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• W2032903792 abstract "Drug resistance is a major challenge to the effective treatment of cancer. We have developed two nanoparticle formulations, cationic liposome-polycation-DNA (LPD) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (MDR) tumors. In this study, we have provided four strategies to overcome drug resistance. First, we formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, we have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, we further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, we delivered a therapeutic siRNA to inhibit MDR transporter. We demonstrated the first evidence of c-Myc siRNA delivered by the LPD-II nanoparticles down-regulating MDR expression and increasing Dox uptake in vivo. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition. This study highlights a potential clinical use for the multifunctional nanoparticles with an effective delivery property and a function to overcome drug resistance in cancer. The activity and the toxicity of LPD- and LPD-II-mediated therapy are compared. Drug resistance is a major challenge to the effective treatment of cancer. We have developed two nanoparticle formulations, cationic liposome-polycation-DNA (LPD) and anionic liposome-polycation-DNA (LPD-II), for systemic co-delivery of doxorubicin (Dox) and a therapeutic small interfering RNA (siRNA) to multiple drug resistance (MDR) tumors. In this study, we have provided four strategies to overcome drug resistance. First, we formed the LPD nanoparticles with a guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammonium chloride, which can induce reactive oxygen species, down-regulate MDR transporter expression, and increase Dox uptake. Second, to block angiogenesis and increase drug penetration, we have further formulated LPD nanoparticles to co-deliver vascular endothelial growth factor siRNA and Dox. An enhanced Dox uptake and a therapeutic effect were observed when combined with vascular endothelial growth factor siRNA in the nanoparticles. Third, to avoid P-glycoprotein-mediated drug efflux, we further designed another delivery vehicle, LPD-II, which showed much higher entrapment efficiency of Dox than LPD. Finally, we delivered a therapeutic siRNA to inhibit MDR transporter. We demonstrated the first evidence of c-Myc siRNA delivered by the LPD-II nanoparticles down-regulating MDR expression and increasing Dox uptake in vivo. Three daily intravenous injections of therapeutic siRNA and Dox (1.2 mg/kg) co-formulated in either LPD or LPD-II nanoparticles showed a significant improvement in tumor growth inhibition. This study highlights a potential clinical use for the multifunctional nanoparticles with an effective delivery property and a function to overcome drug resistance in cancer. The activity and the toxicity of LPD- and LPD-II-mediated therapy are compared. The occurrence of drug resistance is a main impediment to the success of cancer chemotherapy. Cancer cells develop different ways to be resistant to chemotherapy drugs. Overexpression of drug transporter proteins, such as P-glycoprotein (P-gp) 2The abbreviations used are: P-gpP-glycoproteinLPDcationic liposome-polycation-DNALPD-IIanionic liposome-polycation-DNAPEGpolyethylene glycolDoxdoxorubicinsiRNAsmall interfering RNAAAanisamideMDRmultiple drug resistanceROSreactive oxygen speciesVEGFvascular endothelial growth factorDSAAN,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammonium chlorideDOTAP1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propaneDOPA1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphateDCFH-DA2′,7′-dichlorodihydrofluorescein diacetateTEMtransmission electron microscopyTUNELTdT dUTP nick end labelingFITCfluorescein isothiocyanateBoct-butoxycarbonylPBSphosphate-buffered salineAMacetoxymethylesterDAPI4′,6-diamidino-2-phenylindoleILinterleukinMFImean fluorescence intensity. plays a key role in regulating drug resistance. Development of strategies to down-regulate the expression of P-gp or inhibit P-gp function has been the major subject of cancer research. For example, one of the strategies to overcome MDR is to use carriers like nanoparticles to avoid P-gp-mediated drug efflux. Only the drug presenting in the cell membrane can be effluxed out of the cancer cell. The drug delivered by nanoparticles is internalized in the cytoplasm or the lysosome and not pumped out by P-gp (1.Shah N. Chaudhari K. Dantuluri P. Murthy R.S. Das S. J. Drug Target. 