Rapamycin-Loaded Biomimetic Nanoparticles Reverse Vascular Inflammation.

RATIONALE: Through localized delivery of rapamycin via a biomimetic drug delivery system, it is possible to reduce vascular inflammation and thus the progression of vascular disease. OBJECTIVE: Use biomimetic nanoparticles to deliver rapamycin to the vessel wall to reduce inflammation in an in vivo model of atherosclerosis after a short dosing schedule. METHODS AND RESULTS: Biomimetic nanoparticles (leukosomes) were synthesized using membrane proteins purified from activated J774 macrophages. Rapamycin-loaded nanoparticles were characterized using dynamic light scattering and were found to have a diameter of 108±2.3 nm, a surface charge of -15.4±14.4 mV, and a polydispersity index of 0.11 +/ 0.2. For in vivo studies, ApoE-/- mice were fed a high-fat diet for 12 weeks. Mice were injected with either PBS, free rapamycin (5 mg/kg), or rapamycin-loaded leukosomes (Leuko-Rapa; 5 mg/kg) once daily for 7 days. In mice treated with Leuko-Rapa, flow cytometry of disaggregated aortic tissue revealed fewer proliferating macrophages in the aorta (15.6±9.79 %) compared with untreated mice (30.2±13.34 %) and rapamycin alone (26.8±9.87 %). Decreased macrophage proliferation correlated with decreased levels of MCP (monocyte chemoattractant protein)-1 and IL (interleukin)-b1 in mice treated with Leuko-Rapa. Furthermore, Leuko-Rapa-treated mice also displayed significantly decreased MMP (matrix metalloproteinases) activity in the aorta (mean difference 2554±363.9, P=9.95122×10-6). No significant changes in metabolic or inflammation markers observed in liver metabolic assays. Histological analysis showed improvements in lung morphology, with no alterations in heart, spleen, lung, or liver in Leuko-Rapa-treated mice. CONCLUSIONS: We showed that our biomimetic nanoparticles showed a decrease in proliferating macrophage population that was accompanied by the reduction of key proinflammatory cytokines and changes in plaque morphology. This proof-of-concept showed that our platform was capable of suppressing macrophage proliferation within the aorta after a short dosing schedule (7 days) and with a favorable toxicity profile. This treatment could be a promising intervention for the acute stabilization of late-stage plaques.

Lipid-coated superparamagnetic nanoparticles for thermoresponsive cancer treatment.

Poor aqueous solubility, chemical instability, and indiscriminate cytotoxicity have limited clinical development of camptothecin (CPT) as potent anticancer therapeutic. This research aimed at fabricating thermoresponsive nanocomposites that enhance solubility and stability of CPT in aqueous milieu and enable stimulus-induced drug release using magnetic hyperthermia. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and l-α-dipalmitoylphosphatidyl glycerol (DPPG) (1:1, mol/mol) were immobilized on the surface of superparamagnetic Fe3O4 nanoparticles (SPIONs) via high affinity avidin-biotin interactions. Heating behavior was assessed using the MFG-1000 magnetic field generator. Encapsulation efficiency and drug release were quantified by fluorescence spectroscopy. Anticancer efficacy of medicated nanoparticles was measured in vitro using Jurkat cells. The results revealed that drug incorporation did not significantly alter particle size, zeta potential, magnetization, and heating properties of lipid-coated SPIONs. Drug loading efficiency was 93.2 ±â€¯5.1%. Drug release from medicated nanoparticles was significantly faster at temperatures above the lipid transition temperature, reaching 37.8 ±â€¯2.6% of incorporated payload after 12 min under therapeutically relevant hyperthermia (i.e., 42 °C). Medicated SPIONs induced greater cytotoxicity than CPT in solution suggesting synergistic activity of magnetically-induced hyperthermia and drug-induced apoptosis. These results underline the opportunity for thermoresponsive phospholipid-coated SPIONs to enable clinical development of highly lipophilic and chemically unstable drugs such as CPT for stimulus-induced cancer treatment.

