Liposomal Vehicle Research Articles

Macrophage Depletion Ameliorates Glycerol-Induced Acute Kidney Injury in Mice

Background: This study was conducted to elucidate the role of renal macrophages in the development of acute kidney injury (AKI) in a glycerol (Gly)-induced rhabdomyolysis mouse model. Methods: The experimental model of rhabdomyolysis requires injecting 50% Gly (10 ml/kg) intramuscularly into mice. Control mice were injected into the tail vein with the liposomal vehicle. Liposome-encapsulated clodronate (LEC)-only mice were injected with LEC. Gly-only mice were injected with Gly into a hind limb. LEC+Gly-treated mice were injected intravenously with 100 µl of LEC 24 h prior to Gly injection. Mice were sacrificed 24 h after Gly injection. Results: Gly injection increased the serum creatinine level, and induced tubular damage. Renal CD45⁺CD11b⁺Ly6c⁺ or CD45⁺CD11b⁺Ly6c⁺F4/80⁺ macrophages were decreased by pretreatment with LEC in both normal and injured kidneys. Macrophage depletion prevented Gly-induced apoptotic death of tubular epithelial cells by decreasing caspase-9, ERK and p53, while increasing Bcl-2 expression. Expression of the inflammatory mediators NF-κB, MCP-1, ICAM-1, iNOS and COX-2 were also decreased with LEC pretreatment of mice injected with Gly. Conclusion: These results support the hypothesis that depletion of macrophages prevents renal dysfunction by abrogating apoptosis and attenuating inflammation during AKI.

Delivery of lipophilic porphyrin by liposome vehicles: Preparation and photodynamic therapy activity against cancer cell lines

Porphyrin photosensitizers are mostly used components in photodynamic therapy (PDT). The poor solubility of porphyrins in aqueous medium is the problem to be solved for the in vivo applications. The delivery of photosensitizers to the tumor cells using liposome vehicles can help to overcome this problem. In this work, we have first functionalized the protoporphyrin IX with lipophilic oleylamine arms and encapsulated it into 1,2 dioleyl-sn-glycero-phosphatidylcholine (DOPC) liposomes. The appropriate sizes of liposomes are about 140 nm and have the characteristic Soret and Q band absorptions at 405 nm (Soret), 507 nm, 541 nm, 577 nm and 631 nm (Q bands), respectively. In the photodynamic activity studies, the liposomal porphyrins were irradiated with light (375 nm, 10 mW) in the presence of cancer cell lines, HeLa and AGS. We have found that both liposomal porphyrins and oleylamine conjugated porphyrins are much more effective than PpIX. This result can be attributed to the drug delivery characteristic of the liposomes which plays effective role in endocytosis. We also found that, in AGS cells, liposomal PpIX-Ole induced apoptosis more than HeLa cells under light conditions.

Synthesis of a fluorescent analogue of methotrexate lipophilic prodrug

A fluorescent analog of the lipophilic prodrug of antitumor agent methotrexate has been synthesized. The conjugate consists of a residue of rac-1-[13-(Me4-BODIPY-8)tridecanoyl]-2-oleoylglycerol connected to methotrexate by ester bond via β-Ala-N-carbonylmethylene linker (Me4-BODIPY-8,4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl). The probe is designed for incorporation in the membrane of liposomal vehicle to study a mechanism of interaction with tumor cells and an intracellular traffic.

Assembly, characterization, and delivery of quantum dot labeled biotinylated lipid particles.

Lipid nanoparticles composed of mixtures of PEGylated-lipids; cationic and neutral lipids prepared by detergent dialysis can encapsulate biological active molecules and show considerable potential as systemic therapeutic agents. Addition of biotinylated lipids to this formulation allows surface modification of these particles with a suitable ligand or probe conjugated to streptavidin for specific cell targeting. Monitoring long circulating particles and cellular uptake requires stable and bright fluorescent probes. Quantum dots (QDs) constitute a relatively new class of fluorescent probes that overcome the limitations of organic fluorophores in biological imaging applications. Here, a protocol for the encapsulation of QD655 (red) in biotinylated lipid particles (BLPs) prepared by a detergent dialysis technique is presented followed by characterization of the loaded liposomal vehicles. Then, a protocol for BLPs surface modification via biotin-streptavidin linkage with preformed complexes of ligand-QD525 (green) for specific cell targeting of the nanoparticle is detailed. Conditions for cell binding and uptake of two colors QD labeled BLPs as well as basic microscopic settings for confocal live cell imaging are described.