2009; 17: 533-542Crossref PubMed Scopus (108) Google Scholar). Dox-loaded liposomes are able to overcome MDR by increasing Dox uptake in the nuclei and extending retention in the nuclei of the MDR cells (2.Xu D.H. Gao J.Q. Liang W.Q. Pharmazie. 2008; 63: 646-649PubMed Google Scholar, 3.Goren D. Horowitz A.T. Tzemach D. Tarshish M. Zalipsky S. Gabizon A. Clin. Cancer Res. 2000; 6: 1949-1957PubMed Google Scholar). P-glycoprotein cationic liposome-polycation-DNA anionic liposome-polycation-DNA polyethylene glycol doxorubicin small interfering RNA anisamide multiple drug resistance reactive oxygen species vascular endothelial growth factor N,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammonium chloride 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate 2′,7′-dichlorodihydrofluorescein diacetate transmission electron microscopy TdT dUTP nick end labeling fluorescein isothiocyanate t-butoxycarbonyl phosphate-buffered saline acetoxymethylester 4′,6-diamidino-2-phenylindole interleukin mean fluorescence intensity. Small interfering RNA (siRNA) is a promising novel approach of cancer therapy. It offers a new strategy to down-regulate the targeted oncogene for therapeutic intervention. Systemically delivering siRNA to tumors remains a major hurdle in cancer gene therapy (4.Devi G.R. Cancer Gene Ther. 2006; 13: 819-829Crossref PubMed Scopus (292) Google Scholar, 5.Shen Y. IDrugs. 2008; 11: 572-578PubMed Google Scholar). Major problems of siRNA delivery include poor cellular uptake, low stability, and rapid clearance from the systemic circulation. We have developed a cationic lipid containing both a guanidinium and a lysine residue as a cationic head group that can down-regulate mitogen-activated protein kinase (MAPK) signaling, form cationic liposome-polycation-DNA (LPD) to intravenously deliver siRNA to the solid tumor in high efficiency, and achieve a synergistic therapeutic effect with siRNA on a human lung cancer model (6.Chen Y. Sen J. Bathula S.R. Yang Q. Fittipaldi R. Huang L. Mol. Pharm. 2009; 6: 696-705Crossref PubMed Scopus (94) Google Scholar). In this study, we further explored the biological activity of the guanidinium-containing cationic lipid, i.e. N,N-distearyl-N-methyl-N-2-(N′-arginyl) aminoethyl ammonium chloride (DSAA), which may play a role in overcoming drug resistance in tumors. However, nanoparticles containing cationic lipid such as DSAA showed a poor entrapment efficiency of Dox and induced immunotoxicity in mice. To combat these problems, we have further developed the multifunctional LPD-II nanoparticles made with anionic lipids that co-deliver siRNA and Dox into the MDR tumor cells and trigger a synergistic anti-cancer effect. We co-formulated siRNA and Dox in the LPD-II nanoparticles via Dox intercalation into the DNA in the nanoparticles. Both LPD and LPD-II nanoparticles were targeted specifically to the tumor cells by modification with anisamide (AA), a ligand of sigma receptor overexpressed in many human cancer cells. Two different siRNAs, VEGF and c-Myc siRNAs, are selected in this study to achieve the enhanced drug uptake and anti-cancer effect. We hypothesized that therapeutic siRNAs delivered to the MDR cells will down-regulate the target genes and sensitize the tumor cells to the co-delivered Dox, resulting in an enhanced therapeutic activity of the nanomedicine. The experiments were carried out in a xenograft model of the NCI/ADR-RES tumor. DOPA, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Protamine sulfate (fraction X from salmon) and calf thymus DNA were purchased from Sigma-Aldrich (St. Louis, MO). Dox was purchased from IFFECT CHEMPHAR (Hong Kong). Synthetic 19-nucleotide RNAs with 3′-UU overhangs on both sequences were purchased from Dharmacon (Lafayette, CO). For quantitative studies, FITC or cy5.5 was conjugated to 5′ sense sequence. 