Phototoxicity of Liposomal Zn- and Al-phthalocyanine Against Cervical and Oral Squamous Cell Carcinoma Cells In Vitro.

Background Material and Methods Results Conclusions.

Effects of nickel-oxide nanoparticle pre-exposure dispersion status on bioactivity in the mouse lung.

Nanotechnology is emerging as one of the world's most promising new technologies. From a toxicology perspective, nanoparticles possess two features that promote their bioactivity. The first involves physical-chemical characteristics of the nanoparticle, which include the surface area of the nanoparticle. The second feature is the ability of the nanoparticle to traverse cell membranes. These two important nanoparticle characteristics are greatly influenced by placing nanoparticles in liquid medium prior to animal exposure. Nanoparticles tend to agglomerate and clump in suspension, making it difficult to reproducibly deliver them for in vivo or in vitro experiments, possibly affecting experimental variability. Thus, we hypothesize that nanoparticle dispersion status will correlate with the in vivo bioactivity/toxicity of the particle. To test our hypothesis, nano-sized nickel oxide was suspended in four different dispersion media (phosphate-buffered saline (PBS), dispersion medium (DM), a combination of dipalmitoyl-phosphatidyl choline (DPPC) and albumin in concentrations that mimic diluted alveolar lining fluid), Survanta®, or pluronic (Pluronic F-68). Well-dispersed and poorly dispersed suspensions were generated in each media by varying sonication time on ice utilizing a Branson Sonifer 450 (25W continuous output, 20 min or 5 min, respectively). Mice (male, C57BL/6J, 7-weeks-old) were given 0-80 µg/mouse of nano-sized nickel oxide in the different states of dispersion via pharyngeal aspiration. At 1 and 7 d post-exposure, mice underwent whole lung lavage to assess pulmonary inflammation and injury as a function of dispersion status, dose and time. The results show that pre-exposure dispersion status correlates with pulmonary inflammation and injury. These results indicate that a greater degree of pre-exposure dispersion increases pulmonary inflammation and cytotoxicity, as well as decreases in the integrity of the blood-gas barrier in the lung.

Brucine-loaded liposomes composed of HSPC and DPPC at different ratios: in vitro and in vivo evaluation.

OBJECTIVE: The objective of this study is to test the hypothesis that the phase transition temperature (T(m)), the main property of liposomes, can be easily controlled by changing the molar ratio of hydrogenated soy phosphatidylcholine (HSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphacholine (DPPC) after drug encapsulation. MATERIALS AND METHODS: Brucine, an antitumor alkaloid, was encapsulated into the liposomes with different HSPC/DPPC compositions. The T(m)s of the brucine-loaded liposomes (BLs) were determined by differential scanning calorimetry (DSC). Then the physicochemical properties and pharmacokinetics of the BLs with different HSPC/DPPC compositions were investigated and compared. RESULTS: The results of DSC revealed that HSPC and DPPC can combine into one phase. The findings of molecular modeling study suggested that HSPC interacts with DPPC via electrostatic interaction. The molar ratio of HSPC/DPPC influenced the sizes of BLs but had little effect on the entrapment efficiency (EE). The stability of BLs was improved with the increase of the HSPC ratios, especially with the presence of plasma. Following i.v. administration, it was found that AUC values of BLs in vivo were directly related to the HSPC/DPPC ratios of BLs, namely the T(m)s of BLs. DISCUSSION: The behavior of liposomes, especially in vivo pharmacokinetic behavior, can be controlled by the modification of T(m). CONCLUSION: The characterization of BLs in vitro and in vivo had demonstrated that the Tm could be flexibly modified for liposomes composed of both HSPC and DPPC. Using HSPC/DPPC composition may be an efficient strategy to control the T(m), thus control the in vivo pharmacokinetic behavior, of BLs.

