CSF1R inhibitor PLX3397 depletes microglia in Mongolian gerbil Meriones unguiculatus, but not in syrian hamster Mesocricetus auratus.

Microglia are the residential immune cells in the central nervous system. Their roles as innate immune cells and regulators of synaptic remodeling are critical to the development and the maintenance of the brain. Numerous studies have depleted microglia to elucidate their involvement in healthy and pathological conditions. PLX3397, a blocker of colony stimulating factor 1 receptor (CSF1R), is widely used to deplete mouse microglia due to its non-invasiveness and convenience. Recently, other small rodents, including Syrian hamsters (Mesocricetus auratus) and Mongolian gerbils (Meriones unguiculatus), have been recognized as valuable animal models for studying brain functions and diseases. However, whether microglia depletion via PLX3397 is feasible in these species remains unclear. Here, we administered PLX3397 orally via food pellets to hamsters and gerbils. PLX3397 successfully depleted gerbil microglia but had no effect on microglial density in hamsters. Comparative analysis of the CSF1R amino acid sequence in different species hints that amino acid substitutions in the juxtamembrane domain may potentially contribute to the inefficacy of PLX3397 in hamsters.

Methyl vinyl ketone impairs spatial memory and activates hippocampal glial cells in mice.

Memory is a fundamental brain function that can be affected by a variety of external factors including environmental pollutants. One of these pollutants is methyl vinyl ketone (MVK), a hazardous substance found in cigarettes, industrial wastes, and car exhaust. Humans can be exposed to MVK under many circumstances; however, it is unclear whether MVK affects higher-order brain functions such as memory. Here, we examined the memory performances of mice receiving systemic MVK administration. We found that 1 mg/kg of MVK impaired spatial memory. We also showed that 1 mg/kg MVK activated glial cells and altered glial functions in several subregions of the hippocampus, a brain region involved in learning and memory. These results suggest that MVK induces memory deficits and activates glial cells in hippocampal subregions.

Effect of PEG anchor in PEGylation of folate-modified cationic liposomes with PEG-derivatives on systemic siRNA delivery into the Tumor.

In this study, we prepared small interfering RNA (siRNA)/cationic liposome complexes (lipoplexes) modified with folate (FA)-polyethylene glycol (PEG, MW 2000, 3400 or 5000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) to facilitate their uptake into tumor cells via folate receptor (FR), and with PEG1600-cholesterol (PEG1600-Chol) or PEG2000-chondroitin sulfate conjugate (PEG2000-CS), to enhance their systemic stability. Among the FA-PEG-modified siRNA lipoplexes, 0.5 mol% FA-PEG5000-DSPE-modified lipoplexes with 2.5 mol% PEG2000-CS or PEG1600-Chol (LP-0.5F5/2.5P2-CS and LP-0.5F5/2.5P1.6-CL, respectively) exhibited selective growth inhibition of human nasopharyngeal carcinoma KB cells through transduction with polo-like kinase 1 (PLK1) siRNA. Furthermore, the LP-0.5F5/2.5P2-CS and LP-0.5F5/2.5P1.6-CL lipoplexes exhibited decreased agglutination with erythrocytes through PEGylation, and markedly decreased the accumulation of siRNA in murine lungs after systemic injection. Finally, systemic injection of LP-0.5F5/2.5P2-CS and LP-0.5F5/2.5P1.6-CL lipoplexes resulted in accumulation of siRNA in KB tumor xenografts. These findings suggest that PEGylation of FA-PEG5000-DSPE-modified siRNA lipoplexes with PEG2000-CS or PEG1600-Chol might improve their systemic stability without the loss of selective transfection activity in tumor cells.

[Effect of Storage of Tulobuterol Tapes after Package Opening and Liner Peeling on Their Formulation Properties].

Although tulobuterol tape is provided to patients in an inner package, information regarding the stability of the tape after opening the packaging may be requested by patients. This study was performed to generate underlying data on the storage stability after package opening or liner peeling with package opening. Tulobuterol tapes were stored at 25℃, 60% relative humidity (RH); 40℃, 75%RH; or in a refrigerator (2-4℃, 10-30%RH) for 1 day or 3 days. In a peel adhesive strength test after package opening, storage at 25℃, 60%RH had a low effect on the adhesive strength of the tape. Storage after liner peeling with package opening resulted in variable adhesive strength of the tape. Regarding drug release properties, for storage after package opening, the f2 values of tapes stored in the three different conditions were over 50, except for tapes stored at 25℃, 60%RH for 3 days. For the tapes stored at 25℃, 60%RH or 40℃, 75%RH after liner peeling with package opening, the release rate and the ratio of drug released at 24 h may be decreased because the drug content decreased due to drug sublimation. This study suggested that tulobuterol tapes can be stored after package opening at 25℃, 60%RH for 1 d.

