Kohei Tahara

Liposomal diclofenac eye drop formulations targeting the retina: Formulation stability improvement using surface modification of liposomes

An efficient liposomal formulation for targeting the retina was produced as an optimal means of distributing therapeutic agents to the retina. Diclofenac was used as a model compound for liposome encapsulation, and the release rate and distribution to the retina were investigated. The calcium acetate gradient method was found to be the optimal method for encapsulating diclofenac into liposomes. Entrapment efficiency using this method was greater than 97%, whereas conventional hydration method achieved 51.3%. The resultant formulation obtained with the gradient method caused aggregation and/or fusion of liposomes. To avoid inhibition of retinal delivery due to the aggregation of the carrier, surface modification was performed simultaneously with the gradient method. The increase in particle size of the liposomal formulation clearly was inhibited for a long time in the presence of polyvinyl alcohol or its derivative. This observation may be explained by surface modification of the liposomes by physisorption or anchoring effect of polymers on the surface of the lipid bilayer. Furthermore, the sustained release profile of the diclofenac formulation was retained after modification. An in vivo animal study revealed that concentration of the accumulated diclofenac in the retina-choroid was enhanced 1.8-fold by surface-modified liposome entrapment compared to that of the unaltered diclofenac solution.

Pulmonary delivery of elcatonin using surface-modified liposomes to improve systemic absorption: Polyvinyl alcohol with a hydrophobic anchor and chitosan oligosaccharide as effective surface modifiers

The aim of this study was to investigate the feasibility of surface-modified liposomes for pulmonary delivery of a peptide. Chitosan oligosaccharide (oligoCS) and polyvinyl alcohol with a hydrophobic anchor (PVA-R) were used as surface modifiers. The effect of liposomal surface modification on the behavior of the liposomes on pulmonary administration and potential toxicity were evaluated in vitro and in vivo. In an association study with A549 cells, PVA-R modification reduced interaction with A549 cells, whereas oligoCS modification electrostatically enhanced cellular interaction. The therapeutic efficacy of elcatonin (eCT) after pulmonary administration to rats was significantly enhanced and prolonged for 48 h after separate administration with oligoCS- or PVA-R-modified liposomes. oligoCS-modified liposomes adhered to lung tissues and caused opening of tight junctions, which enhanced eCT absorption. On the other hand, PVA-R-modified liposomes induced long-term retention of eCT in the lung fluid, leading to sustained absorption. Consequently, surface modification of liposomes with oligoCS or PVA-R has potential for effective peptide drug delivery through pulmonary administration.

Retinal drug delivery using eyedrop preparations of poly-l-lysine-modified liposomes

The purpose of this study was to develop surface-modified liposomes that enhance the efficiency of eye drop drug delivery to the retina. Various molecular weights and concentrations of the water-soluble cationic polymer poly-L-lysine (PLL) were used to modify the surface of submicronized (100 nm) liposomes. Physicochemical properties of surface-modified liposomes were determined in vitro, and the efficiency of drug delivery to the retina was investigated in vivo. Using coumarin-6 as a model drug and fluorescent marker, we show that liposome surface modification by PLL dramatically increased delivery to mouse retina segments after eye drop administration. However, when PLL of high molecular weight (>30,000) was used at higher concentrations (>0.05%), aggregation of surface-modified liposomes increased particle size and hampered distribution to inner ocular tissues. As a result, the efficiency of drug delivery of these aggregated surface-modified liposomes was the same as unmodified liposomes. The optimal molecular weight and concentration of PLL in drug-delivering liposomes were 15,000-30,000 and 0.005%, respectively. Under these conditions, PLL-modified liposomes were not cytotoxic in corneal or conjunctival cells. In conclusion, surface-modified liposomes have great potential as effective retinal drug delivery carriers in eye drop formulations.