New surfactant with SP-B and C analogs gives survival benefit after inactivation in preterm lambs.

BACKGROUND: Respiratory distress syndrome in preterm babies is caused by a pulmonary surfactant deficiency, but also by its inactivation due to various conditions, including plasma protein leakage. Surfactant replacement therapy is well established, but clinical observations and in vitro experiments suggested that its efficacy may be impaired by inactivation. A new synthetic surfactant (CHF 5633), containing synthetic surfactant protein B and C analogs, has shown comparable effects on oxygenation in ventilated preterm rabbits versus Poractant alfa, but superior resistance against inactivation in vitro. We hypothesized that CHF 5633 is also resistant to inactivation by serum albumin in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Nineteen preterm lambs of 127 days gestational age (term = 150 days) received CHF 5633 or Poractant alfa and were ventilated for 48 hours. Ninety minutes after birth, the animals received albumin with CHF 5633 or Poractant alfa. Animals received additional surfactant if P(a)O(2) dropped below 100 mmHg. A pressure volume curve was done post mortem and markers of pulmonary inflammation, surfactant content and biophysiology, and lung histology were assessed. CHF 5633 treatment resulted in improved arterial pH, oxygenation and ventilation efficiency index. The survival rate was significantly higher after CHF 5633 treatment (5/7) than after Poractant alfa (1/8) after 48 hours of ventilation. Biophysical examination of the surfactant recovered from bronchoalveolar lavages revealed that films formed by CHF 5633-treated animals reached low surface tensions in a wider range of compression rates than films from Poractant alfa-treated animals. CONCLUSIONS: For the first time a synthetic surfactant containing both surfactant protein B and C analogs showed significant benefit over animal derived surfactant in an in vivo model of surfactant inactivation in premature lambs.

Bypassing multidrug resistance in human breast cancer cells with lipid/polymer particle assemblies.

BACKGROUND: Multidrug resistance (MDR) mediated by the overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), remains one of the major obstacles to effective cancer chemotherapy. In this study, lipid/particle assemblies named LipoParticles (LNPs), consisting of a dimethyldidodecylammonium bromide (DMAB)-modified poly(lactic-co-glycolic acid) (PLGA) nanoparticle core surrounded by a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) shell, were specially designed for anticancer drugs to bypass MDR in human breast cancer cells that overexpress P-gp. METHODS: Doxorubicin (DOX), a chemotherapy drug that is a P-gp substrate, was conjugated to PLGA and encapsulated in the self-assembled LNP structure. Physiochemical properties of the DOX-loaded LNPs were characterized in vitro. Cellular uptake, intracellular accumulation, and cytotoxicity were compared in parental Michigan Cancer Foundation (MCF)-7 cells and P-gp-overexpressing, resistant MCF-7/adriamycin (MCF-7/ADR) cells. RESULTS: This study found that the DOX formulated in LNPs showed a significantly increased accumulation in the nuclei of drug-resistant cells relative to the free drug, indicating that LNPs could alter intracellular traffic and bypass drug efflux. The cytotoxicity of DOX loaded-LNPs had a 30-fold lower half maximal inhibitory concentration (IC(50)) value than free DOX in MCF-7/ADR, measured by the colorimetric cell viability (MTT) assay, correlated with the strong nuclear retention of the drug. CONCLUSION: The results show that this core-shell lipid/particle structure could be a promising strategy to bypass MDR.

Phospholipase A(2)-susceptible liposomes of anticancer double lipid-prodrugs.

