Optimization of Drug Loading Procedures and Characterization of Liposomal Formulations of Two Novel Agents Intended for Boron Neutron Capture Therapy (BNCT)

Abstract

The characterization of two liposomal formulations of boronated DNA-interacting agents has been performed. It is shown that the two boronated drugs, WSA-Water Soluble Acridine and WSP-Water Soluble Phenantridine, can be encapsulated within unilamellar sterically stabilized liposomes with high drug-to-lipid ratios (up to 0.50:1 (mol:mol)), using transmembrane pH gradients. The steric stabilization of the liposomes was accomplished by the addition of DSPE-PEG(2000) (PEG-lipid) to DSPC/Cho lipid mixtures and the composition used was DSPC: Cho: DSPE-PEG 55:40:5 (moI%). The loading of the drugs resulted in drug precipitation in the liposomal aqueous core as observed by cryo-transmission electron microscopy (c-TEM). Moreover, it is shown that when pH gradients across the bilayer were used for remote loading of WSP or when ammonium sulfate gradients were used for remote loading of WSA, the formation of small bilayer fragments (discs) was induced. We present compelling evidence that the formation of discs is a consequence of precipitate growth in the liposomal interior. The precipitate growth causes some of the liposomes to rupture resulting in the above mentioned disc-formation and a substantial decrease in trapping efficiency. The in vitro stability of the drug loaded liposomes was excellent, both in buffer and in 25% human serum. For most of the formulations, the release of the drugs was below or around 10% after 24 hours at 37^oC. Furthermore, the influence of initial internal pH and internal buffering capacity on release properties of WSA and WSP were investigated. It is shown that the release profiles of the drugs can be controlled, to a large extent, by varying the composition of the internal liposomal aqueous phase.     

Liposomal Anticancer Drugs as Agents to be used in Combination with other Anticancer Agents: Studies on a Liposomal Formulation with two Encapsulated Drugs

Abstract

When considering the use of combination therapies with liposomal anticancer agents several approaches can be defined. One approach could rely on administration of one liposomal formulation with more than one entrapped cytotoxic drug. This study focuses on an assessment of a liposomal formulation containing vincristine and mitoxantrone. Distearoyl phosphatidylcholine (DSPC)/Cholesterol (Choi) (55:45 molar ratio) liposomes were loaded with vincristine using transmembrane pH gradients. These systems were subsequently incubated with mitoxantrone to effect uptake of the second drug. Retention of both drugs was determined in vitro and in vivo. In vitro drug release indicated >95% retention of mitoxantrone and approximately 75% retention of vincristine when liposomes were prepared with an initial interior pH of 2.0. In vivo results however, demonstrated that greater than 80% of the encapsulated vincristine was released within 1 hour following i.v. administration. The instability of a liposomal formulation containing two anticancer drugs following i.v. administration may be a consequence of a combination of factors including drug-loading induced collapse of the transmembrane pH gradient, loss due to osmotic effects and an associated insertion of serum proteins into the bilayer, as well as the presence of a large biological “sink” which can alter the transbilayer drug gradient in favor of drug release.     

Pulmonary Delivery of Liposomal Drugs

Abstract

This overview will discuss our studies of liposomes aerosols to treat diseases of the lung and will entail (i) formulation and characterization of liposome aerosols, including dry liposome powder aerosols, (ii) modulation of the pharmacokinetic profile of liposomal drugs delivered by aerosol or intratracheal instillation, (iii) liposome-alveolar macrophage interactions in vitro and in vivo, and (iv) safety of liposome aerosols in vivo in mice, sheep and healthy human volunteers. Water-soluble agents can be retained in liposomes during aerosolization with air-pressure nebulizers within certain limitations of liposome composition, size, and operating conditions. Dry powder liposome aerosols have been formulated and deliver water-soluble encapsulated substances efficiently. Pharmacokinetic profiles of liposomal drugs delivered via intratracheal instillation exhibit typical slow release plasma profiles indicating that the carrier is the rate-limiting barrier for release. Accordingly, pulmonary mean residence times are significantly prolonged and systemic concentrations remain low. Liposomes do not inhibit the phagocytic activity of alveolar macrophages in vitro and in vivo, have no apparent histopathologic effects on lung architecture even after chronic administration, and do not alter dynamic compliance, lung resistance, p_aO₂ and p_aCO₂ in awake, unanesthetized sheep and in healthy human volunteers. In conclusion, liposomes are a promising innocuous aerosol delivery system for drugs to achieve prolonged localized drug concentrations in the lung or intracellular drug targeting to alveolar macrophages.     