Theoretical and practical considerations in electrostatic depositioning of charged polymers

ABSTRACTAn interfacial engineering technology, based on the electrostatic deposition of charged polyelectrolytes onto surfaces of oppositely charged templates is reviewed with an emphasis on practical applications in the food, pharmaceutical and personal care industries. On interfaces of disperse systems consecutively deposited polymers provide major advantages in terms of physical and chemical stability of dispersions against superimposed stresses (pH, temperature, ionic strength, freezing, chilling, dehydration, lipid oxidation). The controlled deposition of multiple layers allows for a controlled and triggered release of incorporated functional components. This review highlights the basic principles of the layer‐by‐layer (LbL) electrostatic deposition method as well as some major advantages and drawbacks of this approach. An overview of several systems that can be used as templates for the deposition including emulsion droplets, liposomal vehicles, colloidal aggregates, and planar surfaces is given. Suitable substrates for the deposition are presented with a focus on charged biopolymers such as proteins or polysaccharides since they play an essential role in the formulation and stabilization of food, pharmaceutical and personal care applications. Issues and difficulties associated with implementing the technology on a larger, industrial scale are discussed. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40099.

Use of micro- and nano-sized inertial cavitation nuclei to trigger and map drug release from cavitation-sensitive liposomes

Encapsulation of cytotoxic drugs into liposomes enhances pharmacokinetics and improves passive accumulation in tumors. However, stable liposomes have limited drug release, and thus action, at the target site. This inefficient and unpredictable drug release is compounded by a lack of low-cost, non-invasive methods to map release in real time. We present a new liposomal vehicle that is exclusively triggered by inertial cavitation. Ultrasound exposure of these liposomes in the absence of SonoVue® provided no increase in drug release, whilst with SonoVue® at inertial cavitation pressure levels a substantial (30%) and significant (p < 0.001) increase was observed in vitro. A 16-fold increase in the level of drug release within tumors was similarly observed in the presence of inertial cavitation following intravenous delivery. Passive acoustic mapping of inertial cavitation sources during delivery was also found to correlate strongly with the presence of release. However, variability in tumor perfusion indicated that uneven distribution of micron-sized SonoVue® may limit this approach. Nano-scale cavitation nuclei, which may more readily co-localize with 140 nm liposomes, were thus developed and showed similar cavitation energies to SonoVue® in vitro. These nano-nuclei may ultimately provide a more reliable and uniform way to trigger drug release in vivo.

Enhanced cytotoxicity of optimized liposomal genistein via specific induction of apoptosis in breast, ovarian and prostate carcinomas

Clinical use of genistein against cancer is limited by its extremely low aqueous solubility, poor bioavailability and pharmacokinetics. Based on structural analogy with steroidal compounds, liposomal vehicle compositions were designed and optimized for maximum incorporation of genistein’s flavonoid structure. Model conventional and stealth liposomes of genistein (GenLip) – incorporating unsaturated phospholipids and cholesterol – have demonstrated enhanced drug solubilization (over 350-folds > aqueous drug solution), shelf-life stability, and extended release profile. Owing to effective cellular delivery, preservation of genistein’s antioxidant activity was confirmed through marked neutralization of peroxides via GenLip, in both quantitative and microscopic fluorescent-probe oxidation assays. Furthermore, significant broad-spectrum anticancer efficacy of GenLip, in murine and human cancer cell lines (p < 0.05–0.001), was achieved in a concentration and time-dependent manner – approx. 5–7 lower IC50 values versus all non-incorporated drug controls. Indicative of key pro-apoptotic activity, GenLip produced DNA laddering, with 1/3 of free drug solution content, and resulted in the highest induction level of P53-independent apoptotic pathway markers, compared to all treatments, in our assays (namely, mitochondrial polarization, and caspase-3/7 enzymes). Our proof-of-principle pharmaceutical design of genistein-loaded liposomes shows optimal loading capacity and physico-chemical properties, which improved cellular delivery and specific pro-apototic effectiveness of incorporated drug, against various cancers.