5′-cy5.5- and 5′-FITC-labeled siRNA sequences were also obtained from Dharmacon. The sequence of c-Myc siRNA was 5′-AACGUUAGCUUCACCAACAUU-3′, and the VEGF siRNA was 5′-GCAGAAUCAUCACGAAGUG-3′. The sequence of control siRNA with sequence 5′-AATTCTCCGAACGTGTCACGT-3′ was obtained from Dharmacon. DSAA is a nonglycerol-based guanidine head group containing cationic lipid synthesized in five steps. N-Alkylation by n-octadecyl bromide and subsequent Boc deprotection of mono-Boc-protected ethylene diamine yielded the mixed primary tertiary amine N1,N1-dioctadecylethane-1,2-diamine. Tri-N-tert-butoxycarbonyl protected arginine conjugation to the primary amine group by the conventional 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Quaternization of the tertiary amine group using methyl iodide on the above obtained product gave tri-N-tert-butoxycarbonyl-protected DSAA. To obtain the final product DSAA, Boc group deprotection with trifluoroacetic acid and chloride ion exchange with Amberlyst A 27(Cl−) ion exchange resin were carried out. The resulting compound was characterized by using 1H NMR spectra and liquid secondary ion mass spectrometry. Detailed synthetic procedures and spectral and purity data will be delineated elsewhere. 3S. R. Bathula, Y. Chen, and L. Huang, unpublished observations. NCI/ADR-RES and OVCAR-8 cells were a kind gift from Dr. Russell Mumper (University of North Carolina School of Pharmacy). NCI-ADR/RES is a multidrug-resistant ovarian cancer cell line derived from OVCAR-8 cells (drug-sensitive line) in a poorly documented manner (NCI, National Institutes of Health web site). NCI/ADR-RES cells were maintained in Dulbecco's modified Eagle's medium high glucose with GlutaMAX (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Female athymic nude mice 6–8 weeks of age were purchased from the National Cancer Institute, Frederick, National Institutes of Health. The mice were injected subcutaneously in the right flank with 5 × 106 NCI/ADR-RES cells in 0.1 ml of PBS. All of the work performed on animals was in accordance with and permitted by the University of North Carolina Institutional Animal Care and Use Committee. Calcein-AM has been used as a functional probe for the detection of P-gp activity (7.Holló Z. Homolya L. Hegedüs T. Sarkadi B. FEBS Lett. 1996; 383: 99-104Crossref PubMed Scopus (190) Google Scholar). The cells were treated with lipids and incubated with 1 μmol/liter of calcein-AM for 15 min at 37 °C in Dulbecco's modified Eagle's medium. The cells were resuspended in calcein-AM-free medium, washed twice in PBS, and analyzed using flow cytometry. The uptake calcein-AM in the cells was expressed as: folds of untreated = (MFItreatment − MFIunstained)/(MFIuntreated − MFIunstained). NCI/ADR-RES cells (1 × 106/well) were seeded into 12-well plates. The cells were treated with lipids in serum-containing medium at 37 °C. Then the cells were incubated with 20 μm DCFH-DA (Sigma-Aldrich) in serum-containing medium for 30 min at 37 °C. The cells were quickly washed and immediately analyzed by flow cytometry. LPD nanoparticles were prepared according to the previously described method with slight modifications (8.Cui Z. Han S.J. Vangasseri D.P. Huang L. Mol. Pharmacol. 2005; 2: 22-28Crossref PubMed Scopus (90) Google Scholar). Briefly, cationic liposomes composed of cholesterol and DOTAP or DSAA (1:1 molar ratio) were prepared by thin film hydration followed by membrane extrusion to reduce the particle size. 15 μl of protamine (2 mg/ml), 140 μl of deionized water, and 24 μl of a mixture of siRNA and calf thymus DNA (2 mg/ml) were mixed and kept at room temperature for 10 min before adding 120 μl of cationic liposome (20 mm). The formulations stand at room temperature for 10 min before the addition of DSPE-PEG. The formulations were then mixed with 70 μl of DSPE-PEG or DSPE-PEG-AA (10 mg/ml) and kept at 50–60 °C for 10 min for the attachment of the PEG chains to the surface membrane of the nanoparticles. LPD-II was prepared according to the previously described method with slight modifications (9.Lee R.J. Huang L. J. Biol. Chem. 1996; 271: 8481-8487Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). Briefly, anionic liposomes composed of DOPA, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and cholesterol (2:1:1 molar ratio) were prepared by thin film hydration followed by membrane extrusion to reduce the particle size. To prepare LPD-II, 48 μl of protamine (2 mg/ml), 60 μl of deionized water, and 24 μl of a mixture of siRNA and calf thymus DNA (2 mg/ml) were mixed and kept at room temperature for 10 min before adding 90 μl of anionic liposome (20 mm). LPD-II was left at room temperature for 10 min before the addition of DSPE-PEG. It was then mixed with 54 μl of DSPE-PEG or DSPE-PEG-AA (10 mg/ml) and kept at 50–60 °C for 10 min for the attachment of the PEG chains to the surface membrane of the nanoparticles. TEM images of the LPD-II nanoparticles were acquired by the use of JEOL 100CX II TEM (JEOL, Japan). Briefly, 5 μl of LPD-II nanoparticles was dropped on to a 300 mesh carbon coated copper grid (Ted Pella, Inc., Redding, CA). Excess sample was removed by blotting with a filter paper. The grid was air-dried and viewed in TEM without staining. The scale bar was automatically shown in the image according to the magnification. NCI/ADR-RES cells (1 × 105/well) were seeded in 12-well plates (Corning Inc., Corning, NY) 12 h before experiments. The cells were treated with different formulations at a concentration of 250 nm for 5′-FITC-labeled siRNA or 1.5 μm Dox in serum-containing medium at 37 °C for 4 h. The cells were washed twice with PBS, counterstained with DAPI, and imaged using a Leica SP2 confocal microscope. Dox and siRNA uptake of NCI/ADR-RES cells was also measured by flow cytometry. Briefly, the cells were treated with different formulations at a concentration of 250 nm 5′-FITC-labeled siRNA or 1.5 μm Dox in serum-containing medium at 37 °C for 1 h. The cells were harvested and resuspended at a concentration of 1 × 106 cells/ml. The cells were washed with PBS and analyzed for fluorescence by flow cytometry. NCI/ADR-RES tumor-bearing mice (tumor size, ∼1 cm2) were intravenously injected with siRNA and Dox in different formulations (1.2 mg of siRNA/kg, one injection/day for 2 days). A day after the third injection, the tumors were collected, paraffin-embedded, and sectioned. 7.25-μm-thick sections were immunostained with primary antibodies and visualized by using kits from DakoCytomation. The samples were imaged by using a Nikon Microphot SA microscope or Leica SP2 confocal microscope. NCI/ADR-RES tumor-bearing mice (tumor size, ∼1 cm2) were intravenously injected with siRNA and Dox in different formulations (1.2 mg of siRNA/kg, one injection/day for 2 days). A day after the third injection, the mice were killed, and tumor samples were collected. Total RNA were extracted with the RNeasy® mini kit (Qiagen, Valencia, CA) by following the manufacturer's protocol. cDNA was then prepared in the presence of reverse transcriptase (Promega, Madison, WI). The mRNA levels were determined by an ABI PRISM HT7500 sequence detection system (Applied Biosystems, Foster City, CA) as described previously (10.Song E. Zhu P. Lee S.K. Chowdhury D. Kussman S. Dykxhoorn D.M. Feng Y. Palliser D. Weiner D.B. Shankar P. Marasco W.A. Lieberman J. Nat. Biotechnol. 2005; 23: 709-717Crossref PubMed Scopus (909) Google Scholar). The oligomer pairs used for the amplification of PCR products were AAGCCACAGCATACATCC (forward primer for c-Myc), TTACGCACAAGAGTTCCG (reverse primer for c-Myc), CACCACCAACTACTTAGC (forward primer for glyceraldehyde-3-phosphate dehydrogenase), and GTAGAGGCAGGAATGATG (reverse primer for glyceraldehyde-3-phosphate dehydrogenase). NCI/ADR-RES tumor-bearing mice (tumor size, ∼1 cm2) were intravenously injected with siRNA and Dox in different formulations (1.2 mg of siRNA/kg, one injection/day for 2 days). A day after the third injection, the mice were killed, and the tumor samples were collected. Total protein (40 μg) isolated from the tumors was loaded on a polyacrylamide gel. Tumor lysates were separated on a 10% acrylamide gel and transferred to a polyvinylidene difluoride membrane. The membranes were blocked for 1 h in 5% skim milk and then incubated with polyclonal antibody against c-Myc (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight. The membranes were washed in PBST (PBS with 0.1% Tween 20) three times and then incubated for 1 h with the secondary antibody. The membranes were washed four times and then developed by an enhanced chemiluminescence system according to the manufacturer's instructions (PerkinElmer Life Sciences, Waltham, MA). The intensity of the bands was quantified by Photoshop histogram. Mice with a tumor size of ∼1 cm2 were intravenously injected with cy5.5-labeled siRNA (1.2 mg/kg) and Dox (1.2 mg/kg) in different formulations. 4 h later, the mice were killed, and the tissues were collected, fixed in 10% formalin, and embedded in paraffin. The tumor tissues were sectioned (7.25 μm thick) and imaged using a Leica SP2 confocal microscope. TUNEL staining was performed as recommended by the manufacturer's protocol (Promega, Madison, WI). NCI/ADR-RES tumor-bearing mice were given intravenous injections of the siRNA and Dox formulated in the nanoparticles. 24 h after the second injection, the mice were sacrificed, and the tumors were collected for TUNEL staining. Images from TUNEL-stained tumor sections were captured with Nikon ECLIPSE Ti-U microscopy. NCI/ADR-RES tumor-bearing mice (size, 16–25 mm2) were intravenously injected with different formulations containing siRNA (1.2 mg/kg) or Dox (1.2 mg/kg) once/day for 3 days. Tumor size in the treated mice was measured after treatment. All of the statistical analyses were performed by Student's t test. The data were considered statistically significant when the p value was less than 0.05. Our first strategy is to use a cationic lipid as a P-gp inhibitor as well as a drug carrier. To determine whether the cationic lipid containing a guanidinium residue overcomes MDR, we first studied the uptake of Dox in NCI/ADR-RES cells. NCI/ADR-RES cells were treated with different lipids for different doses. Then cells were treated with the indicated concentration of free Dox. The structures of different lipids used in this study were illustrated in Fig. 1A. As shown in Fig. 1B, incubation with DSAA resulted in a substantial increase in Dox accumulation. The lipids without guanidine groups only caused a slight increase in Dox uptake. Besides, we also evaluated the ability of different lipids to inhibit P-gp using the calcein-AM assay in the resistant and sensitive cell lines (Fig. 1C and supplemental Fig. S1). In drug-resistant NCI/ADR-RES cells, the fluorescence generated from intracellular calcein significantly increased in a dose-dependent manner when cells were treated with DSAA. In contrast, DOTAP or the lipids without guanidinium groups only caused a slight increase in intracellular calcein fluorescence. However, intracellular calcein fluorescence remained only slightly changed after treatment with 10 or 50 μm DSAA for 1 h in the drug-sensitive OVCAR-8 cells (supplemental Fig. S1). Because drug-resistant cells treated with DSAA resulted in significantly enhanced Dox uptake and inhibition of P-gp activity, we further studied the biological functions of DSAA in some detail. The guanidinium head group of DSAA may induce ROS, which down-regulates P-gp expression in MDR cells (11.Hiramatsu M. Mol. Cell Biochem. 2003; 244: 57-62Crossref PubMed Scopus (61) Google Scholar, 12.Cai Y. Lu J. Miao Z. Lin L. Ding J. Cancer Biol. Ther. 2007; 6: 1794-1799Crossref PubMed Scopus (53) Google Scholar). To address this hypothesis, NCI/ADR-RES cells were treated with DSAA or DOTAP at different doses and further incubated with DCFH-DA. The cellular ROS content was detected by using flow cytometry. As shown in Fig. 1D, DSAA could significantly generate ROS in NCI/ADR-RES cells 1 h after treatment. However, the cellular ROS content remained unchanged after treatment with 1, 5, or 10 μm DOTAP for 1 h. Furthermore, MDR expression in MDR cells was significantly suppressed after treatment with 10 or 50 μm DSAA (77 and 73% of untreated control, respectively) (Fig. 1E). These results suggest that ROS induced by DSAA may reverse drug resistance in NCI/ADR-RES cells by suppressing MDR transporter expression and activity. Based on the property of DSAA to enhance Dox uptake in the MDR cells, we further used DSAA as a carrier lipid to deliver Dox and siRNA into NCI/ADR-RES cells. We used AA-targeted LPD nanoparticles containing DSAA to specifically deliver Dox and siRNA to the NCI/ADR-RES cells, which express the sigma receptor (data not shown). As shown in supplemental Fig. S2, A and B), targeted nanoparticles containing DSAA increased both cellular internalization and nuclear uptake of Dox compared with free Dox. The nanoparticles containing DSAA delivered Dox into the cytoplasm of NCI/ADR-RES cells more efficiently than those containing DOTAP. Furthermore, Dox uptake of the cells treated with the targeted nanoparticles was much more than that of cells treated with the nontargeted nanoparticles. Pretreatment or co-incubation of cells with blank-targeted nanoparticles also showed slightly enhanced Dox uptake compared with free Dox. To eliminate the possibility that Dox uptake enhancement was due to apoptosis caused by DSAA, the cells were co-incubated with the caspase-3 inhibitor DEVD-CHO32 and Dox in the targeted nanoparticles containing DSAA. In this treatment, the uptake of Dox was as much as the treatment without the caspase-3 inhibitor (supplemental Fig. S2A). The uptake of Dox in the targeted nanoparticles containing DSAA was further examined in the other drug-resistant cells, H460/RES cells, which also overexpress P-gp. As shown in supplemental Fig. S3, targeted nanoparticles containing DSAA increased Dox uptake compared with free Dox in H460/RES cells. To confirm the ability of DSAA to deliver both Dox and siRNA into NCI/ADR-RES cells, Dox and fluorescently labeled siRNA were co-formulated into different formulations. As shown in Fig. 2A, the uptake of Dox and fluorescently labeled siRNA was much greater in cells treated with the formulation prepared with DSAA than the formulation prepared with DOTAP, and the uptake was ligand-dependent. Some Dox co-localized with siRNA in the cytoplasm, and some translocated to the nucleus after treatment of Dox and FITC-siRNA formulated in the targeted nanoparticles containing DSAA. The results indicate that the nanoparticles containing DSAA efficiently co-delivered siRNA and Dox to the NCI/ADR-RES cells, and the delivery was ligand-specific and formulation lipid-dependent. Our second strategy is to use a therapeutic siRNA to inhibit angiogenesis and sensitize the drug-resistant cells to chemotherapy drugs. To examine the biological activities of siRNA co-formulated with Dox in different formulations in vivo, the VEGF levels in the subcutaneous NCI/ADR-RES tumor were detected by Western blotting 24 h after two daily intravenous injections of VEGF siRNA and Dox in different formulations (Fig. 2B). VEGF expression of the NCI/ADR-RES tumor treated with VEGF siRNA and Dox-containing targeted nanoparticles was significantly suppressed (45% of untreated control) (Fig. 2B), whereas VEGF siRNA and Dox-containing nontargeted nanoparticles and a control siRNA and Dox-containing targeted nanoparticles showed no silencing effect. The results indicate that the nanoparticles containing DSAA could systemically deliver siRNA into the tumor tissue and silence the target protein. Furthermore, the silencing effect was specifically controlled by the targeting ligand. To further study the effect of VEGF siRNA and DSAA on Dox uptake in vivo, tumor-bearing mice were given two daily injections of VEGF siRNA or a control siRNA (1.2 mg/kg) formulated in the targeted nanoparticles containing DSAA or DOTAP. 24 h after the second treatment, free Dox or Dox formulated in the targeted nanoparticles containing DSAA was intravenously injected into tumor-bearing mice. The Dox uptake of NCI/ADR-RES tumor tissue in the tumor-bearing mice was observed 4 h after intravenous injections using confocal microscopy. As shown in Fig. 2D, Dox formulated in the targeted nanoparticles showed enhanced cytosolic delivery in NCI/ADR-RES cells compared with free Dox. Free Dox uptake was higher in the tumor tissues collected from the mice treated with a control siRNA formulated in the DSAA-containing targeted nanoparticles than in those from the mice treated with a control siRNA formulated in the DOTAP-containing targeted nanoparticles. Furthermore, the formulated Dox uptake also increased in the tumor tissues collected from mice treated with a control siRNA in the DSAA-containing targeted nanoparticles rather than those treated with a control siRNA in the DOTAP-containing targeted nanoparticles (Fig. 2C). These results indicate that DSAA reverses drug resistance in NCI/ADR-RES tumors. VEGF inhibitors such as bevacizumab can enhance the permeability of the tumor vasculature, which leads to improved penetration of the free or liposome-encapsulated chemotherapy agents in the tumor (13.Almubarak M. Newton M. Altaha R. J. Oncol. 2008; 2008: 942618Crossref PubMed Google Scholar). We hypothesized that VEGF siRNA may improve the penetration and uptake of free Dox or formulated Dox in the drug-resistant tumor. As shown in Fig. 2D, free Dox uptake was higher in the tumor tissues collected from the mice treated with VEGF siRNA formulated in the DSAA-containing targeted nanoparticles than in those from the mice treated with a control siRNA formulated in the DSAA-containing targeted nanoparticles. Similarly, the formulated Dox uptake was also enhanced in the tumor tissues collected from mice treated with VEGF siRNA in the targeted nanoparticles compared with those treated with a control siRNA in the targeted nanoparticles (Fig. 2C). These results indicate that the uptakes of both free and formulated Dox increased after treatment with VEGF siRNA. To verify the regulatory effects of DSAA and VEGF siRNA on the expression of MDR transporters in vivo, MDR expression in the drug-resistant tumor was examined by immunostaining 24 h after two daily intravenous injections of VEGF siRNA or a control siRNA in different formulations (Fig. 2E). MDR expression of the NCI/ADR-RES tumor treated with VEGF siRNA or a control siRNA in the targeted nanoparticles containing DSAA was partially suppressed (Fig. 2E). A control siRNA delivered by the targeted nanoparticles containing DOTAP showed no effect. Thus, the data indicate that inhibition of the MDR expression mediated by the targeted nanoparticles was formulation lipid-dependent but not siRNA sequence-specific. Taken together, these results demonstrate a combination effect between VEGF siRNA and DSAA in promoting Dox uptake in NCI/ADR-RES tumors. A combination effect between VEGF siRNA and DSAA in promoting cellular apoptosis and tumor growth inhibition was further tested. To examine the therapeutic activities of siRNA and Dox in the different formulations, we stained for the apoptotic markers in NCI/ADR-RES tumors (Fig. 3A). Fig. 3A indicates that ∼8% of NCI/ADR-RES cells treated with VEGF siRNA and Dox in the targeted nanoparticles containing DSAA underwent apoptosis as detected by the TUNEL staining. This value was significantly higher than the cells treated with control siRNA formulated in the targeted nanoparticles containing DSAA or VEGF or control siRNA formulated in the targeted nanoparticles containing DOTAP. To further elucidate the therapeutic effects of Dox and VEGF siRNA in the nanoparticles containing DSAA, tumor growth inhibitory effects were evaluated after treatments of different formulations. Three injections of VEGF siRNA alone in the targeted nanoparticles containing DSAA showed a partial inhibition of tumor growth (p < 0.05 comparing with the untreated control at day 18) (Fig. 3B). Free Dox had no therapeutic effect. A synergistic tumor growth inhibition was observed when mice were treated with Dox and VEGF siRNA co-formulated in the targeted nanoparticles containing DSAA. Thus, targeted nanoparticles loaded with both VEGF siRNA and DSAA could sensitize MDR cells to co-formulated Dox. Furthermore, VEGF siRNA and Dox in the targeted nanoparticles containing DOTAP or in the nontargeted nanoparticles containing DSAA showed less therapeutic effect compared with the treatment of" @default.
• W2032903792 created "2016-06-24" @default.
• W2032903792 creator A5018222046 @default.
• W2032903792 creator A5027835055 @default.
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• W2032903792 date "2010-07-01" @default.
• W2032903792 modified "2023-10-11" @default.
• W2032903792 title "Multifunctional Nanoparticles Delivering Small Interfering RNA and Doxorubicin Overcome Drug Resistance in Cancer" @default.
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• W2032903792 doi "https://doi.org/10.1074/jbc.m110.125906" @default.
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