Fixed Aqueous Layer Thickness Research Articles

To Assess Drug Delivery System Nanocarrier at Industrial Production: Establishment of Liposomal Surface Using Physicochemical Properties

Liposomes have been reported to be useful nanocarrier, however, there are number of challenges to resolve before they can be optimized for drug delivery. Liposomes are taken up by cell in the reticuloendothelial system (RES). Polyethyleneglycol (PEG) modification on the liposomal membrane forms a fixed aqueous layer and thus prevents uptake by the RES. The physicochemical properties of liposomes that are most commonly evaluated particle size and zeta potential are not sufficient indicator of the passive targeting effect by PEG modification. In contrast, the fixed aqueous layer thickness (FALT) around liposomal surface was clear to be regulated to be the utilized action in the body. It was showed that the FALT value of PEG-modified liposomes containing doxorubicin increased with the increase in the molecular weight of PEG. Furthermore, PEG modification with a combination of high- and low- molecular weight PEGs on liposomal membranes showed in optimal results with respect to FALT and a higher antitumor effect. In addition, we designed and synthesized a novel PEG-lipid, different double arms PEG (DDA-PEG), which consisted of two PEG chains of 500 and 2000 in one molecule to develop more useful PEG-modified liposomes. DDA-PEG was found to have superior antitumor activity and was associated with the prevention of tumor metastasis. Furthermore, we sought to (-)-epigallocatechin-3-O-gallate (EGCG) functions as a target ligand of the 67-kDa laminin receptor (67LR), which is expressed on high-grade tumor cells. EGCG-PEG-modified liposome appear to have superior antitumor activity against high 67LR-expressing tumor cells, as the liposomes had dual effects.

Effect of Polymer Phase Transition Behavior on Temperature-Responsive Polymer-Modified Liposomes for siRNA Transfection.

Small interfering RNAs (siRNAs) have been attracting significant attention owing to their gene silencing properties, which can be utilized to treat intractable diseases. In this study, two temperature-responsive liposomal siRNA carriers were prepared by modifying liposomes with different polymers—poly(N-isopropylacrylamide-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAAm-co-DMAPAAm)) and poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) P(NIPAAm-co-DMAAm). The phase transition of P(NIPAAm-co-DMAPAAm) was sharper than that of P(NIPAAm-co-DMAAm), which is attributed to the lower co-monomer content. The temperature dependent fixed aqueous layer thickness (FALT) of the prepared liposomes indicated that modifying liposomes with P(NIPAAm-co-DMAPAAm) led to a significant change in the thickness of the fixed aqueous monolayer between 37 °C and 42 °C; while P(NIPAAm-co-DMAAm) modification led to FALT changes over a broader temperature range. The temperature-responsive liposomes exhibited cellular uptake at 42 °C, but were not taken up by cells at 37 °C. This is likely because the thermoresponsive hydrophilic/hydrophobic changes at the liposome surface induced temperature-responsive cellular uptake. Additionally, siRNA transfection of cells for the prevention of luciferase and vascular endothelial growth factor (VEGF) expression was modulated by external temperature changes. P(NIPAAm-co-DMAPAAm) modified liposomes in particular exhibited effective siRNA transfection properties with low cytotoxicity compared with P(NIPAAm-co-DMAAm) modified analogues. These results indicated that the prepared temperature-responsive liposomes could be used as effective siRNA carriers whose transfection properties can be modulated by temperature.

Tunable Surface Properties of Temperature-Responsive Polymer-Modified Liposomes Induce Faster Cellular Uptake.

Drug delivery by nanoparticle carriers has been limited by inefficient intracellular drug delivery. Temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm)-modified liposomes can release their content following heating. In this study, we synthesized the temperature-responsive polymer poly(N-isopropylacrylamide)-co-N,N′-dimethylaminopropylacrylamide (P(NIPAAm-co-DMAPAAm)) and investigated the properties of liposomes modified with P(NIPAAm-co-DMAPAAm) for intracellular drug carriers. The copolymer displayed a thermosensitive transition at a lower critical solution temperature (LCST) that is higher than body temperature. Above the LCST, the temperature-responsive liposomes started to aggregate and release. The liposomes showed a fixed aqueous layer thickness (FALT) at the surface below the LCST, and the FALT decreased with increasing temperature. Above 37 °C, cytosolic release from the temperature-responsive liposomes was higher than that from the PEGylated liposomes, indicating intracellular uptake. Here, we showed that the tunable surface properties of the temperature-responsive polymer-modified liposomes possibly enabled their dehydration by heating, which likely induced a faster cellular uptake and release. Therefore, the liposomes could be highly applicable for improving intracellular drug-delivery carriers.