Effect of cisplatin containing liposomes formulated by unsaturated chain-containing lipids on gynecological tumor cells

Gynecological tumors are major therapeutic areas of platinum-based anticancer drugs. Here, we report the characterization and in vitro biological assays of cisplatin-containing Egg L-α-phosphatidylcholine liposomes with different amounts of cholesterol. Dynamic light scattering estimated sizes of all obtained liposomes in the 100?nm range that are suitable for in vivo use. On the basis of these data and of the drug loading values, the best formulation has been selected. Stability and drug release properties of platinum-containing liposomes have been verified in serum. The growth inhibitory effects of both liposomal and free drug in a panel of ovarian and breast human cancer cell lines, characterized by a different drug sensitivity, give comparable or better results with respect to free cisplatin drug.     

Ciprofloxacin as Ocular Liposomal Hydrogel

The purpose of this study was to prepare and characterize an ocular effective prolonged-release liposomal hydrogel formulation containing ciprofloxacin. Reverse-phase evaporation was used for preparation of liposomes consisting of soybean phosphatidylcholine (PC) and cholesterol (CH). The effect of PC/CH molar ratio on the percentage drug encapsulation was investigated. The effect of additives such as stearylamine (SA) or dicetyl phosphate (DP) as positive and negative charge inducers, respectively, were studied. Morphology, mean size, encapsulation efficiency, and in vitro release of ciprofloxacin from liposomes were evaluated. For hydrogel preparation, Carbopol 940 was applied. In vitro transcorneal permeation through excised albino rabbit cornea was also determined. Optimal encapsulation efficiency of 73.04 ± 3.06% was obtained from liposomes formulated with PC/CH at molar ratio of 5:3 and by increasing CH content above this limit, the encapsulation decreased. Positively charged liposomes showed superior entrapment efficiency (82.01 ± 0.52) over the negatively charged and the neutral liposomes. Hydrogel containing liposomes with lipid content PC, CH, and SA in molar ratio 5:3:1, respectively, showed the best release and transcorneal permeation with the percentage permeation of 30.6%. These results suggest that the degree of encapsulation of ciprofloxacin into liposomes and prolonged in vitro release depend on composition of the vesicles. In addition, the polymer hydrogel used in preparation ensure steady and prolonged transcorneal permeation. In conclusion, ciprofloxacin liposomal hydrogel is a suitable delivery system for improving the ocular bioavailability of ciprofloxacin.     

Hyaluronic acid modified pH-sensitive liposomes for targeted intracellular delivery of doxorubicin

Context: Surface-modified pH-sensitive liposomal system may be useful for intracellular delivery of chemotherapeutics.

Objective: Achieving site-specific targeting with over-expressed hyaluronic acid (HA) receptors along with using pH sensitive liposome carrier for intracellular drug delivery was the aim of this study.

Materials and methods: Stealth HA-targeted pH-sensitive liposomes (SL-pH-HA) were developed and evaluated to achieve effective intracellular delivery of doxorubicin (DOX) vis–a-vis enhanced antitumor activity.

Results: The in vitro release studies demonstrated that the release of DOX from SL-pH-HA was pH-dependent, i.e. faster at mildly acidic pH ～5, compared to physiological pH ～7.4. SLpH-HA was evaluated for their cytotoxicity potential on CD44 receptor expressing MCF-7 cells. The half maximal inhibitory concentration (IC50) of SL-pH-HA and SL-HA were about 1.9 and 2.5?μM, respectively, after 48?h of incubation. The quantitative uptake study revealed higher localization of targeted liposomes in the receptor positive cells, which was further confirmed by fluorescent microscopy. The antitumor efficacy of the DOX-loaded HA-targeted pH-sensitive liposomes was also verified in a tumor xenograft mouse model.

