Preparation and release properties of arbutin imprinted inulin/polyvinyl alcohol biomaterials.

The main objective of this work was to prepare inulin (INL)/polyvinyl alcohol (PVA) biomaterials imprinted with arbutin (AR) as the target drug. INL from Jerusalem artichoke flour was extracted with hot water extraction method. INL/PVA biomaterials were synthesized with a casting method and a UV curing. The optimal UV curing time and sodium benzoate content were about 10 min and 0.1 wt%, respectively. The biomaterials were characterized by SEM and FT-IR analysis. Mechanical properties of prepared AR imprinted biomaterials were also investigated. AR release was examined with changes of pH at 36.5 °C. The AR release ratio was also investigated using artificial skin. It was found that AR was released constantly for 40 min. Results of drug release mechanism indicated that AR release followed the Fickian diffusion behavior, whereas drug release using artificial skin followed the non-Fickian diffusion behavior. Tyrosinase inhibitory (%) for AR imprinted biomaterials with/without the addition of GL were 58.8% and 79.2%, respectively.

Drinking and water permeability in the Pacific hagfish, Eptatretus stoutii.

Hagfish are osmoconformers, maintaining an internal osmolality that matches their seawater habitats. Hagfish would, therefore, appear to have no physiological need to drink, but previous studies are equivocal regarding whether drinking in hagfish occurs. The current study addressed this knowledge gap, by examining drinking and water permeability in the Pacific hagfish, Eptatretus stoutii. One-third of analysed hagfish were shown to accumulate radiolabelled drinking rate markers (tritiated inulin and polyethylene glycol-4000) in their gut tissues; however, this was attributed to the presence of markers in the blood perfusing the digestive tract, following absorption through paracellular pathways at the gill. No accumulation of marker was observed in hagfish subjected to more dilute (75% seawater) or more concentrated (125% seawater) media. Diffusive water efflux, measured by tritiated water washout, was shown to be very high, with 50% of body water exchanged within 14 to 16 min, depending on exposure salinity. In full-strength seawater, the total exchangeable pool of water was 78% of hagfish mass. We conclude that hagfish do not drink, and their high water permeability is likely to result in rapid osmotic equilibration under circumstances where perturbations may occur.

Drug clearance and arterial uptake after local perivascular delivery to the rat carotid artery.

OBJECTIVES: We attempted to characterize how drug released into the perivascular space enters the arterial wall and how it is cleared from the local environment. BACKGROUND: Drug released into the perivascular space can enter the artery either from the adventitial aspect or from the lumen after absorption by the extraarterial capillaries and mixing within the systemic circulation. Some investigators suggest that this latter mechanism dominates, and they question whether local drug release is synonymous with local deposition. METHODS: We investigated both the pathways by which adventitially released drug is cleared from the perivascular space and those by which drug enters the blood vessel wall. Inulin was used to follow drug release from implanted devices and subsequent entry to the circulation, because of its first-pass urinary excretion. Heparin was used to follow arterial deposition because of its vasoactivity and tissue-binding properties. The different potential pathways of drug entry and egress were systematically removed and the effects on metabolism and deposition determined. RESULTS: Ligature occlusion of the artery did not decrease inulin excretion or heparin deposition. Extravascular wraps designed to shield the device from extramural capillaries reduced inulin excretion rates 10-fold but did not alter heparin deposition into the vessel wall. The deposition of drug after perivascular delivery was 500 times higher than after intraperitoneal administration. CONCLUSIONS: Although almost all the drug released into the perivascular space is cleared through the extravascular capillaries, virtually all the deposited drug diffuses directly from the perivascular space, and little arrives from the endovascular aspect. These data support the view that local drug release leads directly to increased local drug concentration.

Drug interaction and location in liposomes: correlation with polar surface areas.

An important step in liposome characterization is to determine the location of a drug within the liposome. This work thus investigated the interaction of dipalmitoylphosphatidylcholine liposomes with drugs of varied water solubility, polar surface area (PSA) and partition coefficient using high sensitivity differential scanning calorimetry. Lipophilic estradiol (ES) interacted strongest with the acyl chains of the lipid membrane, followed by the somewhat polar 5-fluorouracil (5-FU). Strongly hydrophilic mannitol (MAN) showed no evidence of interaction but water soluble polymers inulin (IN) and an antisense oligonucleotide (OLG), which have very high PSAs, interacted with the lipid head groups. Accordingly, the drugs could be classified as: hydrophilic ones situated in the aqueous core and which may interact with the head groups; those located at the water-bilayer interface with some degree of penetration into the lipid bilayer; those lipophilic drugs constrained within the bilayer.

