Incorporation Of Stearylamine Research Articles

Effect of Surface Charge Density of a w/o/w Emulsion on the Brain Targeting of Levodopa in Rats for the Treatment of Parkinson’s Disease

Background: Amidst levodopa being considered as the “Gold standard” in the treatment of Parkinson’s disease (PD), it still has critical therapeutic issues with its dose regimen and dosage forms leading to severe adverse drug effects, decreased drug efficacy during chronic use, and requires an enforced “drug holiday” in PD patients. Hence, in this study, we designed a novel levodopa and carbidopa water-in-oil-in-water (w/o/w) formulation for bioavailability improvement in the central nervous system (CNS). Methods: The new one-in-one embedment of the w/o/w levodopa and carbidopa emulsion formulation was obtained by a double emulsion technique. The plasma and brain levels following intravenous administration of the emulsions in rats were determined. Results: The incorporation of stearylamine (a cationic surfactant) considerably increased the surface charge density of the emulsion droplets. This formulation exhibited a narrow particle size distribution enabling parenteral administration. The formulation also provided a high drug loading capacity. In in vivo study, this novel formulation significantly increased the bioavailability of levodopa in the CNS (P < 0.001). The strong resistance to desorption (due to higher charge density) and the presence of positive charge on the particles upon dilution may be the main reason for enhanced brain levels of levodopa. Conclusion: Our current formulation F5 may decrease the dose of levodopa, leading to decreased adverse effects and dosing problems, thus appreciably benefit PD patients in the future.

Stability effect of cholesterol-poly(acrylic acid) in a stimuli-responsive polymer-liposome complex obtained from soybean lecithin for controlled drug delivery

The development of polymer-liposome complexes (PLCs), in particular for biomedical applications, has grown significantly in the last decades. The importance of these studies comes from the emerging need in finding intelligent controlled release systems, more predictable, effective and selective, for applications in several areas, such as treatment and/or diagnosis of cancer, neurological, dermatological, ophthalmic and orthopedic diseases, gene therapy, cosmetic treatments, and food engineering.This work reports the development and characterization of a pH sensitive system for controlled release based on PLCs. The selected hydrophilic polymer was poly(acrylic acid) (PAA) synthesized by atom transfer radical polymerization (ATRP) with a cholesterol (CHO) end-group to improve the anchoring of the polymer into the lipid bilayer. The polymer was incorporated into liposomes formulated from soybean lecithin and stearylamine, with different stearylamine/phospholipid and polymer/phospholipid ratios (5, 10 and 20%). The developed PLCs were characterized in terms of particle size, polydispersity, zeta potential, release profiles, and encapsulation efficiency. Cell viability studies were performed to assess the cytotoxic potential of PLCs. The results showed that the liposomal formulation with 5% of stearylamine and 10% of polymer positively contribute to the stabilization of the complexes. Afterwards, the carboxylic acid groups of the polymer present at the surface of the liposomes were crosslinked and the same parameters analyzed. The crosslinked complexes showed to be more stable at physiologic conditions. In addition, the release profiles at different pHs (2–12) revealed that the obtained complexes released all their content at acidic conditions.In summary, the main accomplishments of this work are: (i) innovative synthesis of cholesterol-poly(acrylic acid) (CHO‐PAA) by ATRP; (ii) stabilization of the liposomal formulation by incorporation of stearylamine and CHO-PAA; (iii) new approach for CHO-PAA crosslinking, resulting in more stable PLCs at physiological conditions; (iv) destabilization of PLCs upon slight changes of pH, showing their pH sensitivity; and (v) the PLCs do not exhibit cellular toxicity.