Effects of Antigen-Expressing Immunostimulatory Liposomes on Chemotaxis and Maturation of Dendritic Cells In Vitro and in Human Skin Explants

Antigen-Expressing Immunostimulatory Liposomes (AnExILs) represent a novel DNA vaccination platform based on the production of protein antigens from DNA templates inside liposomes mediated by an in vitro transcription and translation (IVTT) mix. The aim of this study was to analyze the effects of AnExILs on different dendritic cells (DCs) models and to better understand the role of the different components of this formulation on its adjuvanticity. The effect of β-galactosidase-expressing AnExILs on maturation and particle uptake by murine DC cell line, fresh human monocyte-derived DCs or human dermal DCs in skin explants was investigated and compared to the effects of either plain liposomes or IVTT mix alone. AnExILs induced efficient DC chemotaxis and promoted up-regulation of maturation markers on murine DCs, due to the presence of IVTT in the formulation. Furthermore, the amount of active βGal associated with DCs was higher for AnExILs than for free βGal expressed in IVTT or βGal encapsulated into non-adjuvanted liposomes. Most interestingly, the same trend was observed with human DCs. Both IVTT mix and liposomal vehicles were shown to be key components of the AnExIL formulation responsible for its adjuvanticity. AnExILs combine antigen production, adjuvanticity and delivery in one system, and can efficiently activate both murine and human DCs.

Towards boron neutron capture therapy: The formulation and preliminary in vitro evaluation of liposomal vehicles for the therapeutic delivery of the dequalinium salt of bis-nido-carborane

Liposomes of phosphatidylcholine or of dimyristoylphosphatidylcholine that incorporate bis-nido-carborane dequalinium salt are stable in physiologically relevant media and have in vitro toxicity profiles that appear to be compatible with potential therapeutic applications. These features render the structures suitable candidate boron-delivery vehicles for evaluation in the boron neutron capture therapy of cancer.

Lipophilicity of flavonoid complexes with iron(II) and their interaction with liposomes

We studied complex formation of flavonoids quercetin and taxifolin with iron(II) and the complex influence on phase transitions of phospholipid bilayer. UV–Vis spectroscopy revealed that the stoichiometry of flavonoid/iron complexes was equal to 3:2 and 2:1. Molecular modeling and experimental measurements demonstrated the increase of flavonoids lipophilicity after the complex formation. A considerable influence of quercetin–iron complex on Palmitoyl–Oleoyl–Phosphatidylethanolamine transitions from bilayer to hexagonal HII phase was detected by differential scanning calorimetry. The obtained data are related to flavonoid/iron complexes bioavailability, their influence on cell membrane functioning, and should be considered in designing liposomal vehicles for drug and gene delivery.

Clinical developments of chemotherapeutic nanomedicines: polymers and liposomes for delivery of camptothecins and platinum (II) drugs

For the past 40 years, liposomal and polymeric delivery vehicles have been studied as systems capable of modulating the cytotoxicity of small molecule chemotherapeutics, increasing tumor bearing animal survival times, and improving drug targeting. Although a number of macromolecular-drug conjugates have progressed to clinical trials, tuning drug release to maintain efficacy in conjunction with controlling drug toxicity has prevented the clinical adoption of many vehicles. In this article, we review the motivations for and approaches to polymer and liposomal delivery with regard to camptothecin and cisplatin delivery.

Three newly approved analgesics: an update.

Abstract Since 2008, three new analgesic entities, tapentadol immediate release (Nucynta) diclofenac potassium soft gelatin capsules (Zipsor), and bupivacaine liposome injectable suspension (EXPAREL) were granted US Food and Drug Administration (FDA) approval to treat acute pain. Tapentadol immediate-release is a both a mu-opioid agonist and a norepinephrine reuptake inhibitor, and is indicated for the treatment of moderate to severe pain. Diclofenac potassium soft gelatin capsules are a novel formulation of diclofenac potassium, which is a nonsteroidal anti-inflammatory drug (NSAID), and its putative mechanism of action is through inhibition of cyclooxygenase enzymes. This novel formulation of diclofenac allows for improved absorption at lower doses. Liposomal bupivacaine is a new formulation of bupivacaine intended for single-dose infiltration at the surgical site for postoperative analgesia. Bupivacaine is slowly released from this liposomal vehicle and can provide prolonged analgesia at the surgical site. By utilizing NSAIDs and local anesthetics to decrease the transmission of afferent pain signals, less opioid analgesics are needed to achieve analgesia. Since drug-related adverse events are frequently dose related, lower doses from different drug classes may be employed to reduce the incidence of adverse effects, while producing synergistic analgesia as part of a multimodal analgesic approach to acute pain.

Comparison of five different targeting ligands to enhance accumulation of liposomes into the brain

In many different studies nanocarriers modified with targeting ligands have been used to target to the brain. Many ligands have been successful, but it is difficult to compare results from different studies to determine which targeting ligand is the best. Therefore, we selected five targeting ligands (transferrin, RI7217, COG133, angiopep-2, and CRM197) and compared their ability to target liposomes to the brain in vitro and in vivo. In vitro, only CRM197-modified liposomes were able to bind to murine endothelial cells (bEnd.3). Both CRM197 and RI7217-modified liposomes associated with human endothelial cells (hCMEC/D3). In vivo, uptake of targeted liposomes was tested at 12 h after iv injection. For some of the ligands, additional time points of 1 and 6 h were tested. Only the RI7217 was able to significantly enhance brain uptake in vivo at all time points. Uptake in the brain capillaries was up to 10 times higher compared to untargeted liposomes, and uptake in the brain parenchyma was up to 4.3 times higher. Additionally, these results show that many targeting ligands that have been described for brain targeting, do not target to the brain in vivo when coupled to a liposomal delivery vehicle.

Nano-sized Drug Carriers and Key Factors for Lymphatic Delivery

【Specific diseases like cancer and acquired immune deficiency syndrome (AIDS) occur at various organs including lymphatics and spread through lymphatic system. Thus, if therapeutic agents for such diseases are more distributed or targeted to lymphatic system, we can obtain several advantages like reduction of systemic side effect and increase of efficacy. For these reasons, much interest has been focused on the nature of lymphatics and a lot of studies for lymphatic delivery of drugs have been carried out. Because lymphatics consist of single layer endothelium and have high permeability compared with blood capillaries, especially, the studies using nano-sized carriers have been performed. Polymeric nano-particle, liposome, and lipid-based vehicle have been adopted for lymphatic delivery as carriers. According to the administration route and the kind of carrier, the extent of lymphatic delivery efficiency of nano-sized carriers has been changed and influenced by several factors such as size, charge, hydrophobicity and surface feature of carrier. In this review, we summarized the key factors which affect lymphatic uptake and the major features of carriers for achieving the lymphatic delivery. Lymphatic delivery of drug using nano-sized carriers has many fold improved ability of lymphatic delivery compared with that of conventional dosage forms, but it has not shown whole lymph selectivity yet. Even though nano-sized carriers still have the potential and worth to study as lymphatic drug delivery technology as before, full understanding of delivery mechanism and influencing factors, and setting of pharmacokinetic model are required for more ideal lymphatic delivery of drug.】

Anionic long‐circulating liposomes for delivery of radioiodinated antisense oligonucleotides

AbstractLong‐circulating liposomes have been widely used to enhance efficacy of gene therapy. Antisense therapy might increase the efficacy of radiation or chemotherapy; therefore, we undertook to optimize the composition of the liposomal delivery vehicles. The radiolabeling efficiency, radiochemistry purity, and specific radioactivity of radioiodinated antisense oligonucleotides (ASON) were 71.66 ± 7.73, 98.33 ± 0.39%, 4.09 ± 0.11 MBq/nmol. Radioiodinated ASON remained stable in 0.01 M HEPES buffer and human serum even after incubation for 4 hours. Mean diameter of the anionic long‐circulating liposomes (ALCL) was 504 ± 31.76 nm with a polydispersity index (PDI) of 0.107 ± 0.008 before extrusion, 115 ± 8.5 nm with a PDI of 0.103 ± 0.002 after extrusion. The zeta potential of ALCL was −29.23 ± 0.45 mV. ALCL prepared for this study provided 70.28 ± 1.84% encapsulation efficiency. Compared with other liposome formulations, the ALCL mediated enhanced cellular uptake (32.51 ± 1.44%) by MCF‐7 breast cancer cells (p&lt;0.05).Practical applications: Drug delivery systems can in principle provide enhanced efficacy and/or reduced toxicity for anticancer agents. LCL have become a commonly used carrier for gene therapy recently. ALCL are promising for the mediated radiation and antisense therapy for breast cancer which is the leading cause for women.

Abstract 4609: Therapeutic effects of pegylated doxorubicin liposomes in endocrine responsive and unresponsive murine mammary carcinomas

Abstract Doxorubicin is commonly used in the treatment of several cancers, including breast and ovarian cancers. In spite of the development of modern therapeutic strategies, chemotherapy is still the treatment of choice for breast carcinomas resistant to endocrine therapy. The aim of this project was a) to evaluate the effect of doxorubicin in experimental hormone-responsive and unresponsive carcinomas, b) to evaluate if hormone resistance was associated with drug resistance and c) to test endocrine therapy in combination with doxorubicin. The tumors used were ductal metastatic mammary carcinomas expressing high levels of estrogen and progesterone receptors which show different degrees of hormone responsiveness. They have been induced in BALB/c mice by medroxyprogesterone acetate. Free doxorubicin (doxo) and commercial pegylated liposomes containing doxorubicin obtained from Schering-Plough (Cx) or Laboratorios Raffo (Dg), were used. The human ovarian carcinoma cell line SKOV-3, transplanted in nude mice was used as positive control of doxorubicin responsiveness. Eleven days after s.c. transplantation, when tumors were already palpable, mice were treated i.v. with either doxo, Cx or Dg in doses of 18 or 9mg/kg body weight once a week during 1 month. Liposome vehicle was used in controls. High doses of Cx or Dg, inhibited tumor growth, but showed evident signs of toxicity (body weight loss, cardiotoxicity, skin lesions and histopathological evaluation); doxo induced a delay in tumor growth, and more severe signs of toxicity. Interestingly, the lower dose of Cx or Dg had similar therapeutic effects than the higher dose but inducing almost no toxicity. We then selected two mifepristone and 17-β-estradiol-responsive mammary carcinomas, C4-HI and CC4-3-HI, and one variant, C4-2-HI, which is unresponsive to both and we used the same experimental design as explained above. Treatments were started 4 or 11 days after tumor transplantation. The three tumors responded similarly to the chemotherapy. Doxo showed higher side effects than the liposome forms and no differences were registered between Cx or Dg-treated animals. Mice carrying C4-HI tumors of about 50 mm2, were treated with the lower dose of Dg, plus/or the antiprogestin mifepristone (12 mg/kg/day, s.c.) or 5 mg 17-β-estradiol pellets. Combined treatments induced a better therapeutic effect than single treatments (p&amp;lt;0.05). We can conclude that a) no differences were observed between both liposome formulations, b) the impressive therapeutic effects observed with the lower dose of Cx or Dg used, suggests that side effect can be minimized by using lower doses, c) the MPA murine breast cancer model is appropriate for chemotherapeutic drug screening either alone or in combination with endocrine therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4609.

Hereditary inclusion body myopathy: single patient response to GNE gene Lipoplex therapy

Hereditary inclusion body myopathy (HIBM) is an autosomal recessive adult onset myopathy. It is characterized by mutations of the GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) gene. Afflicted patients have no therapeutic options. In preclinical testing, we have previously demonstrated the ability to correct GNE gene function and the safety of delivery of wild type GNE gene using a liposomal delivery vehicle. A single patient (subject #001) with severe HIBM treated by compassionate investigational new drug received four doses of GNE gene Lipoplex via intramuscular injection. GNE transgene expression, downstream induction of sialic acid, safety and muscle function were evaluated. Significant durable improvement in locoregional skeletal muscle function was observed in the injected left extensor carpi radialis longus of #001 in correlation with GNE transgene upregulation and local induction of sialic acid. Other than transient low grade fever and pain at the injection site, no significant toxicity was observed. Proof of principle for manufacturing of 'clinical grade' GNE gene Lipoplex, clinical safety and activity are demonstrated with GNE gene Lipoplex. Further assessment will involve intravenous administration and subsequent phase I trial involving additional but less severely afflicted HIBM patients.

Identification and assessment of permeability enhancing vehicles for transdermal delivery of glucosamine hydrochloride

As an initial step to develop the transdermal delivery system of glucosamine hydrochloride (GL-HCl), the permeation study across the rat skin in vitro was performed to identify the most efficient vehicle with regard to the ability to deliver GL-HCl transdermally. The GL-HCl formulations such as o/w cream, liposome suspension, liposomal gel, and liquid crystalline vehicles were prepared and compared for transdermal flux of GL-HCl. The liquid crystalline vehicles were more effective in increasing the skin permeation of GL-HCl than o/w cream and liposomal vehicles. Of the liquid crystalline vehicles tested, the permeation enhancing ability of the cubic phase was greater than that of the hexagonal phase when the nanoparticle dispersion was used. The skin permeation enhancing ability of the cubic nanoparticles for GL-HCl was further increased by employing both oleic acid and polyethylene glycol 200. Therefore, the cubic liquid crystalline nanodispersion containing oleic acid and PEG 200 can provide a possibility of clinical application of transdermal GL-HCl.

Liposomal 1,25-dihydroxyvitamin D3-3β-bromoacetate is a Stronger Growth-inhibiting Agent than its un-encapsulated Counterpart in Prostate Cancer Cells

Encapsulating cytotoxic drugs in liposomal vehicles allows for the targeting of tumors while protecting the drugs from premature degradation. 1,25-Dihydroxyvitamin D3-3?-bromoacetate (1,25(OH)2D3-3-BE), a vitamin D receptor-alkylating agent inhibits the growth of prostate cancer cells. The aim of the study was to evaluate the efficacy of a liposomal preparation of 1,25(OH)2D3-3-BE versus 1,25(OH)2D3-3-BE in modulating the growth of prostate cancer cells. Results demonstrate that liposomal 1,25(OH)2D3-3-BE is significantly better than 1,25(OH)2D3-3-BE in inhibiting the growth. In addition, liposomal 1,25(OH)2D3-3-BE was found to be stable in human serum. Taken together, results of the studies delineated here suggest a therapeutic potential of liposomal 1,25(OH)2D3-3-BE in prostate cancer.

Evaluation of Efficacy, Biodistribution, and Inflammation for a Potent siRNA Nanoparticle: Effect of Dexamethasone Co-treatment

Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)-based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects. In this study, we characterized a liposomal siRNA delivery vehicle, LNP201, which is capable of silencing an mRNA target in mouse liver by over 80%. The biodistribution profile, efficacy after single and multiple doses, mechanism of action, and inflammatory toxicity are characterized for LNP201. Furthermore, we demonstrate that the glucocorticoid receptor (GR) agonist dexamethasone (Dex) inhibits LNP201-induced cytokine release, inflammatory gene induction, and mitogen-activated protein kinase (MAPK) phosphorylation in multiple tissues. These data present a possible clinical strategy for increasing the safety profile of siRNA-based drugs while maintaining the potency of gene silencing.