Synthesis of diethylenetriaminepentaacetic acid conjugated inulin and utility for cellular uptake of liposomes.

The synthesis, binding of radioactive cations, liposomal encapsulation, and biodistribution of the oxidized-inulin reaction product with ethylenediamine and diethylenetriaminepentaacetic acid (4) are described. The four-step synthesis of the inulin derivative proceeded in a good overall yield of 72%. The complex of the inulin derivative with either 67Ga3+ or 111In3+ was stable in vivo and did not readily distribute into tissues, being excreted primarily in urine after intravenous administration to mice. The liposome-entrapped inulin derivative can be loaded with radioactive heavy metal cations by mobile ionophores in high radiochemical yields of 80-91%. Following the intravenous administration of the liposomal encapsulation of the indium-111-labeled inulin derivative, the entrapped compound had a biodistribution characteristic of liposomes and allowed an estimation of the extent of the intracellular uptake of liposomes. The ability of the inulin derivative to chelate many different types of metals will allow the use of this probe for studying subtle differences in tissue distribution resulting from different drug targeting or delivery protocols in the same animal by multiple labeling techniques. Moreover, the chelate-conjugated inulin permits studies of the applications of drug delivery systems in primates or human subjects by noninvasive techniques such as gamma-scintigraphic or nuclear magnetic resonance imaging methods.

Effect of high glucose on permeability of retinal capillary endothelium in vitro.

PURPOSE: To study the effect of high glucose on the permeability of bovine retinal capillary endothelial cell (BRCEC) monolayers. METHODS: The paracellular permeability of second-passage BRCEC cultured on millipore filters in two chamber transwell inserts was assayed by measuring the peak trans-monolayer electrical resistance and percent equilibration of 14C-inulin 48 hours after it had been added to the luminal chamber. RESULTS: High glucose increased the paracellular permeability of BRCEC monolayers independently of its hypertonic action (5 mM glucose: 154.2 +/- 21.2 and 19.5 +/- 2.4; 30 mM glucose: 134.2 +/- 5.1 [P = 0.01] and 23.5 +/- 2.1 [P = 0.01]; 5 mM glucose + 25 mM mannitol: 168.7 +/- 13.7 ohm.cm2 [P = 0.04] and 19.3% +/- 1.2% 48-hour equilibration of inulin [P = 0.008]). In a separate series of experiments, the authors were unable to show that either aminoguanidine or ponalrestat prevented the effect of high glucose on permeability (30 mM glucose 95.1 +/- 16.7 and 45.4 +/- 5.6; 5 mM glucose: 122.9 +/- 14.2 [P = 0.02] and 36.6 +/- 5.6 [P = 0.001]; 30 mM glucose + aminoguanidine 87.9 +/- 17.5 [P = 0.04] and 75.3 +/- 14.9 [P = 0.6]; 30 mM glucose + ponalrestat 79.9 +/- 12.7 ohm.cm2 [P = 0.1] and 48.2 +/- 2.5% 48-hour equilibration of inulin [P = 0.15]). Ponalrestat did not abrogate the effect of high glucose despite its ability to reduce a high glucose-induced increase in BRCEC intracellular sorbitol levels. CONCLUSIONS: The data are consistent with a role for increased paracellular permeability in breakdown of the blood-retinal barrier in diabetic retinopathy, which appears to be independent of both nonenzymatic glycosylation and the polyol pathway.

Effect of aging and caloric restriction on intestinal permeability.