Effects of PEGylated paclitaxel nanocrystals on breast cancer and its lung metastasis.

As an attractive strategy developed rapidly in recent years, nanocrystals are used to deliver insoluble drugs. PEGylation may further prolong the circulation time of nanoparticles and improve the therapeutic outcome of drugs. In this study, paclitaxel (PTX) nanocrystals (PTX-NCs) and PEGylated PTX nanocrystals (PEG-PTX-NCs) were prepared using antisolvent precipitation augmented by probe sonication. The characteristics and antitumor efficacy of nanocrystals were investigated. The results indicated that the nanocrystals showed rod-like morphology, and the average particle size was 240 nm and 330 nm for PTX-NCs and PEG-PTX-NCs, respectively. The PEG molecules covered the surface of nanocrystals with an 11.54 nm fixed aqueous layer thickness (FALT), much higher than that of PTX-NCs (0.2 nm). PEG-PTX-NCs showed higher stability than PTX-NCs under both storage and physiological conditions. In breast cancer xenografted mice, PEG-PTX-NCs showed significantly better tumor inhibition compared to saline (p < 0.001) and PTX-NC groups (p < 0.05) after intravenous administration. In a model of lung tumor metastasis quantified by the luciferase activity, the PEG-PTX-NCs group showed higher anticancer efficacy not only than saline and PTX-NCs groups, but also than Taxol®, achieving an 82% reduction at the end of the experiment. These studies suggested the potential advantages of PEGylated PTX nanocrystals as alternative drug delivery systems for anticancer therapy.

Change in the Character of Liposomes as a Drug Carrier by Modifying Various Polyethyleneglycol-Lipids

When liposomes, as a superior drug carrier, are injected intravenously, active liposomes as medicines require polyethyleneglycol (PEG) as a modification tool around the liposomal membrane. PEG modification of a liposome forms a fixed aqueous layer, and the trapping by cells of the reticuloendothelial system (RES) is avoided. Hence, PEG-modified liposomes have long circulation in the bloodstream, and passive targeting to tumors has been achieved by PEG modification. We have been studying the passive targeting by liposomes with the expectation of more usefulness. It was proved a correlation between the PEG molecular weight of PEG-modified liposomes and blood circulation time and antitumor effect, too. Liposomes modified by PEG2000 were useful for uptake into tumor cells. We thought that the re-uptake in the liposomal membrane also increased accumulation. Moreover, it was proved that mixing two different PEGs to modify the liposome surface gives a bigger fixed aqueous layer thickness (FALT) around the liposome, giving the liposome strong antitumor activity. Then, we designed a novel PEG-lipid, 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-PEG (different double arms PEG; DDA-PEG), which had two different PEGs (2000 and 500) in one molecule. One of the innovative characteristics of DDA-PEG-modified liposome (DDAL) is that it heightens the contact ability with tumor cells. DDAL may be an effective DDS carrier for solving various PEG dilemmas. It was observed that passive targeting by PEG-modified liposomes had different characteristics by changing PEG length, anchor type or those combination. Thus, it should be applied to liposomes suitable for various diseases.

Enhanced antitumor activity of different double arms polyethyleneglycol-modified liposomal doxorubicin

The effect of polyethyleneglycol (PEG)-modified liposome as a drug carrier has been demonstrated clinically. We designed and synthesized a novel PEG-lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (different double arms PEG, DDA-PEG) which had two PEG chains of 500 and 2000 in one molecule to develop more useful PEG-modified liposome. DDA-PEG-modified liposomal doxorubicin (DDA-LDOX(7.5)) had the biggest fixed aqueous layer thickness (FALT) compared with other PEG-lipid-modified liposomes even if the added amount was a few. It was thought that FALT was the indication of blood circulation time. In DOX uptake in tumor cells, DDA-LDOX(7.5) group increased the DOX concentration in tumor cells because it had contact ability with tumor cells. Hence, DDA-LDOX(7.5) which has long circulation time in the bloodstream and contact ability with tumor cells, also had a strong antitumor effect on mice bearing M5076 ovarian sarcoma cells which were DOX low sensitive cells according to the expression of multidrug resistance protein. Furthermore, this liposome maintained a high DOX concentration in a tumor for a long time. These results indicated that the useful antitumor effect of DDA-LDOX(7.5) against M5076 ovarian sarcoma cells is a promising DDS carrier for therapies against drug resistant tumors.