A novel approach to anticancer drug delivery is presented based on lipid-like liposome-forming anticancer prodrugs that are susceptible to secretory phospholipase A(2) (sPLA(2)) that is overexpressed in several cancer types. The approach provides a selective unloading of anticancer drugs at the target tissues, as well as circumvents the necessity for "conventional" drug loading. In our attempts to improve the performance of the liposomes in vivo, several PEGylated and non-PEGylated liposomal formulations composed of a retinoid prodrug premixed with the sPLA(2)-hydrolyzable DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) were prepared. Besides favorably modifying the physicochemical properties of the liposomes, the incorporation of DPPC and PEG-lipids in the liposomes should substantially enhance the enzymatic activity, as concluded from literature. In addition, one can reap benefits from the presumed permeability enhancing effect of the liberated fatty acids and lysolipids. The size distribution of the prepared liposomes as well as their phase behavior, enzymatic hydrolysis, and cytotoxicity, in the presence and absence of sPLA(2), were determined. The liposomes were around 100nm in diameter and in the gel/fluid coexistence region at 37°C. The enzymatic hydrolysis of the prodrug was pronouncedly accelerated upon the premixing with DPPC, and the hydrolysis was further enhanced by PEGylation. Interestingly, the faster hydrolysis of the prodrug and the released fatty acids and lysolipids from DPPC did not improve the cytotoxicity of the mixture; the effect of combining the prodrug with DPPC was additive and not synergistic. The data presented here question the significance of the permeability enhancing effects claimed for fatty acids and lysolipids at the target cell membrane, and whether these effects can be achieved using physiologically achievable concentrations of fatty acids and lysolipids.

Overcoming cellular multidrug resistance using classical nanomedicine formulations.

Over the past few decades, many different types of nanomedicines have been evaluated, both in vitro and in vivo. In general, nanomedicines are designed to improve the in vivo properties of low-molecular-weight (chemo-) therapeutic drugs, i.e. their biodistribution and the target site accumulation, and to thereby improve the balance between their efficacy and toxicity. A significant number of studies have also addressed the in vitro properties of nanomedicines, showing e.g. their ability to overcome cellular multidrug resistance (MDR). Particularly promising results in this regard have been reported for 'pharmacologically active' carrier materials, such as Pluronics, which are able to directly inhibit drug efflux pumps and other cellular detoxification mechanisms. In the present report, we have set out to evaluate the ability of classical (and pharmacologically inactive) carrier materials to overcome MDR. To this end, four different drug-sensitive and drug-resistant cancer cell lines were treated with increasing concentrations of free doxorubicin, of polymer-bound doxorubicin, of micellar doxorubicin and of liposomal doxorubicin, and resistance indices (IC(50) in resistant cells/IC(50) in sensitive cells) were determined. In addition, the cellular uptake of the four formulations was evaluated using fluorescence microscopy. It was found that the carrier materials did manage to overcome MDR to some extent, but that the overall benefit was quite small; only for polymer-bound doxorubicin in A431 cells, a significant (4-fold) reduction in the resistance index was observed. These findings indicate that the ability of classical nanomedicines to overcome cellular MDR should not be overestimated.

Nanovesicle aerosols as surfactant therapy in lung injury.

Acute lung injury causes inactivation of pulmonary surfactant due to leakage of albumin and other markers. Current surfactants are ineffective in this condition and are instilled intratracheally. Nanovesicles of 300 ± 50 nm composed of nonlamellar phospholipids were developed as pulmonary surfactant aerosols for therapy in acid-induced lung injury. A combination of dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine was used. The size and composition of the nanovesicles were optimized for an improved airway patency in the presence of albumin and serum. In an acid-induced lung injury model in mice, on treatment with nanovesicle aerosols at a dose of 200 mg/kg, the alveolar protein leakage decreased from 8.62 ± 0.97 µg/mL to 1.94 ± 0.74 µg/mL, whereas the airway patency of the bronchoalveolar lavage fluid increased from 0.6 ± 0.0% to 91.7 ± 1.05%. Nanovesicle aerosols of nonlamellar lipids improved the resistance of pulmonary surfactants to inhibition and were promising as a noninvasive aerosol therapy in acute lung injury. FROM THE CLINICAL EDITOR: In acute lung injury, intrinsic surfactants are inactivated via albumin leakage and other mechanisms. Currently existing intratracheal surfactants are ineffective in this condition. The authors demonstrate that novel nanovesicle aerosols of nonlamellar lipids improved the resistance of pulmonary surfactants to inhibition and are promising as a noninvasive aerosol therapy in acute lung injury.