Transdermal iontophoretic delivery of enoxacin from various liposome-encapsulated formulations

The major purpose of this work was to study the effect of various liposome formulations on the iontophoretic transport of enoxacin through excised rat skin. The electrochemical stability of these liposomes was also evaluated. The encapsulation percentage of enoxacin was significantly enhanced after 6 h incubation in an electric field; whereas the fusion of liposomes was inhibited by application of electric current. The results of iontophoretic drug transport showed that the permeability of enoxacin released from liposomes was higher compared with that of free drug. The iontophoretic permeability of enoxacin released from liposomes increased with a decrease in the fatty acid chain length of the phospholipid, which may be due to the different phase transition temperatures of the phospholipids. Incorporation of charged phospholipid resulted in an alteration of the transdermal behavior of enoxacin: the iontophoretic permeation as well as the amount of enoxacin partitioned in skin was greatly reduced after incorporation of stearylamine in liposomes, which can be attributed to the competitive ion effect. The enoxacin released from stratum corneum-based liposomes showed the highest amount of enoxacin partitioned into skin depot. The results of employing cathodal iontophoresis on negative charged liposomes suggested that the liposomal vesicles or phospholipids may carry enoxacin into deeper skin strata via the follicular route.

Effects of charged lipids on the interaction of cholesteryl ester transfer protein with lipid microemulsions

This study reports the effects of charged lipids on the transfer of cholesterlyl-1 pyrene decanoate (Py-CE) between apolipoprotein-free microemulsion particles mediated by cholesteryl ester transfer protein (CETP). The surface charge characteristics of microemulsion particles composed of cholesteryl oleate and egg yolk phosphatidylcholine were altered by incorporating phosphatidyl-serine, oleate or stearylamine into the phosphatidylcholine that forms the surface monolayer of the particle. The transfer of Py-CE was measured continuously by following the decrease in excimer fluorescence that accompanies the transfer of the probe from donor to acceptor particles [Rajaram, Chan and Sawyer (1994) Biochem. J. 304, 423-430]. The inclusion of 20 mol% phosphatidylserine relative to the phospholipid in the surface monolayer of the emulsion caused a 64% decrease in the first-order rate constant describing the transfer. An increase in ionic strength caused a partial reversal of this effects, indicating that electrostatic factors are only partially responsible for the interaction with lipid. Complete inhibition of transfer was observed when 10 mol% sodium oleate was incorporated into the surface monolayer. The incorporation of stearylamine into the emulsion caused a 32% increase in the transfer rate. The binding of CETP to the different emulsion surfaces was also examined using a surface plasmon resonance biosensor. The presence of negatively charged lipid (phosphatidylserine or oleic acid) decreased the rate of association of CETP with the emulsion without a significant change in the dissociation rate constant. The presence of the positively charged lipid stearylamine increased the rate of association of CETP with the lipid surface. It is concluded that a negative surface charge on the monolayer decreases the rate of transfer by decreasing the affinity of CETP for these particles.

Lymphatic delivery and pharmacokinetics of methotrexate after intramuscular injection of differently charged liposome-entrapped methotrexate to rats.

The lymph node targeting ability and pharmacokinetics of methotrexate (MTX) after intramuscular (i.m.) injection of differently charged liposome-entrapped [3H]MTX to rats were evaluated using [3H]MTX as a tracer. Neutral liposomes were prepared with a mixture of phosphatidylcholine, cholesterol and alpha-tocopherol (8:4:0.1, molar ratio). Positively and negatively charged liposomes were also prepared by incorporation of stearylamine (8:4:0.1, molar ratio) and dicetylphosphate (8:4:0.1:1, molar ratio) into neutral liposomes respectively. The encapsulation efficiency (as expressed in terms of radioactivity) in liposomes was increased as alpha-tocopherol was incorporated into the lipid bilayer. The disappearance of [3H]MTX from the i.m. injection site was rapid and essentially complete after 30 min. On the other hand, the disappearance of radioactivity of liposome-entrapped [3H]MTX was much slower when compared to free drug. The area under the drug concentration-time curve (AUC) of liposome-entrapped [3H]MTX in lymph nodes was significantly increased when compared to free [3H]MTX. It suggested that liposomes injected by the i.m. route entered into the lymphatics and only drug released from liposomes diffused directly into the systematic circulation. The liposome-entrapped [3H]MTX by i.m. route was markedly localized in the lymph nodes. The concentration of MTX-equivalents in regional lymph node after i.m. injection of liposome-entrapped [3H]MTX was > 100-350 fold higher when compared to the plasma concentration. These values are more than 10-20 fold higher compared to the i.m. injection of free [3H]MTX. The positively charged liposomes were more localized in lymph nodes compared to neutral and negatively charged ones. While liposomes injected by i.v. route were localized in liver, spleen and lung compared to free [3H]MTX, it was evident that i.m. administration of liposomes resulted in enhanced localization of MTX in the lymphatic system but decreased deposition in kidney, liver and other non-targeting tissues compared to free [3H]MTX. The targeting ability and carrier properties of liposome-entrapped anticancer drugs with varying surface charge, lipid compositions and route of administration are of significant importance to alter biodistribution in chemotherapy.

