Novel antiangiogenic therapy targeting biglycan using tumor endothelial cell-specific liposomal siRNA delivery system.

Tumor blood vessels play important roles in tumor progression and metastasis. Targeting tumor endothelial cells (TECs) is one of the strategies for cancer therapy. We previously reported that biglycan, a small leucine-rich proteoglycan, is highly expressed in TECs. TECs utilize biglycan in an autocrine manner for migration and angiogenesis. Furthermore, TEC-derived biglycan stimulates tumor cell migration in a paracrine manner leading to tumor cell intravasation and metastasis. In this study, we explored the therapeutic effect of biglycan inhibition in the TECs of renal cell carcinoma using an in vivo siRNA delivery system known as a multifunctional envelope-type nanodevice (MEND), which contains a unique pH-sensitive cationic lipid. To specifically deliver MEND into TECs, we incorporated cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) into MEND because αV ß3 integrin, a receptor for cRGD, is selective and highly expressed in TECs. We developed RGD-MEND-encapsulating siRNA against biglycan. First, we confirmed that MEND was delivered into OS-RC-2 tumor-derived TECs and induced in vitro RNAi-mediated gene silencing. MEND was then injected intravenously into OS-RC-2 tumor-bearing mice. Flow cytometry analysis demonstrated that MEND was specifically delivered into TECs. Quantitative RT-PCR indicated that biglycan was knocked down by biglycan siRNA-containing MEND. Finally, we analyzed the therapeutic effect of biglycan silencing by MEND in TECs. Tumor growth was inhibited by biglycan siRNA-containing MEND. Tumor microenvironmental factors such as fibrosis were also normalized using biglycan inhibition in TECs. Biglycan in TECs can be a novel target for cancer treatment.

Simulation of Stimuli-Responsive and Stoichiometrically Controlled Release Rate of Doxorubicin from Liposomes in Tumor Interstitial Fluid.

PURPOSE: To simulate the stimuli-responsive and stoichiometrically controlled doxorubicin (DOX) release from liposomes in in vivo tumor interstitial fluid (TIF), the effect of ammonia concentration and pH on the DOX release from liposomes in human plasma at 37°C was quantitatively evaluated in vitro and the release rate was calculated as a function of ammonia concentration and pH. METHODS: Human plasma samples spiked with DOX-loaded PEGylated liposomes (PLD) or Doxil®, containing ammonia (0.3-50 mM) at different pH values, were incubated at 37°C for 24 h. After incubation, the concentration of encapsulated DOX in the samples was determined by validated solid-phase extraction (SPE)-SPE-high performance liquid chromatography. RESULTS: Accelerated DOX release (%) from liposomes was observed as the increase of ammonia concentration and pH of the matrix, and the decrease of encapsulated DOX concentration. The release rate was expressed as a function of the ammonia concentration and pH by using Henderson-Hasselbalch equation. CONCLUSIONS: The DOX release from PLD in TIF was expressed as a function ammonia concentration and pH at various DOX concentrations. Further, it was found that the DOX release from liposomes in a simulated TIF was more than 15 times higher than in normal plasma.

Development of liposomal anticancer drugs.

Liposomes are drug delivery systems that can alter the pharmacokinetic properties of compounds. The adverse effects of anticancer agents are a limiting factor for cancer chemotherapy, therefore, liposomal formulations have the potential to improve the therapeutic efficacy of anticancer agents by enhancing their accumulation in tumors and reducing non-selective distribution to normal tissues, which is known as the enhanced permeability and retention effect. To develop a liposomal anticancer agent as a drug product, its formulation must be designed to ensure its quality until it is administered to patients and to exert maximum potency in clinical use rather than in animal experiments. The chemical stability and physicochemical stability of the ingredients are key factors in the design of liposomal formulations. Drug release rates are critical factors in the therapeutic efficacy of liposomal drug products because the encapsulated drug has no pharmacological activity, and only released drug can exert antitumor/toxic activities. Liposomes should maintain the drug in a stable state in the circulation and then promptly release it after accumulation in the target tissue in order to achieve a sufficient drug concentration. To understand the profile of the formulation and to guarantee the quality of drug product, a reliable analytical method that can determine the released and encapsulated drugs in biological fluids is required. Simple online solid phase extractions of the released and encapsulated drugs using a column-switching HPLC system meet the requirements and this system enables accurate in vitro release testing and in vivo pharmacokinetic evaluation. This review introduces the process of liposomal drug product development from various viewpoints.