The apolipoprotein A-I mimetic peptide ETC-642 exhibits anti-inflammatory properties that are comparable to high density lipoproteins.

OBJECTIVES: Mimetic peptides of apolipoprotein A-I (apoA-I) present a new strategy for promoting the biological activity of high density lipoproteins (HDL). This study aimed to compare the anti-inflammatory effects of ETC-642, a new apoA-I mimetic peptide, with discoidal reconstituted HDL (rHDL). METHODS: New Zealand White rabbits (n=42) received daily infusions of saline, rHDL or discoidal complexes of an amphipathic peptide, ETC-642 (1-30 mg/kg), prior to insertion of non-occlusive carotid collars. Human coronary artery endothelial cells (HCAECs) were pre-incubated with ETC-642 or rHDL before TNF-α stimulation. Monocyte adhesion was investigated by pre-incubating HCAECs with rHDL or ETC-642, stimulating with TNF-α and incubating with THP-1 monocytes. RESULTS: Infusion of ETC-642 resulted in dose-dependent reductions of collar-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the artery wall (p<0.05). Pre-incubation of HCAECs with ETC-642 and rHDL reduced TNF-α-induced THP-1 monocyte adhesion (p<0.01). Furthermore, ETC-642 and rHDL treatment reduced TNF-α induced mRNA levels of inflammatory markers VCAM-1, fractalkine, MCP-1 and the p65 subunit of NF-κB (p<0.05). CONCLUSION: These studies demonstrate that ETC-642 exhibits anti-inflammatory properties that are comparable to apoA-I both in vivo and in vitro and that these effects are mediated via the NF-κB signaling pathway.

Correlation between in vivo pharmacokinetics, intratumoral distribution and photodynamic efficiency of liposomal mTHPC.

Foslip is a recently designed third generation photosensitiser based on unilamellar dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) liposomal formulations of meta-tetra(hydroxyphenyl)chlorine (mTHPC). The present study investigates Foslip behaviour and its photodynamic efficiency in EMT6 xenografted nude mice at different times following i.v. administration of 0.3 mg kg(-1) mTHPC in a Foslip formulation. Plasma pharmacokinetics and biodistribution were studied by high performance liquid chromatography and were described by a three compartments analysis with half-lifes of 0.13, 4.31 and 35.7 h. The highest tumour to muscle ratios were observed at 6 and 15 h post-administration. Intratumoral distribution was carried out using two photon excitation confocal microscopy. Progressive efflux from the vascular compartment was noted in favour of tumour parenchyma, which was almost completed at 15 h. The best tumour response was obtained for a drug-light interval of 6 h, interval for which mTHPC was present in both endothelial and parenchyma cells. Tumour and plasma concentrations however were far below their maximal values. Based on these observations, we assume that the presence of mTHPC in both vasculature and tumour cells is required for optimal PDT efficacy.

The effect of hyaluronic acid and phospholipid based lubricants on friction within a human cartilage damage model.

