Liposome-based Drug Delivery Systems Research Articles

Optimization of selected liposome-encapsulated diketopiperazines

Response surface methodology (RSM) is an effective way to optimize various processes and formulations were investigators are able to determine how variables influence the formulation by assessing interaction effects in order to obtain an optimum formulation. Cyclic dipeptides have been shown to inhibit tumor cells but problems with their physicochemical characteristics have limited further development of these compounds clinically. The aim was to optimize a liposome-based drug delivery system incorporating cyclo(His-Gly) and cyclo(His-Ala) using response surface methodology. Liposomes, containing varying compositions of phosphatidylcholine, cholesterol, stearylamine, vitamin E and PEG2000-phosphatidylethanolamine were prepared according to the thin film hydration and extrusion method. Drugs were passively encapsulated and separated from free drug using Sephadex G50 columns. RSM, utilizing a central composite rotatable design (CCRD) was used to assess the influence of selected variables on encapsulation, uptake, membrane leakage, polydispersity and zeta potential. A quadratic model was used to fit data for all responses. The most significant interaction was between cholesterol and stearylamine content with cellular uptake, indicating that these parameters cannot be altered independently of each other. Optimization of the formulation yielded stable liposomes with an average encapsulation of 0.123 mg drug/mg lipid.

Application of liposomes in drug development--focus on gastroenterological targets.

Over the past decade, liposomes became a focal point in developing drug delivery systems. New liposomes, with novel lipid molecules or conjugates, and new formulations opened possibilities for safely and efficiently treating many diseases including cancers. New types of liposomes can prolong circulation time or specifically deliver drugs to therapeutic targets. This article concentrates on current developments in liposome based drug delivery systems for treating diseases of the gastrointestinal tract. We will review different types and uses of liposomes in the development of therapeutics for gastrointestinal diseases including inflammatory bowel diseases and colorectal cancer.

Analysis of gemcitabine liposome injection by HPLC with evaporative light scattering detection

Gemcitabine liposome injection (i.e., stealth liposomes) has facilitated the targeting of gemcitabine for cancer treatment. We systemically reviewed liposome-based drug-delivery systems, which can improve pharmacokinetics, reduce side effects, and potentially increase tumor uptake, for pancreatic cancer therapy. A novel liposomal formulation, which allows for higher tumor-targeting efficiencies and can be used in current clinical trials to treat this challenging disease, has gained great popularity and attention. In this work, a simple, rapid high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of N-(carbonyl-methoxypolyethylene glycol 2000)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt and neutral colipids cholesterol and hydrogenated soy phosphatidylcholine or distearoyl phosphatidylcholine. Because of the poor ultraviolet absorbance of the lipids, evaporative light-scattering detection (ELSD) was used to monitor the separation. The separation was carried out on a YMC-Pack column (YMC Co., Ltd., Kyoto, Japan). Lipids were eluted using binary linear gradients starting from a mixture of 80% A and 20% B to 100% B in 10 minutes, followed by a 6-minute plateau at 100% B, where A is chloroform/isopropyl alcohol/diethylamine/trifluoroacetic acid (TFA) (50:50:0.01:0.0025) and B is chloroform/isopropyl alcohol/H2O/diethylamine/TFA (41:50:9:0.01:0.0025). The mobile phase composition was then changed back to initial solvent mixture in 1 minute, and the column was equilibrated for 13 minutes before every subsequent run. Then, 0.025% (v/v) TFA was added into the mobile phase to enhance the retaining of the stealth lipids. This newly developed method enabled direct analysis of liposomes without solvent lipid extraction and was validated to be linear, precise, accurate, specific, and sensitive. The method has been successfully employed in a wide range of lipid-based formulation screening, process development, and stability testing. Further, we describe the simple, rapid HPLC-ELSD method for the simultaneous determination of all the lipids and active pharmaceutical ingredients in various liposome-based drug formulations. The method can quantitate all the lipids of active targeting liposomes, which bond with folic acid.