Effect of Doxorubicin Release Rate From Polyethylene Glycol-Modified Liposome on Anti-tumor Activity in B16-BL6 Tumor-Bearing Mice.

To investigate the effect of doxorubicin (DOX) release rates from polyethylene glycol (PEG)-liposomes on the anti-tumor activity, several in-vitro and in-vivo studies were performed by utilizing three types of DOX-PEG-liposomes showing the slow (L-Slow), middle (L-Mid) and fast (L-Fast) release rates of DOX. L-Mid provided the highest anti-tumor activity in B16-BL6 tumor-bearing mice, although the largest amount of DOX distribution into the tumor tissue was observed in L-Slow-administered mice and the lowest was in L-Fast-administered mice. To elucidate the reason for this discrepancy, DOX distribution into cancer cells constituting the tumor tissue was determined and the highest DOX distribution into cancer cells was observed in L-Mid-administered mice. These results clearly indicate that the adequate drug release rate from liposome should make it possible to deliver the substantial amounts of drugs into cancer cells, leading to the actual anti-tumor activity.

Dicetyl phosphate-tetraethylenepentamine-based liposomes for systemic siRNA delivery.

Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.

Size separation and size determination of liposomes.

We developed a method for separating liposomes by size and determining their average diameters. Liposomes with different average diameters were separated on a monolithic silica capillary column, and the size of the liposomes corresponding to each peak was determined online with a dynamic light scattering detector coupled to the capillary liquid chromatography system. The calculated diameters for the separated liposomes were similar to the diameter values measured in batch mode. We demonstrate that this combination of a monolithic capillary column and light scattering detection could be used for size separation of liposomes and could provide more details about average diameters than batch-mode analysis.

MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion.

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.

Characteristics of gastrointestinal absorption of DX-9065a, a new synthetic anticoagulant.

DX-9065a, a newly synthesized anticoagulant that selectively inhibits factor Xa, is a zwitterion and has characteristics of high water solubility and low lipophilicity. We predicted the fraction absorbed (Fa) of DX-9065a to be approximately 15-35% in humans, based on the boundary layer theory using the intestinal perfusion method in rats. However, human oral bioavailability was 2-3% in clinical trials, and the result of actual human bioavailability was lower than that of the predicted Fa. Thus, in this report, the reason for low oral bioavailability of DX-9065a was examined by in vitro and in vivo experiments. The factors affecting oral bioavailability of DX-9065a were not the hepatic first-pass effect, degradation of the drug in intestinal fluid, nor the interaction of the drug with the intestinal mucin. Furthermore, no effect of P-gp efflux was observed. Oral absorption of the drug in rats with bile duct ligation was significantly higher than that in normal rats with bioavailability of 17 and 3%, respectively. It was confirmed that bile acids inhibited DX-9065a absorption because DX-9065a interacted with bile acids to form insoluble complexes. The results suggest that the complex formation of DX-9065a with bile acids in the intestinal tract is an important factor affecting absorption of DX-9065a.

The gene-silencing effect of siRNA in cationic lipoplexes is enhanced by incorporating pDNA in the complex.

Efficient delivery is a key issue in translating interference RNA technology into a feasible therapy. The efficiency of carrier systems used for this technology is commonly tested by co-transfection, i.e. simultaneous transfection with an exogenous gene and with the siRNA. Two approaches can be distinguished: (1) with the two transfectants in the same carrier complex (siRNA/pDNA/carrier) and (2) with the two transfectants in different carrier complexes (pDNA/carrier and siRNA/carrier). The process to prepare the nucleic acid(s)-carrier complexes and the transfection procedure may affect the effectiveness of the gene-silencing process. In this study, two preparation methods were compared, namely the co-preparation of an siRNA/pDNA/liposome lipoplex (Method I) and the separate preparation of an siRNA/liposome lipoplex and a pDNA/liposome lipoplex (Method II). siRNA in the lipoplex produced by Method I showed a stronger gene-silencing effect than that in the lipoplexes prepared by Method II. There was no significant difference between the two methods in the amount of siRNA delivered to cells. Cellular entry and intracellular trafficking of siRNA/pDNA/liposome lipoplex is likely to differ from those of the separate lipoplexes. When in Method II non-transcriptional pDNA was included in the complex with siRNA, the gene-silencing effect was significantly enhanced. If and to what extent the experimental design is suitable to quantify RNA interference remains to be demonstrated.