The lubricating abilities of different formulations of high molecular weight hyaluronic acid (HA), dipalmitoyl phosphatidylcholine (DPCC) and mixtures of both HA and DPCC were assessed in an in vitro model. Levels of start-up friction were determined using an osteoarthritis (OA) damaged human cartilage model set within a specially designed friction rig. To examine the long term benefits of HA, the extent of penetration of HA into cartilage tissue was investigated using fluorescently labelled HA and confocal microscopy. It was found that in this model, all formulations of HA and the majority of DPCC lubricants reduced friction (HA 5 and 10 mg ml(-1), DPPC 200 mg ml(-1) reductions of 51.9%, 46.7% and 46.5% respectively), compared to a Ringers solution control. Lubrication was found not to be concentration dependent for HA formulations, but concentration was key for DPCC lubrication (100 mg ml(-1) reduced friction by only 15.9%). By combining HA and DPCC (HA/DPPC; 5 mg ml(-1)/100 mg ml(-1) and 10 mg ml(-1)/200 mg ml(-1)), a further improvement was noted (69.5% and 61.9%, respectively) as the mean levels of friction were reduced by up to a further 17% than the most effective individual formulation (HA 5 mg ml(-1)). Penetration of HA into bovine cartilage by up to 300 microm from the surface was observed over a 48 h period. It was observed that HA specifically targeted the chondrocytes as it was primarily found within the lacunae surrounding the cells.

Exogenous surfactant therapy for ARDS.

Regardless of the cause, a common pathophysiological feature of patients with acute respiratory distress syndrome is a dysfunction of the endogenous surfactant system. Although exogenous surfactant therapy has proven to be an effective treatment for neonatal respiratory distress syndrome, no similar current effective therapy exists for patients with acute respiratory distress syndrome. This is mainly due to the complexity of the lung injury that is involved with this disorder. Results from clinical trials, to date, have failed to show an improvement in patient survival after administration of exogenous surfactant; however, ongoing and future research efforts suggest that this therapy may eventually be feasible.

Surfactant kinetics in preterm infants on mechanical ventilation who did and did not develop bronchopulmonary dysplasia.

OBJECTIVE: To characterize surfactant kinetics in vivo in two groups of premature infants on different levels of mechanical ventilation and at different risk of developing bronchopulmonary dysplasia. DESIGN: Controlled observational study in two independent groups of infants. SETTING: Neonatal intensive care unit. PATIENTS: Thirteen preterm infants (26 +/- 0.5 wks, birth weight 801 +/- 64 g) on high ventilatory setting and who finally all developed bronchopulmonary dysplasia (MechVentBPD), and eight (26 +/- 0.5 wks, birth weight 887 +/- 103 g) who had minimal or no lung disease and of whom none developed bronchopulmonary dysplasia (MechVentNoBPD). MEASUREMENTS AND MAIN RESULTS: Endotracheal 13C-labeled dipalmitoyl-phosphatidylcholine was administered and subsequent measurements of the 13C enrichment of surfactant-disaturated phosphatidylcholine (DSPC) from serial tracheal aspirates were made by gas chromatography-mass spectrometry. We calculated disaturated phosphatidylcholine pharmacokinetic variables in terms of half-life and apparent pool size from the enrichment decay curves over time. DSPC concentration from tracheal aspirates was expressed as milligrams/milliliter epithelial lining fluid (ELF-DSPC). Data are presented as mean +/- se. In MechVentBPD infants vs. MechVentNoBPD, ELF-DSPC was much reduced, 2.9 +/- 0.6 vs. 9.4 +/- 3.0 mg/mL ELF (p =.03), half-life was shorter, 19.4 +/- 2.8 vs. 42.5 +/- 6.3 hrs (p =.002), and apparent pool size larger, 136 +/- 21 vs. 65.8 +/- 16.0 mg/kg (p =.057). In MechVentBPD, apparent DSPC pool size positively correlated with mean airway pressure x Fio(2) and inversely correlated with ELF-DSPC. ELF-DSPC was inversely correlated with mean airway pressure x Fio(2). No significant correlations were found in the MechVentNoBPD group. CONCLUSIONS: MechVentBPD infants showed profound alteration of surfactant kinetics compared with preterm infants with minimal lung disease, and these alterations were correlated with severity of ventilatory support.

Oestradiol skin delivery from ultradeformable liposomes: refinement of surfactant concentration.