Pharmacokinetics and tissue distribution of methotrexate after intravenous injection of differently charged liposome-entrapped methotrexate to rats

Liposome-entrapped [ 3H]MTX was prepared by modification of reverse-phase evaporation vesicle (REV) methods. Neutral liposomes were prepared with a mixture of phosphatidylcholine (PC), cholesterol (CH) and α-tocopherol (α-T) (8:4:0.1, molar ratio). Positively and negatively charged liposomes were also prepared by incorporation of stearylamine (SA) (8:4:0.1:1, molar ratio) and dicetyl phosphate (DCP) (8:4:0.1:1, molar ratio) into neutral liposomes, respectively. The release profiles of [ 3H]MTX from liposomes in the presence of rat plasma were the same shape regardless of surface charges, showing initial fast release followed by much slower release. The release profiles of [ 3H]MTX from liposomes were dependent on the surface charge of liposomes. Negatively charged liposomes showed much greater [ 3H]MTX release compared to positively charged and neutral liposomes. The enhanced in vitro release of [ 3H]MTX from negatively charged liposomes in the presence of rat plasma may be due to the interaction of incorporated lipid compositions of liposomes with plasma components, resulting in a change in drug release through the lipid bilayers by destabilization of the liposomal membranes. After intravenous (i.v.) injection of free and liposome-entrapped [ 3H]MTX to rats, the elimination of [ 3H]MTX from the blood stream showed biphasic patterns indicating rapid declining disposition up to 30 min followed by a slower elimination phase. Liposome-entrapped [ 3H]MTX maintained a higher and longer plasma concentration, and mainly intact form in plasma compared to free [ 3H]MTX for 2 h. Liposome-entrapped [ 3H]MTX enhanced bioavailability in plasma due to the retardation of drug release and protection of drug clearance by lipid bilayers of liposomes. Negatively charged liposome-entrapped drug was cleared more rapidly from the blood, resulting from the rapid uptake by liver to a greater extent, possibly via the reticuloendothelial system (RES), since liposome-entrapped drug cannot be eliminated by the kidney, the main eliminating organ for MTX. The tissue distribution of liposome-entrapped [ 3H]MTX was widely different when compared to free [ 3H]MTX. There was a markedly increased uptake of drug in spleen from neutral and negatively charged liposomes. Negatively charged liposomes also increased localization of drug in liver, lung and lymph nodes compared to neutral and positively charged liposomes at 2 h after i.v. injection to rats. From these findings, liposomes containing anticancer drugs would appear to be an effective carrier system for targeting to these sites. Although liposome-entrapped [ 3H]MTX was widely distributed in tissues without the exact role of the liposomal surface charge being known, it was evident that the surface charge of liposomes altered the biodistribution and membrane permeation of drug.

Monensin intercalation in liposomes: effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells

Monensin, a car☐ylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied. Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells. Liposomal monensin)(1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin. This observed effect is highly dependent on the liposomal lipid composition. The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charged vesicles. This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium. Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum. The uptake of 125I-gelonin from neutral vesicles is significantly higher (∼ 2.0-fold) than that from the negative vesicles. The uptake from positive vesicles is highly dependent on the concentration of stearylamine. The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin. However, monensin in positive vesicle has no effect on it. These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.

The effect of blood on the uptake of liposomal lipid by perfused rat liver

Liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC) and its mixtures with phosphatidylinositol (PI) and stearylamine. The absorption of the liposomes by perfused rat liver has been studied as a function of blood level (0-7% haematocrit). It has been found that the rate constant for uptake of liposomes (perfusion constant, kp) is markedly reduced by addition of blood to the perfusate particularly in the haematocrit range 0-3%. The perfusion constant is dependent on the liposome composition and decreases with incorporation of PI and increases with incorporation of stearylamine into DPPC liposomes, but is independent of the initial size of the liposomes in the range of weight-average diameter from 40-400 nm. The possible effects of blood components on the liposomes and their subsequent absorption by the liver are discussed.