Preparation and evaluation of fast-dissolving films of etilefrine hydrochloride for practical buccal dosing.

Etilefrine hydrochloride (ET) is an important drug in the treatment of hypotension, and parenteral injections and oral tablets are the conventional dosage forms. However, parenteral injections may cause abnormally high plasma levels as well as pain and necrosis, and oral tablets undergo first-pass metabolism. Although fast-dissolving buccal tablets were previously reported, the initial absorption rate was a little slow and the plasma levels were varied extensively. Recently, many films have been developed as novel dosage forms. Therefore, in the present study, film dosage forms containing ET were produced using water-soluble polymers and glycerin (GLY) as excipients to obtain a practical buccal dosage form. Films composed of ET, GLY, and sodium alginate (AL) exhibited good physical characteristics and rapid release in vitro (more than 70% at 2 min). The compacted AL film containing 2 mg ET (1 × 1 cm) exhibited rapid absorption (>19 ng/mL at 0.5 h), maintained an effective plasma level (>7 ng/mL) for a long time period (0.5-4 h), and had an adequate plasma concentration-time profile with a smaller standard error (<15.3 ng/mL). These results suggest that the present compacted buccal film is a superior dosage form of ET for practical use.

Targeting potential of alginate-glycyl-prednisolone conjugate nanogel to inflamed joints in rats with adjuvant-induced arthritis.

The efficacy of alginate-glycyl-prednisolone conjugate nanogel (AL-GP-NG) was previously reported to be better than that of prednisolone (PD) alone in arthritic rats. In the present study, novel AL-GP-NG was prepared and its targeting potential was investigated. AL-GP-NG with a PD content of 6.3% (w/w) was obtained and had a slightly larger submicron size and similar zeta potential to that of the previous nanogel. Drug release profiles and pharmacokinetic features were similar to those of the previous nanogel. AL-GP-NG showed prolonged release at weakly acidic and neutral pH and the good systemic retention of total (free + conjugated) PD after an intravenous (i.v.) injection in rats. In animal studies using normal and adjuvant-induced arthritic rats, the distribution of total PD was examined after an i.v. injection. AL-GP-NG achieved a markedly higher drug concentration at inflamed joints than PD alone. Furthermore, ALGP-NG showed specific drug localisation to inflamed joints in arthritic rats, but not in normal rats. Furthermore, specific drug localisation to the joints by AL-GP-NG persisted. Collectively, these results demonstrated the good targeting potential of AL-GP-NG to inflamed joints, suggesting its suitability for the treatment of arthritis.

Preparation and evaluation of conjugate nanogels of glycyl-prednisolone with natural anionic polysaccharides as anti-arthritic delivery systems.

Although prednisolone (PD) is used as an anti-arthritis drug due to its rapid and strong anti-inflammatory potential, its frequent and large dosing often brings about adverse effects. Therefore, targeting therapy has attracted increasing attention to overcome such adverse effects. In the present study, nanogels (NGs) composed of macromolecule-PD conjugates were developed as a novel targeting delivery system, and their anti-inflammatory potential was examined. Conjugates were prepared by carbodiimide coupling between glycyl-prednisolone (GP) and the natural anionic polysaccharides, alginic acid (AL) and hyaluronic acid (HA). NGs were produced by the evaporation of organic solvent from the conjugate solution. The obtained NGs, named AL-GP-NG and HA-GP-NG, respectively, were examined for particle characteristics, in vitro release, pharmacokinetics, and in vivo efficacy. Both NGs were several hundred nanometers in size, had negative zeta potentials, and several % (w/w) drug contents. They released PD gradually at pH 7.4 and 6. They exhibited fairly good retention in the systemic circulation. In the efficacy examination using rats with adjuvant-induced arthritis, both NGs showed the stronger and more prolonged suppression of paw inflammation than PD alone. These suggested that the present NGs should be possibly useful as anti-arthritis targeting therapeutic systems.

Effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro gene­silencing effects and siRNA biodistribution in mice.