Discussion: DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 receptor interaction. The major side-effect of conventional DOX formulation, i.e. cardiotoxicity was also estimated by measuring serum enzyme levels of LDH and CPK and found to be minimized with developed formulation. Overall, HA targeted pH-sensitive liposomes were significantly more potent than the non-targeted liposomes in cells expressing high levels of CD44.

Conclusion: Results strongly implies the promise of such liposomal system as an intracellular drug delivery carrier developed for potential anticancer treatment.     

Ultrasound-enhanced bevacizumab release from echogenic liposomes for inhibition of atheroma progression

Context: Bevacizumab (BEV) is a monoclonal antibody to vascular endothelial growth factor (VEGF) that ameliorates atheroma progression by inhibiting neovascularization.

Objective: We aimed to determine whether BEV release from echogenic liposomes (BEV-ELIP) could be enhanced by color Doppler ultrasound (US) and whether the released BEV inhibits VEGF expression by endothelial cells in vitro.

Materials and methods: BEV-ELIP samples were subjected to 6?MHz color Doppler ultrasound (MI?=?0.4) for 5?min. We assessed release of BEV with a direct ELISA and with fluoresceinated BEV (FITC-BEV) loaded into ELIP by the same method. Human umbilical vein endothelial cell (HUVEC) cultures were stimulated to express VEGF by 10?nM phorbol-12-myristate 13-acetate (PMA). Cell-associated VEGF levels were determined using a cell-based ELISA.

Results: Overall, US caused an additional 100?µg of BEV to be released or exposed per BEV-ELIP aliquot within 60?min BEV-ELIP treated with US inhibited VEGF expression by 90% relative to non-treated controls and by 70% relative to BEV-ELIP without US. Also, US-treated BEV-ELIP inhibited HUVEC proliferation by 64% relative to untreated controls and by 45% relative to BEV-ELIP without US.

Discussion and conclusion: We have demonstrated that BEV-ELIP retains its VEGF-binding activity in a liposomal formulation and that clinical Doppler US can significantly increase that activity, both by releasing free BEV and by enhancing the surface exposure of the immunoreactive antibody.     

Efficacy of Gel Formulations Containing free and Liposomal Foscarnet in a Murine Model of Cutaneous HSV-1 Infection

Abstract

The efficacy of gel formulations containing free and liposomal foscarnet has been evaluated in a murine model of cutaneous Herpes simplex virus type-1 infection. Both formulations were applied topically 3 times daily for 4 days and initiated 24 h post-infection. The penetration of liposomes incorporated into the gel in infected skin tissues was better than that of liposomes dispersed in buffer. Therein, their localization mostly matched that of viral antigen detected by immunoperoxydase staining. Despite these facts, the efficacy of gel formulations of both free and liposomal foscarnet in preventing the development of a zosteriform rash in mice was similar. Electron microscopic examination revealed that liposomes incorporated into the gel formed aggregates together with the micelles of gel. Diffusion studies showed that liposomes were trapped within these aggregates and were hardly able to diffuse across a polycarbonate membrane. In addition, although the liposomes were shown to be highly stable in vitro, the formation of these aggregates destabilized their membrane resulting in a premature release of foscarnet from liposomes. The efficacy of both gel formulations was higher than that of solutions of free or liposomal foscarnet suggesting that the gel formulation is a suitable matrix for the delivery of drugs. Thus, strategies aimed at reducing the interaction of liposomes with the gel could be a convenient approach to improve the efficacy of liposome-encapsulated drug over the free drug.     