Tumor targeting of doxorubicin by anti-MT1-MMP antibody-modified PEG liposomes.

Immunoliposomes are potent carriers for targeting of therapeutic drugs to specific cells. Membrane type-1 matrix metalloproteinase (MT1-MMP), which plays an important role in angiogenesis, is expressed on angiogenic endothelium cells as well as tumor cells. Then, the MT1-MMP might be useful as a target molecule for tumor and neovascularity. In the present study, we addressed a utility of antibodies against the MT1-MMP as a targeting ligand of liposomal anticancer drug. Fab' fragments of antibody against the MT1-MMP were modified at distal end of polyethylene glycol (PEG) of doxorubicin (DXR)-encapsulating liposomes, DXR-sterically stabilized immunoliposomes (DXR-SIL[anti-MT1-MMP(Fab')]). Modification with the antibody significantly enhanced cellular uptake of DXR-SIL[anti-MT1-MMP(Fab')] into the HT1080 cells, which highly express MT1-MMP, compared with the non-targeted liposomes (DXR-stealthliposomes (DXR-SL)), suggesting that MT1-MMP antibody (Fab') is a potent targeting ligand for the MT1-MMP expressed cells. In vivo systemic administration of DXR-SIL[anti-MT1-MMP(Fab')] into the tumor-bearing mice showed significant suppression of tumor growth compared to DXR-SL. This is presumably due to the active targeting of immunoliposomes for tumor and neovascularity. However, tumor accumulation of DXR-SIL[anti-MT1-MMP(Fab')] and DXR-SL were comparable, suggesting that both liposomal formulations accumulated in tumor via enhanced permeation and retention (EPR) effect, but not via targeting to the MT1-MMP expressed on both the endothelial and tumor cells. It appears that the enhanced antitumor activity of DXR-SIL[anti-MT1-MMP(Fab')] resulted from acceleration of cellular uptake of lioposomes owing to the incorporated antibody after extravasation from capillaries in tumor.

Targeted delivery of anticancer drugs to tumor vessels by use of liposomes modified with a peptide identified by phage biopanning with human endothelial progenitor cells.

As tumor angiogenic vessels are critical for tumor growth and express different molecules on their surface from those on normal vessels, these vessels are expected to be an ideal target for anticancer drug delivery systems. It was previously reported that endothelial progenitor cells (EPCs) are involved in angiogenesis, tumor growth, and metastasis, and that EPCs show gene expression patterns similar to those of tumor endothelial cells. In the present study, a tumor vessel-targeting peptide, ASSHN, was identified from a phage-display peptide library by in vitro biopanning with human EPCs (hEPCs) and in vivo biopanning using angiogenesis model mice prepared by the dorsal air sac method. Phage clones displaying ASSHN peptide showed a marked affinity for hEPCs in vitro, and also for tumor vessels in vivo. PEGylated liposomes modified with the ASSHN peptide (ASSHN-Lip) were designed and prepared for the delivery of anticancer agents. Confocal images showed that ASSHN-Lip clearly bound to hEPCs in vitro and tumor vessels, and also showed extravasation from the vessels. The administration of doxorubicin-encapsulated ASSHN-Lip into Colon26 NL-17-bearing mice significantly suppressed tumor growth compared with doxorubicin-encapsulated PEGylated liposomes. These results suggest that the delivery of anticancer agents with ASSHN-Lip could be useful for targeted cancer therapy.

In vitro and in vivo evaluation of novel cationic liposomes utilized for cancer gene therapy.

Advanced peritoneal carcinomatoses is very difficult to treat. We have explored the potential therapeutic application of gene therapy using cationic liposomes in this disease. The lacZ gene was introduced in vitro into ovarian and endometrial cancer cells using cationic liposomes. The transfection efficiency was similar to that of commercially available liposomes in serum-free medium (11.0-20.9% vs. 5.4-26.0%). In serum-containing medium, the efficiency was 1.9-18.1%, which is comparable with the efficiency in serum-free medium. However, the efficiency of commercial liposomes decreased drastically to between 0.1% and 4.7% in the serum-containing medium. When cultured cells were transfected with the herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV) was added, the anti-tumor effect of GCV was 47-640 times greater than when the same experiment was performed with lacZ gene. Evaluation of anti-tumor effect was performed with the MTT assay. In vivo, the HRA and mEIIL ascitic mice were treated with HSV-tk gene and GCV using the peritoneal route, a significant prolongation of the mean survival time was observed by Kaplan-Meier analysis (16-18 days and 15-30 days, respectively, p < 0.05). These results indicate a potential role for gene therapy in the treatment of advanced intraperitoneal carcinomatoses using the novel cationic liposomes.

