Bioadhesive Liposomes Research Articles

Preparation, Optimization and Characterization of Bovine Lactoferrin‐loaded Liposomes and Solid Lipid Particles Modified by Hydrophilic Polymers Using Factorial Design

Bioadhesive liposomes and solid lipid particles (SLPs) modified by pectin and chitosan for oral administration of bovine lactoferrin (bLf) were prepared using a 2(4) full-factorial design to identify the key formulation variables influencing particle size and drug entrapment efficiency (EE). Netlike structures of the polymer-particle mixture consisting of a polymeric network in which multiple particles were imbedded were observed by scanning electron microscopy (SEM). Chemical stability of bLf after encapsulation into pectin- and chitosan-modified liposomes and SLPs was confirmed by Fourier transform infrared spectra (FTIR). Bovine lactoferrin was located within phospholipid bilayer, whereas in SLPs bLf was within the matrix. The crystalline nature of bLf after encapsulation was investigated by differential scanning calorimetry (DSC) of drug-loaded particles, indicating amorphous dispersion of bLf in the polymer-lipid matrix of pectin- and chitosan-modified liposomes and SLPs. In vivo pharmacokinetic investigation of bLf in pectin- and chitosan-modified liposomes and SLPs showed prolonged mean residence time (MRT) of bLf in rat blood and increased the relative bioavailability (Fbio %) by 1.95- to 2.69-fold compared with free bLf. The developed carrier systems are considered to be promising vehicles for oral delivery.

Mucoadhesive liposomes for intranasal immunization with an avian influenza virus vaccine in chickens

The aim of this study was to characterize a nasally delivered bioadhesive liposome using an inactivated H5N3 virus as a model antigen. Bioadhesive liposomes were developed using tremella (T) or xanthan gum (XG) as the bioadhesive polysaccharide. Using chickens as the target animal, we evaluated whether delivery of a bioadhesive liposomal influenza vaccine via a mucosal site of infection could improve vaccine effectiveness. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cytotoxicity assays demonstrated that T, XG and liposomes were non toxic to chicken spleen macrophages. Enzyme-linked immunosorbent assay (ELISA) was used to determine the adjuvant effect of the bioadhesive liposomal-vaccines. Chickens immunized with a low dose (200 μL) of bioadhesive liposomal influenza vaccine had significantly higher mucosal and serum antibody levels ( P < 0.05). In addition, liposomes mixed with a low-viscosity bioadhesive gel used for nasal delivery resulted in superior antibody responses compared with liposomes mixed with a high-viscosity gel ( P < 0.05). This suggest that a low-viscosity gel mixed with liposomes is more suitable for nasal delivery, and that chickens elicit higher mucosal secretory immunoglobulin A (s-IgA) and serum IgG after two vaccinations.

Liposomal dexamethasone–diclofenac combinations for local osteoarthritis treatment

Conventional chronic and acute treatments for osteoarthritis (OA) are by oral NSAIDs (such as diclofenac) and intra-articular injected glucocorticosteroids (such as dexamethasone). In free form, diclofenac and dexamethasone generate severe adverse effects with risks of toxicity. To reduce these drawbacks, we investigated local injections of liposomal formulations for diclofenac and dexamethasone (each alone, and their combination). Bioadhesive liposomes carrying hyaluronan (HA-BAL) or collagen (COL-BAL) as their surface-anchored ligand were used for the task. Each drug alone or their combination showed high efficiency encapsulations (≥80%) and performance as slow-release depots (half-lives in the range of 1–3 days under the fastest conditions). Employing RIA and immunoblot assay techniques, it was verified that the encapsulated drugs retained their biological activities: inhibitions of Cyclooxygenases enzyme-activity (diclofenac) and of Cyclooxygenases protein-expression (dexamethasone). Using live-animal MRI, a single intra-articular injection of each liposome-drug(s) formulation sufficed to reduce knee joint inflammation in OA rats over a time span of 17 days, HA-BAL better than COL-BAL. The most effective treatment was by the combination of both drugs in HA-BAL, a single dose reducing the inflammation volume down to 12.9% from initial over that time span. We find all three HA-BAL formulations worthy of further studies.

Development of micro- and nanosystems for drug delivery

Methods for preparing colloidal delivery systems for drugs of different chemical structures have been developed and optimized. Proteins were encapsulated in bioadhesive biodegradable starch microparticles and liposomes from negatively charged and zwitter-ionic soybean phospholipids. Proteins and a poorly watersoluble anticancer drug-tamoxifen-were encapsulated in nanoparticles based on the amphiphilic graft block copolymer dextran-poly(ɛ-caprolactone). In vitro release studies showed sustained release of proteins and tamoxifen in different media.