Incorporation of dimethoxycurcumin into charged liposomes and the formation kinetics of fractal aggregates of uncharged vectors

Abstract

Dimethoxycurcumin (DMC) is a lipophilic analog of curcumin found in Curcuma longa Linn., which is known to possess significant activity against various cancer cell lines. The purpose of this study was to develop suitable liposomal formulations in order to overcome DMC’s poor water solubility and to study the aggregation kinetic profile using the fractal analysis. DMC was incorporated into liposomal formulations composed of DPPC, DPPC:DPPG:chol (9:1:1 molar ratio) and DPPC:DODAP:chol (9:1:1 molar ratio) liposomes. Light scattering techniques were used to elucidate the physicochemical parameters of the liposomal formulations with and without DMC. The structural characteristics of the incorporated molecule were found to be crucial and promote the aggregation mechanism depending also on the liposomes’ composition. The results of our study contribute to the overall scientific efforts to prepare efficient carriers for DMC and could be a useful tool in order to study more efficiently the kinetics of the aggregation process of the liposomal carriers.     

Design and characterization of multifaceted lyophilized liposomal wafers with promising wound healing potential

Various dressings are available to heal chronic wounds which many times fail to achieve the expected results. To overcome some of their drawbacks, formulation of a novel dressing; lyophilized liposomal wafers having better wound healing potential has been proposed in the present study. The drug incorporated in the formulation is gatifloxacin (GTX) which is a fourth-generation fluoroquinolone antibiotic having in vitro activity against both Gram-negative and Gram-positive bacteria. The formulation was designed in three stages where at first liposomes were prepared, the liposomes were converted to gel using chitosan and lastly this gel was lyophilized to form liposomal wafers. Liposomes were prepared by varying the concentration of lipid and cholesterol and evaluated for particle size, entrapment efficiency, in vitro cumulative release, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Liposomes were converted to liposomal gel using chitosan and evaluated for texture, clarity, viscosity, spreadibility and in vitro drug release. Finally, this liposomal batch was subjected to lyophilization to convert it to liposomal wafers and subjected to SEM, differential scanning calorimetric, X-ray diffraction and drug release studies. The in vivo studies were carried out on Wistar rats where wound healing potential of the wafers was confirmed by histopathological evaluation.     

Novel vaginal drug delivery system: deformable propylene glycol liposomes-in-hydrogel

Deformable propylene glycol-containing liposomes (DPGLs) incorporating metronidazole or clotrimazole were prepared and evaluated as an efficient drug delivery system to improve the treatment of vaginal microbial infections. The liposome formulations were optimized based on sufficient trapping efficiencies for both drugs and membrane elasticity as a prerequisite for successful permeability and therapy. An appropriate viscosity for vaginal administration was achieved by incorporating the liposomes into Carbopol hydrogel. DPGLs were able to penetrate through the hydrogel network more rapidly than conventional liposomes. In vitro studies of drug release from the liposomal hydrogel under conditions simulating human treatment confirmed sustained and diffusion-based drug release. Characterization of the rheological and textural properties of the DPGL-containing liposomal hydrogels demonstrated that the incorporation of DPGLs alone had no significant influence on mechanical properties of hydrogels compared to controls. These results support the great potential of DPGL-in-hydrogel as an efficient delivery system for the controlled and sustained release of antimicrobial drugs in the vagina.     

Enhanced Neutrophil Stimulation by Phorbol Ester Encapsulated in pH-Sensitive Liposomes

Abstract

Phorbol 12-myristate 13-acetate (PMA) and arachidonic acid (AA) are both hydrophobic stimulators for superoxide release by guinea pig neutrophils. However AA incorporated into liposomes is no longer an effective stimulator. In contrast, PMA incorporated into liposomes is more effective in neutrophil stimulation than free PMA. the ED₅₀ of superoxide release was 3.1 × 10^?8M, and 4.0 × 10^?10 M for free PMA and liposomes composed of egg phosphatidylethanolamine (PE) /AA/ PMA (molar ratio 7:2:1), respectively. PMA incorporated into PE/AA liposomes could also shorten the lag period of superoxide release in a concentration-dependent fashion. the enhanced stimulation activity of PMA in liposomes was correlated with the enhanced liposome uptake by neutrophils, probably via phagocytosis. Weak bases and a proton ionophore inhibited superoxide release by cells stimulated with either free or liposomal PMA. these results suggested that free PMA attached to cell membranes might be endocytosed and stimulate the superoxide-generating systems via an endocytic compartment(s). Since liposomes effectively deliver the contents into the compartments, liposomal PMA may thus be a potent stimulator for neutrophils. This hypothesis is further supported by the observation that pH-sensitive liposomes, which are active in the acidic endocytic compartments, are more effective carriers for PMA than the conventional pH-insensitive liposomes.     

