2-DSPE confers the highest stealth properties to liposomes (∼31-fold lower cell association than naked liposomes) with respect to all PEGylating agents tested. However, the pharmacokinetic studies showed that the use of cholesterol as anchoring group yields PEGylated liposomes with longer permeance in the circulation and higher systemic bioavailability among the tested formulations. Liposomes decorated with mPEG114-Chol had 3.2- and ∼2.1-fold higher area under curve (AUC) than naked liposomes and branched (mPEG114)2-DSPE-coated liposomes, respectively, which reflects the high stability of this coating agent. By comparing the PEGylating agents with same size, namely, linear 5 kDa PEG derivatives, linear mPEG114-DSPE yielded coated liposomes with the best in vitro stealth performance. Nevertheless, the in vivo AUC of liposomes decorated with linear mPEG114-DSPE was lower than that obtained with liposomes decorated with linear mPEG114-Chol. Computational molecular dynamics modeling provided additional insights that complement the experimental results.

Topical anesthetic analgesic therapy using the combination of ropivacaine and dexmedetomidine: hyaluronic acid modified long-acting nanostructured lipid carriers containing a skin penetration enhancer.

PURPOSE: Hyaluronic acid-poly(ethylene glycol)-distearoyl phosphoethanolamine (HA-PEG-DSPE) modified and tocopheryl polyethylene glycol 1000 succinate (TPGS) contained nanostructured lipid carriers (NLCs) were prepared loading ropivacaine and dexmedetomidine to improve the topical anesthetic analgesic anesthesia efficiency. METHODS: NLCs were prepared by the solvent diffusion method. The average particle size, zeta potential, release behavior, and cytotoxicity of the NLCs were tested. Ex vivo skin permeation was studied using a Franz diffusion cell mounted with depilated rat skin. Local anesthesia antinociceptive efficiency was evaluated by rat tail flick latency study in vivo. RESULTS: NLCs have sizes of about 100 nm, with negative zeta potentials. All the NLCs formulations were found to be significantly less cytotoxic than free drugs at equivalent concentrations. The cumulative amount of drugs penetrated through rat skin from NLCs was 2.0-4.7 folds higher than that of the drugs solution. The in vivo anesthesia antinociception study displayed that NLCs showed stronger and longer anesthesia antinociceptive effect when compared with single drugs loaded NLCs and drugs solution even at a lower dosage of drugs. CONCLUSION: The results demonstrated that the HA modified, TPGS contained, dual drugs loaded NLCs could perform a synergistic effect and may reduce the amount of drugs, which can lower the toxicity of the system and at the meanwhile, increase the anesthesia antinociceptive efficiency.

Selectivity Enhancement of Paclitaxel Liposome Towards Folate Receptor-Positive Tumor Cells by Ligand Number Optimization Approach.

The present work aims to develop folate-targeted paclitaxel liposome (F-PTX-LIP), which will selectively target tumor cells overexpressing folate receptor (FR) and leave normal cells. Liposomes were prepared by thin-film hydration method followed by post-insertion of synthesized ligand 1,2-distearoyl-sn-glycero-phosphoethanolamine-polyethyleneglycol 2000-folic acid (DSPE-PEG2000-FA) on the outer surface of the liposome. The synthesized ligand was evaluated for in vivo acute toxicity in Balb/c mice. Developed liposomal formulations were characterized using transmission electron microscopy (TEM) and small-angle neutron scattering (SANS). We have investigated the effect of ligand number on cell uptake and cytotoxicity by confocal laser scanning microscopy (CLSM), competitive inhibition and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Compared to lung adenocarcinoma cells (A549), uptake in human ovarian carcinoma cells (SKOV3) was 2.2- and 1.2-fold higher for liposome with 480 and 240 ligand number respectively. Competitive inhibition experiment shows that prior incubation of SKOV3 cells with free folic acid significantly reduced the cell uptake of F-PTX-LIP with 480 ligand number (480 F-PTX-LIP) by 2.6-fold. 480 F-PTX-LIP displays higher cytotoxicity than free drug and PTX liposome. Moreover, it specifically targets the cells with higher folate receptor expression. Optimized 480 F-PTX-LIP formulation can be potentially useful for the treatment of folate receptor-positive tumors.

In vivo activation of PEGylated long circulating lipid nanoparticle to achieve efficient siRNA delivery and target gene knock down in solid tumors.