Correlation of Fixed Aqueous Layer Thickness Around PEG-modified Liposomes with In Vivo Efficacy of Antitumor Agent-containing Liposomes

Liposomes are recognized as useful drug carriers, but have some problems to overcome. Liposomes are easily opsonized with serum proteins (opsonization) and taken up by the reticuloendothelial system (RES) cells, such as spleen and liver. Polyethyleneglycol (PEG) modification on the liposomal membrane forms a fixed aqueous layer and thus prevents opsonization and uptake by the RES. Our research indicates clearly that the electrical potential distributions near the membrane surfaces were different between doxorubicin (DOX)-containing liposomes with and without a PEG coating. Moreover, the value of the fixed aqueous layer thickness (FALT) around the liposome, formed by PEG modification, correlates with the circulation time and antitumor effect in a murine model. In this review, we introduce the observation that measurement of FALT as a physical characteristics is a useful method for demonstrating the antitumor effect of antitumor agent-containing PEG-modified liposomes. The use of this technique may preclude the performance of certain in vivo experiments. Our approach using FALT enables the rapid and reliable development of PEG-modified liposome formulations.

Effect of Octreotide–Polyethylene Glycol(100) Monostearate Modification on the Pharmacokinetics and Cellular Uptake of Nanostructured Lipid Carrier Loaded with Hydroxycamptothecine

A new conjugate, octreotide-polyethylene glycol(100) monostearate (OPMS), was developed for the enhancement of targeting delivery of hydroxycamptothecine (HCPT) loaded in nanostructured lipid carrier (NLC). 2 × 10(-3) and 5 × 10(-3) mmol of OPMS were respectively used to modify NLC so that the targeted nanocarriers with low and high ligand density were obtained. For comparison, the pegylated NLCs without octreotide were prepared by adding equal molar amounts of polyethylene glycol(100) monostearate (PGMS). The relation between the modification levels and properties of various NLCs were studied in vivo and in vitro. At a high modification level, a slower release rate of HCPT and the more stable nanocarriers was achieved. At the same time, the fixed aqueous layer thickness (FALT) and average surface density of PEG chains (SD(PEG)) was increased, but the distance (D) between two neighboring PEG grafting sites became narrower. The in vivo pharmacokinetic study in healthy rat indicated that the modified NLCs had a longer circulation than NLC (P < 0.05) due to pegylation effect and OPMS modified NLCs had larger MRT and AUC(0-t) than that of PGMS modified NLCs at the same modification level. Furthermore, the florescence microscopy observation also showed the targeting effect of octreotide modification on somatostatin receptors (SSTRs) of tumor cell (SMMC-7721). The uptake of SMMC-7721 was much more than that of normal liver cell (L02) for OPMS modified NLC, and the highest uptake was observed for 5 × 10(-3) mmol of OPMS modified one. No obvious difference was found among the L02 uptake of OPMS modified NLCs and NLC, but their uptake was higher than that of PGMS modified NLCs. All the results indicated that the OPMS highly modified NLCs would improve the effect of antitumor therapy by inhibiting the degradation, evading RES and enhancing the drug uptake of tumor cells.

Abstract 1443: Dual effect of novel polyethyleneglycol modified liposome on antitumor activity and liver metastasis