The aims of this study were to refine ultradeformable liposomes for oestradiol skin delivery and to evaluate Span 80 and Tween 80 as edge activators compared with sodium cholate. Vesicles containing phosphatidylcholine (PC) mixed with edge activators and oestradiol were prepared. Entrapment efficiency and vesicle size were determined. Interactions between activators and vesicles were investigated using differential scanning calorimetry. Transepidermal permeation of oestradiol from vesicles was studied compared to saturated aqueous control in vitro. The maximum flux (J(max)) and its time (T(max)) were calculated from the flux curves and skin deposition was assessed. The compositions of refined formulations were predicted, liposomes prepared, and tested against control. Entrapment efficiency depended on PC concentration with some contribution from sodium cholate and Tween 80. Vesicle sizes ranged from 124 to 135 nm. Edge activators interacted with lipid bilayers and disrupted packing. The refined edge activator concentrations in PC vesicles were 14.0, 13.3 and 15.5% w/w for sodium cholate, Span 80 and Tween 80, respectively; they increased J(max) by 18, 16 and 15-fold and skin deposition by 8, 7 and 8-fold compared with control. Ultradeformable vesicles thus improved skin delivery of oestradiol compared to control and Span 80 and Tween 80 were equivalent to sodium cholate as edge-activators.

Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing water-soluble prodrugs of paclitaxel.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, used clinically for the treatment of ovarian and breast cancer. Due to its aqueous insolubility it is administered dissolved in ethanol and Cremophor EL (polyethoxylated castor oil), which has serious side effects. In order to eliminate this vehicle, in previous work we entrapped paclitaxel in conventional and in polyethylene glycol coated liposomes. However, in neither formulation did we obtain satisfactory entrapment efficiency. In this study we increased the paclitaxel concentration entrapped in liposomes by incorporating different water-soluble prodrugs, such as the 2'-succinyl, 2'-methylpyridinium acetate and 2'-mPEG ester paclitaxel derivatives, in the lipid vesicles. Liposomes containing 2'-mPEG (5000)-paclitaxel showed the best performance in terms of stability, entrapment efficiency and drug concentration (6.5 mgml(-1)). The in vitro cytotoxic activity of this liposomal prodrug was similar to that of the parent drug. The pharmacokinetic parameters for the free and for the liposomal prodrugs fitted a bi-exponential plasma disposition. The most important change in pharmacokinetic values of the prodrug vs. the free drug liposomal formulations was t(1/2)beta, plasma lifetime, which was longer in liposomes containing 2'-mPEG (5000)-paclitaxel.

PD 098059, an inhibitor of ERK1 activation, attenuates the in vivo invasiveness of head and neck squamous cell carcinoma.

Increased mortality of patients with oral cancer largely reflects the local and regional spread of the disease. The invasiveness of these tumours requires hydrolases which are regulated through AP-1-dependent transcriptional mechanisms. Since the amount/activity of transcription factors bound to the AP-1 motif are regulated partly through the extracellular signal-regulated kinases (ERK1/ERK2), we determined the effect of PD 098059, an inhibitor of ERK1/ERK2 activation, on the in vivo invasiveness of a human squamous cell carcinoma cell line (UM-SCC-1) derived from the oral cavity. We utilized the floor of mouth musculature consisting of the mylohyoid, geniohyoid and genioglossus muscle (which are sequentially arranged), as a natural barrier to assess tumour spread in vivo in the nude mouse. Mice were inoculated with tumour cells superficial to the mylohyoid muscle. After 18 days, tumours were injected with either empty liposomes (control) or liposomes containing 5 microM PD 098059 and, after an additional 22 days, the jaws of mice examined histologically. Highly infiltrative tumours, which had penetrated the genioglossus muscle, were evident in 10/12 control mice. In contrast, in 9/12 mice in which the tumours were injected with PD 098059, tumours did not extend beyond the mylohyoid or geniohyoid muscles. Tumours penetrated bone nutrient canals in 7/12 control mice but in only 3/12 PD 098059-treated mice. Neurotropism, characteristic of aggressive oral squamous cell carcinoma, was evident in 6/12 control mice but was completely abolished (0/12 mice) in the PD 098059-treated mice. Using a staging system based on the muscle layer involved, neurotropism, as well as bone involvement, we found the inhibition of invasion to be statistically significant (P < 0.01). The reduced invasiveness of the PD 098059-liposome-treated oral cancers was associated with diminished 92-kDa type IV collagenase and ERK1/ERK2 activities but was not a consequence of a slower tumour growth rate. This is the first study to demonstrate reduced in vivo invasiveness of a malignancy brought about by an inhibitor of ERK1/ERK2 activation. These results raise the exciting possibility that second generation PD 098059 congeners may reduce the spread of the disease in patients afflicted with oral cancers.