Drug delivery to the ocular posterior segment using lipid emulsion via eye drop administration: Effect of emulsion formulations and surface modification

This work explored submicron-sized lipid emulsion as potential carriers for intraocular drug delivery to the posterior segment via eye drops. The effects of physicochemical properties of lipid emulsion on drug delivery were evaluated in vivo using mice. Different formulations of submicron-sized lipid emulsions were prepared using a high pressure homogenization system. Using coumairn-6 as a model drug and fluorescent marker, fluorescence could be observed in the retina after administration of the lipid emulsion. The fluorescence intensity observed after administration of medium chain triglycerides containing the same amount of coumarin-6 was much lower than that observed after administration of lipid emulsions. The inner oil property and phospholipid emulsifier did not affect the drug delivery efficiency to the retina. However, compared with unmodified emulsions, the fluorescence intensity in the retina increased by surface modification using a positive charge inducer and the functional polymers chitosan (CS) and poloxamer 407 (P407). CS-modified lipid emulsions could be electrostatically interacted with the eye surface. By its adhesive property, poloxamer 407, a surface modifier, possibly increased the lipid emulsion retention time on the eye surface. In conclusion, we suggested that surface-modified lipid emulsions could be promising vehicles of hydrophobic drug delivery to the ocular posterior segment.

Surface Modification of Liposomes Using Polymer-Wheat Germ Agglutinin Conjugates to Improve the Absorption of Peptide Drugs by Pulmonary Administration

In this study, we investigated the feasibility of a system based on liposomal surface modification with a novel mucoadhesive polymer-lectin conjugate for the pulmonary delivery of therapeutic peptides and proteins. We covalently attached wheat germ agglutinin (WGA), a ligand that specifically interacts with alveolar epithelial cells, to carbopol (CP), a mucoadhesive polymer, using the carbodiimide method and then evaluated the efficacy and potential toxicity of CP-WGA surface-modified liposomes in vivo and in vitro. In association studies, CP-WGA modification enhanced the interaction with A549 lung epithelial cells compared with unmodified or CP-modified liposomes. This increased association was dependent on temperature and the surface concentration of free WGA. These results suggested synergy of WGA and CP, and retention of the biological cell binding activity of WGA, leading to improved liposome-cell interactions. Moreover, improvement of liposomal bioadhesion to lung epithelia significantly enhanced and prolonged the therapeutic efficacy of calcitonin, a model peptide drug, without any evidence of toxicity, following administration of calcitonin-loaded CP-WGA-modified liposomes. Hence, surface modification of liposomes with CP-WGA has potential for effective pulmonary administration of peptides.

Real-time in vivo imaging of surface-modified liposomes to evaluate their behavior after pulmonary administration

Our previous study demonstrated that surface modification of liposomes using polyvinyl alcohol with a hydrophobic anchor (PVA-R) achieved sustained absorption from the lung after pulmonary administration and prolonged the pharmacological effects of the model peptide drug. In the present study, the behavior of PVA-R-modified liposomes in the lung and whole body was monitored using a real-time in vivo imaging system. Subsequently, the influence of surface modification with PVA-R on liposomal behavior in lung tissue was examined. Indocyanine green (ICG) was used as a near-infrared label of PVA-R-modified liposomes and was used to observe their dynamic behavior using non-invasive in vivo imaging (IVIS® imaging system) after pulmonary administration to rats. PVA-R-modified submicron-sized liposomes (ssLips) induced long-term retention in the lung compared with unmodified liposomes. Moreover, liposome association with alveolar macrophages (NR8383) was decreased by PVA-R modification in vitro. Therefore, PVA-R modification may prevent rapid elimination of ssLips by macrophages, thereby increasing retention in the lung.