Lipophilic prodrug conjugates allow facile and rapid synthesis of high-loading capacity liposomes without the need for post-assembly purification

Abstract

Dihydropyridopyrazoles are simplified synthetic analogues of podophyllotoxin that can effectively mimic its molecular scaffold and act as potent mitotic spindle poisons in dividing cancer cells. However, despite nanomolar potencies and ease of synthetic preparation, further clinical development of these promising anticancer agents is hampered due to their poor aqueous solubility. In this article, we developed a prodrug strategy that enables incorporation of dihydropyridopyrazoles into liposome bilayers to overcome the solubility issues. The active drug was covalently connected to either myristic or palmitic acid anchor via carboxylesterase hydrolyzable linkage. The resulting prodrugs were self-assembled into liposome bilayers from hydrated lipid films using ultrasound without the need for post-assembly purification. The average particle size of the prodrug-loaded liposomes was about 90?nm. The prodrug incorporation was verified by differential scanning calorimetry, spectrophotometry and gel filtration reaching maximum at 0.3 and 0.35 prodrug/lipid molar ratios for myristic and palmitic conjugates, respectively. However, the ratio of 0.2 was used in the particle size and biological activity experiments to maintain long-term stability of the prodrug-loaded liposomes against phase separation during storage. Antiproliferative activity was tested against HeLa and Jurkat cancer cell lines in vitro showing that the liposomal prodrug retained antitubulin activity of the parent drug and induced apoptosis-mediated cancer cell death. Overall, the established data provide a powerful platform for further clinical development of dihydropyridopyrazoles using liposomes as the drug delivery system.     

Liposomes as an ocular delivery system for acetazolamide: In vitro and in vivo studies

The purpose of this study was to formulate topically effective controlled release ophthalmic acetazolamide liposomal formulations. Reverse-phase evaporation and lipid film hydration methods were used for the preparation of reversephase evaporation (REVs) and multilamellar (MLVs) acetazolamide liposomes consisting of egg phosphatidylcholine (PC) and cholesterol (CH) in the molar ratios of (7∶2), (7∶4), (7∶6), and (7∶7) with or without stearylamine (SA) or dicetyl phosphate (DP) as positive and negative charge inducers, respectively. The prepared liposomes were evaluated for their entrapment efficiency and in vitro release. Multilamellar liposomes entrapped greater amounts of drug than REVs liposomes. Drug loading was increased by increasing CH content as well as by inclusion of SA. Drug release rate showed an order of negatively charged > neutral > positively charged liposomes, which is the reverse of the data of drug loading efficiency. Physical stability study indicated that approximately 89%, 77%, and 69% of acetazolamide was retained in positive, negative, and neutral MLVs liposomal formulations up to a period of 3 months at 4°C. The intraocular pressure (IOP)-lowering activity of selected acetazolamide liposomal formulations was determined and compared with that of plain liposomes and acetazolamide solution. Multilamellar acetazolamide liposomes revealed more prolonged effect than REVs liposomes. The positively charged and neutral liposomes exhibited greater lowering in IOP and a more prolonged effect than the negatively charged ones. The positive multilamellar liposomes composed of PC:CH:SA (7:4:1) molar ratio showed the maximal response, which reached a value of −7.8±1.04 mmHg after 3 hours of topical administration. Published: January 5, 2007     

Speaker Abstracts

Animal Experiments—An Essential Component for the Development of Liposomal Anticancer Agents

During several years, in our institute more than a dozen of established or novel anticancer compounds have been encapsulated in liposomes and their pharmacological behavior has been tested in in vitro and in vivo experimental models.

It was revealed, that for each substance a tailored liposomal system had to be developed. Animal experiments designed to determine both the antitumor activity and side effects of liposomal in comparison to the free drugs have shown that in the majority of cases a benefit for the vesicular formulation could be obtained. In 7/12 liposomal compounds tested (Bleomycin, Daunorubicin, Cisplatin, Carboplatin, Cyclophosphamide, CCNU, Alkylphospholipids) a substantial decrease of toxicity, mainly due to changed pharmacokinetic data could be observed. The therapeutic efficacy could be increased by use of liposomes for Bleomycin, Taxol, and Mitoxantrone while in other examples no change (Daunorubicin, Methotrexate, TNF) or even a decrease of activity (Cisplatin, Cyclophosphamide, CCNU) was registered.

Carboplatin is one example in which by liposomal encapsulation the pharmcological properties were decisively changed. While the free drug leads to leuko- and thrombopenia, the Carboplatin-liposomes (CPL) revealed after only one i.p. or i.v. injection into mice a substantial and long-standing leukocytosis. That effect was paralleled by a release of cytokines from macrophages into the serum, an increased number of peripheral blood stem cells and colony forming activity. The anticancer activity of carboplatin was remarkably improved especially in breast cancer xenografts by using the liposomal formulation. We hypothesise that CPL of specific size and constitution are efficiently taken up by macrophages/monocytes. That leads to the induction of growth factors inducing secondarily a stimulation of haematopoiesis.

Another example is the encapsulation of Tamoxifen (Tam), an antiestrogen used mainly as first line therapy in estrogen receptor positive breast cancer. Tamoxifen-containing LUVETs prepared from egg phosphocholine, dicetylphosphate and an alkylphospholipid (OPP) had a higher in vitro cytotoxicity both in Tam-sensitive and –resistant breast cancer lines. In vivo testing in xenografts with inherited or acquired Tam-resistance showed that in 2/4 models resistance could be overcome by an oral treatment with appropriate liposomes.

These both examples impressively document that only by inclusion of a consequent in vivo testing procedure the surprising pharmacological effects of liposomal anticancer agents can be revealed.     

Topical delivery and in vivo antileishmanial activity of paromomycin-loaded liposomes for treatment of cutaneous leishmaniasis

The present study aimed to evaluate the potential of liposomes loaded with paromomycin (PA), an aminoglycoside antibiotic associated with poor skin penetration, for the topical treatment of cutaneous leishmaniasis (CL). Fluid liposomes were prepared and characterized for particle size, zeta potential, and drug entrapment. Permeation studies were performed with two in vitro models: intact and stripped skin. The antileishmanial activity of free and liposomal PA was evaluated in BALB/c mice infected by Leishmania (L.) major. Drug entrapment ranged from 10 to 14%, and the type of vesicle had little influence on this parameter. Particle size and polydispersity index of the vesicles composed by phosphatidylcholine (PC) and PC/cholesterol (Chol) ranged from of 516 to 362?nm and 0.7 to 0.4, respectively. PA permeation across intact skin was low, regardless of the formulation tested, while drug penetration into skin (percent of the applied dose) from PC (7.2?±?0.2%) and PC/Chol (4.8?±?0.2%) liposomes was higher than solution (1.9?±?0.1%). PA-loaded liposomes enhanced in vitro drug permeation across stripped skin and improved the in vivo antileishmanial activity in experimentally infected mice. Our findings suggest that the liposomes represent a promising alternative for the topical treatment of CL using PA.     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

Preparation and Evaluation of Thermosensitive Liposomal Hydrogel for Enhanced Transcorneal Permeation of Ofloxacin

Ofloxacin, available as ophthalmic solution, has two major problems: first, it needs frequent administration every 4 hours or even every 1 hour to treat severe eye infection; second, there is formation of white crystalline deposit on cornea due to its pH-dependent solubility, which is very low at pH of corneal fluid. In order to provide a solution to previous problems, ofloxacin in this study is prepared as topically effective in situ thermosensitive prolonged release liposomal hydrogel. Two preparation procedures were carried out, leading to the formation of multilamellar vesicles (MLVs) and reverse-phase evaporation vesicles (REVs) at pH 7.4. Effects of method of preparation, lipid content, and charge inducers on encapsulation efficiency were studied. For the preparation of in situ thermosensitive hydrogel, chitosan/β-glycerophosphate system was synthesized and used as carrier for ofloxacin liposomes. The effect of addition of liposomes on gelation temperature, gelation time, and rheological behaviors of the hydrogel were evaluated. In vitro transcorneal permeation was also determined. MLVs entrapped greater amount of ofloxacin than REVs liposomes at pH 7.4; drug loading was increased by including charge-inducing agent and by increasing cholesterol content until a certain limit. The gelation time was decreased by the addition of liposomes into the hydrogel. The prepared liposomal hydrogel enhances the transcorneal permeation sevenfold more than the aqueous solution. These results suggested that the in situ thermosensitive ofloxacin liposomal hydrogel ensures steady and prolonged transcorneal permeation, which improves the ocular bioavailability, minimizes the need for frequent administration, and decreases the ocular side effect of ofloxacin.     