Lipids, curvature, and nano‐medicine

The physical properties of the lamellar lipid-bilayer component of biological membranes are controlled by a host of thermodynamic forces leading to overall tensionless bilayers with a conspicuous lateral pressure profile and build-in curvature-stress instabilities that may be released locally or globally in terms of morphological changes. In particular, the average molecular shape and the propensity of the different lipid and protein species for forming non-lamellar and curved structures are a source of structural transitions and control of biological function. The effects of different lipids, sterols, and proteins on membrane structure are discussed and it is shown how one can take advantage of the curvature-stress modulations brought about by specific molecular agents, such as fatty acids, lysolipids, and other amphiphilic solutes, to construct intelligent drug-delivery systems that function by enzymatic triggering via curvature.Practical applications: The simple concept of lipid molecular shape and how it impacts on the structure of lipid aggregates, in particular the curvature and curvature stress in lipid bilayers and liposomes, can be exploited to construct liposome-based drug-delivery systems, e.g., for use as nano-medicine in cancer therapy. Non-lamellar-forming lysolipids and fatty acids, some of which may be designed to be prodrugs, can be created by phospholipase action in diseased tissues thereby providing for targeted drug release and proliferation of molecular entities with conical shape that break down the permeability barrier of the target cells and may hence enhance efficacy.

Targeted delivery of a novel group of site-directed transglutaminase inhibitors to the liver using liposomes: a new approach for the potential treatment of liver fibrosis

Liver fibrosis and its end-stage disease cirrhosis are a main cause of mortality and morbidity worldwide. Thus far, there is no efficient pharmaceutical intervention for the treatment of liver fibrosis. Liver fibrosis is characterized by excessive accumulation of the extracellular matrix (ECM) proteins. Transglutaminase (TG)-mediated covalent cross-linking has been implicated in the stabilization and accumulation of ECM in a number of fibrotic diseases. Thus, the use of tissue TG2 inhibitors has potential in the treatment of liver fibrosis. Recently, we introduced a novel group of site-directed irreversible specific inhibitors of TGs. Here, we describe the development of a liposome-based drug-delivery system for the site-specific delivery of these TG inhibitors into the liver. By using anionic or neutral-based DSPC liposomes, the TG inhibitor can be successfully incorporated into these liposomes and delivered specifically to the liver. Liposomes can therefore be used as a potential carrier system for site-specific delivery of the TG2 inhibitors into the liver, opening up a potential new avenue for the treatment of liver fibrosis and its end-stage disease cirrhosis.

Targeted SAINT-O-Somes for improved intracellular delivery of siRNA and cytotoxic drugs into endothelial cells

In non-phagocytic cells such as endothelial cells, processing of liposomes and subsequent release of drug content is often inefficient due to the absence of professional processing machinery, which limits pharmacological efficacy. We therefore developed a liposome based drug delivery system with superior intracellular release characteristics. The design was based on long circulating conventional liposomes that were formulated with a cationic amphiphile, 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chlorid (SAINT-C18). These so-called SAINT-O-Somes had a diameter of 100 nm, were as stable as conventionally formulated liposomes, and showed superior release of their content at pH conditions that liposomes encounter when they are endocytosed by cells. Attachment of anti-E-selectin specific antibodies to the distal end of surface grafted poly(ethylene glycol) resulted in immuno-SAINT-O-Somes that were as efficiently taken up by inflammation activated endothelial cells as conventional anti-E-selectin specific immunoliposomes. More importantly, intracellular release of calcein encapsulated in these targeted SAINT-O-Somes was 10 fold higher as compared to the release of calcein from conventional liposomes. For intracellular delivery siRNA into activated endothelial cells, formulation with SAINT-C18 was a necessity to induce a specific down-regulation of gene expression of VE-cadherin. Additionally, targeted doxorubicin loaded SAINT-O-Somes decreased endothelial cell viability significantly more than targeted conventional doxorubicin liposomes. SAINT-O-Somes therefore represent a new class of lipid based particles with superior drug release characteristics that can be applied for the efficacious intracellular delivery of hydrophilic drugs including siRNA.

Evaluation of pH-responsive liposomes containing amino acid-based zwitterionic lipids for improving intracellular drug delivery in vitro and in vivo