Abstract [Purpose] We synthesized polyethyleneglycol-lipid with different double arms (DDA-PEG) as the novel modification material on liposomal membrane, and reported that doxorubicin (DOX) contained DDA-PEG modified liposome (DDA-LDOX) had superior properties (cytotoxicity and DOX uptake into tumor cells). On the other hand, it was suggested that fixed aqueous layer thickness (FALT) around the liposomal membrane was concerned with long circulation in blood or antitumor effect. In 100th AACR meeting (2009), we reported the connection with the FALT and the biological factor (amount of DOX uptake / IC50 of cell cytotoxicity) in vitro. In this study, DDA-LDOX had been studied the antitumor activity on primary tumor in vivo. Moreover, another activity of DDA-LDOX was examined against liver metastasis as the risk factor in cancer therapy. [Methods] Liposome (DSPC/cholesterol/DSPG/PEG/DOX = 100:100:60:15:18 μmol) was prepared according to the method of Bangham. PEG2000 modified liposome and PEG2000/PEG500 modified liposome was expressed as Single-LDOX and Mixed-LDOX, respectively. Zeta potentials of liposomes were measured by the change of NaCl concentration in buffer and FALT was calculated. In the study of antitumor activity to primary tumor, M5076 ovarian sarcoma cells were transplanted into the backs of mice, and each liposomes were injected intravenously at a dose of 2.5 mg/kg on 19th, 22nd and 25th day after tumor inoculation. The mice were sacrificed after 48 hr from last administration, and the tumor was removed and weighed. In the study of anti-metastasis activity, M5076 ovarian sarcoma cells were inoculated in tail vein of mice. These mice were injected various liposomes at a dose of 2.5 mg/kg (i.v.) on 6th, 9th and 12th day after tumor inoculation. After 48 hr from last administration, the liver was removed and weighed, and score of liver metastasis was recorded (0-5 stage, 0: non-metastasis). [Results and discussion] In the result of antitumor activity, the tumor weight after Single-LDOX treatment was reduced to 65.5% of control level (1.13±0.45 g). In contrast, after DDA-LDOX treatment, it was reduced to 36.4% of control level. In anti-metastasis activity, the score of hepatic metastasis was DOX sol &amp;gt; plain-LDOX &amp;gt; Single-LDOX = DDA-LDOX. The inhibitory effect on liver metastasis by Single-LDOX and DDA-LDOX were obviously. The metastasis score correlated with the liver weight, namely, inhibition of metastasis by DDA-LDOX was supported from liver weight. DOX concentration in the liver was plain-LDOX &amp;gt; Single-LDOX = DDA-LDOX &amp;gt; DOX sol. Thus, we speculated that weak effect of plain-LDOX was caused by DOX accumulation into hepatic normal cell. In this study, the character of Single-LDOX was similar to DOXIL®, namely, it was suggested that DDA-LDOX increase the therapeutic efficacy, compared with DOXIL®. In conclusion, we expected that DDA-LDOX had the dual effect on antitumor activity and liver metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1443.

Synthesis and application of poly(ethylene glycol)–cholesterol (Chol–PEG m) conjugates in physicochemical characterization of nonionic surfactant vesicles

Vesicles possessing poly(ethylene glycol) (PEG) chains on their surface have been described as a blood-persistent drug delivery system with potential applications for intravenous drug administration. In this research with different molecular weights (400–10,000 g/mol) of PEG, a series of Chol–PEG m conjugates were generated by the DCC ( N, N′-dicyclohexylcarbodiimide, DCC)/(4-dimethylaminopyridine, 4-DMAP) esterification method, and confirmed by FT-IR and 1H NMR spectrum. Then their properties in aqueous solution were studied by electron microscopy images, associative behavioral and systematic tensiometric studies over a wide concentration range. In order to elucidate the application of this Chol–PEG m in vesicles, conventional nonionic surfactant vesicles (niosomes) composed of span 60 and cholesterol were prepared and the influence of various hydrophilic chains of the Chol–PEG m conjugates was investigated. Results indicated that all the niosomes prepared, with or without Chol–PEG m composition were similar in micrograph with diameter between 120 nm and 180 nm. The fixed aqueous layer thickness (FALT) around niosomes increased as Chol–PEG m chain length increase, particularly in the Chol–PEG 10,000 modified niosomes with 9.33 ± 0.67 nm. In vitro release experiments indicated that release rate of nimodipine from Chol–PEG m modified niosomes was enhanced. Chol–PEG m modified niosomes showed greater accumulative release than that of plain niosomes over a period of 24 h. These studies have shed some light on the suitability of Chol–PEG m containing niosome preparation.