Ultrasound enhances liposome-mediated gene transfection.

Previous studies have shown that some series of liposomes, usually containing cationic lipids, are useful tools for gene introduction into cells. To investigate the effect of ultrasound (US) on liposome-mediated transfection, three types of liposomes (designated L1, L2 and L3, in the order of increasing transfection efficiency) containing O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl) diethanolamine chloride, dioleoylphosphatidylethanolamine, and/or cholesterol at varying ratios, were used in this study. HeLa cells were treated with liposome-DNA complexes containing luciferase genes for 2 h before sonication. Optimal US condition for the enhancement was determined to be 0.5 W/cm2, 1 MHz continuous wave for 1 min and was above threshold for inertial cavitation based on EPR detection of free radicals. Luciferase expressions 24 h after the treatments were significantly increased by sonication to 2.4 fold with L1, and 1.7 fold with L2. However, with L3, which showed the highest level of expression among the liposomes, significant but minimal enhancement was observed when sonication was done 15 min after the DNA-L3 treatment, suggesting that efficiency of the liposome also determines the proper timing for sonication. The 2 h pre-sonication incubation with liposome-DNA complexes for L1 and L2 (30 min for L3) required to attain enhancement, suggests that US works to enhance transfection only after cells had enough DNA uptake.

Behavior of mucoadhesive nanoparticles having hydrophilic polymeric chains in the intestine.

The behavior of nanoparticles having surface hydrophilic poly(N-isopropylacrylamide), poly(N-vinylacetamide), poly(vinylamine) or poly(methacrylic acid) chains in the intestine was examined. The permeability of salmon calcitonin (sCT) from the mucosal to serosal side of the everted jejunum was enhanced in the presence of nanoparticles. This enhancement, which correlated with the amount of sCT incorporated in nanoparticles, disappeared completely after removal of the mucous layer. When fluorescein isothiocyanate-dextran (FD-4) was used instead of sCT, its permeability through the everted jejunum with and without mucous layer was not enhanced by any nanoparticles, because FD-4 was not incorporated in nanoparticles at all. These findings indicated that the accumulation of nanoparticles incorporating sCT in the mucous layer resulted in the enhancement of sCT permeability. Nanoparticles with poly(N-isopropylacrylamide) or poly(vinylamine) on their surfaces did not cause any damage to the intestinal mucosa. Also, none of the nanoparticles opened the tight junction of the intestinal membrane. It was concluded that mucoadhesion of nanoparticles incorporating sCT to the gastrointestinal mucosa contributed to the absorption enhancement of sCT.

Influence of dose and animal species on accelerated blood clearance of PEGylated liposomal doxorubicin.

We recently demonstrated that Doxil loses its long-circulating properties when injected repeatedly at doses below 2 mg/m(2) in dogs. In studies using other animal species, PEGylated liposomal doxorubicin has been reported not to induce the accelerated blood clearance (ABC) phenomenon. We investigated the issue of whether Doxil can elicit the ABC phenomenon in several species. In minipigs, the ABC phenomenon was induced at 2 mg/m(2). In other animal species, the ABC phenomenon was not observed at higher doses (>2 mg/m(2)), but was observed at much lower doses (0.2 mg/m(2)). The pharmacokinetic profile of a second dose of Doxil reflected the circulating anti-PEG IgM level induced by the first dose. The ABC phenomenon was not observed at the clinically recommended DXR dose (20 mg/m(2)) in any animal species. These results indicate that Doxil can cause the ABC phenomenon in all animals tested, the extent of induction was dependent on the first dose of Doxil, and a higher Doxil dose lessened the ABC phenomenon. The current study results suggest that a careful study design including selection of animal species is important for preclinical studies using PEGylated liposomal formulations even if they contain anticancer drugs that suppress the host immune response.

Suppression in mice of immunosurveillance against PEGylated liposomes by encapsulated doxorubicin.