Rapid determination of the encapsulation efficiency of a liposome formulation using column-switching HPLC

The feasibility of a rapid automated method for determination of the encapsulation efficiency (EE) of a liposome formulation using a column-switching HPLC system was confirmed by employing several types of liposome formulations containing doxorubicin (DXR). A suspension of DXR liposome was injected directly into an online solid-phase extraction (SPE) system comprising a Diol SPE column and an ODS SPE column connected in series. Free (not encapsulated) DXR was trapped on the Diol SPE column, whereas encapsulated DXR was eluted without interaction. The eluted encapsulated DXR was trapped on the ODS SPE column after being extracted from the inner phase of the liposome by mixing with an organic solvent. Trapped free and encapsulated DXR were eluted sequentially and analyzed separately by gradient HPLC. The time taken by this automated method was only 25min, whereas conventional methods such as ultracentrifugation are time consuming and labor intensive. Validation results and comparison with ultracentrifugation suggested that our method was sufficiently accurate and sensitive to be used to evaluate EE of a liposome formulation without complicated pretreatment.

Nanomedical system for nucleic acid drugs created with the biodegradable nanoparticle platform

Nanomedical applications of biodegradable poly(DL-lactide-co-glycolide) (PLGA) nanoparticles (NPs) developed are discussed in this review. A surface-functionalized PLGA NP platform for drug delivery was established to encapsulate a number of macromolecular drugs such as peptides and nucleic acids as well as low-molecular-weight drugs by the emulsion solvent diffusion method. The interaction of PLGA NPs with cells and tissues could be controlled by changing the surface properties of NPs, suggesting their potential utility for the intracellular drug delivery of nucleic acid-based drugs. Furthermore, orally administered NF-κB decoy oligonucleotide-loaded CS-PLGA NPs are also useful in treating experimental colitis. These approaches using surface-modified PLGA NPs could be able to open new possibilities for nucleic acid-based drug delivery via noninvasive administration method.

Topical Use of Angiopoietin-like Protein 2 RNAi-loaded Lipid Nanoparticles Suppresses Corneal Neovascularization

Corneal neovascularization (CNV) is a sight-threatening condition that is encountered in various inflammatory settings including chemical injury. We recently confirmed that angiopoietin-like protein 2 (ANGPTL2) is a potent angiogenic and proinflammatory factor in the cornea, and we have produced a single-stranded proline-modified short hairpin anti-ANGPTL2 RNA interference molecule that is carried in a lipid nanoparticle (ANGPTL2 Li-pshRNA) for topical application. In this study, we have further examined the topical delivery and anti-ANGPTL2 activity of this molecule and have found that fluorescence-labeled ANGPTL2 Li-pshRNA eye drops can penetrate all layers of the cornea and that ANGPTL2 mRNA expression was dramatically inhibited in both epithelium and stroma at 12 and 24 hours after administration. We also examined the inhibitory effect of ANGPTL2 Li-pshRNA on CNV in a mouse chemical injury model and found that the area of angiogenesis was significantly decreased in corneas treated with ANGPTL2 Li-pshRNA eye drops compared to controls. Together, these findings indicate that this modified RNA interference agent is clinically viable in a topical formulation for use against CNV.

Characterization of insulin-loaded liposome using column-switching HPLC.

We evaluated the drug-encapsulation state of insulin (INS)-loaded liposome using a novel column-switching HPLC system that can automatically separate unloaded drug from encapsulated drug by hydrophobic interaction. When the INS-loaded liposome was dispersed in water (pH 7.4), the encapsulation efficiency (EE) obtained by the column-switching HPLC system was consistent with that obtained by a conventional ultracentrifugation method. However, the INS-loaded liposome dispersed in 0.1% acetic acid (pH 3.3) showed disagreement between the EEs obtained by both methods. Considering the results of particle size, zeta potential, and transmission electron microscope (TEM) observations, we hypothesized that the column-switching HPLC method was able to distinguish INS adsorbed onto the liposome surface from the encapsulated INS, although an ultracentrifugation method precipitated the adsorbed INS onto the liposome surface along with the encapsulated INS. Therefore, the novel column-switching HPLC system may be a more accurate and useful technique for characterization and optimization of the INS-loaded liposome formulation.