Cyclooxygenase inhibition by diclofenac formulated in bioadhesive carriers

Adverse effects and gastrointestinal toxicity limit the use of Diclofenac, a frequently-used NSAID for treatments of rheumatic disorders and other chronic inflammatory diseases. Diclofenac-carrier formulations may alleviate adverse effects, increase efficacy and allow local administration. We report here our first step, biophysical and biochemical investigations of Diclofenac formulated in our previously-developed bioadhesive liposomes carrying hyaluronan (HA-BAL) or collagen (COL-BAL) on their surface. Both liposome types encapsulated Diclofenac at high efficiency, encapsulated doses reaching 13mg drug/ml, and performed as sustained-release Diclofenac depots, half-lives of drug release (under fastest conditions) ranging from 1 to 3days. Therapeutic activity of liposomal Diclofenac was evaluated in CT-26 cells that possess the CD44 hyaluronan receptors and integrins, and are a bench-mark for intracellular COX enzymes. HA-BAL and COL-BAL showed high cellular-affinity that was 40 fold and 6 fold over that of regular liposomes. Free, and liposome-encapsulated, Diclofenac showed similar activities. For example: 2–3nM Diclofenac given to intact cells generated COX-inhibition levels in the range of 60–70% for free drug and for encapsulated drug in COL-BAL and in HA-BAL. We propose these novel Diclofenac formulations possess key physicochemical and biochemical attributes for task performance, meriting the next step into in vivo studies.

Lipsome-formulated enzymes for organophosphate scavenging: Butyrylcholinesterase and Demeton-S

Butyrylcholinesterase-encapsulating bioadhesive liposomes are investigated as prophylactic scavengers of organophosphates for local administration to skin, eyes, airways, and lungs—gates through which organophosphates penetrate living systems. The systems were optimized with respect to: encapsulation efficiency; type of bioadhesive ligand bound to liposomes (collagen or hyaluronan); ligand density at the liposomal surface; retention of encapsulated-enzyme activity; protection of encapsulated enzyme from proteolysis; and scavenging the model organophosphate Demeton-S (DS). Monolayers of PC-12 cells were selected for feasibility testing based on: high affinity binding of the bioadhesive liposomes—Δ G 0 release upon binding ranged from −9 to −12 kcal/mol ligand; ability to mimic an organophosphate attack upon intact cells and measuring its impact on intracellular acetylcholinesterase. Under attack, unprotected cells lost 80–90% of intracellular enzyme activity. The loss was reduced to 20–30% for protected cells (pre-treated with the formulations), at the expense of liposomal Butyrylcholinesterase. These results support our prophylactic approach.

Hyaluronan is a key component in cryoprotection and formulation of targeted unilamellar liposomes

Lyophilized unilamellar liposomes (ULV), the dosage form of choice for shelf-life, revert upon reconstitution to the larger multilamellar liposomes (MLV), which is detrimental to the many carrier-mediated therapies that require small particles. High doses of sugars such as trehalose, sucrose and others, included in the original formulations for cryoprotection, were shown to prevent the conversion to MLV. In this study we set out to test whether hyaluronan (HA), the surface-bound ligand in our previously developed targeted bioadhesive liposomes (BAL), can also act as a cryoprotectant. The studies included structural and physicochemical characterization of original and reconstituted hyaluronan-ULV (HA-ULV). For each HA-ULV, similar regular ULV (RL-ULV) served as controls. Four properties were tested: particle size, zeta potential, encapsulation efficiency and half-life of drug release (τ1/2), for three drugs—chloramphenicol (CAM), vinblastine (VIN) and mitomycin C (MMC). Encapsulation efficiencies of the original systems were quite alike for similar RL-ULV and HA-ULV ranging from 25% to 70%. All systems acted as sustained-release drug depots, τ1/2 ranging from 1.3 to 5.3 days. Drug species and lipid composition were the major determinants of encapsulation and release magnitudes. By all tests, as anticipated, lyophilization generated significant changes in the reconstituted RL-ULV: 17-fold increase in diameter; tripling of zeta potential; 25–60% drop in encapsulation efficiencies; 25–30% decrease in τ1/2. In contrast, the reconstituted HA-ULV retained the same dimensions, zeta potentials, encapsulation efficiencies and τ1/2 of the original systems. These data clearly show HA to be a cryoprotectant, adding another clinically relevant advantage to HA-BAL. We propose that, like the sugars, HA cryoprotects by providing substitute structure-stabilizing H-bonds.