We developed a lipid nanoparticle formulation (LNPK15) to deliver siRNA to a tumor for target gene knock down. LNPK15 is highly PEGylated with 3.3% 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-(polyethylene glycol-2000) (PEG-DSPE) and shows a long duration: the half-lives of siRNA in LNPK15 were 15.2 and 27.0h in mice and monkeys, respectively. Although LNPK15 encapsulating KRAS-targeting siRNA (LNPK15/KRAS) had very weak KRAS gene knock down activity in MIA PaCa-2 cells in vitro, LNPK15/KRAS showed a strong anti-tumor efficacy in MIA PaCa-2 tumor xenograft mice after intravenous administration at 5mg/kg twice weekly. KRAS mRNA and protein knock down was observed in tumor tissue, suggesting on-target anti-tumor efficacy. In order to elucidate the in vitro-in vivo discrepancy, we performed ex vivo knock down assay using serum samples obtained after intravenous administration of LNPK15/KRAS to mice and monkeys. The collected samples were added to MIA PaCa-2 cells, and KRAS gene knock down was evaluated after a 24-h incubation period. The knock down efficacy was weak (≈20%) with serum samples at initial sampling point (2h), and it became much stronger (∼90%) with serum samples at later time points. Lipid composition of LNPK15 in the serum samples was also investigated. Among the five lipids incorporated in LNPK15, PEG-DSPE was degraded more rapidly than siRNA and the other lipids in both mice and monkeys. In vitro lipase treatment of LNPK15/KRAS also hydrolyzed PEG-DSPE and enhanced knock down activity. From these results, it was concluded that LNPK15 acquires increased knock down activity after undergoing PEG-DSPE hydrolysis in vivo, and that is the key mechanism to achieve both long circulation and potent knock down efficiency. We also proposed an in vitro assay system using lipase for quality control of LNP to ensure biological activity.

Anti-RelA siRNA-Encapsulated Flexible Liposome with Tight Junction-Opening Peptide as a Non-invasive Topical Therapeutic for Atopic Dermatitis.

Small interfering RNA (siRNA) has been proposed as a novel treatment for atopic dermatitis (AD) because it suppresses sequence-specific mRNA expression. Indeed siRNA-based therapy achieves an almost complete cure with fewer side effects than currently available treatments. However, the tight junctions in the granular layer of the epidermis in the atopic skin are barriers to siRNA delivery. We previously reported the potential clinical utility of AT1002, a peptide that opens tight junctions. In the present study, we evaluated a topical siRNA-based therapy for AD using AT1002 in combination with a flexible liposome. The 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/cholesteryl hemisuccinate (CHEMS) liposome was chosen as a carrier for siRNA because of its highly flexible structure and permeability. We prepared siRNA-encapsulated DOPE/CHEMS liposomes and examined their physical properties, safety, uptake into RAW264.7 cells, and topical application in healthy and AD-affected skin. We then assessed the efficacy of anti-nuclear factor-kappa B (NF-κB) (RelA) siRNA (siRelA)-encapsulated DOPE/CHEMS liposomes with AT1002 in AD model mice. The siRNA-DOPE/CHEMS liposomes were absorbed significantly better than siRNA alone and they enhanced siRNA skin penetration without toxicity. Moreover, siRelA-DOPE/CHEMS liposomes with AT1002 alleviated AD symptoms and reduced the levels of inflammatory cytokines in AD model mice. Therefore, the combination of AT1002 and DOPE/CHEMS liposomes could be a dermally applied RNA interference therapeutic system for effective RNA delivery and AD treatment.

Cell cytotoxicity, immunostimulatory and antitumor effects of lipid content of liposomal delivery platforms in cancer immunotherapies. A comprehensive in-vivo and in-vitro study.

Liposome is one of the promising technologies for antigen delivery in cancer immunotherapies. It seems that the phospholipid content of liposomes can act as immunostimulatory molecules in cancer immunotherapy. In the present study, the immunological properties of different phospholipid content of liposomal antigen delivery platforms were investigated. To this aim, F1 to F4 naïve liposomes (without tumor-specific loaded antigens) of positively charged DOTAP/Cholesterol/DOPE (4/4/4 mol ratio), negatively charged DMPC/DMPG/Cholesterol/DOPE (15/2/3/5), negatively charged DSPC/DSPG/Cholesterol/DOPE (15/2/3/5) and PEGylated HSPC/mPEG2000-DSPE/Cholesterol (13/110) liposomal compositions were administered in mice bearing C26 colon carcinoma to assess tumor therapy. Moreover, In-vitro studies were conducted, including cytotoxicity assay, serum cytokines measurements, IFN-γ and IL-4 ELISpot assay, T cells subpopulation frequencies assay. The liposomes containing DOTAP and DOPE (F1 liposomes) were able to stimulate cytotoxic T lymphocytes signals such as IFN-γ secretions. In parallel, the aforementioned phospholipids stimulated secretion of IL-4 and IL-17 cytokines from T helper cells. However, these liposomes did not improve survival indices in mice. As conclusion, DOTAP and DOPE contained liposomes (F1 liposomes) stimulate a mixture of Th1 and Th2 immune responses in a tumor-specific antigens-free manner in mice bearing C26 colon carcinoma. Therefore, phospholipid composition of liposomes merits consideration in designing antigen-containing liposomes for cancer immunotherapy.

Design of a Novel PEGylated Liposomal Vector for Systemic Delivery of siRNA to Solid Tumors.

A small interfering RNA (siRNA) delivery system using dioleylphosphate-diethylenetriamine conjugate (DOP-DETA)-based liposomes (DL) was assessed for systemic delivery of siRNA to tumors. DL carrying siRNA capable of inducing efficient gene silencing with low doses of siRNA were modified with polyethylene glycol (PEG-DL/siRNA) for systemic injection of siRNA. The biodistribution of DL and siRNA in the PEG-DL/siRNA was studied by using radiolabeled DL and fluorescence-labeled siRNA, respectively. DL in the PEG-DL/siRNA showed a high retention in the plasma, accumulation in the tumor, and low accumulation in the liver and spleen after intravenous injection. The in vivo effects of PEGylation were observed only when distearoylphosphatidylethanolamine (DSPE)-PEG but not distearoylglycerol (DSG)-PEG were used. This result suggests that the electrostatic interaction between lipid molecules on the surface of PEG-DL/siRNA was a critical determinant for the in vivo effect of PEGylation. When PEG-DL/siRNA (0.1 mg/kg siRNA) was intravenously injected into tumor-bearing mice, in vivo gene silencing was observed in subcutaneous tumors. These results indicate that PEG-DL/siRNA designed in this study is a promising formulation for systemic use of siRNA.

Dual functionalized liposomes for efficient co-delivery of anti-cancer chemotherapeutics for the treatment of glioblastoma.

Glioblastoma is a hostile brain tumor associated with high infiltration leading to poor prognosis. Anti-cancer chemotherapeutic agents have limited access into the brain due to the presence of the blood brain barrier (BBB). In this study, we designed a dual functionalized liposomal delivery system, surface modified with transferrin (Tf) for receptor mediated transcytosis and a cell penetrating peptide-penetratin (Pen) for enhanced cell penetration. We loaded doxorubicin and erlotinib into liposomes to enhance their translocation across the BBB to glioblastoma tumor. In vitro cytotoxicity and hemocompatibility studies demonstrated excellent biocompatibility for in vivo administration. Co-delivery of doxorubicin and erlotinib loaded Tf-Pen liposomes revealed significantly (p < 0.05) higher translocation (~15%) across the co-culture endothelial barrier resulting in regression of tumor in the in vitro brain tumor model. The biodistribution of Tf-Pen liposomes demonstrated ~12 and 3.3 fold increase in doxorubicin and erlotinib accumulation in mice brain, respectively compared to free drugs. In addition, Tf-Pen liposomes showed excellent antitumor efficacy by regressing ~90% of tumor in mice brain with significant increase in the median survival time (36 days) along with no toxicity. Thus, we believe that this study would have high impact for treating patients with glioblastoma.

Nanoscale Lipophilic Prodrugs of Dexamethasone with Enhanced Pharmacokinetics.

The encapsulation of glucocorticoids, such as dexamethasone, in nanoparticles (NPs) faces two main issues: a low drug loading and the destabilization of the nanoparticle suspension due to drug crystallization. Here, we successfully formulated a prodrug of dexamethasone, dexamethasone palmitate (DXP), into nanoparticles stabilized by the sole presence of distearoyl- sn-glycero-3-phosphoethanolamine- N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG2000). Two formulation processes, nanoprecipitation and emulsion-evaporation, allowed the formation of stable nanoparticles. By adjusting the drug/lipid ratio and the DXP concentration, nanoparticles of DXP (DXP-NPs) with a size between 130 and 300 nm can be obtained. Owing to the presence of DSPE-PEG2000, a high drug entrapment efficiency of 98% w/w was reached for both processes, corresponding to a very high equivalent dexamethasone drug loading of around 50% w/w in the absence of crystallization upon storage at 4 °C. The anti-inflammatory activity of DXP-NPs was preserved when incubated with macrophages activated with lipopolysaccharide. Pharmacokinetics parameters were evaluated after intravenous (IV) injection of DXP-NPs to healthy mice. The release of DXM from DXP-NPs in plasma was clearly controlled up to 18 h compared with the free drug, which was rapidly eliminated from plasma after administration. In conclusion, a novel type of nanoparticle combining the advantages of prodrugs and nanoparticles was designed, easy to produce with a high loading efficiency and leading to modified pharmacokinetics and tissue distribution after IV administration.