Prothrombin activation on phospholipid membranes with positive electrostatic potential.

The conversion of prothrombin into thrombin, which is a crucial reaction in hemostatic plug formation, is greatly stimulated by phospholipids plus calcium ions. It has been proposed that phospholipid surfaces which promote blood coagulation should have a negative surface charge [Bangham, A. D. (1961) Nature (London) 192, 1197-1198]. However, the experiments that led to this proposal were carried out with one kind of anionic phospholipid (dicetyl phosphate). Here we report that membranes, which contain phosphatidylserine (PS) as the anionic phospholipid, can be made positively charged by incorporation of stearylamine and still exhibit almost full procoagulant and prothrombin-converting activity. This suggests that electrostatic forces contribute negligibly to the binding of coagulation factors to PS-containing membranes. Introduction of stearylamine in membranes containing phosphatidyl-beta-lactate (PLac) causes considerable inhibition of their prothrombin-converting activity. Since PLac and PS only differ by the presence of an amino group in the polar head group, the much higher procoagulant activity of PS-containing vesicles is indicative of an important function of the amino group of PS in the interaction with coagulation factors. We propose that the association of coagulation factors with PS-containing membranes results from complex formation between Ca2+ ions and ligands supplied by the protein and by PS molecules. The ability to form such a complex may well explain why cell membranes with PS have such excellent procoagulant properties.

The absorption of phospholipid vesicles by perfused rat liver depends on vesicle surface charge

Two types of sonicated vesicle have been prepared from dipalmitoylphosphatidylcholine (DPPC) by incorporation of phosphatidylinositol (PI) to give negatively charged vesicles and stearylamine to give positively charged vesicles. The absorption of the vesicles by rat liver has been investigated by perfusion techniques. A steady state of vesicle absorption is rapidly established in approx. 2 min and the initial rates of absorption decrease with PI content of the vesicles and increase with stearylamine content. In the steady state, the uptake of vesicles by the liver is similarly dependent on vesicle charge, being inhibited by PI and enhanced by incorporation of stearylamine in the vesicles. Fractionation of the liver into subcellular fractions following perfusion showed that most of the vesicular lipid could be found associated with a nuclear (plus plasma membrane) fraction. The suppression of vesicle absorption by PI may be of value as a means of bypassing the liver in relation to the use of vesicles as a delivery system.

Interaction of bovine brain phospholipid exchange protein with liposomes of different lipid composition

The major phospholipid exchange protein from bovine brain catalyzes the transfer of phosphatidylinositol and phosphatidylcholine between rat liver microsomes and sonicated liposomes. The effect of liposomal lipid composition on the transfer of these phospholipids has been investigated. Standard liposomes contained phosphatidylcholine-phosphatidic acid (98:2, mol%); in general, phosphatidylcholine was substituted by various positively charged, negatively charged, or zwitterionic lipids. The transfer of phosphatidylinositol was essentially unaffected by the incorporation into liposomes of phosphatidic acid, phosphatidylserine, or phosphatidylglycerol (5–20 mol%) but strongly depressed by the incorporation of stearylamine (10–40 mol%). Marked stimulation (2–4-fold) of transfer activity was observed into liposomes containing phosphatidylethanolamine (2–40 mol%). The inclusion of sphingomyelin in the acceptor liposomes gave mixed results: stimulation at low levels (2–10 mol%) and inhibition at higher levels (up to 40 mol%). Cholesterol slightly diminished transfer activity at a liposome cholesterol/phospholipid molar ratio of 0.81. Similar effects were noted for the transfer to phosphatidylcholine from microsomes to these various liposomes. Compared to standard liposomes, the magnitude of K m tended to increase for liposomes which depressed phospholipid transfer and to decrease for those which stimulated; little change was observed in the values of V. Single phospholipid liposomes of phosphatidylinositol were inhibitory when added to standard liposomes.