Polyethylene glycol (PEG)­modifications (PEGylations) of cationic liposome/small interfering RNA complexes (siRNA lipoplexes) can enhance their systemic stability. The present study determined the effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro gene­silencing effects and siRNA biodistribution after intravenous injection. Three types of dialkyl or trialkyl cationic lipids were used in the current study for the preparation of cationic liposomes. Additionally, various PEGylated siRNA lipoplexes that contained PEG­1,2­distearoyl­sn­-glycero­-3­phosphoethanolamine (DSPE), PEG­1,2­distearoyl­rac­glycero­3­-methylpolyoxyethylene (DSG), PEG­cholesterol (PEG­Chol) and PEG­chondroitin sulfate conjugate (PEG­CS) were prepared. The results revealed that PEGylation of siRNA lipoplexes with PEG­DSPE strongly decreased gene­silencing effects in cells. In contrast, those with PEG­DSG, PEG­Chol and PEG­CS did not largely decrease gene-silencing effects. However, regardless of the PEG­derivative type, PEGylation of siRNA lipoplexes decreased their agglutination with erythrocytes. Furthermore, intravenous injection of PEGylated siRNA lipoplexes markedly decreased the accumulation of siRNA in the lungs, regardless of the type of PEG­derivative. However, non­PEGylated siRNA lipoplexes accumulated mainly in the lungs regardless of the siRNA lipoplex cationic lipid type. The results indicated that PEGylation of siRNA lipoplexes with PEG­DSG, PEG­Chol and PEG­CS may improve systemic stability without losing transfection activity by PEGylation.

Preliminary Investigation of the Usability Characteristics Required for Wound Management Products by Semi-Structural Interview of Medical Staff.

Consideration of drug usability characteristics is important during the design process. Although many wound management products have been developed in recent years, there are few studies on their usability. We investigated the needs and characteristics of wound management products required by medical professionals, so as to consider these in future development projects. Semi-structured interviews were conducted in a group of healthcare professionals. Interview responses were analyzed based on thematic analysis. Four themes common to all facilities were secondary wounds, adaptability of materials, convenience, and physicochemical properties. Economic efficiency of medical care was found to be considered only at the hospital, and quality of life of patients was found to be considered only at the home palliative care clinic. Requirements for wound management products can be affected by participants' roles and their facility settings. However, there were needs common to all fields that all wound management products should aim to incorporate.

Formulation Development of Mucoadhesive Microparticle-Laden Gels for Oral Mucositis: An In Vitro and In Vivo Study.

In order to relieve pain due to oral mucositis, we attempted to develop mucoadhesive microparticles containing indomethacin (IM) and gel preparations with IM microparticles that can be applied to the oral cavity. The mucoadhesive microparticles were prepared with a simple composition consisting of IM and polyvinyl alcohol (PVA). Two kinds of PVA with different block properties were used, and microparticles were prepared by heating-filtration and mixing-drying. From the X-ray powder diffraction patterns, differential scanning calorimetry thermograms, and morphological features of the IM microparticles, IM should exist as polymorphic forms in the microparticles. Rapid drug release properties were observed in the IM microparticles. Increased drug retention was observed in IM microparticles containing PVA, and the IM-NK(50) gel, using a common block character PVA and heating-filtration, showed good long-term drug retention properties. In vivo experiments showing significantly higher drug concentrations in the oral mucosa were observed with IM microparticles prepared by heating-filtration, and the IM-NK(50) gel maintained significantly higher drug concentrations in the oral mucosa. From these results, the IM-NK(50) gel may be useful as a preparation for relieving oral mucositis pain.

Preparation of orally fast-dissolving tablets of etilefrine hydrochloride to achieve efficient absorption.

Etilefrine hydrochloride (ET) is commonly used in the treatment of hypotension in dosage forms of oral tablets and parenteral injections. However, oral tablets only temporarily achieve high plasma levels and have low bioavailability (BA), while intravenous injections may cause pain and necrosis around administration sites. In an attempt to overcome these limitations, the buccal delivery of ET using oral droplets has been investigated. In this study, a buccal tablet as an alternative dosage form was developed for practical use. Buccal tablets were prepared by the direct compression method with sodium alginate (AL) and mannitol (MA) as excipients. Their disintegration and in vitro drug release were rapid (more than 50% being released after 3 min). Furthermore, effective plasma levels (> 5-7 ng/mL) were reached within 0.5 h of buccal administration in rats. The systemic absorption of these tablets was similar to that of buccal droplets. Therefore, the ET buccal tablets developed herein have potential as an alternative dosage form for hypotension therapy.

Development of Morin-Loaded Nanoemulsions Containing Various Polymers; Role of Polymers in Formulation Properties and Bioavailability.

Emulsions for oral delivery are not suitable for sustained drug absorption because such preparations diffuse rapidly in the gastrointestinal (GI) tract after oral administration. In order to generate sustained drug absorption and increase oral bioavailability, various polymers were added to a morin (MO) nanoemulsion to improve retention in the GI tract and alter the surface properties of oil droplets in the nanoemulsion. The influence of these polymers on the formulation properties was investigated. The area under the blood concentration-time curve (AUC) and the mean residence time (MRT) after oral administration of the nanoemulsions were measured, and the influence of the polymers on bioavailability was investigated. Chitosan (Chi) addition MO nanoemulsion (MO-Chi nanoemulsion) showed the highest AUC and MRT. MO-Chi nanoemulsion increased retention in the GI tract because of the relatively higher viscosity and high affinity between mucin and Chi covering the oil droplets. Furthermore, MO-Chi nanoemulsion could maintain the drug in oil droplets by suppression of drug release through the polymer hydration layer, and sustained drug release achieved continuous drug absorption. Nanoemulsions with sodium carboxymethylcellulose and poly-γ-glutamic acid potassium salt showed the next highest AUC and MRT after MO-Chi nanoemulsion. From these results, it was suggested that by increasing the viscosity of the nanoemulsion, there was high affinity between the added polymer and mucin, and sustained drug release was useful for enhancing the bioavailability of the polymer-containing nanoemulsions.

Nanogels of a Succinylated Glycol Chitosan-Succinyl Prednisolone Conjugate: Release Behavior, Gastrointestinal Distribution, and Systemic Absorption.

Recently, the potential of nanoparticles (NPs) in ulcerative colitis (UC) therapy has been increasingly demonstrated. Namely, anionic NPs have been found to be accumulated efficiently to the UC damaged area due to epithelial enhanced permeability and retention (eEPR) effect. Previously, a novel anionic nanogel system (NG(S)) was prepared, and evaluated for the efficacy and toxicity. In the present study, release behaviors and biodistribution were investigated in detail to elucidate the functional mechanisms. Rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ulcerative colitis (UC) were used as biomodels. In vitro release was examined with or without the contents of the cecum or distal colon. Gastrointestinal distribution and plasma concentrations were investigated after the intragastric administration of 10 mg prednisolone (PD) eq./kg. At pH 1.2 and 6.8, release behaviors were slow, but controlled. Overall release was not markedly different irrespective of coexistence of intestinal contents. In in vivo studies, a large amount of PD was distributed in the lower parts of the gastrointestinal tract 6 and 12 h after administration with NG(S). PD accumulated well in the colonic parts, and prolonged release was noted. The systemic absorption of PD with NG(S) was hardly found. NG(S) concentrated the drug in the colon and showed controlled release. These behaviors were considered to lead to the previously reported good results, promotion of effectiveness and suppression of toxic side effects.

Effects of cationic lipids in cationic liposomes and disaccharides in the freeze-drying of siRNA lipoplexes on gene silencing in cells by reverse transfection.

RNA interference is a promising technology to inhibit the production of target proteins, and screening with synthetic small interfering RNA (siRNA) libraries has become a crucial research tool used to study gene function in cells. Reverse (Rev) transfection with freeze-dried siRNA/cationic liposome complexes (siRNA lipoplexes) can simplify and speed up siRNA transfection without the preparation of siRNA lipoplexes just before transfection. In this study, we examined the effects of cationic lipids in cationic liposomes and disaccharides in freeze-drying of siRNA lipoplexes on gene silencing in cells by Rev-transfection. We used three types of cationic cholesterol derivatives and three types of dialkyl or trialkyl cationic lipids for the preparation of cationic liposomes, and we prepared six types of freeze-dried siRNA lipoplexes in the presence of trehalose or sucrose solution in multi-well plates. Increasing concentrations of trehalose or sucrose included during freeze-drying of siRNA lipoplexes resulted in increased gene silencing activity upon Rev-transfection. Strong gene silencing activity was observed regardless of the type of cationic lipid in cationic liposomes when siRNA lipoplexes were freeze-dried with the disaccharides at concentrations of more than 25 mM or 100 mM. In addition, siRNA lipoplexes freeze-dried with 100 mM trehalose or sucrose showed long-term (1 month) stability without apparent loss of gene silencing activity. These findings suggested that Rev-transfection with freeze-dried siRNA lipoplexes may have potential applications in the screening of gene function using siRNA libraries.

Gene delivery into hepatic cells with ternary complexes of plasmid DNA, cationic liposomes and apolipoprotein E-derived peptide.

Cationic liposomes containing a cationic lipid, such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), have often been used for the transduction of plasmid DNA (pDNA) in vivo. However, such liposomes induce gene expression primarily in the lungs after intravenous injection. To improve the delivery of cationic liposomes/pDNA complexes (pDNA lipoplexes) to the liver by intravenous administration, the current study synthesized two apolipoprotein E (ApoE)-derived peptides, dApoE-R9 and ApoE-F-R9, for liver targeting via certain ApoE receptors, including the low-density lipoprotein receptor. Ternary complexes of pDNA, cationic liposomes and ApoE-R9 peptide were also prepared. After in vitro transfection, ternary complexes with DOTAP/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposomes exhibited high transfection activity in HepG2 cells compared with DOTAP/cholesterol (Chol) liposomes. In particular, ternary complexes with dApoE-R9 exhibited high transfection activity in cells compared with ApoE-F-R9. However, in vivo transfection studies revealed that ternary complexes with DOTAP/DOPE liposomes and dApoE-R9 did not increase gene expression in the liver compared with DOTAP/DOPE lipoplexes. In contrast, ternary complexes with DOTAP/Chol liposomes and dApoE-R9 increased gene expression in the liver compared with DOTAP/Chol lipoplexes. The results demonstrated that the in vivo optimal liposomal formulation in ternary complexes with ApoE-R9 peptide for liver delivery were different from those that were in vitro.

Novel Xyloglucan Sheet for the Treatment of Deep Wounds: Preparation, Physicochemical Characteristics, and in Vivo Healing Effects.

In the present study, a novel wound dressing made of xyloglucan (Xyl)-sucrose (Suc) hydrogel was developed for the treatment of deep wounds including pressure ulcers. The dressing was prepared by casing an aqueous solution of Xyl and sugar and then warming, and a hydrogel sheet was obtained. The in vitro characteristics of these sheets, such as their strength, extensibility, water content, adhesion potential, and water absorption, were examined. The strength, Young's modulus, and adhesion strength of the sheets were greater when they had a lower water content. Furthermore, adhesion and gradual water absorbability were similar to those of commercial dressings. These in vitro features suggest that Xyl sheets possess the physicochemical properties required for wound dressings. In the in vivo experiment, a Xyl sheet made from a mixture of 3.0% (w/v) Xyl solution and 33.3% (w/w) Suc, which displayed moderate strength and water content, was selected and compared with gauze, commercial polyurethane film, and Xyl/Suc (1 : 2) hydrogel using a rat deep wound model caused by serious frostbite. Wound healing rates based on reductions in wound areas were the best in the order of the sheet > hydrogel > commercial film > gauze. The sheet resulted in better wound surface states than the other preparations by improving the conditions. Thus, the potential applicability of Xyl sheets to the treatment of deep wounds was demonstrated.

Nanogels of Succinylated Glycol Chitosan-Succinyl Prednisolone Conjugate: Preparation, In Vitro Characteristics and Therapeutic Potential.

A novel anionic nanogel system was prepared using succinylated glycol chitosan-succinyl prednisolone conjugate (S-GCh-SP). The nanogel, named NG(S), was evaluated in vitro and in vivo. S-GCh-SP formed a nanogel via the aggregation of hydrophobic prednisolone (PD) moieties and the introduced succinyl groups contributed to the negative surface charge of the nanogel. The resultant NG(S) had a PD content of 13.7% (w/w), was ca. 400 nm in size and had a ζ-potential of -28 mV. NG(S) released PD very slowly at gastric pH and faster but gradually at small intestinal pH. Although NG(S) was easily taken up by the macrophage-like cell line Raw 264.7, it did not decrease cell viability, suggesting that the toxicity of the nanogel was very low. The in vivo evaluation was performed using rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. NG(S) and PD alone were not very effective at 5 mg PD eq./kg. However, NG(S) at 10 mg PD eq./kg markedly suppressed colonic damage, whereas PD alone did not. Furthermore, thymus atrophy was less with NG(S) than with PD alone. These results demonstrated that NG(S) is very safe, promotes drug effectiveness and has low toxicity. NG(S) has potential as a drug delivery system for the treatment of ulcerative colitis.