Rational design of amphiphile-based drug carriers and sterically stabilized carriers

Abstract

This paper describes the parameters recommended for rational design of amphiphile-based drug carriers. The main advantage of a carrier is its ability to modify the pharmacokinetics and biodistribution of the drug, so that the drug level at the target is sufficient for therapeutic benefits. Three parameters are described. Two of them, the drug-to-carrier partition coefficient (Ky_iC) and the rate of drug release from the carrier (k_ff), are related to drug-carrier interactions; the third one is the rate of carrier clearance (k_c). We demonstrate that carrier performance for drugs associated with the carrier amphiphile(s) is determined to a large extent by K_c, while for drugs encapsulated in the aqueous phase of the carrier it is important that k_off will be similar to k_c These conclusions are based on two examples: (i) Amphotericin B as a drug associated with five dosage forms which represent different types of amphiphile-based carriers: micelles (Fungizone), stable micelle-like disks (Amphocil), a complex with phospholipids (ABPLC), liposomes (AmBisome), and a submicronized emulsion, (ii) Liposomal doxorubicin which consisted of either doxorubicin associated with the membrane of negatively-charged, fluid oligolamellar liposomes (L-DOX) or doxorubicin loaded by an ammonium sulfate gradient into small, unilamellar, rigid liposomes having steric stabilizing lipid grafted in their lipid bilayer, (S-DOX). To better understand what contributes to k, we also describe the effect of bilayer acyl chain composition and the role of precipitation of the drug inside the liposomes.     

Caprylate-Conjugated Cisplatin for the Development of Novel Liposomal Formulation

Cisplatin, first (platinum) compound to be evolved as an anticancer agent, has found its important place in cancer chemotherapy. However, the dose-dependent toxicities of cisplatin, namely nephrotoxicity, ototoxicity, peripheral neuropathy, and gastrointestinal toxicity hinder its widespread use. Liposomes can reduce the toxicity of cisplatin and provide a better therapeutic action, but the low lipid solubility of cisplatin hinders its high entrapment in such lipid carrier. In the present investigation, positively charged reactive aquated species of cisplatin were complexed with negatively charged caprylate ligands, resulting in enhanced interaction of cisplatin with lipid bilayer of liposomes and increase in its encapsulation in liposomal carrier. Prepared cisplatin liposomes were found to have a vesicular size of 107.9 ± 6.2 nm and zeta potential of −3.99 ± 3.45 mV. The optimized liposomal formulation had an encapsulation efficiency of 96.03 ± 1.24% with unprecedented drug loading (0.21 mg cisplatin / mg of lipids). The in vitro release studies exhibited a pH-dependent release of cisplatin from liposomes with highest release (67.55 ± 3.65%) at pH 5.5 indicating that a maximum release would occur inside cancer cells at endolysosomal pH. The prepared liposomes were found to be stable in the serum and showed a low hemolytic potential. In vitro cytotoxicity of cisplatin liposomes on A549 lung cancer cell line was comparable to that of cisplatin solution. The developed formulation also had a significantly higher median lethal dose (LD₅₀) of 23.79 mg/kg than that of the cisplatin solution (12 mg/kg). A promising liposomal formulation of cisplatin has been proposed that can overcome the disadvantages associated with conventional cisplatin therapy and provide a higher safety profile.Key Words: cisplatin, complexation, cytotoxicity, LD₅₀, liposome