We developed pH-responsive liposomes containing synthetic glutamic acid-based zwitterionic lipids and evaluated their properties both in vitro and in vivo with the aim of constructing an efficient liposome-based systemic drug delivery system. The glutamic acid-based lipids; 1,5-dihexadecyl N-glutamyl- l-glutamate ( L1) and 1,5-dihexadecyl N, N-diglutamyl-lysyl- l-glutamate ( L2) were synthesized as a pH-responsive component of liposomes that respond to endosomal pH. The zeta potential of liposomes containing L1 or L2 was positive when the solution pH was below 4.6 or 5.6, respectively, but negative at higher pH values. The pH-responsive liposomes showed improved fusogenic potential to an endosome-mimicking anionic membrane at acidic pH, where the zeta potential of the liposomes was positive. We then prepared doxorubicin (DOX)-encapsulating liposomes containing L1 or L2, and clarified by confocal microscopic studies that the contents were rapidly transferred into both the cytoplasm and nucleus. Release of DOX from the endosomes mediated by the pH-responsive liposomes dramatically inhibited cancer cell growth. The L2-liposomes were slightly more effective than L1-liposomes as a drug delivery system. Intravenously injected L2-liposomes displayed blood persistence comparable to that of conventional phospholipid (PC)-based liposomes. Indeed, the antitumor efficacy of L2-liposomes was higher than that of PC-based liposomes against a xenograft breast cancer tumor in vivo. Thus, the high performance of L2-liposomes results from both efficient intracellular drug delivery and comparable blood persistence in comparison with the conventional PC-based liposomes in vitro and in vivo.

Near‐IR Remote Release from Assemblies of Liposomes and Nanoparticles

Set free by light: Near-IR (NIR) laser-initiated remote release of fluorescent dye from complexes of liposome-gold-nanoparticle aggregates is demonstrated (see fluorescence images). Complexes of the desired size are shown to be a viable approach to the construction of vesicle-based drug-delivery systems with light-triggered remote release characteristics. This opens up a new method to manipulate liposome-based drug-delivery systems in a biocompatible way by using the near-IR spectral range.

Liposomes as targeted drug delivery systems in the treatment of breast cancer

Solid tumors such as breast cancer have historically provided many challenges to anti-cancer therapy. Therapeutic hurdles to drug penetration in solid tumors include heterogeneous vascular supply and high interstitial pressures within tumor tissue, particularly in necrotic zones, lower pH and presence of leaky vasculature leading to reduced therapeutic response. Liposome based drug delivery systems offer the potential to enhance the therapeutic index of anti-cancer agents, either by increasing the drug concentration in tumor cells and/or by decreasing the exposure in normal tissues exploiting enhanced permeability and retention effect phenomenon and by utilizing targeting strategies. This review discusses recent trends in liposome-based drug delivery system both for diagnosis and treatment of breast cancer.

Secreted phospholipase A 2 as a new enzymatic trigger mechanism for localised liposomal drug release and absorption in diseased tissue

Polymer-coated liposomes can act as versatile drug-delivery systems due to long vascular circulation time and passive targeting by leaky blood vessels in diseased tissue. We present an experimental model system illustrating a new principle for improved and programmable drug-delivery, which takes advantage of an elevated activity of secretory phospholipase A 2 (PLA 2) at the diseased target tissue. The secretory PLA 2 hydrolyses a lipid-based proenhancer in the carrier liposome, producing lyso-phospholipids and free fatty acids, which are shown in a synergistic way to lead to enhanced liposome destabilization and drug release at the same time as the permeability of the target membrane is enhanced. Moreover, the proposed system can be made thermosensitive and offers a rational way for developing smart liposome-based drug delivery systems. This can be achieved by incorporating specific lipid-based proenhancers or prodestabilisers into the liposome carrier, which automatically becomes activated by PLA 2 only at the diseased target sites, such as inflamed or cancerous tissue.

Interaction of N-myristoyldimyristoylphosphatidylethanolamine with dimyristoylphosphatidylcholine investigated by differential scanning calorimetry: binary phase diagram

The temperature-composition phase diagram was derived for hydrated, binary mixtures of N-myristoyldimyristoylphosphatidylethanolamine ( N-14 DMPE) and dimyristoylphosphatidylcholine by high sensitivity differential scanning calorimetry. Gel phase immiscibility was detected in mixtures containing up to 20 mol% N-14 DMPE and there was no evidence for compound formation between the two components. In the fluid phase nearly complete miscibility is indicated by the calorimetric data. These results are relevant to understanding the role of N-acylphosphatidylethanolamines in the stress combating responses of organisms and in their application to developing liposome-based drug delivery systems.