Intestinal permeability is increased in several disorders such as Crohn's disease or rheumatoid arthritis. Since aging leads to alteration of many biological functions, the effect of aging on intestinal permeability was studied by measuring the intestinal permeability in aging rats gavaged with different size permeability probes--mannitol, polyethylene glycol (PEG) 400, and inulin. In rats fed with control diet, there was a significant increase in intestinal permeability to medium size probes PEG 400 (14.8 +/- 0.4 and 21.0 +/- 1.1% at 3 and 28 months respectively, p less than .01) and mannitol (3.41 +/- 0.4 and 5.3 +/- 0.5% at 3 and 28 months, respectively, p less than .01). Intestinal permeability of the large macromolecule inulin did not change (0.42 +/- 0.03 and 0.38 +/- 0.02% at 3 and 28 months, respectively) with aging. There was no correlation between weight of the rats and their intestinal permeability. Because dietary caloric restriction has been found to prolong the life span, retard deterioration of several biological functions, and affect intestinal absorptive functions, we examined the effect of lifelong calorie restriction on intestinal permeability changes. Lifelong calorie-restricted diet did not affect age-related change in intestinal permeability. We conclude that intestinal permeability of medium size probes increases with aging and that lifelong caloric restriction does not prevent this change. We speculate that age-associated deterioration in intestinal barrier functions could permit increased systemic absorption of lumenal antigens and could perhaps contribute to the genesis of antigen-related age-associated diseases.

Villous trophoblasts cultured on semi-permeable membranes form an effective barrier to the passage of high and low molecular weight particles.

An effective in vitro model of the placental villous syncytium cultured on semi-permeable substrata is essential for studies of infectious pathogen transmission from mother to fetus. Current models using amniotic membranes or thinner artificial membranes show significant leakage, suggesting disruption of tight junctions or the presence of gaps between syncytial units. Such disruption and discontinuity of trophoblast cultures are probably the result of high stromal cell contamination, poor viability and lack of proliferation in culture. We have successfully cultured confluent layers of tight-junctioned syncytium on semi-permeable insert membranes using highly viable purified cytotrophoblasts and an alternating multiple seeding and differentiation technique. Using criteria including transepithelial diffusion of high and low molecular weight substances, electrical resistance and directional secretion of the matrix metalloproteinase, MMP-9, we demonstrate that these cultures form effective and functional physical barriers that can be maintained for up to 1 month.

Tracer technique to measure in vivo chemical transport rates within an implantable cell transplantation device.

An in vivo tracer technique that uses radiolabeled insulin as the tracer molecule has been developed to assess the rate of chemical transport between the cell transplantation chamber of an implantable bioartificial device and the host's circulatory system. The device considered here employs site-directed neovascularization of a porous matrix to induce capillary growth adjacent to an immunoisolated cell implantation chamber. This device design is being investigated as a vehicle for therapeutic cell transplantation, with the advantages that it allows the cells to perform their therapeutic function without the danger of immune rejection and it avoids damaging contact of blood flow with artificial surfaces. A pharmacokinetic model of the mass transport between the implantation chamber, the vascularized matrix, and the body has been devised to allow proper analysis and understanding of the experimental tracer results. Experiments performed in this study have been principally directed at evaluation of the tracer model parameters, but results also provide a quantitative measure of the progression of capillary growth into a porous matrix. Measured plasma tracer levels demonstrate that chemical transport rates within the implanted device increase with the progression of matrix vascular ingrowth. Agreement between the fitted model curves and the corresponding measured concentrations at different levels of capillary ingrowth demonstrate that the model provides a realistic representation of the actual capillary-mediated transport phenomena occurring within the device.

The intracerebral distribution of BCNU delivered by surgically implanted biodegradable polymers.

The local concentration and distribution of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) within normal brain tissue were studied following surgical implantation of biodegradable polymer containing BCNU in New Zealand White rabbits. Cylindrical discs of poly(bis(p-carboxyphenoxy)-propane:sebacic acid) copolymer in a 20:80 formulation were made containing [3H]-inulin or [3H]-BCNU labeled in the methylene hydrogens of the chloroethyl groups. These were implanted in the brains of 56 New Zealand White rabbits. The animals were sacrificed 3, 7, 14, or 21 days later and the brains were rapidly removed, frozen, and prepared for quantitative autoradiography. Autoradiographs from coronal sections bisecting the polymer were analyzed to determine both the proportion of the brain section exposed to the tracer and the local drug concentrations as a function of distance from the polymer. Tritiated BCNU was also injected directly into the brains of eight additional rabbits, and local brain concentrations were studied over time. The results of this study demonstrate that approximately 50% of the area of the brain sections was exposed to radiolabeled compound 3 days after BCNU-polymer implantation, 15% at 7 days, and less than 10% at 14 and 21 days. Polymer discs containing 600 micrograms BCNU generated 6 mM concentrations of BCNU in brain tissue 10 mm from the polymer at 3 and 7 days. Pharmacological studies demonstrated that approximately 25% of the tritium label was associated with intact BCNU 3 days following polymer implantation. Radiolabeled inulin delivered by polymer remained dispersed throughout the ipsilateral hemisphere for 14 days. Direct injection of [3H]-BCNU into brain parenchyma resulted in widely distributed tracer at 1 and 3 hours with rapid disappearance thereafter. It is concluded that local delivery of BCNU to brain tissue with this polymeric drug delivery system results in sustained high local concentrations of BCNU which may be of value in the treatment of patients with brain tumors.