Spray of phospholipid powder reduces peritoneal adhesions in rabbits.

BACKGROUND: The possible effects of peritoneal dialysis and a combination of two exogenous phospholipids, dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG), on experimentally induced intraperitoneal adhesion formation in rabbits were compared. METHODS: Fifty New Zealand rabbits equally divided in five groups underwent a midline laparotomy to create a right iliac fossa 5 x 1 cm parietal peritoneal defect and a matching defect over the adjacent large bowel. In 10 control rabbits (group I) the abdominal wound was closed without any further intervention. Twenty rabbits forming groups II and III underwent two sessions of peritoneal dialysis, one following abdominal closure and the second 24 h later, through a catheter placed at surgery. Rabbits in group III received an intraperitoneal injection of DPPC and PG after each session of dialysis. In 10 animals (group IV) a DPPC gel was applied to the defect over the large bowel and in 10 animals (group V) the peritoneal cavity was sprayed with a 'puff of DPPC:PG (7:3) powder prior to abdominal closure. All the animals were killed a week after the laparotomy to assess the extent of adhesion formation. RESULTS: The formation of adhesions was reduced in all the groups compared to the controls but a statistically significant difference was observed only in the group receiving the intraperitoneal 'puff' of DPPC:PG powder. CONCLUSION: A combination of DPPC and PG sprayed as a 'puff' intraperitoneally reduces experimentally induced peritoneal adhesions in rabbits.

Protective effect of liposomal alpha-tocopherol against bleomycin-induced lung injury.

The clinical use of bleomycin in the treatment of squamous cell carcinomas, lymphomas and testicular tumours has been limited by its toxic effects, the most serious being pulmonary injury. The present study was undertaken to investigate whether alpha-tocopherol, incorporated in liposomes and delivered directly to the lung, could offer protection against bleomycin-induced lung damage and fibrosis in the rat. Animals were administered, intratracheally, plain liposomes (composed of dipalmitoylphosphatidylcholine, DPPC) or alpha-tocopherol-containing liposomes (8 mg alpha-tocopherol/kg body weight) and 30 min later, were exposed to bleomycin sulphate (4 units/kg body weight) by intratracheal instillation; treated animals were killed 21 days later. Results of this study showed that lungs of animals treated with bleomycin were seriously damaged, as evidenced by significant histological changes and increases in lung weight, lipid peroxidation, myeloperoxidase activity and hydroxyproline content as well as decreases in lung angiotensin converting enzyme (ACE) and alkaline phosphatase (AKP) activities. Pretreatment of rats with plain liposomes alone did not alter significantly the bleomycin-induced changes of all parameters examined. In contrast, pretreatment of rats with alpha-tocopherol liposomes 30 min prior to bleomycin administration resulted in little or no histological changes and conferred a significant protection against bleomycin-induced changes in edema, lipid peroxidation, hydroxyproline content, and ACE, AKP and myeloperoxidase activities. Results of this study suggested that pretreatment of rats with alpha-tocopherol liposomes can provide a significant protection against bleomycin-induced lung injury.