Liposomes Promote Pulmonary Glucocorticoid Delivery

The beneficial effects of glucocorticoids in treating pulmonary inflammatory disorders are complicated by systemic adverse effects. Thus, a possible reduction in dosage and dosing frequency would be advantageous, particularly for patients requiring high doses of the drug. We believe that this can be achieved by developing formulations that increase the retention of glucocorticoids in the lung and a liposome-based drug delivery system may be useful. In the present study, we examined the pulmonary delivery of a liposomal glucocorticoid formulation. Male adult rats were intratracheally instilled with free [3H]dexamethasone (DEX) or [l4C]liposome-entrapped [3H]dexamethasone (L-DEX) (800 μg DEX/kg body weight) and animals were killed at different times within a 72-h treatment period. Pulmonary retention of [3H]DEX in animals instilled with free DEX was found to be approximately 1.5% of the administered dose 4 h post-instillation, with no radioactivity detectable 24 h post-instillation. Liposome encapsulation of the drug altered the pulmonary retention of DEX with about 34% and 8% of radioactivity remaining in the lung at 4 and 24 h post-instillation, respectively. The intratracheal instillation of free DEX or L-DEX reduced the number of leukocytes in peripheral blood to a similar extent (50% of control values) at 4h. However, unlike free-DEX-treated animals whose leukocyte counts returned to control levels by 24 h, the circulating leukocyte counts of L-DEX-treated animals remained depressed in the same period. Furthermore, DEX-induced changes in ACTH levels were less evident in animals treated with the liposomal formulation than those treated with free DEX. Our data suggest that the administration of liposome-entrapped DEX has the distinct advantage of enhancing the anti-inflammatory activity of the drug and therefore, possibly reducing its need for frequent administration.

Simultaneous measurement of liposome extravasation and content release in tumors.

The success of liposome-based drug delivery systems for tumor targeting relies on maximum extravasation of liposomes into tumor interstitium, as well as optimal release of contents from the liposomes once within the tumor Liposome extravasation and content release are two separate processes that can be individually or jointly manipulated so a method is needed to monitor these two processes independently and simultaneously. In this report, we describe a method to measure liposome extravasation and content release in tumor tissues growing in a rat skinfold window chamber preparation. Mixtures of liposomes containing either doxorubicin or calcein, both of which are fluorescent, and liposomes surface-labeled with rhodamine were injected intravenously. Fluorescent, light intensities in a tumor region in two fluorescent channels were measured using an image-processing system. Light intensities of plasma from blood samples were also measured using this system. These measurements were used to calculate the amounts of liposomes and released contents in both plasma and tumor interstitium. The calculations were based on the fact that the liposome surface labels and contents emit fluorescent light at different wavelengths and when encapsulated, the contents fluorescence is self-quenched. The model included equations to account for fluorescent light "cross-contamination" by the two fluorochromes as well as equations relating the measured fluorescent light intensities to the amounts of liposomes and released contents. This method was applied to three situations in which liposome extravasation and content release were manipulated in different, predictable ways. Our results indicate that this method can perform simultaneous independent and quantitative measurements of liposome extravasation and content release. This method can potentially be used to study drug delivery of other carrier systems in vivo.

Antitumor effect of liposome-incorporated camptothecin in human malignant xenografts.

The nuclear enzyme topoisomerase I (topo I) has been recently recognized as the target for the anticancer drug camptothecin (CPT; NSC 94600) and its derivatives. This drug has been reported to display effective antitumor effects on a variety of human tumor models xenografted in nude mice. However clinical studies of sodium CPT have revealed that the open-ring form of the drug is a poor inhibitor of topo I and much less potent antitumor agent than CPT lactone. However, the insolubility of CPT lactone makes it difficult to devise a suitable formulation for further clinical testing. In view of these observations, we report here the successful incorporation of CPT into a liposome-based drug delivery system (LCPT) composed of DPPC:Sph:CHOL:PI (2.4:6.6:1.0:0.05 M ratio) that can be used as a suitable formulation for clinical testing of the drug. Higher incorporation efficiency was observed when the total phospholipids:drug ratio = 40 and the cholesterol content = 1%. Image analysis of the CPT-containing liposomes with freeze-fracture electron microscopy has indicated that CPT significantly increased the interlamellar space of the vesicles as a result of its intercalation between lipid bilayers. This has occurred with no major disruptive effects on the bilayer structure. The in vitro drug release study in human serum was characterized by an initial rapid loss-of 50% of contents during 4 h, followed by a slow leakage of the remaining 50% of the total drug over a 20 h period. When tested for its antitumor activity on nude mice xenografted with human malignant melanoma and breast carcinoma, LCPT displayed effective antitumor activity with minimal host toxicity. For example, single i.m. injection of LCPT at 10 mg/kg has produced complete tumor regression to nude mice xenografted with CLO breast carcinoma. Likewise, similar results were obtained with the nude mice xenografted with human malignant BRO cells melanoma. These results appear to suggest that i.m. administration of liposome-incorporated CPT has considerable potential for the treatment of human neoplastic diseases, especially lymph node metastases.