Progress in the development of nonviral gene delivery vectors continues to be hampered by low transfection activity and toxicity. Here we proposed to develop a lipid prodrug based on a polyamine analogue bisethylnorspermine (BSP) that can function dually as gene delivery vector and, after intracellular degradation, as active anticancer agent targeting dysregulated polyamine metabolism. We synthesized a prodrug of BSP (LS-BSP) capable of intracellular release of BSP using thiolytically sensitive dithiobenzyl carbamate linker. Biodegradability of LS-BSP contributed to decreased toxicity compared with nondegradable control L-BSP. BSP showed a strong synergistic enhancement of cytotoxic activity of TNF-related apoptosis-inducing ligand (TRAIL) in human breast cancer cells. Decreased enhancement of TRAIL activity was observed for LS-BSP when compared with BSP. LS-BSP formed complexes with plasmid DNA and mediated transfection activity comparable to DOTAP and L-BSP. Our results show that BSP-based vectors are promising candidates for combination drug/gene delivery.
gene delivery; plasmid DNA; cationic lipid; bisethylnorspermine; TRAIL
In this manuscript, we have reported a novel 2D fingerprint-based artificial neural network QSAR (FANN-QSAR) method in order to effectively predict biological activities of structurally diverse chemical ligands. Three different types of fingerprints, namely ECFP6, FP2 and MACCS, were used in FANN-QSAR algorithm development, and FANN-QSAR models were compared to known 3D and 2D QSAR methods using five data sets previously reported. In addition, the derived models were used to predict GPCR cannabinoid ligand binding affinities using our manually curated cannabinoid ligand database containing 1699 structurally diverse compounds with reported cannabinoid receptor subtype CB2 activities. To demonstrate its useful applications, the established FANN-QSAR algorithm was used as a virtual screening tool to search a large NCI compound database for lead cannabinoid compounds and we have discovered several compounds with good CB2 binding affinities ranging from 6.70 nM to 3.75 μM. To the best of our knowledge, this is the first report for a fingerprint-based neural network approach validated with a successful virtual screening application in identifying lead compounds. The studies proved that the FANN-QSAR method is a useful approach to predict bioactivities or properties of ligands and to find novel lead compounds for drug discovery research.
QSAR; Artificial neural networks; molecular fingerprints; bioactivity prediction; cannabinoid; CB2; virtual screening
This study assesses the pharmacokinetics (PK) and pharmacodynamics (PD) of Nrf2-mediated increased expression of Phase II drug metabolizing enzyme (DME) and antioxidant enzymes which represents an important component of cancer chemoprevention in rat lymphocytes following intravenous (i.v.) administration of an anti-cancer phytochemical sulforaphane (SFN)
SFN was administered intravenously to four groups of male Sprague-Dawley JVC rats each group comprising four animals. Blood samples were drawn at selected time points. Plasma were obtained from half of the blood samples and analyzed using a validated LC-MS/MS method. Lymphocytes were collected from the remaining blood samples using Ficoll-Paque™ Plus centrifuge medium. Lymphocyte RNAs were extracted, converted to cDNA, and quantitative real-time PCR analyses were performed and fold changes were calculated against those at time zero for the relative expression of Nrf2-target genes of phase II DME/antioxidant enzymes. PK-PD modeling was conducted based on Jusko’s indirect response model (IDR) using GastroPlus™ and Bootstrap Method.
SFN plasma concentration declined biexponentially and the pharmacokinetic parameters were generated. Rat lymphocyte mRNA expression levels showed no change for GSTM1, SOD, NF-κB, UGT1A1, or UGT1A6. Moderate increases (2-5 folds) over the time zero were seen for HO-1, Nrf2, and NQO1, and significant increase (> 5 folds) for GSTT1, GPx1, and Maf. PK-PD analyses using GastroPlus™ and Bootstrap method provided reasonable fitting for the PK and PD profiles and parameter estimates.
Our present study shows that SFN could induce Nrf2-mediated phase II DME/antioxidant mRNA expression for NQO1, GSTT1, Nrf2, GPx, Maf, and HO-1 in rat lymphocytes after i.v. administration, suggesting that Nrf2-mediated mRNA expression in lymphocytes may serve as surrogate biomarkers. The PK-PD IDR model simultaneously linking the plasma concentrations of SFN and the PD response of lymphocyte mRNA expression is valuable for quantitating Nrf2 mediated effects of SFN. This study may provide a conceptual framework for future clinical PK-PD studies of dietary cancer chemopreventive agents in human.
sulforaphane; pharmacokinetics; pharmacodynamics; lymphocyte; phase II genes; Nrf2
Therapeutic proteins have emerged as a significant class of pharmaceutical agents over the past several decades. The potency, rapid elimination, and systemic side-effects have prompted the need of spatiotemporally controlled release for proteins maybe more than any other active therapeutic molecules. This work examines the release of two model protein compounds, bovine serum albumin (BSA) and an anti-integrin antibody (AI), from electrospun polycaprolactone (PCL) nanofiber mats. The anti-integrin antibody was chosen as a model of antibody therapy; in particular, anti-integrin antibodies are a promising class of therapeutic molecules for cancer and angiogenic diseases. The release kinetics were studied experimentally and interpreted in the framework of a recently published theory of desorption-limited drug release from non-degrading—or very slowly degrading—fibers. The results are consistent with a protein release mechanism dominated by desorption from the polymer surface, while the polycaprolactone nanofibers are not degrading at an appreciable rate.
Nanofiber; protein; modeling; controlled release
In our recent studies exploring the biophysical characteristics of resistant cell lipids, and the role they play in drug transport, we demonstrated the difference of drug-resistant breast cancer cells from drug-sensitive cells in lipid composition and biophysical properties, suggesting that cancer cells acquire a drug-resistant phenotype through the alteration of lipid synthesis to inhibit intracellular drug transport to protect from cytotoxic effect. In cancer cells, epigenetic changes (e.g., DNA hypermethylation) are essential to maintain this drug-resistant phenotype. Thus, altered lipid synthesis may be linked to epigenetic mechanisms of drug resistance. We hypothesize that reversing DNA hypermethylation in resistant cells with an epigenetic drug could alter lipid synthesis, changing the cell membrane’s biophysical properties to facilitate drug delivery to overcome drug resistance. Herein we show that treating drug-resistant breast cancer cells (MCF-7/ADR) with the epigenetic drug, 5-aza-2′-deoxycytidine (decitabine), significantly alters cell lipid composition and biophysical properties, causing the resistant cells to acquire biophysical characteristics similar to those of sensitive cell (MCF-7) lipids. Following decitabine treatment, resistant cells demonstrated increased sphingomyelinase activity, resulting in a decreased sphingomyelin level that influenced lipid domain structures, increased membrane fluidity, and reduced P-glycoprotein expression. Changes in the biophysical characteristics of resistant cell lipids facilitated doxorubicin transport and restored endocytic function for drug delivery with a lipid-encapsulated form of doxorubicin, enhancing the drug efficacy. In conclusion, we have established a new mechanism for efficacy of an epigenetic drug, mediated through changes in lipid composition and biophysical properties, in reversing cancer drug resistance.
Cancer cell membrane; Sphingomyelin; Drug resistance; Membrane rigidity; P-glycoprotein; Cancer therapy; Epigenetic
Helicobacter pylori (H. pylori) infection with its vast prevalence is responsible for various gastric diseases including gastritis, peptic ulcers, and gastric malignancy. While effective, current treatment regimens are challenged by a fast declining eradication rate due to the increasing emergence of H. pylori strains resistant to existing antibiotics. Therefore there is an urgent need to develop novel antibacterial strategies against H. pylori. In this study, we developed a liposomal nanoformulation of linolenic acid (LipoLLA) and evaluated its bactericidal activity against resistant strains of H. pylori. Using a laboratory strain of H. pylori, we found that LipoLLA was effective in killing both spiral and coccoid forms of the bacteria via disrupting bacterial membranes. Using a metronidazole-resistant strain of H. pylori and seven clinically isolated strains, we further demonstrated that LipoLLA eradicated all strains of the bacteria regardless of their antibiotic resistance status. Furthermore, under our experimental conditions, the bacteria did not develop drug resistance when cultured with LipoLLA at various sub-bactericidal concentrations, whereas they rapidly acquired resistance to both metronidazole and free linolenic acid (LLA). Our findings suggest that LipoLLA is a promising antibacterial nanotherapeutic to treat antibiotic-resistant H. pylori infection.
Helicobacter pylori; Bacterial infections; Free fatty acid; Linolenic acid; Liposome
The extracellular enzyme matrix metalloproteinase-9 (MMP-9) is overexpressed in atherosclerotic plaques and in metastatic cancers. The enzyme is responsible for rupture of the plaques and for the invasion and metastasis of a large number of cancers. The ability of ultrasonic excitation to induce thermal and mechanical effects has been used to release drugs from different carriers. However, majority of these studies were performed with low frequency ultrasound (LFUS) at kHz frequencies. Clinical usage of LFUS excitations will be limited due to harmful biological effects. Herein, we report our results on the release of encapsulated contents from substrate lipopeptide incorporated echogenic liposomes triggered by recombinant human MMP-9. The contents release was further enhanced by the application of diagnostic frequency (3 MHz) ultrasound. The echogenic liposomes were successfully imaged employing a medical ultrasound transducer (4 – 15 MHz). The conditioned cell culture media from cancer cells (secreting MMP-9) released the encapsulated dye from the liposomes (30 – 50%) and this release is also increased (50 – 80%) by applying diagnostic frequency ultrasound (3 MHz) for 3 minutes. With further developments, these liposomes have the potential to serve as multimodal carriers for triggered release and simultaneous ultrasound imaging.
Echogenic liposomes; MMP-9 triggered release; Ultrasound triggered release
The transfer of genetic material into cells using non-viral vectors offers unique potential for therapeutics; however, the efficacy of delivery depends upon a poorly understood, multistep pathway, limiting the prospects for successful gene delivery. Mechanistic insight into DNA association and release has been hampered by a lack of atomic resolution structural and dynamic information for DNA-polymer complexes (polyplexes). Here, we report a dendrimer-based polyplex system containing poly(ethyleneglycol) (PEG) arms that is suitable for atomic-level characterization by solution NMR spectroscopy. NMR chemical shift, linewidth, and proton transverse relaxation rate measurements reveal that free and dendrimer-bound polyplex DNA exchange rapidly relative to the NMR timescale (< millisecond). The dendrimers retain a high degree of mobility in the polyplex, whereas the DNA shows restrained mobility, suggesting that the polyplex is a highly dynamic complex with a rapidly exchanging dendrimer atmosphere around a more rigid DNA framework.
Polyplexes; polyplex NMR spectroscopy; DNA dynamics; PAMAM dendrimer dynamics; polyplex DNA release
Ribavirin is frontline treatment for hepatitis C virus infection. To determine the role of nucleoside transporters in the intestinal absorption of orally administered ribavirin, we perfused the intestines of Ent1(−/−) and wild-type mice, in situ, with [3H] ribavirin (20, 200 and 5000 μM) in the presence and absence of sodium. The decrease in luminal ribavirin concentration over 30 minutes was measured at 5-minute intervals. Blood samples were collected approximately every 10 minutes. Ribavirin plus phosphorylated metabolite concentrations (hereafter referred to as ribavirin) were determined in tissue, blood and plasma by HPLC fractionation and scintillation counting. There was no significant difference between wild-type and Ent1(−/−) mice in intestinal loss of ribavirin at any ribavirin concentration studied. Perfusions without sodium drastically reduced the intestinal loss of ribavirin in both wild-type and Ent1(−/−) mice. After 20 μM ribavirin perfusions, Ent1(−/−) intestinal tissue contained 8-fold greater ribavirin than wild-type mice (p<0.01). Ribavirin concentrations in the wild-type intestinal tissue were 70-fold higher after 200 vs. 20 μM perfusions (p<0.001), indicating saturation of intestinal ribavirin efflux and possibly other processes as well. Ribavirin plasma concentrations were significantly higher in wild-type mice (2.7-fold) vs. Ent1(−/−) mice at 30 minutes after the 20 μM perfusion (p<0.01). These results suggest that, at lower intestinal concentrations of ribavirin, concentrative and equilibrative nucleoside transporters are important in the intestinal absorption of ribavirin. At higher intestinal concentrations, these transporters are saturated and other processes in the intestine (transport and/or metabolism) play an important role in the absorption of ribavirin.
Ribavirin; nucleoside transporter; hepatitis C; absorption; perfusion
Most diagnosed early stage breast cancer cases are treated by lumpectomy and adjuvant radiation therapy, which significantly decreases the locoregional recurrence but causes inevitable toxicity to normal tissue. By using a technique of preparing liposomes carrying technetium-99m (99mTc), rhenium-186 (186Re), or rhenium-188 (188Re) radionuclides, as well as chemotherapeutic agents, or their combination, for cancer therapy with real time image-monitoring of pharmacokinetics and prediction of therapy effect, this study investigated the potential of a novel targeted focal radiotherapy with low systemic toxicity using radioactive immunoliposomes to treat both the surgical cavity and draining lymph nodes in a rat breast cancer xenograft positive surgical margin model. Immunoliposomes modified with either panitumumab (anti-EGFR), or bevacizumab (anti-VEGF) were remote loaded with 99mTc diagnostic radionuclide, and injected into the surgical cavity of female nude rats with positive margins post lumpectomy. Locoregional retention and systemic distribution of 99mTc-immunoliposomes were investigated by nuclear imaging, stereofluorescent microscopic imaging and gamma counting. Histopathological examination of excised draining lymph nodes was performed. The locoregional retention of 99mTc-immunoliposomes in each animal was influenced by the physiological characteristics of surgical site of individual animals. Panitumumab- and bevacizumab-liposome groups had higher intracavitary retention compared with the control liposome groups. Draining lymph node uptake was influenced by both the intracavitary radioactivity retention level and metastasis status. Panitumumab-liposome group had higher accumulation on the residual tumor surface and in the metastatic lymph nodes. Radioactive liposomes that were cleared from the cavity were metabolized quickly and accumulated at low levels in vital organs. Therapeutic radionuclide-carrying specifically targeted panitumumab- and bevacizumab- liposomes have increased potential compared to non-antibody targeted liposomes for post-lumpectomy focal therapy to eradicate remaining breast cancer cells inside the cavity and draining lymph nodes with low systemic toxicity.
breast cancer; targeted therapy; immunoliposomes; intracavitary injection; metastasis
Griffithsin (GRFT) is a lectin that has been shown to inhibit HIV infection by binding to high mannose glycan structures on the surface of gp120, and is among the most potent HIV entry inhibitors reported so far. However, important biochemical details on the antiviral mechanism of GRFT action remain unexplored. In order to understand the role of the three individual carbohydrate-binding sites (CBS) in GRFT, mutations were made at each site (D30A, D70A, and D112A), and the resulting mutants were investigated. NMR studies revealed that each GRFT variant was folded but showed significant peak movement on the carbohydrate-binding face of the protein. The wild-type and each point mutant protein appeared as tight dimers with a Kd below 4.2 µM. Mutation of any individual CBS on GRFT reduced binding of the protein to mannose, and ELISA assays revealed a partial loss of ability of each GRFT point mutant to bind gp120, with a near-complete loss of binding by the triple mutant D30A/D70A/D112A GRFT. A more quantitative surface plasmon resonance (SPR) examination showed a rather small loss of binding to gp120 for the individual GRFT point mutants (KD: 123 to 245 pM range versus 73 pM for wild-type GRFT), but dramatic loss of the triple mutant to bind gp120 derived from R5 and X4 strains (KD > 12 nM). In contrast to the 2- to 3-fold loss of binding to gp120, the single CBS point mutants of GRFT were significantly less able to inhibit viral infection, exhibiting a 26- to 1900-fold loss of potency, while the triple mutant was at least 875 fold less effective against HIV-1 infection. The disparity between HIV-1 gp120 binding ability and HIV inhibitory potency for these GRFT variants indicates that gp120 binding and virus neutralization do not necessarily correlate, and suggests a mechanism that is not based on simple gp120 binding.
Griffithsin (GRFT); lectin; HIV entry inhibitor; microbicide; carbohydrate binding site (CBS); nuclear magnetic resonance (NMR); mannose titration; analytical ultracentrifugation; surface plasmon resonance (SPR)
This review summarizes the in vivo assessment--preliminary, preclinical, and clinical--of chemotherapeutics derived from camptothecin or a derivative. Camptothecin is a naturally occurring, pentacyclic quinoline alkaloid that possesses high cytotoxic activity in a variety of cell lines. Major limitations of the drug, including poor solubility and inactivity at physiological conditions, prevent full clinical utilization. Camptothecin remains at equilibrium in an active lactone form and inactive hydrolyzed carboxylate form. The active lactone binds to DNA topoisomerase I cleavage complex, believed to be the single site of activity inhibiting DNA religation, resulting in apoptosis. A series of small molecule camptothecin derivatives have been developed that increase solubility, lactone stability and bioavailability to varying levels of success. A number of macromolecular agents have also been described wherein camptothecin(s) are covalently appended or non-covalently associated with the goal of improving solubility and lactone stability, while taking advantage of the tumor physiology to deliver larger doses of drug to the tumor with lower systemic toxicity. With the increasing interest in drug delivery and polymer therapeutics, additional constructs are anticipated. The goal of this review is to summarize the relevant literature for others interested in the field of camptothecin-based therapeutics, specifically in the context of biodistribution, dosing regimens, and pharmacokinetics with the desire of providing a useful source of comparative data. To this end, only constructs where in vivo data is available are reported. The review includes published reports in English through mid-2009.
Camptothecins; topoisomerase I inhibitors; polymer therapeutics; in vivo; cancer therapy
Immunostimulatory sequences (ISS) are short DNA sequences containing unmethylated CpG dimers that have multiple effects on the host immune system, including the ability to stimulate antigen-specific cytotoxic T lymphocytes (CTLs) and drive Th1-type immune responses. Listeriolysin O (LLO)-containing pH-sensitive liposomes have been shown to efficiently deliver macromolecules to the cytosol of APCs and efficiently stimulate CTLs. We hypothesized that encapsulating ISS-oligodeoxyribonucleotides (ODNs) in this delivery system would enhance the cell-mediated immune response and skew Th1-type responses in protein antigen-based vaccination utilizing LLO-liposomes. In vitro studies indicated that co-encapsulation of ISS in LLO-liposomes engendered activation of the NF-κB pathway while maintaining the efficient cytosolic delivery of antigen mediated by the co-encapsulated LLO. Antigen-specific CTL responses monitored by using the model antigen ovalbumin (OVA) in mice were enhanced when mice were immunized with OVA and ISS-ODN-containing LLO-liposomes compared with those immunized with either OVA-containing LLO-liposomes or OVA-ISS conjugates. The enhanced immune responses were of the Th1-type as monitored by the robust OVA-specific IgG2a induction and the OVA CD8 peptide-stimulated IFN-γ secretion. Our study suggests that including ISS-ODN in LLO-containing pH-sensitive liposomes yields a vaccine delivery system that enhances the cell-mediated immune response and skews this response toward the Th1-type.
liposome; listeriolysin O; ISS-ODN
The emergence and re-emergence of bacterial strains that are resistant to current antibiotics reveals the clinical need for new agents that possess broad-spectrum antibacterial activity. Furthermore, bacteriophobic coatings that repel bacteria are important for medical devices, as the lifetime, reliability, and performance of implant devices are hindered by bacterial adhesion and infection. Dendrimers, a specific class of monodisperse macromolecules, have recently shown potential to function as both antibacterial agents as well as antimicrobial surface coatings. This review discusses the limitations with currently used antibacterial agents and describes how various classes of dendrimers, including glycodendrimers, cationic dendrimers, anionic dendrimers, and peptide dendrimers, have the potential to improve upon or replace certain antibiotics. Furthermore, the unexplored areas in this field of research will be mentioned to present opportunities for additional studies regarding the use of dendrimers as antimicrobial agents.
Dendrimer; dendritic polymers; bacteria; antimicrobial; infection; antibacterial agents; coatings; carriers; drug delivery
The chemokine receptor 4 (CXCR4), which is over-expressed in many types of cancer, is an emerging target in the field of molecular imaging and therapeutics. The CXCR4 binding of several peptides, including the cyclic Ac-TZ14011, has already been validated. In this study mono-, di- and tetrameric Ac-TZ14011-containing dendrimers were prepared and functionalized with a multimodality hybrid label, consisting of a Cy5.5-like flourophore and a DTPA chelate. Confocal microscopy revealed that all three dendrimers could target CXCR4 in vitro. The unlabeled dimer and tetramer had a slightly lower affinity for CXCR4 than the unlabeled monomer. However, when labeled with the multimodal label the CXCR4 affinity of the dimer and tetramer was significantly higher compared to the labeled monomer. On top of that, biodistribution studies revealed that the additional peptides in the dimer and tetramer reduced nonspecific tissue binding. Thus, multimerization of the cyclic Ac-TZ14011 peptide reduces the negative influence of the multimodal label on the receptor affinity and the biodistribution.
CXCR4; peptides; dendrimers; multimodal hybrid imaging; fluorescence; SPECT/CT
Amphotericin B (AmB), a well-known polyene antifungal agent displays a marked tendency to self-associate and, as a consequence, exhibits very poor solubility in water. The therapeutic index of AmB is low, and is associated with significant dose-related nephrotoxicity, as well as acute, infusion-related febrile reactions. Reports in the literature indicate that that toxicity of AmB may be related to the physical state of the drug. Reaction of AmB in dimethylformamide with bis(dimethylaminopropyl)carbodiimide yielded an unexpected N-alkylguanidine/N-acylurea bis-adduct of AmB which was highly water soluble. The absorption spectrum of the AmB derivative in water indicated excellent monomerization, and the anti-fungal activities of reference AmB and its water-soluble derivative against C. albicans were found to be virtually identical. Furthermore, the water-soluble adduct is significantly less active in engaging TLR4 which would suggest that the adduct may be less proinflammatory.
Amphotericin B; solubility; toxicity; aggregation; carbodiimide; antifungal; toll-like receptors
Neovascularization (NV) in the cornea is a major cause of vision impairment and corneal blindness. Hemangiogenesis and lymphangiogenesis induced by inflammation underlie the pathogenesis of corneal NV. The current mainstay treatment, corticosteroid, treats the inflammation associated with corneal NV, but is not satisfactory due to such side effects as cataract and the increase in intraocular pressure. It is imperative to develop a novel therapy that specifically targets the hemangiogenesis, lymphangiogenesis and inflammation pathways underlying corneal NV. Histone deacetylase inhibitors (HDACi) have been in clinical trials for cancer and other diseases. In particular, HDACi suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. The functional mechanism of SAHA in cancer and especially in corneal NV remains unclear. Here, we show that topical application of SAHA inhibits neovascularization in an alkali-burn corneal injury model. Mechanistically, SAHA inhibits corneal NV by repressing hemangiogenesis, inflammation pathways and previously overlooked lymphangiogenesis. Topical SAHA is well tolerated on the ocular surface. In addition, the potency of SAHA in corneal NV appears to be comparable to the current steroid therapy. SAHA may possess promising therapeutic potential in alkali-burn corneal injury and other inflammatory neovascularization disorders.
corneal neovascularization; HDAC; HDAC inhibitor; hemangiogenesis; lymphangiogenesis; inflammation; alkali-burn injury
The objective of this study was to enhance oral mucosal permeation of fenretinide by co-incorporation of propylene glycol (PG) and menthol in fenretinide/Eudragit® RL PO mucoadhesive patches. Fenretinide is an extremely hydrophobic chemopreventive compound with poor tissue permeability. Co-incorporation of 5-10 wt% PG (mean Js = 16-23 μg cm−2 h−1; 158-171 μg fenretinide/g tissue) or 1-10 wt% PG + 5 wt% menthol (mean Js = 18-40 μg cm−2 h−1; 172-241 μg fenretinide/g tissue) in fenretinide/Eudragit® RL PO patches led to significant ex vivo fenretinide permeation enhancement (p < 0.001). Addition of PG above 2.5 wt% in the patch resulted in significant cellular swelling in the buccal mucosal tissues. These alterations were ameliorated by combining both enhancers and reducing PG level. After buccal administration of patches in rabbits, in vivo permeation of fenretinide across the oral mucosa was greater (~43 μg fenretinide/g tissue) from patches that contained optimized permeation enhancer content (2.5 wt% PG + 5 wt% menthol) relative to permeation obtained from enhancer-free patch (~ 17 μg fenretinide/g tissue) (p < 0.001). In vitro and in vivo release of fenretinide from patch was not significantly increased by co-incorporation of permeation enhancers, indicating that mass transfer across the tissue, and not the patch, largely determined the permeation rate control in vivo. As a result of its improved permeation and its lack of deleterious local effects, the mucoadhesive fenretinide patch co-incorporated with 2.5 wt% PG + 5 wt% menthol represents an important step in the further preclinical evaluation of oral site-specific chemoprevention strategies with fenretinide.
fenretinide; oral mucosa; local drug delivery; mucoadhesive patch; permeation enhancement; propylene glycol; menthol
Multidrug resistance (MDR) is a major cause of failure in cancer chemotherapy. Tocopheryl polyethylene glycol 1000 succinate (TPGS) has been extensively explored for the treatment of MDR in cancer because of its ability to inhibit P-glycoprotein. Here, we have established multifunctional nanoparticles (MFNPs) using a single-molecule modification of TPGS, which can deliver a hydrophobic drug, paclitaxel (PTX), and a hydrophilic drug, fluorouracil (5-FU), and overcome MDR in cancer. Our data indicated that, when delivered into a PTX-resistant cell line using MFNPs, the combination of PTX and 5-FU was more cytotoxic than each agent individually.
multidrug resistance; TPGS; paclitaxel; multifunctional nanoparticles
We examined the mechanism of accumulation of charged polynuclear platinum complexes (PPCs), based on analogy of polyarginine interactions with the cell surface heparan sulfate proteoglycan (HSPG) family of protein-linked glycosoaminoglycan polysaccharides (GAGs). GAGS such as heparan sulfate (HS) and chondroitin sulfate (CS) mediate the cellular entry of many charged molecules. Fluorescence microscopy and flow cytometry showed that PPCs, but not the neutral cisplatin or oxaliplatin, blocked the cellular entry of TAMRA-R9 (a nonarginine peptide, R9) coupled to the TAMRA fluorescent label 5-(and 6-)carboxytetramethylrhodamine) in Chinese Hamster Ovary (CHO), human colon carcinoma (HCT116), and osteosarcoma (SAOS-2) cells. Furthermore, detection of platinum accumulation in wt CHO, mutant CHO-pgsD-677 (lacking HS), and CHO-pgsA (lacking HS/CS) cells confirms that HSPG-mediated interactions are an important mechanism for PPC internalization, but not so for uncharged cisplatin and oxaliplatin. Endocytosis inhibitor studies show that macropinocytosis, a mechanism of cell entry for heparan sulfate GAGs and arginine-rich peptides, is important in the cellular accumulation of “non-covalent” TriplatinNC, and to a lesser degree, the covalently-binding BBR3464. Clathrin-mediated endocytosis, however, was not involved in either case. Overall the results suggest a new proteoglycan-mediated mechanism for cellular accumulation of PPCs not shared by cisplatin or oxaliplatin. The results have significant implications for rational design of platinum antitumor drugs with distinct biological profiles in comparison to the clinically-used agents as well as expanding the chemotypes for HS proteoglycan-dependent receptors.
Triplatinum compounds; cellular accumulation; cisplatin resistance; heparan sulfate; glycosaminoglycans
Poly(amidoamine) (PAMAM) dendrimers have been proposed for a variety of biomedical applications and are increasingly studied as model nanomaterials for such use. The dendritic structure features both modular synthetic control of molecular size and shape and presentation of multiple equivalent terminal groups. These properties make PAMAM dendrimers highly functionalizable, versatile single-molecule nanoparticles with a high degree of consistency and low polydispersity. Recent nanotoxicological studies showed that intravenous administration of amine-terminated PAMAM dendrimers to mice was lethal, causing a disseminated intravascular coagulation-like condition. To elucidate the mechanisms underlying this coagulopathy, in vitro assessments of platelet functions in contact with PAMAM dendrimers were undertaken. This study demonstrates that cationic G7 PAMAM dendrimers activate platelets and dramatically alter their morphology. These changes to platelet morphology and activation state substantially altered platelet function, including increased aggregation and adherence to surfaces. Surprisingly, dendrimer exposure also attenuated platelet-dependent thrombin generation, indicating that not all platelet functions remained intact. These findings provide additional insight into PAMAM dendrimer effects on blood components and underscore the necessity for further research on the effects and mechanisms of PAMAM-specific and general nanoparticle toxicity in blood.
nanotoxicity; PAMAM dendrimers; platelet activation; biocompatibility; thrombin generation
With the recent growth of the global monoclonal antibody market, ultrasensitive techniques are required for rapid analysis of possible immunogenic residues, such as galactose-α-1,3-galactose (α-1,3-Gal) on therapeutic proteins expressed in murine or CHO cell lines. We report a capillary electrophoretic approach in conjunction with exoglycosidase digestion for structural elucidation of N-linked IgG glycans containing the above immunogenic epitope. The method uses commercially available reagents and instrumentation, thus making the described methodology readily available for implementation and validation within the biotechnology industry. The method was first evaluated using polyclonal mouse IgG N-glycans which are known to contain α-1,3-Gal containing epitopes. High reproducibility in migration time enabled determination of GU values for five α-1,3-Gal containing structures. The method was successfully applied to the analysis of a NCI reference standard monoclonal antibody and two development phase monoclonal antibodies. The limit of detection and limit of quantitation were 1 and 2 µg of intact protein IgG starting material, respectively, further indicating the high sensitivity of the described method.
