DPPC-coated lipid nanoparticles as an inhalable carrier for accumulation of resveratrol in the pulmonary vasculature, a new strategy for pulmonary arterial hypertension treatment.

In this study, we investigated the feasibility of dipalmitoylphosphatidylcholine-coated lipid nanoparticles (DPPC-LNs) as a carrier for preferential accumulation into lungs of Resveratrol (Res), a potentially promising drug for the treatment of pulmonary arterial hypertension (PAH). Res-loaded DPPC-LNs were prepared following a thin film hydration-ultrasonic dispersion technique using glyceryl monostearate as lipid core. DPPC can reduce the interactions between nanoparticles and pulmonary surfactant. The optimal formulation was prepared and characterized for physicochemical properties, storage stability and in vitro release profiles. The optimal formulation was evaluated for uptake by pulmonary arterial smooth muscle cells (PASMCs) using fluorescence microscopy. The efficacy of Res-loaded DPPC-LNs in reducing hyperplasia was tested in 5-HT induced proliferated PASMCs. The drug absorption profiles upon intratracheal administration were monitored in healthy rats. Optimized spherical DPPC-LNs - with mean size of 123.7 nm, zeta potential of -19.4 mV and entrapment efficiency of 94.40% - exhibited an 80% cumulative drug release over 48 h. Fluorescence microscopic study revealed an time-dependent enhancement of cellular uptake of Rh123-labeled DPPC-LNs by PASMCs. PASMC proliferation induced by 5-HT was significantly inhibited by Res-loaded DPPC-LNs. Optimized DPPC-LNs appeared to be safe when incubated with PASMCs. Besides, plasma and lung tissue data analysis indicated higher value of accumulation after intratracheal administration of Res-loaded DPPC-LNs in comparison with the intravenously dosed Res solution, indicating longer retention of Res in the lungs and their slower entry to the systemic blood circulation. DPPC-LNs could be a viable delivery system for site-specific treatment of PAH.

Polymer-Lipid Microparticles for Pulmonary Delivery.

Toward engineering approaches that are designed to optimize the particle size, morphology, and mucoadhesion behavior of the particulate component of inhaler formulations, this paper presents the preparation, physicochemical characterization, and preliminary in vitro evaluation of multicomponent polymer-lipid systems that are based on "spray-drying engineered" α-lactose monohydrate microparticles. The formulations combine an active (budesonide) with a lung surfactant (dipalmitoylphosphatidylcholine) and with materials that are known for their desirable effects on morphology (polyvinyl alcohol), aerosolization (l-leucine), and mucoadhesion (chitosan). The effect of the composition of formulations on the morphology, distribution, and in vitro mucoadhesion profiles is presented along with "Calu-3 cell monolayers" data that indicate good cytocompatibility and also with simulated-lung-fluid data that are consistent with the therapeutically useful release of budesonide.

PLGA/DPPC/trimethylchitosan spray-dried microparticles for the nasal delivery of ropinirole hydrochloride: in vitro, ex vivo and cytocompatibility assessment.

In the present study we investigated polymer-lipid microparticles loaded with ropinirole hydrochloride (RH) for nasal delivery. RH microparticles were further evaluated by means of scanning electron microscopy (SEM), ζ-potential measurements, Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and x-ray diffraction (XRD). In vitro release studies were performed in simulated nasal electrolyte solution (SNES) pH5.5 at 35°C. Ex vivo permeation studies were conducted across sheep nasal mucosa. Cytocompatibility was tested in cultured human airway epithelial cells (Calu-3). SEM studies revealed spheroid microparticles in the range of 2.09µm to 2.41µm. The presence of trimethylchitosan (TMC) induced a slight shift towards less negative ζ-potential values. Surface chemistry (XPS) revealed the presence of dipalmitoylphospatidylcholine (DPPC) and poly(lactic-co-glycolic acid) (PLGA) onto microparticles' surface, further corroborating the FT-IR and XRD findings. In vitro release studies showed that the microparticle composition can partly modulate the release of RH. Ex vivo studies demonstrated a 2.35-folded enhancement of RH permeation when RH was co-formulated with TMC of low molecular weight, compared to the control. All formulations tested were found to be non-toxic to cells. The results suggest that polymer-lipid microparticles may be a promising carrier for the nasal delivery of RH.

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.

Biocompatibility of lipid-protein-sugar particles containing bupivacaine in the epineurium.

Novel lipid-protein-sugar particles (LPSPs) are potentially biocompatible because they are composed of naturally occurring ingredients and their expected tissue dwell times are relatively short. In this research, we used histological sections to study tissue reaction to LPSPs (4.4-microm median diameter) when used for sciatic nerve block in the rat. As a reference, we compared LPSPs to 60-microm median diameter poly(lactic-co-glycolic) acid (PLGA) microspheres (110,000 MW PLGA, glycolic/lactic ratio 65:35). Four days after injection, both particle types produced acute inflammation within the confines of the injectate, inflammation in adjacent tissues, and myotoxicity. Bupivacaine-free particles did not display myotoxicity, and inflammation in adjacent tissues was reduced. At 2 weeks, inflammation from LPSPs had almost disappeared, whereas PLGA microspheres had a foreign-body giant cell reaction until at least 8 weeks after injection. In contrast, 3.6-microm median diameter, 20,000-MW PLGA microspheres produced a primarily histiocytic reaction 2 weeks after injection. In summary, the LPSPs and PLGA microspheres studied herein have excellent biocompatibility, but tissue reaction to the former is of much shorter duration. Myotoxicity and inflammation of surrounding tissue is largely attributed to bupivacaine. Foreign-body giant cells may be attributed to particle size rather than a specific reaction to PLGA.

Phospholipid surfactant adsorption by respirable quartz and in vitro expression of cytotoxicity and DNA damage.

Respirable-sized quartz was treated with a saline dispersion of dipalmitoyl phosphatidylcholine (DPPC), a primary component of pulmonary surfactant, to model the adsorption of phospholipid surfactant onto quartz dust following particle deposition in the bronchoalveolar region of the lung. Control and surfactant-treated dusts were used to challenge lavaged rat pulmonary macrophages in vitro over a 1-week period, to determine the effects of adsorbed surfactant on the expression of quartz cytotoxicity and genotoxicity. DNA damage was determined by the single cell gel electrophoresis 'comet' assay. Untreated quartz induced DNA damage, increasing with dose and with time of incubation of dust with macrophages over a 5 day period. DPPC treatment of quartz suppressed DNA damage through 1 day of macrophage challenge. DNA damage then increased over a 5 day period, to approximately half the positive control (untreated quartz) values. Cytotoxicity was measured by trypan blue dye exclusion and by the Live-Dead fluorescence assay for cell viability. Cytotoxicity of surfactant-treated quartz measured one day after challenge of lavaged macrophages was suppressed to values near those of the negative controls, and then increased over a 1 week incubation period to levels near those expressed by native quartz positive controls. Quartz similarly treated with dioleoyl phosphatidylcholine mixed with DPPC substituted in one acyl group with a boron-containing fluorescent chromophore was used with confocal microscopy to measure particle-associated fluorescent surfactant in cells. Approximately half of the fluorescence intensity was lost over a 1 week period following challenge of lavaged macrophage. Results are discussed in terms of a model of restoration of quartz particle surface toxicity as prophylactic surfactant is removed from particle surface by cellular enzymatic digestion processes.

Artificial reconstituted pulmonary surfactant in prevention and treatment of respiratory distress syndrome in neonates.

AIM: To test an artificial reconstituted pulmonary surfactant (APS) for prevention and treatment of respiratory distress syndrome (RDS). METHODS: A membrane-formed method combined with supersonic dispersing was used to prepare APS. A pulsating bubble surface tension measurement was established to compare surface properties of APS with natural pulmonary surfactant (NPS). A preliminary clinical trial was made for prevention and treatment of RDS. RESULTS: The APS reduced surface tension from 44.0 mN/m to < 1.0 mN/m in vitro. The changes of APS lipid contents were < 5% of labeled content at 37 degrees C. Clinical trial showed that the APS prevented RDS in 20/20 and cured RDS in 2/2 premature neonates. CONCLUSION: The APS had good surface properties similar to NPS.

Antinociception and side effects of L- and D-dipalmitoylphosphatidyl choline liposome-encapsulated alfentanil after spinal delivery in rats.

We have observed that spinal liposome administration in the rat resulted in in an allodynia evoked by light touch. We later determined that liposomes composed of D-isomer phospholipids were essentially non-toxic. This study examines the effects of alfentanil encapsulated in liposomes made from the natural L-isomer and synthetic D-isomer of dipalmitoyl phosphatidyl choline on antinoceiception, side effects, and algogenic behaviour. Both unilamellar and multilamellar liposomes were studied. Rats prepared with chronic intrathecal catheters received intrathecal injections of alfentanil (5 or 50 micrograms) in saline or encapsulated in liposomes composed of either L- or D-isomers of dipalmitoyl phosphatidyl choline (DPPC) in unilamellar or multilamellar liposome formulations. Antinociception was measured using the hot plate test (52.5 degrees). Side effects were measured by catalepsy, corneal responses, pinna response, righting reflex, and paw step. Allodynia was measured by lightly stroking the animal's back. Intrathecal alfentanil in saline or in the liposomes produced a dose-dependent increased latency in the hot plate response. Encapsulation of alfentanil in the liposomes produced a significant decrease in the loss of corneal, paw step and righting reflex and a slight decrease in catalepsy and loss of the pinna response. There was no significant difference between liposome preparations in preventing side effects. L-multilamellar-DPPC produced allodynia in 100% of the animals whereas significantly less allodynia was observed with the other preparations. This study indicates that liposomal preparations can significantly enhance the therapeutic ratio of a lipid soluble opioid after spinal delivery. However, the choice of lipids for the formulation of liposomes intended for spinal drug delivery must be considered since the L-isomer and larger lipid load of multilamellar liposomes have a direct spinal effect leading to alledynia. Previous studies have in fact shown that spinal lysolecithin can yield focal demyelination.

Nitrogen-filled liposomes as a vascular US contrast agent: preliminary evaluation.

Liposomes with a mean diameter of 1-2 microns were made to entrap nitrogen gas and tested as an ultrasound (US) contrast agent. The gas-filled liposomes, or Aerosomes (ImaRx Pharmaceutical, Tucson) were tested in vitro for size, stability, reflectivity, and acoustic characterization, and were tested in vivo for acute toxicity in mice and for cardiac imaging in rabbits after intravenous injection. Aerosomes have much greater reflectivity and higher attenuation than do standard liposomes and retain their acoustic properties after storage in aqueous media for several months. The interpolated median lethal dose of Aerosomes is approximately 2.5 mmol of lipid per kilogram, and the imaging dose is under 5 mumol of lipid per kilogram, yielding a potential therapeutic index of over 500 to 1. Postcontrast US images showed sustained enhancement of all four cardiac chambers as well as enhancement in the aorta, vena cava, and hepatic veins. Aerosomes hold promise as a contrast agent for cardiac and blood-pool imaging. Further work is in progress to characterize and develop this novel US contrast agent.

Influence of phospholipid/amphotericin B ratio and phospholipid type on in vitro renal cell toxicities and fungicidal activities of lipid-associated amphotericin B formulations.

We studied the influence of the lipid/amphotericin B (AMB) ratio and the phospholipid type on the in vitro renal cell toxicity and antifungal efficacy of lipid-associated AMB (L-AMB). L-AMB was prepared at one of two different lipid/AMB ratios (1 and 40) by incubating AMB with empty small unilamellar vesicles, made from one of three different phospholipids: dipalmitoyl-, dimirystoyl-, and distearoylphosphatidylcholine (DPPC, DMPC, and DSPC, respectively). Renal cell toxicity, investigated through an assessment of the Na-dependent uptake of phosphate by proximal tubular cells, and fungicidal effect against Candida albicans were studied after 1 h of treatment at 37 degrees C. The amount of unbound AMB present in each L-AMB formulation was studied by use of circular dichroism. At a lipid/AMB ratio of 40, the three lipidic formulations were not toxic for renal cells but were less effective against C. albicans than AMB; however, DSPC-AMB, which contained 50% unbound AMB, was more effective against C. albicans than DPCC-AMB or DMPC-AMB, containing 0 and 13% unbound AMB, respectively. At a lipid/AMB ratio of 1, the antifungal effects of L-AMB and AMB were similar, whatever the phospholipid used, but only DMPC-AMB remained highly protective against AMB renal cell toxicity, despite the presence of the same amount of unbound AMB (50%) in DMPC-AMB and DPPC-AMB. We conclude that the in vitro activities and renal cell toxicities of different L-AMB formulations are influenced by the phospholipid type and the lipid/AMB ratio. The optimal ratio depends on the phospholipid itself. At a lipid/AMB ratio of 40, the antifungal activity depends mainly on the amount of unbound AMB in the formulation. At a lipid/AMB ratio of 1, the renal cell toxicity also depends on the fluidity of the phospholipid.