Characterization of Novel Mixed Polyethyleneglycol Modified Liposomes

In our previous paper, mixed polyetheleneglycol (PEG) modification of liposomes by a mixture of 1-monomethoxypolyethyleneglycol-2,3-distearoylglycerol (PEG-DSG) with short polyoxyethylene chain and PEG-DSG with long one was shown to increase fixed aqueous layer thickness (FALT) around the liposomal membrane, and this was useful in vivo. In this study, we investigated the characterization of mixed PEG modification of liposomes with different anchors (PEG2000-DSG and PEG2000-cholesterol (CHO)). When the liposomes was modified by a mixture of PEG2000-DSG and PEG2000-CHO, FALT was increased compared to that of each single PEG-lipids modification and the most suitable mix modification (PEG2000-DSG:PEG2000-CHO = 3:1) showed a maximum FALT. This phenomenon was speculated to be based on the difference in the insertion state of the PEG anchor unit in the liposomal membrane. PEG-CHO-modified liposomes (single or mixed PEG-modified liposomes) were easily incorporated into the liposomal membranes compared with that of single PEG-DSG-modified liposomes. Namely, it was considered that the cholesterol anchor as a single chain was able to be easily introduced, compared with the DSG anchor as two chains, and induced some interaction with both PEG modification. In conclusion, it is expected that novel PEG-modified liposomes with PEG2000-DSG and PEG2000-CHO (3:1) had superior physicochemical properties.

Characterization and cytotoxicity of mixed polyethyleneglycol modified liposomes containing doxorubicin

Liposomes are recognized as one of the useful drug carriers, but have many problems to overcome before their clinical application. Liposomes, bonding peculiarly with serum protein (opsonization), are taken up by reticuloendothelial system (RES) cells in the liver and spleen. It is known that polyethyleneglycol (PEG) modification of the liposome surface induces the formation of a fixed aqueous layer around the liposomes due to the interaction between the PEG-polymer and water molecule, and thus prevents the attraction of opsonins. Namely, PEG-modified liposomes are able to escape trapping by the RES cells, and have a prolonged circulation time. In this study, the effects of different anchors with the same PEG molecular weight on the cell uptake and cytotoxicity of mixed PEG-modified liposomal doxorubicin (DOX) were examined. The fixed aqueous layer thickness (FALT) of liposomes covered with mixtures of PEG-molecules which differ in their chain length were increased, compared to that of the single PEG2000-modified liposome. Mixed PEG-modification of liposomes with different anchors (PEG2000-(1-monomethoxypolyethyleneglycol-2,3-distearoylglycerol (DSG): cholesterol (CHO) = 1:1)-modified liposome) led to an increase in the FALT, compared to that of each single PEG-modification. The uptake of DOX into Ehrlich ascites carcinoma cells by the liposomes covered with PEG-CHO was higher than the other liposomes. Thus, liposomes covered with PEG-DSG and PEG-CHO have an enhanced cytotoxicity. In conclusion, it was confirmed that mix-modified liposomes using PEG-lipid with different anchors were superior.

Pharmacokinetics and tissue distribution of etoposide delivered in long circulating parenteral emulsion

Purpose: The aim of the study is to ascertain the influence of pegylation of parenteral emulsion (PE) on their long circulating property.Methods: Etoposide encapsulated parenteral emulsion (EPE) was prepared using soybean oil, egg lecithin and cholesterol. Etoposide encapsulated long circulating parenteral emulsion (PEG–EPE) was prepared using PEG (2000)-DSPE as a stealth agent. The effect of monovalent and divalent electrolytes on the stability of EP was assessed by measuring the fixed aqueous layer thickness (FALT) and flocculation rate. Pharmacokinetics and tissue distribution pattern of PE following i.v. (bolus) were assessed in Wistar rats and Swiss albino mice.Results: FALT of PEG–EPE was larger than that of EPE. In case of PEG–EPE, as the concentration of pegylated lipid (PEG) increased from 0.15 to 0.45% w/v the flocculation rate decreased asymptomatically in the presence of monovalent and divalent electrolytes. The increased circulation time of PEG–EPE (0.3%) after intravenous injection to rats confirms the presence of FALT around globules. PEG–EPE showed improved pharmacokinetic parameters with 5.5 times higher AUC than etoposide commercial formulation (ETP). Tissue distribution results show that etoposide levels in all tissues except in brain and heart were lower in case of PEG–EPE than ETP. The percentage of tumor growth suppression rate (%T/C) in Lewis lung carcinoma bearing mice was 63.23, 62.83 and 33.78% in EPE, PEG–EPE and ETP treated mice, respectively. The improved activity of PEG–EPE is due to enhanced permeability and retention effect (EPR).Conclusion: Encapsulation of etoposide in PEG-coated PE produced improved pharmacokinetic profile than that of EPE and ETP.

In vivo tumor targeting of tumor necrosis factor-α-loaded stealth nanoparticles: Effect of MePEG molecular weight and particle size

The aim of this study is to reveal the influence of methoxypolyethyleneglycol (MePEG) molecular weight and particle size of stealth nanoparticles on their in vivo tumor targeting properties. Three sizes (80, 170 and 240 nm) of poly methoxypolyethyleneglycol cyanoacrylate-co- n-hexadecyl cyanoacrylate (PEG-PHDCA) nanoparticles loading recombinant human tumor necrosis factor-α (rHuTNF-α) were prepared at different MePEG molecular weights (MW = 2000, 5000 and 10,000) using double emulsion method. The opsonization in mouse serum was evaluated by Coomassie brilliant blue staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Phagocytosis was evaluated by incubating 125I-rHuTNF-α-loaded nanoparticles with mouse macrophages (RAW264.7). The pharmacokinetics, biodistribution and tumor targeting studies were performed in S-180 tumor-bearing mice. Higher MePEG molecular weight provided thicker fixed aqueous layer thickness (FALT) and smaller particle size offered higher surface MePEG density. The serum protein adsorption and phagocytic uptake were markedly decreased for the nanoparticles with higher MePEG molecular weight or smaller size. The particles (80 nm) made of PEG 5000-PHDCA, possessing a thicker FALT (5.16 nm) and a shortest distance (0.87 nm) between two neighboring MePEG chains, showed the strongest capacity of decreasing protein adsorption and phagocytic uptake. These particles extended the half-life of rHuTNF-α in S-180 tumor-bearing mice by 24-fold (from 28.2 min to 11.33 h), elevated the rHuTNF-α peak concentration in S-180 tumors by 2.85-fold and increased the area under the intratumoral rHuTNF-α concentration curve by 7.44-fold. The results of the present study showed PEG-PHDCA nanoparticles with higher MePEG molecular weight and smaller particle size could achieve higher in vivo tumor targeting efficiency.

The Characterization and Cytotoxicity of Mixed PEG-DSG Modified Liposomes

It is known that polyethyleneglycol (PEG) modification of the liposome surface leads to the formation of a fixed aqueous layer around the liposomes due to interaction between the PEG polymer and water molecules, which prevents the attraction of opsonins. When a combination of PEG-distearolyglycerol (PEG-DSG) whose characteristics are remarkably different is used, interaction between molecules occurs, leading to increased fixed aqueous layer thickness (FALT). From this speculation, we studied the effect of both modification of PEG900-DSG and PEG2000-DSG modified liposome on FALT, cell uptake and biodistribution. The FALT of mixed PEG modified liposome increased, compared to that of each single PEG modified liposome. In this mixed modification, maximum FALT was shown at liposome modified by added PEG-2000:PEG-900=2:1. This most suitable additional ratio was equal to actual incorporated ratio. On the other hand, cell uptake of mixed modified liposome containing doxorubicin (DOX) was similar with that of PEG2000 modified liposome. Furthermore, mixed PEG modification of liposome was tendency to increase cytotoxicity, compared to that of other modifications. After DOX contained liposome treatment, DOX distribution in the tumor and antitumor activity of DOX increase by mixed PEG modification. In conclusion, it was suggested that mixed PEG liposome (PEG-2000:PEG-900=2:1) was useful for cancer chemotherapy.

Study on the characterization of mixed polyethyleneglycol modified liposomes containing doxorubicin

Previously, we suggested that mixed polyethyleneglycol (PEG)-modification of liposomes with mixture of 1-monomethoxypolyethyleneglycol–2,3-distearoylglycerol (PEG–DSG) with short and long polyoxyethylene chains led to increasing fixed aqueous layer thickness (FALT) and was useful in vivo. FALT is expected to be one of the important factors that influence the pharmacokinetics of liposomes. In this study, we investigated the connection between FALT and some parameters in vitro. In both mixed and single PEG-modified liposomes, the incorporation ratios of PEG–DSGs were the same, the residual amount of PEG–DSGs with serum proteins was not affected by molecular weight. Consequently, the effective properties in vivo of mixed PEG-modification may be the increase in the absolute amount of PEG–DSGs on the liposome membranes, decrease of slipping out action taken PEG–DSGs with long polyoxyethylene chains by serum protein, and thus maintenance of FALT. While, it has been suggested that mixed PEG-modified liposomes are effective in vitro, too, because of the lower leakage and same level of doxorubicin (DOX) uptake as plain liposomal DOX.

Effects of mixed polyethyleneglycol modification on fixed aqueous layer thickness and antitumor activity of doxorubicin containing liposome

Polyethyleneglycol (PEG) has often been used for the modification of liposomes, but it is difficult to insert PEG on the surface of liposomes, and the effects of modification are not marked enough. In this study, we examined the fixed aqueous layer thickness (FALT) of single or mixed PEG (molecular weight, 340, 500, 900, 2000)-modified doxorubicin (DOX) liposomes, and physical character and biological properties of these liposomes. On single PEG-modification, as the PEG-molecular weight increased, FALT also increased, but the ratio of the increase was reduced. While on modification by a mixture of PEG2000 and PEG with a short polyoxyethylene chain (PEG340 or PEG500), FALT increased compared with the single PEG2000-modified value. Moreover, when liposomes were modified by mixture of PEG2000 and PEG500, we recognized the most suitable mixed rate (PEG2000, 500=2:1), showed the maximum FALT. On the other hand, in vivo, as increase of FALT, DOX concentrations increased in the plasma and in the tumor, decreased in the liver. Furthermore, liposomes with remarkable increase of FALT showed enhancement of antitumor activity. As a result, the connection among increase of FALT and improvement of circulation in blood, the involvement of antitumor activity of DOX of these liposomes was suggested.

Physicochemical specification of drug carrying liposomes for the quality control in the industrial production

Animal data of liposomal drugs in a laboratory environment are often not reproduced on industrial scale products. One of the most important reasons is in the fact that the colloid-chemical properties of the liposomes manufactured on a small scale are not reproduced in a large scale production. In this review, colloid chemical characterization of liposomes is introduced, fixed aqueous layer thickness (FALT) as an example, and its importance in a quality control of a liposomal product in an industrial scale production is shown. Methoxy-polyethylenglycol-diacylgricerol (PEG-DAG) with varying PEG chain length and acyl chains were synthesized, FALT of liposomes coated with PEG-DAG determined and tissue distribution in tumor bearing mice studied. The higher incorporation ratio of PEG-DAG into liposomal membrane was observed with PEG-DAG with short acyl chains (myristoyl) and a small PEG molecular weight (1000). The easier to be incorporated, the easier to be stripped in the serum. The disposition data in the mice well reflected the colliod chemical and in vitro data of the PEG liposomes. Galactosyl-carbonyl-propionyl-polyethyleneglycol-diacylglycerol (Gal-PEG-DAG) with oxyethylene number, n=10, 20 and 40 were synthesized. The exposure of galactosy residue beyond the fixed aqueous layer of liposomes coated with Gal-PEG-DAG was monitored by RCA lectin induced agglutination, half life in the blood and organ distribution after i.v. injection into rats determined and intrahepatic distribution studied. Only the liposomes containing Gal-PEG 10DAG aggregated with the lectin, indicating that only with this derivative the galactose group was adequately exposed. The Gal-PEG 10-DAG liposomes were cleared from plasma with a half life of 0.3 h. The plasma elimination could be attributed entirely to increased uptake by the liver. The increased liver uptake was almost entirely attributed to increased uptake by the non-parenchymal cell. Incorporation of PEG-DSPE in the Gal-PEG 10DAG liposomes caused (1) a 3-fold increase in blood circulation time, (2) a small but significant decrease in hepatic uptake after 20 h and (3) a significant shift in intrahepatic distribution in favor of the hepatocytes, comparable to that of the control liposomes. In conclusion, therapeutic efficacy and safety of liposomes can be controlled by their colloid chemical, more exactly, surface chemical properties. By setting up reasonable quality control specifications of the properties in the laboratory and examining the specifications satisfied in upscaling, the efficacy and safety are reproduced in a large scale product.

An importance of colloid chemical characterization of liposomes for DDS in a large scale production

Efficacy and safety data of liposomal drugs in a laboratory environment are often not reproduced on an industrial production scale. This is largely due to the fact that the colloid-chemical properties of the liposomes manufactured on a small scale are not reproduced in large scale production. Though the size and the electric charge of liposomes are measured and are adequately specified in relation to the bio-distributions in most developments of liposomes (1), uniformity of lipid components, exposure of bio-chemically important functional groups on the outer surface of liposomes (2), fixed aqueous layer thickness (FALT), number of the lipid bilayers, etc., are dependent upon the scale of production. Nevertheless these properties are not always exactly specified. Uniformity, especially of the functional groups on the membrane surface can be assessed chemically or bio-chemically with fractionated samples, and FALT can be easily determined through electro-chemical means (3). In this review, colloid chemical characterization of liposomes is introduced, FALT as an example, and its importance in a quality control of a liposomal product in an industrial scale production is shown. Methoxy-polyethyleneglycol-diacylglycerol (PEG-DAG) with varying PEG chain length and acyl chains were synthesized, FALT of liposomes coated with PEG-DAG determined and tissue distribution in tumor bearing mice. The higher incorporation ratio of PEG-DAG into liposomal membrane was observed with PEG-DAG with short acyl chains (myristoyl) and a small PEG molecular weight (1000). The easier to incorporate, the easier to be stripped in the serum. The disposition data in the rats well reflected the colloid chemical and in vitro data of the PEG liposomes. Galactosyl-carbonyl-propionyl-polyethyleneglycol-diacylglycerol (Gal-PEG-DAG) with oxyethylene number, n = 10, 20 and 40 were synthesized. The exposure of the galactose residue beyond the fixed aqueous layer of liposomes coated with Gal-PEG-DAG was monitored by a lectin, Ricinus communis agglutinin (RCA) induced agglutination, the half life in the blood after i.v. injection into rats, organ distribution determined and intrahepatic distribution studied. Only the liposomes containing the Gal-PEG10-DAG aggregated with the lectin, indicating that only with this derivative the galactose group was adequately exposed. The Gal-PEG10-DAG liposomes were cleared from the plasma with a half life of 0.3 h. The plasma elimination could be attributed entirely to increased uptake by the liver. The increased liver uptake was almost entirely attributed to increased uptake by the non-parenchymal cell. Incorporation of PEG-DSPE in to the Gal-PEG10-DAG liposomes caused 1) a three-fold increase in blood circulation time, 2) a small but significant decrease in hepatic uptake after 20 h and 3) a significant shift in intrahepatic distribution in favor of the hepatocytes, comparable to that of the control liposomes. In conclusion, therapeutic efficacy and safety of liposomes can be controlled by their colloid chemical, more exactly, surface chemical properties. By setting up reasonable quality control specification of the properties in laboratory and examining the specifications satisfied in upscaling, the efficacy and safety are reproduced in a large scale product.

Effect of sterical stabilization on macrophage uptake in vitro and on thickness of the fixed aqueous layer of liposomes made from alkylphosphocholines

A serious problem using liposomes for therapeutic purposes is the fast removal from blood circulation by components of the reticuloendothelial system (RES) most likely after opsonization of the vesicles. This study was performed to quantify the reduction in macrophage uptake in vitro of sterically stabilized liposomes (PEG-liposomes) prepared from hexadecylphosphocholine, cholesterol and poly(ethylene glycol 2000) distearoylphosphoethanolamine (PEG 2000DSPE) for the first time. The uptake was determined using HPC-liposomes of different defined size (125, 250 and 1000 nm) without and with sterical stabilization by incorporating 5 mol% of PEG 2000DSPE. HPTS was used as fluorescence marker allowing the discrimination between general uptake and the part of liposomes internalized into the low pH-compartment (Daleke, L.D., Hong, K. and Papahadjopoulos, D. (1990) Biochim. Biophys. Acta 1024, 352–366). Liposomal uptake by J774 mouse macrophage-like cells was time-dependent. Both the uptake and internalization were clearly reduced for PEG-liposomes compared to plain liposomes. Sterical stabilization reduced the general uptake of liposomes in vitro by more than 50% and the internalization by about 50–60%. PEG-liposomes additionally showed a delay in internalization into the macrophages during the first 6 h. Size of used liposomes had only a minor influence on liposomal uptake but highest concentration of lipid was found for large multilammelar vesicles (MLV). The fixed aqueous layer thickness (FALT) was determined by zeta potential measurements of plain and sterically stabilised HPC-liposomes (100 nm) in solutions of different ion concentrations. The calculation of the thickness was based on the linear correlation between ln ζ (zeta-potential) and κ (Debye Hückel-Parameter). FALT was calculated and found to be for plain HPC-liposomes 0.83 ± 0.17 nm and for PEG-HPC-liposomes 3.57 ± 0.17 nm. Exchange of the HPC by an alkylphospholipid with different head group has no or only minor effect (PEG-OPP-liposomes 3.44 ± 0.31 nm). Thus the reduced uptake of HPC-LUVET correlates with an increased thickness of the fixed aqueous layer around these liposomes and could support the hypothesis that the thickness is an important property responsible for preventing opsonization and resulting finally in a reduced macrophage uptake.