PEGylated liposomes (PEG-lip) can escape from recognition by immune system and show a longer half-life in the blood than non-PEGylated liposomes. In this study, we investigated the influence of injected PEG-lip encapsulating doxorubicin (PEG-lip-DOX) on the biodistribution of subsequently injected PEG-lip in mice. PEG-lip-DOX, free doxorubicin or empty PEG-lip were initially injected into BALB/c mice via a tail vein, and 3days later [(3)H]-labeled PEG-lip ([(3)H] PEG-lip) were injected into these same mice. At 24h after the injection, the distribution of [(3)H] PEG-lip in the liver and spleen was significantly reduced in the PEG-lip-DOX group compared with that in the free doxorubicin or PEG-lip group. Consequently, the plasma concentration of [(3)H] PEG-lip was significantly elevated by the pretreatment with PEG-lip-DOX. Altered pharmacokinetics was observed at least until 72h after the injection of [(3)H] PEG-lip. The influence of the injected PEG-lip-DOX on the pharmacokinetics of the subsequently injected [(3)H] PEG-lip was clearly observed from 1 to 14days, and slightly observed on days 21 and 28, after the injection of the PEG-lip-DOX. Flow cytometric analysis showed that the number of liver Kupffer cells was significantly reduced after the treatment with PEG-lip-DOX. On the other hand, a similar alteration in the distribution of the subsequently injected [(3)H] PEG-lip was observed in immunodeficient mice such as BALB/c nu/nu and severe combined immunodeficiency (SCID) mice. These findings suggest that immune cells including liver Kupffer cells responsible for recognizing PEG-lip were selectively damaged by the encapsulated doxorubicin in PEG-lip injected initially, which damage led to prolongation of the half-life of subsequently injected [(3)H] PEG-lip in the blood.

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.

Accelerated blood clearance of PEGylated liposomes containing doxorubicin upon repeated administration to dogs.

The accelerated blood clearance phenomenon involving anti-PEG IgM production has been recognized as an important issue for the design and development of PEGylated liposomes. Here, we show that empty PEGylated liposomes and Doxil, PEGylated liposomes containing doxorubicin, both caused anti-PEG IgM production and thereby a rapid clearance of the second and/or third dose of Doxil in Beagle dogs in a lipid-dose, inverse-dependent manner. It appears that the pharmacokinetic profile of the second and third administration of Doxil reflected the presence of anti-PEG IgM circulating in the blood. Doxil plus an excess amount of empty PEGylated liposomes rather enhanced the production of anti-PEG IgM compared to Doxil of the same doxorubicin dose. During sequential administration, increasing the lipid dose of Doxil in each dose by the addition of empty PEGylated liposomes strongly attenuated the magnitude of the ABC phenomenon during the effectuation phase of a second and third dose of Doxil. Our results suggest that the pre-clinical study of anti-cancer drug-containing PEGylated liposomes with dogs must be carefully designed and performed with monitoring of the anti-PEG IgM and liposomal drugs circulating in the blood.

Agitation during lipoplex formation harmonizes the interaction of siRNA to cationic liposomes.

We recently demonstrated that agitation during lipoplex formation (vorLTsiR) improves the gene knockdown effect of siRNA because the resultant decrease in lipoplex size leads to an enhanced uptake by cells. In furthering this line of research, the present study was focused on the interaction of siRNA to cationic liposomes during lipoplex preparation. A fluorescence resonance energy transfer (FRET) study indicated that the application of agitation in the presence of siRNA effectively reorganized positively charged lipids (DC-6-14 and DOPE) in an order that effectively promoted further electrostatic interaction between the negatively charged phosphate backbone of siRNA and the positively charged lipids in the cationic liposome membrane. A circular dichroism (CD) study indicated that the agitation did not bring about a change in the A-form helix of siRNA, therefore the interactions between the lateral anionic groups of siRNA - responsible for the characteristic bands of the A-form helix - and cationic liposomes were effectively promoted. Factorial design coupled with response surface methodology was used to statistically analyze the influence of vortex speed and time and siRNA dose on the in vitro gene knockdown effects of siRNA-lipoplex that were spontaneously formulated (spoLTsiR) along with that formulated under agitation (vorLTsiR). The analysis indicated that vortex speed plays the most important role in enhancing the gene knockdown effect of siRNA among the three variables, although all three are important. It was concluded that the high energy transmitted by applying agitation during lipoplex formation harmonized the interaction of siRNA to positively charged lipids (DC-6-14 and DOPE) in cationic liposomes, resulting in a superior gene knockdown efficacy of vorLTsiR compared to spoLTsiR. Our study suggests that the preparation procedure is one of the critical factors in producing the enhanced gene knockdown effect of siRNA.

Agitation during lipoplex formation improves the gene knockdown effect of siRNA.

The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA.