Showing papers in "Molecular Pharmaceutics in 2009"
TL;DR: The translation from concept to clinic for the first experimental therapeutic to provide targeted delivery of synthetic, small interfering RNA (siRNA) in humans is described and the designed features of this experimental therapeutic are described.
Abstract: Experimental therapeutics developed to exploit RNA interference (RNAi) are now in clinical studies. Here, the translation from concept to clinic for the first experimental therapeutic to provide targeted delivery of synthetic, small interfering RNA (siRNA) in humans is described. This targeted, nanoparticle formulation of siRNA, denoted as CALAA-01, consists of a cyclodextrin-containing polymer (CDP), a polythethylene glycol (PEG) steric stabilization agent, and human transferrin (Tf) as a targeting ligand for binding to transferrin receptors (TfR) that are typically upregulated on cancer cells. The four component formulation is self-assembled into nanoparticles in the pharmacy and administered intravenously (iv) to patients. The designed features of this experimental therapeutic are described, and their functions illustrated.
958 citations
TL;DR: Interestingly, SNP exhibited good anti-inflammatory properties as indicated by concentration-dependent inhibition of marker enzymes (matrix metalloproteinase 2 and 9), which clearly indicate that silver nanoparticles could provide a safer alternative to conventional antimicrobial agents in the form of a topical antimicrobial formulation.
Abstract: Silver is an effective antimicrobial agent with low toxicity, which is important especially in the treatment of burn wounds where transient bacteremia is prevalent and its fast control is essential. Drugs releasing silver in ionic forms are known to get neutralized in biological fluids and upon long-term use may cause cosmetic abnormality, e.g., argyria and delayed wound healing. Given its broad spectrum activity, efficacy and lower costs, the search for newer and superior silver based antimicrobial agents is necessary. Among the various options available, silver nanoparticles have been the focus of increasing interest and are being heralded as an excellent candidate for therapeutic purposes. This report gives an account of our work on development of an antimicrobial gel formulation containing silver nanoparticles (SNP) in the size range of 7−20 nm synthesized by a proprietary biostabilization process. The typical minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against ...
545 citations
TL;DR: Different model membranes, drug-lipid interactions and their significance, studies of model membrane interactions with nanocarriers, and how biophysical interaction studies with lipid model membranes could play an important role in drug discovery and drug delivery are discussed.
Abstract: The transport of drugs or drug delivery systems across the cell membrane is a complex biological process, often difficult to understand because of its dynamic nature. In this regard, model lipid membranes, which mimic many aspects of cell-membrane lipids, have been very useful in helping investigators to discern the roles of lipids in cellular interactions. One can use drug-lipid interactions to predict pharmacokinetic properties of drugs, such as their transport, biodistribution, accumulation, and hence efficacy. These interactions can also be used to study the mechanisms of transport, based on the structure and hydrophilicity/hydrophobicity of drug molecules. In recent years, model lipid membranes have also been explored to understand their mechanisms of interactions with peptides, polymers, and nanocarriers. These interaction studies can be used to design and develop efficient drug delivery systems. Changes in the lipid composition of cells and tissue in certain disease conditions may alter biophysical interactions, which could be explored to develop target-specific drugs and drug delivery systems. In this review, we discuss different model membranes, drug-lipid interactions and their significance, studies of model membrane interactions with nanocarriers, and how biophysical interaction studies with lipid model membranes could play an important role in drug discovery and drug delivery.
456 citations
TL;DR: Combination PTX and CUR therapy was very effective in enhancing the cytotoxicity in wild-type and resistant cells by promoting the apoptotic response, and has significant promise in the clinical management of refractory diseases, especially in ovarian cancer.
Abstract: Development of multidrug resistance (MDR) against a variety of conventional and novel chemotherapeutic agents is a significant challenge in effective cancer therapy. Over the last several years, we have focused on a multimodal therapeutic strategy to overcome tumor MDR by enhancing the delivery efficiency to the tumor mass and lowering the apoptotic threshold by modulation of the intracellular signaling mechanisms. In this study, we have examined augmentation of therapeutic efficacy upon coadministration of paclitaxel (PTX) and curcumin (CUR), an inhibitor of nuclear factor kappa B (NFkappaB) as well as a potent down-regulator of ABC transporters, in wild-type SKOV3 and drug resistant SKOV3(TR) human ovarian adenocarcinoma cells. PTX and CUR were encapsulated in flaxseed oil containing nanoemulsion formulations. The results showed that the encapsulated drugs were effectively delivered intracellular in both SKOV3 and SKOV3(TR) cells. CUR administration was shown to inhibit NFkappaB activity and down regulate P-glycoprotein expression in resistant cells. Combination PTX and CUR therapy, especially when administered in the nanoemulsion formulations, was very effective in enhancing the cytotoxicity in wild-type and resistant cells by promoting the apoptotic response. Overall, this cotherapy strategy has significant promise in the clinical management of refractory diseases, especially in ovarian cancer.
450 citations
TL;DR: This review discusses the new targeting strategies with recent examples, which utilize the environmental stimuli to activate the nanocarriers, and traditional strategies for tumor-targeted nanoccarriers, which are briefly discussed with an emphasis on their achievements and challenges.
Abstract: One of the main goals of nanomedicine is to develop a nanocarrier that can selectively deliver anticancer drugs to the targeted tumors. Extensive efforts have resulted in several tumor-targeted nanocarriers, some of which are approved for clinical use. Most nanocarriers achieve tumor-selective accumulation through the enhanced permeability and retention effect. Targeting molecules such as antibodies, peptides, ligands, or nucleic acids attached to the nanocarriers further enhance their recognition and internalization by the target tissues. While both the stealth and targeting features are important for effective and selective drug delivery to the tumors, achieving both features simultaneously is often found to be difficult. Some of the recent targeting strategies have the potential to overcome this challenge. These strategies utilize the unique extracellular environment of tumors to change the long-circulating nanocarriers to release the drug or interact with cells in a tumor-specific manner. This review ...
421 citations
TL;DR: This review examines some of the key barriers that affect the biodistribution of oligonucleotides both in molecular form and when they are associated with nanocarriers.
Abstract: Attaining the full therapeutic utility of antisense and siRNA oligonucleotides will require understanding of the biological barriers that stand between initial administration of these drugs and their final actions within cells. This review examines some of the key barriers that affect the biodistribution of oligonucleotides both in molecular form and when they are associated with nanocarriers. An understanding of the biological processes underlying these barriers will aid in the design of more effective delivery systems.
287 citations
TL;DR: It is concluded that magnetite nanoparticles coated with triblock copolymers containing PEO tail lengths of above 2 kDa are biocompatible and appropriate for in vivo application.
Abstract: Magnetic targeting is useful for intravascular or intracavitary drug delivery, including tumor chemotherapy or intraocular antiangiogenic therapy. For all such in vivo applications, the magnetic drug carrier must be biocompatible and nontoxic. In this work, we investigated the toxic properties of magnetic nanoparticles coated with polyethylenoxide (PEO) triblock copolymers. Such coatings prevent the aggregation of magnetic nanoparticles and guarantee consistent magnetic and nonmagnetic flow properties. It was found that the PEO tail block length inversely correlates with toxicity. The nanoparticles with the shortest 0.75 kDa PEO tails were the most toxic, while particles coated with the 15 kDa PEO tail block copolymers were the least toxic. Toxicity responses of the tested prostate cancer cell lines (PC3 and C4-2), human umbilical vein endothelial cells (HUVECs), and human retinal pigment epithelial cells (HRPEs) were similar. Furthermore, all cell types took up the coated magnetic nanoparticles. It is concluded that magnetite nanoparticles coated with triblock copolymers containing PEO tail lengths of above 2 kDa are biocompatible and appropriate for in vivo application.
265 citations
TL;DR: In tumor-bearing mice, the higher dose of tax produces greater and more sustained tumor shrinkage and tumor cell apoptosis, and the results thus begin to address mechanisms for how nonspherical carriers deliver both imaging agents and anticancer therapeutics to solid tumors.
Abstract: Shape effects of synthetic carriers are largely unexplored in vivo, although recent findings suggest that flexible filaments can persist in the circulation even if microns in length. Here, to bette...
264 citations
TL;DR: This review provides an overview of the current delivery strategies for synthetic siRNA, focusing on the targeted, self-assembled nanoparticles which show potential to become a useful and efficient tool in cancer therapy.
Abstract: RNA interference (RNAi) as a mechanism to selectively degrade mRNA (mRNA) expression has emerged as a potential novel approach for drug target validation and the study of functional genomics. Small interfering RNAs (siRNA) therapeutics has developed rapidly and already there are clinical trials ongoing or planned. Although other challenges remain, delivery strategies for siRNA become the main hurdle that must be resolved prior to the full-scale clinical development of siRNA therapeutics. This review provides an overview of the current delivery strategies for synthetic siRNA, focusing on the targeted, self-assembled nanoparticles which show potential to become a useful and efficient tool in cancer therapy.
256 citations
TL;DR: PEG addition improves dispersion, neutralize charge, and enhance particle diffusion in cervical mucus in a manner strongly dependent on polymer MW and density, which improves the understanding of, and confirm a possible direction for, the rational design of effective carriers for mucosal drug/vaccine delivery.
Abstract: Drug delivery to mucosal epithelia is severely limited by the mucus gel, which is a physical diffusion barrier as well as an enzymatic barrier in some sites. Loading of drug into polymer particles can protect drugs from degradation and enhance their stability. To improve efficacy of nanoparticulate drug carriers, it has been speculated that polymers such as poly(ethylene)glycol (PEG) incorporated on the particle surface will enhance transport in mucus. In the present study, we demonstrate the direct influence of PEG on surface properties of poly(lactic-co-glycolic)acid (PLGA) nanoparticles (d = 170 ± 57 nm). PEG of various molecular weights (MW = 2, 5, 10 kDa) were incorporated at a range of densities from 5 – 100% on the particle surface. Our results indicate PEG addition improves dispersion, neutralize charge, and enhance particle diffusion in cervical mucus in a manner strongly dependent on polymer MW and density. Diffusion of PEGylated particles was 3 – 10× higher than unmodified PLGA particles. These findings improve the understanding of, and confirm a possible direction for, the rational design of effective carriers for mucosal drug/vaccine delivery.
255 citations
TL;DR: Results of this study coherently suggest that PLGA NPs are not readily taken up by cells, but deliver the payload to cells by extracellular drug release and/or direct drug transfer to contacting cells, which are contrasted with the prevalent view.
Abstract: We reexamined the cellular drug delivery mechanism by poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) to determine their utility and limitations as an intracellular drug delivery system. First, we prepared PLGA NPs which physically encapsulated Nile red (a hydrophobic fluorescent dye), in accordance with the usual procedure for labeling PLGA NPs, incubated them with mesothelial cells, and observed an increase in the intracellular fluorescence. We then prepared NPs from PLGA chemically conjugated to a fluorescent dye and observed their uptake by the mesothelial cells using confocal microscopy. We also used coherent anti-Stokes Raman scattering (CARS) microscopy to image cellular uptake of unlabeled PLGA NPs. Results of this study coherently suggest that PLGA NPs (i) are not readily taken up by cells, but (ii) deliver the payload to cells by extracellular drug release and/or direct drug transfer to contacting cells, which are contrasted with the prevalent view. From this alternative standpoint, we analyzed cytotoxicities of doxorubicin and paclitaxel delivered by PLGA NPs and compared with those of free drugs. Finally, we revisit previous findings in the literature and discuss potential strategies to achieve efficient drug delivery to the target tissues using PLGA NPs.
TL;DR: Solutol HS 15 micelles are suitable nanocarriers for ICG which improve the optical properties and stability of the dye, and encapsulation efficiency of ICG was high at 95%.
Abstract: Indocyanine green (ICG) is a fluorescence dye that is widely used for near-infrared imaging. Application of this dye is limited by its numerous disadvantageous properties in aqueous solution, including its concentration-dependent aggregation, poor aqueous stability in vitro and low quantum yield. Additionally, ICG is highly bound to nonspecific plasma proteins, leading to rapid elimination from the body with a half-life of 3-4 min. In this study, encapsulation of ICG within various micellar systems was investigated with the aim of overcoming these limitations. The aggregation behavior of different aqueous ICG formulations was studied using cryogenic transmission electron microscopy (cryo-TEM) and absorption spectroscopy. The micellar systems were characterized by their optical properties, particle size distribution, zeta potential and hemolytic activity. Encapsulation efficiency was determined using analytical ultracentrifugation. The best results were achieved for ICG encapsulated within aqueous Solutol HS 15 micelles. This formulation exhibited a lower aggregation behavior, a 3-fold increased quantum yield and high aqueous stability (over 4 weeks) compared to free aqueous ICG. The micelles were found to have an average diameter of 12 nm and a zeta potential close to zero (-2.1 +/- 1.7 mV). Encapsulation efficiency of ICG was high at 95%. The formulation did not display significant hemolytic activity. Consequently, Solutol HS 15 micelles are suitable nanocarriers for ICG which improve the optical properties and stability of the dye.
TL;DR: It was concluded that, when an amorphous molecular level solid dispersion containing a hydrophobic drug and hydrophilic polymer is subjected to moisture, drug crystallization can occur via one of two routes: crystallization from the plasticized one-phaseSolid dispersion, or crystallized from a plasticized drug-rich amorphously phase in a two-phasesolid dispersion.
Abstract: The objective of this study was to investigate the phase behavior of amorphous solid dispersions composed of a hydrophobic drug and a hydrophilic polymer following exposure to elevated relative humidity. Infrared (IR) spectroscopy, differential scanning calorimetry (DSC) and moisture sorption analysis were performed on five model systems (nifedipine-poly(vinylpyrrolidone) (PVP), indomethacin-PVP, ketoprofen-PVP, droperidol-PVP, and pimozide-PVP) immediately after production of the amorphous solid dispersions and following storage at room temperature and elevated relative humidity. Complete miscibility between the drug and the polymer immediately after solid dispersion formation was confirmed by the presence of specific drug-polymer interactions and a single glass transition (T(g)) event. Following storage at elevated relative humidity (75-94% RH), nifedipine-PVP, droperidol-PVP, and pimozide-PVP dispersions formed drug-rich and polymer-rich amorphous phases prior to crystallization of the drug, while indomethacin-PVP and ketoprofen-PVP dispersions did not. Drug crystallization in systems exhibiting amorphous-amorphous phase separation initiated earlier ( or=46 days at 94% RH). Evidence of moisture-induced amorphous-amorphous phase separation was observed following storage at as low as 54% RH for the pimozide-PVP system. It was concluded that, when an amorphous molecular level solid dispersion containing a hydrophobic drug and hydrophilic polymer is subjected to moisture, drug crystallization can occur via one of two routes: crystallization from the plasticized one-phase solid dispersion, or crystallization from a plasticized drug-rich amorphous phase in a two-phase solid dispersion. In the former case, the polymer is still present in the same phase as the drug, and can inhibit crystallization to a greater extent than the latter scenario, where the polymer concentration in the drug phase is reduced as a result of the amorphous-amorphous phase separation. The strength of drug-polymer interactions appears to be important in influencing the phase behavior.
TL;DR: In this article, the authors investigated the mechanisms by which a cationic lipid carrier mediates siRNA delivery into mammalian cells and showed that approximately 95% of siRNA lipoplexes enter the cells through endocytosis and persist in endolysosomes for a prolonged period of time.
Abstract: A key challenge for therapeutic application of RNA interference is to efficiently deliver synthetic small interfering RNAs (siRNAs) into target cells that will lead to the knockdown of the target transcript (functional siRNA delivery). To facilitate rational development of nonviral carriers, we have investigated by imaging, pharmacological and genetic approaches the mechanisms by which a cationic lipid carrier mediates siRNA delivery into mammalian cells. We show that approximately 95% of siRNA lipoplexes enter the cells through endocytosis and persist in endolysosomes for a prolonged period of time. However, inhibition of clathrin-, caveolin-, or lipid-raft-mediated endocytosis or macropinocytosis fails to inhibit the knockdown of the target transcript. In contrast, depletion of cholesterol from the plasma membrane has little effect on the cellular uptake of siRNA lipoplexes, but it abolishes the target transcript knockdown. Furthermore, functional siRNA delivery occurs within a few hours and is gradually inhibited by lowering temperatures. These results demonstrate that although endocytosis is responsible for the majority of cellular uptake of siRNA lipoplexes, a minor pathway, probably mediated by fusion between siRNA lipoplexes and the plasma membrane, is responsible for the functional siRNA delivery. Our findings suggest possible directions for improving functional siRNA delivery by cationic lipids.
TL;DR: A PSMA-targeted ligand is developed that delivers attached imaging and therapeutic agents selectively to PCa cells without targeting normal cells and could allow detection, staging, monitoring, and treatment of PCa with improved accuracy and efficacy.
Abstract: Prostate cancer (PCa) is a major cause of mortality and morbidity in Western society today. Current methods for detecting PCa are limited, leaving most early malignancies undiagnosed and sites of metastasis in advanced disease undetected. Major deficiencies also exist in the treatment of PCa, especially metastatic disease. In an effort to improve both detection and therapy of PCa, we have developed a PSMA-targeted ligand that delivers attached imaging and therapeutic agents selectively to PCa cells without targeting normal cells. The PSMA-targeted radioimaging agent (DUPA−99mTc) was found to bind PSMA-positive human PCa cells (LNCaP cell line) with nanomolar affinity (KD = 14 nM). Imaging and biodistribution studies revealed that DUPA−99mTc localizes primarily to LNCaP cell tumor xenografts in nu/nu mice (% injected dose/gram = 11.3 at 4 h postinjection; tumor-to-muscle ratio = 75:1). Two PSMA-targeted optical imaging agents (DUPA−FITC and DUPA−rhodamine B) were also shown to efficiently label PCa cells a...
TL;DR: The history of uncovering and defining this interplay with a primary emphasis on studies from the laboratory is reviewed, followed by the human transporter-enzyme interaction studies.
Abstract: Two decades ago the importance of transporter-enzyme interplay and its effects on drug bioavailability and hepatic disposition were first recognized. Here we review the history of uncovering and defining this interplay with a primary emphasis on studies from our laboratory. We review the early 1990s oral bioavailability studies that found that the highly lipophilic, poorly water-soluble cyclosporine formulation on the market at that time did not have an absorption problem, but rather a gut metabolism problem. This led to studies of the interactive nature of CYP3A and P-glycoprotein in the intestine, and investigations of this interplay using cellular systems and isolated perfused rat organ studies. Studies investigating uptake transporter-enzyme interactions using cellular, perfused rat liver and intact rats are reviewed, followed by the human transporter-enzyme interaction studies. Work characterizing the rate limiting processes in the drug transporter-metabolism alliance is then addressed, ending with a review of areas of the interplay that require further studies and analysis.
TL;DR: In vivo toxicity studies of this formulation demonstrated that conjugates remain to be optimized for therapeutic application and high in vitro biocompatibility and efficient and sequence-specific gene silencing was found at > or =25 nM siRNA, comparable to the corresponding electrostatic polyplexes.
Abstract: Extracellular stability of electrostatically formed siRNA polyplexes is a significant concern in the delivery process. To overcome the risk of polyplex dissociation in the extracellular environment, siRNA was covalently incorporated into a pH- and redox-responsive polymer conjugate. The novel siRNA conjugate consists of polylysine (PLL) as RNA binding and protecting polycation, polyethylene glycol (PEG) as solubilizing and shielding polymer, the lytic peptide melittin masked by dimethylmaleic anhydride (DMMAn) removable at endosomal pH, and the siRNA attached at the 5'-end of the sense strand via a bioreducible disulfide bond. The purified siRNA conjugate was stable in the presence of the polyanion heparin at conditions where the analogous electrostatic siRNA polyplexes disassemble. Only the combination of heparin plus a reducing agent such as glutathione triggered the release of siRNA from the conjugate. High in vitro biocompatibility (absence of cytotoxicity or hemolytic activity at neutral pH) and efficient and sequence-specific gene silencing was found at > or =25 nM siRNA, comparable to the corresponding electrostatic polyplexes. In vivo toxicity studies of this formulation demonstrated that conjugates remain to be optimized for therapeutic application.
TL;DR: The preliminary in vivo knockdown experiment suggests that PEG-PEI/siRNA complexes are promising nanomedicines for pulmonary siRNA delivery and indicated that P EGylated nanocarriers are more suitable for lung application.
Abstract: This study describes the physicobiological characterization of PEI- and PEG-PEI polyplexes containing partially 2'-OMe modified 25/27mer dicer substrate siRNAs (DsiRNAs) and their in vivo behavior regarding biodistribution and systemic bioavailability after pulmonary application as well as their ability to knock down gene expression in the lung. Biophysical characterization included circular dichroism of siRNA in polyplexes, condensation efficiency of polymers and in vitro stability. After in vivo application, biodistribution and kinetics of radiolabeled polyplexes were quantified and recorded over time in three-dimensional SPECT images and by end point scintillation counting. The influence on lung tissue and on the humoral and cellular immunosystem was investigated, and finally knockdown of endogenous gene expression in the lung was determined qualitatively. While all of the polymers used in our study were proven to effectively condense siRNA, stability of the complexes depended on the PEG grafting degree. Interestingly, PEI 25 kDa, which showed the least interaction with mucin or surfactant in vitro, performed poorly in vivo. Our nuclear imaging approach enabled us to follow biodistribution of the instilled nanocarriers over time and indicated that PEGylated nanocarriers are more suitable for lung application. While moderate proinflammatory effects were attributed to PEI25k-PEG(2k)(10) nanocarriers, none of the treatments caused histological abnormalities. Our preliminary in vivo knockdown experiment suggests that PEG-PEI/siRNA complexes are promising nanomedicines for pulmonary siRNA delivery. These results encouraged us to further investigate possible adverse effects and to quantify in vivo gene silencing in the lung after intratracheal instillation of PEG-PEI/siRNA complexes.
TL;DR: The micelle formulation effectively suppressed the growth of existing MDR tumors in mice for at least 50 days by three iv injections at a 3-day interval at a dose of 10 mg of DOX/kg, and was superior to its first generation, which targeted pH 6.8 and folate receptor.
Abstract: The second generation of pH-sensitive micelles composed of poly(l-histidine-co-l-phenlyalanine(16 mol %))(MW:5K)-b-PEG(MW:2K) and poly(l-lactic acid)(MW:3K)-b-PEG(MW:2K)-folate (80/20 wt/wt %) was ...
TL;DR: Anti-VEGF siRNA/PEI-HA complex was thought to be applied successfully as target specific antiangiogenic therapeutics for the treatment of diseases in the tissues with HA receptors, such as liver cancer and kidney cancer.
Abstract: Hyaluronic acid (HA) plays important biological roles in tissue integrity, angiogenesis, wound healing, and cell motility through the interaction with receptors on cell membranes. In this work, we investigated the effect of HA modification on the receptor-mediated endocytosis labeling HA derivatives with quantum dots (QDots). HA-QDot conjugates with a degree of modification less than ca. 25 mol % appeared to be more efficiently taken up to B16F1 cells by HA receptor mediated endocytosis than QDots alone. On the basis of bioimaging study, polyethyleneimine, PEI-HA conjugate with 24.2 mol % PEI content was developed as a target specific intracellular delivery carrier of siRNA. The siRNA/PEI-HA complex exhibited higher gene silencing efficiency in B16F1 cells with HA receptors than siRNA/PEI complex. Anti-PGL3-Luc siRNA/PEI-HA complex appeared to silence PGL3-Luc gene in the range of 50%-85% depending on the serum concentration up to 50 vol %. According to in vivo biodistribution test, siRNA/PEI-HA complex accumulated mainly in the tissues with HA receptors such as liver, kidney, and tumor. Furthermore, intratumoral injection of anti-VEGF siRNA/PEI-HA complex resulted in an effective inhibition of tumor growth by the HA receptor mediated endocytosis to tumor cells in C57BL/6 mice. Considering all these results, anti-VEGF siRNA/PEI-HA complex was thought to be applied successfully as target specific antiangiogenic therapeutics for the treatment of diseases in the tissues with HA receptors, such as liver cancer and kidney cancer.
TL;DR: Collectively, these high affinity, PSMA-specific radioimaging agents demonstrate significant potential for use in localizing prostate cancer masses, monitoring response to therapy, detecting prostate cancer recurrence following surgery, and selecting patients for subsequent PSma-targeted chemotherapy.
Abstract: The high mortality and financial burden associated with prostate cancer can be partly attributed to a lack of sensitive screening methods for detection and staging of the disease. Guided by in silico docking studies using the crystal structure of PSMA, we designed and synthesized a series of PSMA-targeted 99mTc−chelate complexes for imaging PSMA-expressing human prostate cancer cells (LNCaP cell line). Of the six targeted radioimaging agents synthesized, three were found to bind LNCaP cells with low nanomolar affinity. Moreover, the same three PSMA-targeted imaging agents were shown to localize primarily to LNCaP tumor xenografts in nu/nu mice, with an average of 9.8 ± 2.4% injected dose/g tissue accumulating in the tumor and only 0.11% injected dose/g tissue retained in the muscle at 4 h postinjection. Collectively, these high affinity, PSMA-specific radioimaging agents demonstrate significant potential for use in localizing prostate cancer masses, monitoring response to therapy, detecting prostate cance...
TL;DR: Lipoplexes containing a hyaluronic acid-dioleoylphosphatidylethanolamine (HA-DOPE) conjugate mediated good transfection on CD44 expressing cell lines in culture.
Abstract: Lipoplexes containing a hyaluronic acid−dioleoylphosphatidylethanolamine (HA−DOPE) conjugate were designed to target the CD44 receptor on breast cancer cells. Cationic liposomes composed of a mixture of [2-(2,3-didodecyloxypropyl)hydroxyethyl]ammonium bromide (DE) and dioleoylphosphatidylethanolamine (DOPE) with or without HA−DOPE were prepared, characterized, and used to form a complex with plasmid DNA pCMV-luc. Lipoplexes displayed a negative zeta potential and a mean diameter between 250−300 nm. Cytotoxicity and transfection efficiency of the lipoplexes were determined on the MDA-MB-231and MCF-7 breast cancer cell lines. Cytotoxicity was not modified by the presence of HA−DOPE. However HA−DOPE increased the level of transfection on CD44-expressing MDA-MB-231 cells compared to the MCF-7 line, which expresses very low levels of CD44. The transfection on the MDA-MB-231 cells was highly inhibited by anti-CD44 Hermes-1 antibody but not by the nonspecific anti-ErbB2 antibody. In conclusion, cationic liposome...
TL;DR: The overall results suggested that the hydrophobically modified PEIs provide a promising delivery strategy for siRNA therapeutic applications.
Abstract: This study was conducted to formulate a nonviral delivery system for the delivery of small interfering RNA (siRNA) to B16 melanoma cells in vitro. For this purpose, oleic and stearic acid modified derivatives of branched polyethylenimine (PEI) were prepared and evaluated. The hydrophobically modified polymers increased siRNA condensation up to 3 folds as compared to the parent PEI. The modified PEIs exhibited up to 3-fold higher siRNA protection from degradation in fetal bovine serum as compared to the parent PEI. The formulated complexes were shown to enter B16 cells in a time-dependent fashion, reaching over 90% of the cells after 24 h, as compared to only 5% of the cells displaying siRNA uptake in the absence of any carrier. A proportional reduction in siRNA cell uptake was observed with reduced polymeric content in the formulations. When used to deliver various doses of siRNA to B16 cells, the modified PEIs were superior or comparable to some of the commercially available transfection agents; the hydrophobically modified polymers gave 3-fold increased siRNA delivery than the parent PEI, approximately 5-fold higher delivery than jetPEI and Metafectene, a comparable delivery to Lipofectamine 2000, but a 1.6-fold decreased delivery compared to INTERFERin, which was the most efficient reagent in our hands. Using an siRNA specific for integrin alpha(v), a dose-dependent decrease in integrin alpha(v) levels was demonstrated in B16 cells by flow cytometry, revealing a more pronounced reduction of integrin alpha(v) levels for oleic- and stearic-acid modified PEIs. The overall results suggested that the hydrophobically modified PEIs provide a promising delivery strategy for siRNA therapeutic applications.
TL;DR: Biodistribution studies clearly demonstrated that PEG( 4) linkers are particularly useful for improving the tumor uptake and clearance kinetics of (99m)Tc-2PEG(4)-dimer and ( 99m) tricine-3PEG (4-dimer) from noncancerous organs.
Abstract: This report describes the synthesis of two cyclic RGD (Arg-Gly-Asp) conjugates, HYNIC-2PEG(4)-dimer (HYNIC = 6-hydrazinonicotinyl; 2PEG(4)-dimer = E[PEG(4)-c(RGDfK)](2); and PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and HYNIC-3PEG(4)-dimer (3PEG(4)-dimer = PEG(4)-E[PEG(4)-c(RGDfK)](2)), and evaluation of their (99m)Tc complexes [(99m)Tc(HYNIC-2PEG(4)-dimer)(tricine)(TPPTS)] ((99m)Tc-2PEG(4)-dimer: TPPTS = trisodium triphenylphosphine-3,3',3''-trisulfonate) and [(99m)Tc(HYNIC-3PEG(4)-dimer)(tricine)(TPPTS)] ((99m)Tc-3PEG(4)-dimer) as novel radiotracers for imaging integrin alpha(v)beta(3) expression in athymic nude mice bearing U87MG glioma and MDA-MB-435 breast cancer xenografts. The integrin alpha(v)beta(3) binding affinities of RGD peptides were determined by competitive displacement of (125)I-c(RGDyK) on U87MG glioma cells. It was found that the two PEG(4) linkers between RGD motifs in HYNIC-2PEG(4)-dimer (IC(50) = 2.8 +/- 0.5 nM) and HYNIC-3PEG(4)-dimer (IC(50) = 2.4 +/- 0.7 nM) are responsible for their higher integrin alpha(v)beta(3) binding affinity than that of HYNIC-PEG(4)-dimer (PEG(4)-dimer = PEG(4)-E[c(RGDfK)](2); IC(50) = 7.5 +/- 2.3 nM). Addition of extra PEG(4) linker in HYNIC-3PEG(4)-dimer has little impact on integrin alpha(v)beta(3) binding affinity. (99m)Tc-2PEG(4)-dimer and (99m)Tc-3PEG(4)-dimer were prepared in high yield with >95% radiochemical purity and the specific activity of >10 Ci/mumol. Biodistribution studies clearly demonstrated that PEG(4) linkers are particularly useful for improving the tumor uptake and clearance kinetics of (99m)Tc-2PEG(4)-dimer and (99m)Tc-3PEG(4)-dimer from noncancerous organs. It was also found that there was a linear relationship between the tumor size and radiotracer tumor uptake expressed as %ID (percentage of the injected dose) in U87MG glioma and MDA-MB-435 breast tumor models. The blocking experiment showed that the tumor uptake of (99m)Tc-2PEG(4)-dimer is integrin alpha(v)beta(3)-mediated. In the metabolism study, (99m)Tc-2PEG(4)-dimer had high metabolic stability during its excretion from renal and hepatobiliary routes. (99m)Tc-3PEG(4)-dimer also remained intact during thee excretion from the renal route, but, had approximately 30% metabolism during the excretion from the hepatobiliary route. Planar imaging studies in U87MG glioma and MDA-MB-435 breast tumor models showed that the tumors of approximately 5 mm in diameter could be readily visualized with excellent contrast. Thus, (99m)Tc-3PEG(4)-dimer is a very promising radiotracer for the early detection of integrin alpha(v)beta(3)-positive tumors, and may have the potential for noninvasive monitoring of tumor growth or treatment efficacy.
TL;DR: A novel polymer conjugate of CPT using a core-functionalized, symmetrically PEGylated poly(l-lysine) (PLL) dendrimer showed superior efficacy in murine and human colon carcinoma tumor models when compared with no treatment or treatment with irinotecan.
Abstract: Polymer conjugates of camptothecin (CPT) have been pursued as a solution to the difficulties present in treating cancers with CPT and its derivatives. Covalent attachment of CPT to a polymer can improve solubility, increase blood circulation time, enhance tumor uptake, and significantly improve efficacy of the drug. In this report, we describe a novel polymer conjugate of CPT using a core-functionalized, symmetrically PEGylated poly(l-lysine) (PLL) dendrimer. The PEGylated dendrimer consisted of a lysine dendrimer functionalized with aspartic acid, which was used as an attachment site for poly(ethylene glycol) (PEG) and CPT. The final conjugate had a molecular weight of 40 kDa and was loaded with 4−6 wt % CPT. Polymer-bound CPT was shown to have a long blood circulation half-life of 30.9 ± 8.8 h and a tumor uptake of 4.2 ± 2.3% of the injected dose/g of tissue, compared to free CPT in which less than 1% was retained in the blood after 30 min and had a tumor accumulation of 0.29 ± 0.04% of the injected dos...
TL;DR: No particular lipid emerged as the ideal substituent for transgene expression, although linoleic acid appeared to be superior to other lipid substituents, and no correlation was evident between the level of substitution and DNA delivery efficiency of the polymers.
Abstract: This study was conducted in order to develop amphiphilic, low molecular weight polymeric carriers for nonviral gene delivery. Caprylic, myristic, palmitic, stearic, oleic and linoleic acids were grafted onto the 2 kDa polyethylenimine (PEI) and properties critical for gene delivery were investigated using 293T and bone marrow stromal cells. The extent of lipid substitution on the polymers was controlled by the lipid:PEI feed ratio during the synthesis. The toxicity of the native and lipid-substituted 2 kDa PEI was relatively lower than the 25 kDa PEI, although lipid substitution generally increased the toxicity of the polymers in vitro. Lipid substitution reduced the ability of the polymers to complex DNA, as well as the stability of final complexes, as measured by heparin-induced dissociation. Once fully complexed to a plasmid DNA, however, the lipid-substituted polymers increased the plasmid DNA delivery to the cells. In 293T cells, the lipid-substituted polymers displayed a transfection ability that was equivalent to highly effective 25 kDa PEI, but without the toxic effect associated with the latter polymer. Among the lipids explored, no particular lipid emerged as the ideal substituent for transgene expression, although linoleic acid appeared to be superior to other lipid substituents. No correlation was evident between the level of substitution and DNA delivery efficiency of the polymers, and as little as 1 lipid substitution per PEI was effective in transforming the ineffective 2 kDa PEI into an effective carrier. The current structure-function studies are providing important clues about the properties critical for gene delivery and providing carriers effective for nonviral plasmid delivery.
TL;DR: This study finds that the innate immune response to densely functionalized, oligonucleotide-modified nanoparticles is significantly less when compared to a lipoplex carrying the same DNA sequence.
Abstract: The immune response of macrophage cells to internalized polyvalent nucleic acid-functionalized gold nanoparticles has been studied. This study finds that the innate immune response (as measured by interferon-beta levels) to densely functionalized, oligonucleotide-modified nanoparticles is significantly less (up to a 25-fold decrease) when compared to a lipoplex carrying the same DNA sequence. The magnitude of this effect is inversely proportional to oligonucleotide density. It is proposed that the enzymes involved in recognizing foreign nucleic acids and triggering the immune response are impeded due to the local surface environment of the particle, in particular high charge density. The net effect is an intracelluar gene regulation agent that elicits a significantly lower cellular immune response than conventional DNA transfection materials.
TL;DR: For these high-solubility low-permeability P-GP substrates, P-gp limits in vivo intestinal absorption in the distal segments of the small intestine; however P- gp plays a minimal role in the proximal intestinal segments due to significant lower P- GP expression levels in this region.
Abstract: The purpose of this study was to investigate the role of P-gp efflux in the in vivo intestinal absorption process of BCS class III P-gp substrates, i.e. high-solubility low-permeability drugs. The in vivo permeability of two H2-antagonists, cimetidine and famotidine, was determined by the single-pass intestinal perfusion model in different regions of the rat small intestine, in the presence or absence of the P-gp inhibitor verapamil. The apical to basolateral (AP−BL) and the BL−AP transport of the compounds in the presence or absence of various efflux transporters inhibitors (verapamil, erythromycin, quinidine, MK-571 and fumitremorgin C) was investigated across Caco-2 cell monolayers. P-gp expression levels in the different intestinal segments were confirmed by immunoblotting. Cimetidine and famotidine exhibited segmental dependent permeability through the gut wall, with decreased Peff in the distal ileum in comparison to the proximal regions of the intestine. Coperfusion of verapamil with the drugs sign...
TL;DR: Results from an extended treatment group indicated that the survival of the brain tumor-bearing rats could be further significantly enhanced, indicating that an extended chemotherapy with topotecan liposomes modified with TAM and WGA would be beneficial for treatment.
Abstract: Chemotherapy of brain tumors remains a big challenge owing to the low drug transport across the blood-brain barrier (BBB), multidrug resistance (MDR), and poor penetration into the tumor tissue. We developed a novel dual-targeting liposomal carrier that enabled drug to transport across the BBB and then target the brain tumor. In the dual-targeting liposomal carrier, tamoxifen (TAM) was incorporated into the lipid bilayer membrane of liposomes and wheat germ agglutinin (WGA) was conjugated to the liposomes' surface. Topotecan was then loaded into the above liposomes. In vitro, topotecan liposomes modified with TAM and WGA were applied to the glioma cells, BBB model, and avascular C6 glioma spheroids, respectively. In vivo, they were systemically administered via vein to brain C6 glioma-bearing rats. In view of the microtiter tetrazolium (MTT) results, topotecan liposomes modified with TAM and WGA exhibited a significant inhibitory effect compared to unmodified topotecan liposomes, suggesting that TAM plus WGA contributed strong drug delivery effects into the brain tumor cells after direct drug exposure. In the experiments of drug transport across the BBB model following drug exposure to tumor cells, topotecan liposomes modified with TAM and WGA exhibited the most robust dual-targeting effects: crossing the BBB and then targeting brain tumor cells. Similar strong activity was found in the reduction of C6 glioma tumor spheroid volume and in the apoptosis of the spheroids. In the brain tumor-bearing rats, the dual-targeting effects of topotecan liposomes modified with TAM and WGA could be evidently observed, resulting in a significant improvement in the overall survival of the brain tumor-bearing rats compared with free topotecan and topotecan liposomes. Moreover, results from an extended treatment group indicated that the survival could be further significantly enhanced, indicating that an extended chemotherapy with topotecan liposomes modified with TAM and WGA would be beneficial for treatment. The dual-targeting effects in vivo of topotecan liposomes modified with TAM and WGA could be related to an enhanced effect by TAM via inhibiting efflux of MDR proteins in the BBB and the brain tumor, and an enhanced effect by WGA via endocytosis in the BBB and in the brain tumor. In conclusion, topotecan liposomes modified with TAM and WGA significantly improve topotecan transport across the blood-brain barrier and the survival of brain tumor-bearing animals, showing dual-targeting effects. These findings would encourage further developments of noninvasive therapy for brain tumor.
TL;DR: The results indicate a great promise of the multifunctional carrier EHCO for systemic and targeted delivery of therapeutic siRNA to treat human diseases with RNAi.
Abstract: In this study, novel peptide-targeted delivery systems were developed for systemic and targeted delivery of therapeutic siRNA based on a multifunctional carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinylhistinyl-1-aminoethyl)propionamide] (EHCO), which showed pH-sensitive amphiphilic cell membrane disruption. EHCO formed stable nanoparticles with siRNA. Targeted siRNA delivery systems were readily formed by surface modification of the nanoparticles. PEGylation of the siRNA/EHCO nanoparticles significantly reduced nonspecific cell uptake. The incorporation of a bombesin peptide or RGD peptide via a PEG spacer resulted in receptor-mediated cellular uptake and high gene silencing efficiency in U87 cells. Fluorescence confocal microscopic studies demonstrated that EHCO/siRNA nanoparticles and PEG modified EHCO/siRNA nanoparticles were able to facilitate endosomal escape of the siRNA delivery systems. Systemic administration of a therapeutic anti-HIF-1alpha siRNA with the peptide-targeted delivery systems resulted in significant tumor growth inhibition than a nontargeted delivery system or free siRNA via intravenous injection in nude mice bearing human glioma U87 xenografts. The results indicate a great promise of the multifunctional carrier EHCO for systemic and targeted delivery of therapeutic siRNA to treat human diseases with RNAi.