Selective uptake by cancer cells of liposomes coated with polysaccharides bearing 1-aminolactose.

We investigated the selective uptake of liposomes chemically modified by polysaccharides-cholesterol derivatives with 1-aminolactose (lactose) in two human hepatoma cell lines (HUH7 and Alexander), a human colon cancer cell line (FCC) and a human lung cancer cell line (KNS). The uptakes of the labeled liposomes alone (conventional liposomes), those with cholesterol pullulan (CHP) and with lactose (lactose CHP) were compared in four cancer cells and normal rat hepatocytes after 3 hours of incubation. The radioactivities of the lactose CHP were 4.4, 4, 3.4 and 4.4 times greater than those of CHP in HuH7, Alexander, FCC and KNS cells, respectively, after 3 hours of incubation. All the above differences were statistically significant (p < 0.01). No statistically significant differences were seen in the case of hepatocytes. Thus, cancer cells have a common affinity with lactose CHP liposomes, however, these mechanisms appear to have no connection with the galactose-specific asialoglycoprotein receptors of hepatocytes.

Application of liposomes as potential cutaneous drug delivery systems. In vitro and in vivo investigation with radioactively labelled vesicles.

The potential application of liposomes as dermal delivery systems was investigated, with regard to vesicle composition and size. Liposomes were made up of phospholipids or skin lipids, referred to as phospholipid-based liposomes and stratum corneum lipid-based liposomes, respectively. A stripping procedure from stratum corneum to dermis by means of adhesive tape was carried out to evaluate the extent of accumulation in the superficial layers of the skin. The various liposomes were radiolabelled both in the bilayer structures with [3H]cholesterol, [14C]dipalmitoylphosphatidylcholine and [14C]palmitic acid, depending on vesicle type, and in the aqueous compartments with [14C]inulin. Inulin absorption and elimination was also evaluated. Stratum corneum lipid-based liposomes could permeate the stratum corneum to a greater extent than phospholipid-based liposomes. Stratum corneum lipid-based liposomes could deliver a greater amount of aqueous radiolabelled marker ([14C]inulin) to the deeper skin strata (epidermis and dermis), while avoiding systemic absorption and, hence, organ distribution and renal elimination of [14C]inulin. Another important parameter in determining the extent of absorption is the vesicle size: the greater the mean size of liposomes, the poorer the permeation through stratum corneum layers. When fluid liposomes made up of unsaturated lecithins were used, a percutaneous absorption was obtained instead of dermal delivery. Stratum corneum lipid-based unilamellar liposomes may be suitable devices for dermal delivery.

Characterization of in vivo immunoliposome targeting to pulmonary endothelium.

Two rat monoclonal antibodies, 34A and 201B, which specifically bind to a surface glycoprotein (gp112) of the pulmonary endothelial cell surface, have been coupled to unilamellar liposomes of approximately 0.25 microns in diameter. The 34A- and 201B-liposomes (monoclonal antibodies 273-34A and 411-201B, respectively), but not antibody-free liposomes and liposomes coupled to 14, a nonspecific monoclonal antibody, accumulate efficiently (approximately 30% injected dose) in the lung of mice which have been injected via the tail vein. Immunoliposome targeting to lung is demonstrated both by using a 125I-labeled lipid marker and an entrapped water-soluble marker. Lung accumulation of 34A-liposomes is completely blocked by a preincubation of free antibody 34A, but not antibody 14, indicating that the immunoliposome accumulation at the target site is immunospecific. Time course studies have revealed that 34A-liposomes bind to lung antigens within 1 min after injection, indicating that the target binding takes place during the first few passages of immunoliposomes through the lung capillary bed. Unbound immunoliposomes are taken up by liver and spleen within 3-5 min after injection. The level of lung accumulation increases significantly as the protein:lipid ratio of the immunoliposome increases. Approximately 50% of injected dose is accumulated in lung for 34A-liposomes, with an average of 935 antibody molecules per liposome. Immunoliposomes of larger size accumulate in lung more significantly than those of smaller size. Injection with higher doses also enhances the level of lung accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Technical and clinical evaluation of a new asymmetric polysulfone membrane (Biosulfane).