Physicochemical Evaluation of a Stability-Driven Approach to Drug Entrapment in Regular and in Surface-Modified Liposomes

The traditional mode of encapsulating drugs in liposomes poses risks to drug stability, especially when recognition agents are attached to the liposomal surface to obtain targeted liposomes. To reduce such risks, we devised a simple, novel method to entrap drugs in liposomes, consisting of (i) preparation and lyophilization of drug-free regular and surface-modified liposomes and (ii) drug encapsulation in the course of liposome reconstitution through rehydration in an aqueous solution of the drug. In this paper, we report physicochemical studies in which we compared regular and surface-modified liposomes made by this novel approach (denoted N-liposomes) to respective liposomes made by the traditional mode (denoted T-liposomes). The studies were performed with fluorescein, sucrose, histidine, mitomycin C (MMC), and chloramphenicol (CAM) encapsulated (each) in regular and in bioadhesive liposomes, the latter having hyaluronic acid as the surface-bound ligand. Our major findings are as follows: (1) The drug-specific encapsulation efficiencies spanning the range of 10–90% were, excepting sucrose, either similar in the N- and T-liposomes or better in the N- than in the T-liposomes, for both regular and bioadhesive liposomes. (2) For all liposome types and methods of preparation, fluorescein, histidine, and MMC did not adsorb to the liposomal surface. Sucrose and MMC did adsorb to the liposomal surface irrespective of the liposome preparation mode, sucrose favoring bioadhesive over regular liposomes and MMC having the opposite trend. (3) For both regular and bioadhesive liposomes, the mechanism of drug efflux from the N-liposomes was found to be governed by a single rate constant, as previously found for the T-liposomes. The magnitudes obtained, ranging from 3.5(±0.2) × 10−3 to 400(±17) × 10−3 h−1, were always drug specific and occasionally also liposome type (i.e., regular or bioadhesive) specific. For MMC and CAM, the novel approach rendered liposomes with improved sustained release. The results reported here attest, overall, to the potential of this novel approach, meriting further investigations. Studies currently underway with MMC indicate N-liposomes also have functional advantages.

Hyaluronic Acid-Modified Bioadhesive Liposomes as Local Drug Depots: Effects of Cellular and Fluid Dynamics on Liposome Retention at Target Sites

Bioadhesive liposomes, in which hyaluronic acid is the surface-anchored bioadhesive ligand, are being testedin vitroin order to evaluate their bioadhesivity. The first part of the test, binding to monolayers of cells modeling thein vivodesignated sites under static conditions, was reported in a previous communication. This communication reports the results of the second and third parts of the test, which consist of evaluating the retention of bound liposomes in the face of tissue-related events such as cell migration, proliferation, and death, and under fluid flow. Thein vivo-designated binding sites for the bioadhesive liposomes were modeled, as before, by monolayers of the A431 cell line served. A setup for perfusing a culture flask containing a monolayer of cells was devised for measuring the retention under fluid flow. The major findings are: (1) Over a selected tested period of 28 h, the cell cultures mimicked the tissue-related events described above, whether they did or did not receive a dose of liposomes. Over the same period and throughout these events, the bioadhesive liposomes remained bound at equilibrium-like levels, in the range of 0.03 ng lipid/105cells. (2) Fluid flowed over a cell monolayer dosed with bioadhesive liposomes swept away part of the dose. This loss was due mostly to that fraction of the dose which was in excess of the binding capacity of the monolayer, and occurred over the first 15 min of flow. Thereafter, the cell monolayer retained the bound liposomes, at equilibrium-like levels and with no further loss, even for the longest flow period tested (45 min of continuous flow) and under a flow rate of 0.64 ml/min. This study, together with previous results, shows that the hyaluronic acid-modified liposomes meet all parts of the bioadhesivity test. We therefore find merit in their further investigation.

Liposome-mediated drug targeting in topical and regional therapies.

Liposome-mediated drug targeting is reviewed in four major categories of topical and regional therapies: wounds and burns, ocular, intraperitoneal, and pulmonary. A survey of the data in the field is preceded by definitions of carrier-mediated drug targeting, in particular for topical and regional treatments. The ability of liposomes to meet essential requirements for task performance and liposome surface-modification as the major approach to endow liposomes with targeting abilities are reviewed. Analysis of current findings in the field shows that (1) most studies explored regular liposomes that were unable to meet the essential requirements for targeting and (2) in vivo drug targeting in topical and regional therapies has been achieved rarely and seldom attempted, yet there are encouraging indications from a few studies that using surface-modified liposomes such targeting is feasible. Both established and novel liposomal systems attest to this feasibility and point out future directions. The former can be found by revisiting immunoliposomes that were initially designed for systemic administration but might well fit topical and regional cases. The latter is exemplified by bioadhesive liposomes, designed specifically for topical/regional therapies. It is concluded that careful implementation of such approaches could be successful for the achievement of liposome-mediated drug targeting in topical and regional therapies.

Bioadhesive, collagen-modified liposomes: molecular and cellular level studies on the kinetics of drug release and on binding to cell monolayers

Liposomes, modified by covalently-anchoring collagen to their surface, were investigated for their abilities to be bioadhesive and to act as sustained-release drug carriers. These bioadhesive liposomes have the potential to induce significant improvements in topical and regional therapies. The major findings for uni- (ULV) and multilamellar (MLV) bioadhesive liposomes are: (a) Both ULV and MLV release small molecular weight drugs over prolonged periods. For example, rate constants of ( 6 ± 0.5) · 10 −3 and ( 2.6 ± 0.8) · 10 −3 h −1, were obtained for the release of vinblastine and fluconazole, respectively, from collagen-ULV. (b) For a given drug, that rate constant can be shifted (up or down) by the choice of liposome type and collagen-surface density and the latter, if high enough, lead to the formation of an additional liposome-associated drug reservoir. (c) Using monolayers of the A431 cell line to model the in vivo targets, the bioadhesive (but not the regular) liposomes were found to bind with high affinity to the monolayers. For example, equilibrium dissociation constants of 6.3(±3) μM and 2.7(±0.5) μM were determined for bioadhesive MLV and ULV, respectively, with corresponding saturation occupancies of 3.7(±1) and 4.0(±0.2) pmoles liposomal collagen/ monolayer of 10 5 cells. (d) Following the retention of bioadhesive MLV at A431 monolayers for 24 h, it was found that: at 4°C, 24 h did not suffice to reach equilibrium, but at 37°C equilibrium binding was obtained within 3–5 h and there was quantitative liposome retention (per viable monolayer) thereafter. It is concluded that these liposomes are bioadhesive sustained-release carriers, as desired, meriting further cellular and in vivo studies.

Bioadhesive liposomes as topical drug delivery systems: molecular and cellular studies

Regular liposomes were modified by the covalent anchoring of bioadhesive ligands to the liposomal surface. The ligands are macromolecules capable of binding to membrane-embedded receptors or to components of the extracellular matrix. These modified liposomes have the potential, as bioadhesive drug delivery systems, for topical and local therapies. The first step in developing such delivery systems is reported here for three types of liposomes with epidermal growth factor (EGF), gelatin or collagen as the surface-anchored ligands. Each of these ligands was crosslinked, using glutaraldehyde, to amine residues on the liposomal surface, the latter supplied by the inclusion of phosphatidylethanolamine in the liposome formulation. The kinetics of drug release from EGF-modified liposomes encapsulating vinblastine and from gelatin-modified liposomes encapsulating progesterone or fluconazole were studied. A single rate constant (0.05 h −1), was found for the diffusion of encapsulated vinblastine from the EGF-modified and from the nonbioadhesive liposomes. Rate constants for the diffusion of encapsulated progesterone or fluconazole were similar from the gelatin-modified and from the nonbioadhesive liposomes. Typical magnitudes obtained are 0.04 and 0.024 h −1, for progesterone and fluconazole, respectively. An additional liposome-associated drug pool was found for the gelatin-modified systems, which may be attributed to drug entrapped within the gelatin layer at the liposomal surface. Typical magnitudes obtained for the rate constants of drug diffusion from this pool are 0.4 and 0.7 h −1, for progesterone and fluconazole, respectively. Binding studies were performed using monolayers of the A431 cell line. The present bioadhesive liposomes showed significant binding whereas nonbioadhesive liposomes did not bind at all. The magnitudes obtained for the dissociation constants ( Kd) are in the range of: 0.5 nM, 800 μg/ml and 400 μg/ml for the bioadhesive liposomes carrying EGF, collagen or gelatin, respectively. The data suggest that these bioadhesive liposomes can meet the requirements essential for site-adherent sustained release drug depots.