therapeutic antibody; monoclonal antibody; alpha galactose; immunogenic epitopes; capillary electrophoresis; exoglycosidase digestion; glycomics
Our aim was to explore the involvement of the transcriptional suppressor GCF2 in silencing RhoA, disorganization of the cytoskeleton, mislocalization of MRP1, and sensitivity to anti-cancer agents as an upstream gene target in cancer therapy. Increased expression of GCF2 was found in human cisplatin-resistant cells, and overexpression in GCF2-transfected cells results in loss of RhoA expression and disruption of the actin-filamin network. In consequence, the membrane transporter MRP1 was internalized from the cell surface into the cytoplasm, rendering cells sensitive to doxorubicin by more than 10-fold due to increased accumulation of doxorubicin in the cells. The GCF2 transfectants also showed reduced accumulation of cisplatin and increased resistance. siRNA targeted to GCF2 suppressed the expression of GCF2 in cisplatin-resistant cells, re-activated RhoA expression, and restored the fine structure of actin microfilaments. MRP1 was also relocated to the cell surface. siRNA targeted to RhoA increased resistance 3-fold in KB-3-1 and KB-CP.5 cells. These data for the first time demonstrate a novel complex regulatory pathway downstream from GCF2 involving the small GTPase RhoA, actin/filamin dynamics, and membrane protein trafficking. This pathway mediates diverse responses to cytotoxic compounds, and also provides a molecular basis for further investigation into the pleiotropic resistance mechanism at play in cisplatin-resistant cells.
chemotherapy; trafficking; cytoskeleton; cisplatin resistance; doxorubicin; cisplatin
Although siRNA techniques have been broadly applied as a tool for gene knockdown, substantial challenges remain in achieving efficient delivery and in vivo efficacy. In particular, the low efficiency of target gene silencing in vivo is a critical limiting step to the clinical application of siRNA therapies. Poly(amidoamine) (PAMAM) dendrimers are widely used as carriers for drug and gene delivery; however, in vivo siRNA delivery by PAMAM dendrimers remains to be carefully investigated. In this study, the effectiveness of G5 and G6 PAMAM dendrimers with 8% of their surface amines conjugated to MPEG-5000 was studied for siRNA delivery in vitro and for intramuscular in vivo delivery in mice. The results from the PEG-modified dendrimers were compared to the parent dendrimers as well as Lipofectamine 2000 and INTERFERin. Both PEG-modifed dendrimers protect the siRNA from being digested by RNase and gave high transfection efficiency for FITC-labeled siRNA in the primary vascular smooth muscle cells (VSMC) and mouse peritoneal macrophages. The PEG-modified dendrimers achieved knockdown of both plasmid (293A cells) and adenovirus-mediated green fluorescence protein (GFP) expression (Cos7 cells) in vitro with efficiency similar to that shown for Lipofectamine 2000. We further demonstrated in vivo that intramuscular delivery of GFP-siRNA using PEG-modified dendrimer significantly suppressed GFP expression in both transiently adenovirus infected C57BL/6 mice and in GFP transgenic mice.
PEG-conjugated PAMAM dendrimer; cytotoxicity; RNase stability; in vitro and intramuscular siRNA delivery; gene silencing efficiency
We have developed FINDSITEX, an extension of FINDSITE, a protein threading based algorithm for the inference of protein binding sites, biochemical function and virtual ligand screening, that removes the limitation that holo protein structures (those containing bound ligands) of a sufficiently large set of distant evolutionarily related proteins to the target be solved; rather, predicted protein structures and experimental ligand binding information are employed. To provide the predicted protein structures, a fast and accurate version of our recently developed TASSERVMT, TASSERVMT-lite, for template-based protein structural modeling applicable up to 1000 residues is developed and tested, with comparable performance to the top CASP9 servers. Then, a hybrid approach that combines structure alignments with an evolutionary similarity score for identifying functional relationships between target and proteins with binding data has been developed. By way of illustration, FINDSITEX is applied to 998 identified human G-protein coupled receptors (GPCRs). First, TASSERVMT-lite provides updates of all human GPCR structures previously modeled in our lab. We then use these structures and the new function similarity detection algorithm to screen all human GPCRs against the ZINC8 non-redundant (TC<0.7) ligand set combined with ligands from the GLIDA database (a total of 88,949 compounds). Testing (excluding GPCRs whose sequence identity > 30% to the target from the binding data library) on a 168 human GPCR set with known binding data, the average enrichment factor in the top 1% of the compound library (EF0.01) is 22.7, whereas EF0.01 by FINDSITE is 7.1. For virtual screening when just the target and its native ligands are excluded, then the average EF0.01 reaches 41.4. We also analyze off-target interactions for the 168 protein test set. All predicted structures, virtual screening data and off-target interactions for the 998 human GPCRs are available at http://cssb.biology.gatech.edu/skolnick/webservice/gpcr/index.html.
TASSERVMT; FINDSITE; GPCR modeling; template-based modeling; virtual screening