Preparation of Chondroitin Sulfate-Glycyl-Prednisolone Conjugate Nanogel and Its Efficacy in Rats with Ulcerative Colitis.

A conjugate between chondroitin sulfate (CS) and glycyl-prednisolone (GP), named CS-GP, was produced by carbodiimide coupling at a high GP/CS ratio. CS-GP was not water-soluble and gave a nanogel (NG) in aqueous solution. Two types of nanogels, NG(I) and NG(II), with prednisolone (PD) contents of 5.5 and 21.1% (w/w), respectively, were obtained. They had particle sizes of approximately 280 and 570 nm, respectively, and showed negative ζ-potentials of approximately -40 mV. The PD release rate was slower in the nanogels than in a solution of CS-GP with a PD content of 1.4% (w/w). The PD release rate was slower in NG(II) than in NG(I), and was elevated at pH 7.4 than at pH 6.8. NG(II) was applied in vivo to rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis, and its therapeutic efficacy and pharmacokinetic features were investigated. The therapeutic efficacy of NG(II) was slightly better than that of PD alone. Drug delivery to the lower intestines was enhanced with NG(II). The CS-GP nanogel has potential as a potent DDS for the treatment of ulcerative colitis.

Development of microparticles coated with poly-γ-glutamic acid to improve oral absorption of a poorly water-soluble drug.

Novel microparticles coated with poly-γ-glutamic acid (PGA) were developed to improve the oral absorption of indomethacin (IM), a poorly water-soluble drug. Microparticles containing γ-IM (IMbulk-PGA) or crystal polymorph α-IM (IMpolymorph-PGA) were prepared. Additionally, microparticles were prepared containing α-IM without PGA (IMpolymorph without PGA). IMbulk-PGA and IMpolymorph-PGA exhibited better drug retention properties on mucin disks. Drug release rates from IMpolymorph-PGA and IMpolymorph without PGA were higher than from IM bulk powder, and drug release from IMbulk-PGA was also improved. Drug release from IMbulk-PGA could be improved with the use of Tween 80. In addition, PGA may influence the ionization of IM or affect specific molecular interactions. After the microparticles were administered orally to mice, IMbulk-PGA and IMpolymorph-PGA increased the plasma drug concentration more rapidly compared with IM bulk powder, but IMpolymorph without PGA did not increase the plasma drug concentration. It was considered that IMbulk-PGA and IMpolymorph-PGA rapidly reached the intestinal membrane through the mucus layer and IM was absorbed quickly. Because IMbulk-PGA and IMpolymorph-PGA showed a rapid increase in plasma drug concentration, IMbulk-PGA and IMpolymorph-PGA could be useful preparations to improve the gastrointestinal absorption of IM. Furthermore, IMbulk-PGA may maintain higher plasma drug concentrations than IMpolymorph-PGA.

Effect of Cationic Lipid Type in Folate-PEG-Modified Cationic Liposomes on Folate Receptor-Mediated siRNA Transfection in Tumor Cells.

In this study, we examined the effect of cationic lipid type in folate (FA)-polyethylene glycol (PEG)-modified cationic liposomes on gene-silencing effects in tumor cells using cationic liposomes/siRNA complexes (siRNA lipoplexes). We used three types of cationic cholesterol derivatives, cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol), N-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide (OH-Chol), and cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol), and we prepared three types of FA-PEG-modified siRNA lipoplexes. The modification of cationic liposomes with 1-2 mol % PEG-lipid abolished the gene-silencing effect in human nasopharyngeal tumor KB cells, which overexpress the FA receptor (FR). In contrast, FA-PEG-modification of cationic liposomes restored gene-silencing activity regardless of the cationic lipid type in cationic liposomes. However, the optimal amount of PEG-lipid and FA-PEG-lipid in cationic liposomes for selective gene silencing and cellular uptake were different among the three types of cationic liposomes. Furthermore, in vitro transfection of polo-like kinase 1 (PLK1) siRNA by FA-PEG-modified liposomes exhibited strong cytotoxicity in KB cells, compared with PEG-modified liposomes; however, in in vivo therapy, intratumoral injection of PEG-modified PLK1 siRNA lipoplexes inhibited tumor growth of KB xenografts, as well as that of FA-PEG-modified PLK1 siRNA lipoplexes. From these results, the optimal formulation of PEG- and FA-PEG-modified liposomes for FR-selective gene silencing might be different between in vitro and in vivo transfection.