First generation asymmetric polysulfone membranes had high hydraulic permeability (kf = 40 ml/h/mmHg/sqm) but a low diffusive permeability due to the hydrophobic nature and wall thickness of 75-100 microns. We have tested a new polysulfone membrane with a wall thickness of 40 microns in a series of in vitro and in vivo dialysis session experiments. The new "Biosulfane" membrane presented a Kf of 45.8 with constant performance up to 240 mins. The koA was 760 and the clearance value at 350 ml/min of Qb in hemodiafiltration was 255 ml/min for urea, 210 for creatinine, 225 for phosphate, 76 for inulin. In high flux dialysis the clearances were similar except for inulin which was 32% lower due to the lower convection amount. Beta-2 microglobulin clearance was 22 ml/min in high flux dialysis and 37 in hemodiafiltration. Solute sieving coefficients were close to 1 for the majority of the studied solutes in a wide range of molecular weights and slight variations were observed for charged solutes due to Donnan's effect. The sieving for Inulin was 0.96 while that for Beta-2 microglobulin was not measurable due to a large molecule adsorption on the inner structure of the fibres. The good performances of this membrane are probably due to reduced wall thickness and a consequent improvement in diffusive permeability to small size solutes.

Correlation of beta-2-microglobulin concentration changes to changes of distribution volume.

Plasma and ultrafiltrate beta-2-microglobulin (B2M) concentrations were determined during hemofiltration (HF) and hemodialysis (HD) in order to evaluate elimination kinetics of B2M. Calculations were done on the basis of plasma-water-concentrations (PWC). Elimination of B2M during HF follows first order kinetics (r = 0.97) and the volume of B2M distribution was calculated to be 17 +/- 2% of body weight. This reflects extracellular volume (ECV). Changes of ECV in HD were induced by weight loss and further provoked by fluid shifts from intra- to extracellular volume and vice versa induced by varying dialysate sodium concentration. These ECV changes were followed by determining inuline in plasma and total dialysate. Changes of B2M concentration correlate well to changes of ECV (r = 0.98). Thus intratreatment concentration changes of B2M in cuprophane dialysis reflect simultaneous changes of B2M distribution volume. This does not exclude the possibility of B2M generation stimulated by dialysis, but proving such effects in vivo will be difficult because of multiple variants, that must be controlled.

Transport functions in a bioartificial kidney under uremic conditions.

A bioartifical kidney, a confluent monolayer of renal tubular cells attached to a permselective synthetic membrane, may some day substitute for the failing renal function. In such a device, the cells would have to exhibit transport properties when exposed to uremic plasma. To test the feasibility of a bioartificial kidney, LLC-PK1 cells derived from the proximal tubule were grown to confluence on microporous, cellulose nitrate membranes and transepithelial transport of inulin, glucose and tetraethylammonium (TEA) was investigated with standard tissue culture medium and with the hemofiltrate obtained from a uremic patient. Inulin, which is neither reabsorbed nor secreted by renal epithelial cells, did not diffuse through the LLC-PK1 monolayer in either tissue culture fluid or hemofiltrate. Glucose was preferentially reabsorbed and TEA was preferentially secreted in both media. In hemofiltrate, LLC-PK1 cells reabsorbed glucose at the same rate as in tissue culture fluid but secreted TEA at a significantly higher rate. This difference was related to the pH of the fluids since it was abolished by correction to the same pH value. For both solutes, the passive transepithelial leakage was significantly lower in hemofiltrate. We conclude that LLC-PK1 cells retain their reabsorbtive and secretory transport characteristics under uremic conditions.

Disposition of 3H-cholesteryl ether labeled liposomes following intravenous administration to mice: comparison with an encapsulated 14C-inulin as aqueous phase marker.

In this study, the blood clearance and organ distribution of intravenously administered liposomes (distearoyl phosphatidylcholine [DSPC] and cholesterol in a ratio of 3:1) was evaluated by utilizing 3H-cholesteryl ether as the lipid phase marker. Also, the ability of liposomes, as a drug delivery system, to alter the distribution and retention of encapsulated agents was investigated by comparing the distribution of intravenously administered free and liposome-encapsulated 14C-inulin within a 48-hr postadministration period. Intravenously administered DSPC liposomes were distributed in all organs examined with the highest levels of 3H-cholesteryl ether and 14C-inulin present in the liver and spleen; peak levels occurred 3 hr postadministration (71.86% +/- 9.39 versus 77.67% +/- 10.30, respectively, in the liver and 5.05% +/- 1.07 versus 5.36% +/- 1.09, respectively, in the spleen) declining gradually during the remaining experimental period. The lowest levels of 3H-cholesteryl ether and 14C-inulin following administration of liposome-encapsulated inulin were found in the lung, kidney, and heart. The area under curve showed much more accumulation of 14C-inulin (6-fold higher) in the body following administration of the liposome-encapsulated drug than the free drug. The ratio of 3H to 14C following administration of liposome-encapsulated inulin was constant throughout the entire observation period, suggesting that the disposition of inulin acquired both the carrier's rate of clearance and tissue distribution. These results indicated that following intravenous administration of liposome-encapsulated inulin, the majority of the radioactively labeled formulation was retained by the organs of the reticuloendothelial system (liver and spleen); liposomes greatly enhanced the retention of inulin in the body; and the liposomal formulation did not destabilize and subsequently did not release the encapsulated inulin to the tissues and organs.

The effect of different lipid components on the in vitro stability and release kinetics of liposome formulations.

Liposomes are colloidal carriers that form when certain (phospho)lipid molecules are hydrated in an aqueous media with some energy input. The ideal liposome formulation with optimum stability will improve drug delivery by decreasing the required dose and increasing the efficacy of the entrapped drug at the target organ or tissue. The most important parameter of interest in this article was to compare the efficacy of three different liposomes formulated with DSPC, DMPC, and DPPC, all saturated neutral phospholipids with different acyl chain lengths and transition temperatures. DMPC has a phase transition temperature (Tc) below 37 degrees C, whereas the other two phospholipids possess Tcs above the physiological temperature. These lipids were then added to a cholesterol concentration of 21% to optimize the stability of the vesicles. The liposomes were prepared by a sonication and incubated in phosphate buffered saline (PBS) at 4 degrees C and 37 degrees C. The encapsulation efficiency, initial size, and drug retention of the vesicles were tested over a 48-hr period employing radiolabeled inulin as a model drug. The phase transition temperature of liposomes, which depends on the Tc of the constituent lipids, was an important factor in liposome stability. Of all the liposomes tested, the greatest encapsulation efficiency was found for the DSPC liposomes (2.95%) that also had the greatest drug retention over 48 hr at both 4 degrees C (87.1 +/- 6.8%) and 37 degrees C (85.2 +/- 10.1%), none of these values being significantly different from time zero. The lowest drug retention was found for DMPC liposomes for which a significant difference in drug retention was seen after only 15 min at both 4 degrees C (47.3 +/- 6.9%) and 37 degrees C (53.8 +/- 4.3%). The DPPC liposomes showed a significant difference in drug retention after 3 hr at 4 degrees C (62.1 +/- 8.2%) and after 24 hr at 37 degrees C (60.8 +/- 8.9%). Following the initial drop at certain time intervals a plateau was reached for all of the liposome formulations after which no significant difference in drug retention was observed. In conclusion, liposomes with higher transition temperatures appear to be more stable in PBS either at 4 degrees C or 37 degrees C, indicating that the increase in acyl chain length (and therefore transition temperature) is directly proportional to stability.

Intracellular delivery of membrane-impermeable hydrophilic molecules to a hepatoblastoma cell line by asialoglycoprotein-labeled liposomes.

BACKGROUND AND PURPOSE: Mammalian hepatic receptors are specific for the terminal D-galactose of desialylated glycoproteins. This study used asialofetuin-labeled liposomes (AF-liposomes) to target hepatoma cells using this receptor mechanism, and investigated their efficiency in intracellular delivery of membrane-impermeable hydrophilic molecules to a hepatoblastoma cell line (HepG2). METHODS: Inulin was used as a model molecule. Blank liposomes consisting of phosphatidylcholine:phosphatidic acid:cholesterol in 6:1:6 molar ratio were prepared. Palmitoyl-asialofetuin was anchored onto the blank liposomes using a detergent dialysis method. 3H-inulin was entrapped in the AF-liposomes by dehydration (freeze-drying) and rehydration followed by freeze-thaw sonication. Plain liposomes (N-liposomes) were prepared by the same process but without AF. HepG2 cells were incubated for 3 hours with free 3H-inulin, N-liposomal 3H-inulin, AF-liposomal 3H-inulin, or free AF. The cellular uptake of 3H-inulin and the cell viability were then determined. Uptake of AF-liposomes in a non-hepatoma cell line, NIH3T3, was also studied for comparison. RESULTS: Cellular uptake of free inulin was negligible while uptake of liposomal inulin, either in N-liposomes or in AF-liposomes, was significant (p < 0.01). The uptake of AF-liposomal inulin was significantly higher than that of N-liposomal inulin in HepG2 cells but not in NIH3T3 cells. Free AF and blank AF-liposomes inhibited the HepG2 cell uptake of AF-liposomal inulin. CONCLUSIONS: These results indicate that AF-liposomes enhanced intracellular delivery of a membrane-impermeable hydrophilic drug into hepatoma cells by a receptor mechanism. AF-liposomes are a potential drug carrier for intracellular delivery of membrane-impermeable hydrophilic drugs to HepG2 cells.

Low-visibility light-intensity laser-triggered release of entrapped calcein from 1,2-bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine liposomes is mediated through a type I photoactivation pathway.

We recently reported on the physical characteristics of photo-triggerable liposomes containing dipalmitoylphosphatidylcholine (DPPC), and 1,2-bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC(8,9)PC) carrying a photo agent as their payload. When exposed to a low-intensity 514 nm wavelength (continuous-wave) laser light, these liposomes were observed to release entrapped calcein green (Cal-G; Ex/Em 490/517 nm) but not calcein blue (Cal-B; Ex/Em 360/460 nm). In this study, we have investigated the mechanism for the 514 nm laser-triggered release of the Cal-G payload using several scavengers that are known specifically to inhibit either type I or type II photoreaction pathways. Liposomes containing DPPC:DC(8,9)PC: distearoylphosphatidylethanolamine (DSPE)-polyethylene glycol (PEG)-2000 (86:10:04 mole ratio) were loaded either with fluorescent (calcein) or nonfluorescent ((3)H-inulin) aqueous markers. In addition, a non-photo-triggerable formulation (1-palmitoyl-2-oleoyl phosphatidylcholine [POPC]:DC(8,9)PC:DSPE-PEG2000) was also studied with the same payloads. The 514 nm wavelength laser exposure on photo-triggerable liposomes resulted in the release of Cal-G but not that of Cal-B or (3)H-inulin, suggesting an involvement of a photoactivated state of Cal-G due to the 514 nm laser exposure. Upon 514 nm laser exposures, substantial hydrogen peroxide (H2O2, ≈100 µM) levels were detected from only the Cal-G loaded photo-triggerable liposomes but not from Cal-B-loaded liposomes (≤10 µM H2O2). The Cal-G release from photo-triggerable liposomes was found to be significantly inhibited by ascorbic acid (AA), resulting in a 70%-80% reduction in Cal-G release. The extent of AA-mediated inhibition of Cal-G release from the liposomes also correlated with the consumption of AA. No AA consumption was detected in the 514 nm laser-exposed Cal B-loaded liposomes, thus confirming a role of photoactivation of Cal-G in liposome destabilization. Inclusion of 100 mM K3Fe(CN)6 (a blocker of electron transfer) in the liposomes substantially inhibited Cal-G release, whereas inclusion of 10 mM sodium azide (a blocker of singlet oxygen of type II photoreaction) in the liposomes failed to block 514 nm laser-triggered Cal-G release. Taken together, we conclude that low-intensity 514 nm laser-triggered release of Cal-G from photo-triggerable liposomes involves the type I photoreaction pathway.