Formulation optimization of lyophilized aptamer-gold nanoparticles: Maintained colloidal stability and cellular uptake.

Anti-nucleolin (NCL) aptamer AS1411 is the first anticancer aptamer tested in clinical trials. Gold nanoparticles (AuNP) have been widely exploited for various biomedical applications due to their unique functional properties. In this study, we evaluated the colloidal stability and targeting capacity of AS1411-funtionalized AuNP (AuNP/NCL-Apt) against MCF-7 breast cancer cell line before and after lyophilization. Trehalose, mannitol, and sucrose at various concentrations were evaluated to determine their cryoprotection effects. Our results indicate that sucrose at 10 % (w/v) exhibits the best cryoprotection effect and minimal AuNP/NCL-Apt aggregation as confirmed by UV-Vis spectroscopy and dynamic light scattering (DLS) measurements. Moreover, the lyophilized AuNP/NCL-Apt at optimized formulation maintained its targeting and cytotoxic functionality against MCF-7 cells as proven by the cellular uptake assays utilizing flow cytometry and confocal laser scanning microscopy (CLSM). Quantitative PCR (qPCR) analysis of nucleolin-target gene expression also confirmed the effectiveness of AuNP/NCL-Apt. This study highlights the importance of selecting the proper type and concentration of cryoprotectant in the typical nanoparticle lyophilization process and contributes to our understanding of the physical and biological properties of functionalized nanoparticles upon lyophilization.

Preparation, Optimization and In vitro Evaluation of Doxorubicin-loaded into Hyaluronic Acid Coated Niosomes Against Breast Cancer.

Doxorubicin (DOX) is widely used against solid tumors. Niosomes are self-assembled nanocarriers of non-ionic surfactants. DOX loaded into cationic niosomes (DOX-Nio) was prepared via thin film hydration method. DOX-Nio was then decorated with a hyaluronic acid (DOX-HA-Nio) via electrostatic interaction. DOX-Nio and DOX-HA-Nio displayed a particle size of 120.0±1.02 and 182.9±2.3 nm, and charge of + 35.5±0.15 and -15.6±0.25 mV, respectively, with PDI < 0.3. DOX-HA-Nio showed a good stability regarding size and charge over 4 weeks at 4 °C and maintain their integrity after lyophilization. HPLC results showed a 94.1±4.2 % encapsulation efficiency of DOX with good entrapment and slow, prolonged DOX release even after 48 hrs. Cell viability assay showed an IC50 of 14.26 nM for the DOX-HA-Nio against MCF-7 cell line with micromolar IC50 results against CD-44 negative cell lines (NIH/3T3). DOX-HA-Nio was proven to be an effective, targeted nanocarrier for DOX against MCF-7 cell line.

Biological Performance of Primary Dental Pulp Stem Cells Treated with Gold Nanoparticles.

Gold nanoparticles (AuNPs) are one of the most stable nanoparticles that have been prevalently used as examples for biological and biomedical applications. Herein, we evaluate the effect of AuNPs on the biological processes of dental pulp stem cells derived from exfoliated deciduous teeth (SHED). Two different shapes of PEGylated AuNPs, rods (AuNR-PEG) and spheres (AuNS-PEG), were prepared and characterized. SHED cells were treated with different concentrations of AuNR-PEG and AuNS-PEG to determine their effect on the stemness profile of stem cells (SCs), proliferation, cytotoxicity, cellular uptake, and reactive oxygen species (ROS), for cells cultured in media containing-fetal bovine serum (FBS) and serum-free media (SFM). Our results showed that both nanoparticle shapes maintained the expression profile of MSC surface markers. Moreover, AuNS-PEG showed a stimulatory effect on the proliferation rate and lower toxicity on SHED, compared to AuNR-PEG. Higher concentrations of 0.5-0.125 nM of AuNR-PEG have been demonstrated to cause more toxicity in cells. Additionally, cells treated with AuNPs and cultured in FBS showed a higher proliferative rate and lower toxicity when compared to the SFM. For cellular uptake, both AuNS-PEG and AuNR-PEG were uptaken by treated cells efficiently. However, cells cultured in SFM media showed a higher percentage of cellular uptake. For ROS, AuNR-PEG showed a significant reduction in ROS at lower concentrations (<0.03 nM), while AuNS-PEG did not show any significant difference compared to the control untreated cells. Thus, our results give evidence about the optimum concentration and shape of AuNPs that can be used for the differentiation of stem cells into specific cell lineages in tissue engineering and regenerative medicine.

Synergistic combination of doxorubicin with hydralazine, and disulfiram against MCF-7 breast cancer cell line.

Disulfiram and hydralazine have recently been reported to have anti-cancer action, and repositioned to be used as adjuvant in cancer therapy. Chemotherapy combined with other medications, such as those that affect the immune system or epigenetic cell profile, can overcome resistance with fewer adverse effects compared to chemotherapy alone. In the present study, a combination of doxorubicin (DOX) with hydrazine (Hyd) and disulfiram (Dis), as a triple treatment, was evaluated against wild-type and DOX-resistant MCF-7 breast cancer cell line. Both wild-type MCF-7 cell line (MCF-7_WT) and DOX-resistant MCF-7 cell line (MCF-7_DoxR) were treated with different combination ratios of DOX, Dis, and Hyd followed by measuring the cell viability using the MTT assay. Synergism was determined using a combination index, isobologram analysis, and dose-reducing index. The anti-proliferation activity and mechanism of the triple combination were investigated by apoptosis analysis. The results showed a reduction in the IC50 values of DOX in MCF-7_WT cells (from 0.24 µM to 0.012 µM) and MCF-7_DoxR cells (from 1.13 µM to 0.44 µM) when treated with Dis (0.03µM), and Hyd (20µM) combination. Moreover, The triple combination DOX/Hyd/Dis induced significant apoptosis in both MCF-7_WT and MCF-7_DoxR cells compared to DOX alone. The triple combination of DOX, Dis, and Hyd showed a synergistic drugs combination to decrease the DOX dose needed to kill both MCF-7_WT and MCF-7_DoxR cancer cells and enhanced chemosensitivity to DOX.

Micafungin effect on Pseudomonas aeruginosa metabolome, virulence and biofilm: potential quorum sensing inhibitor.

The prevalence of antibiotic resistance in Pseudomonas aeruginosa places a heavy burden on the health care sectors urging the need to find alternative, non-antibiotic strategies. The interference with the P. aeruginosa quorum sensing (QS) system represents a promising alternative strategy to attenuate the bacterial virulency and its ability to form biofilms. Micafungin has been reported to impede the pseudomonal biofilm formation. However, the influences of micafungin on the biochemical composition and metabolites levels of P. aeruginosa have not been explored. In this study, the effect of micafungin (100 µg/mL) on the virulence factors, QS signal molecules and the metabolome of P. aeruginosa was studied using exofactor assay and mass spectrometry-based metabolomics approaches. Furthermore, confocal laser scanning microscopy (CLSM) using the fluorescent dyes ConA-FITC and SYPRO® Ruby was used to visualize micafungin disturbing effects on the pseudomonal glycocalyx and protein biofilm-constituents, respectively. Our findings showed that micafungin significantly decreased the production of various QS-controlled virulence factors (pyocyanin, pyoverdine, pyochelin and rhamnolipid), along with a dysregulation in the level of various metabolites involved in QS system, lysine degradation, tryptophan biosynthesis, TCA cycle, and biotin metabolism. In addition, the CLSM examination showed an altered matrix distribution. The presented findings highlight the promising role of micafungin as a potential quorum sensing inhibitor (QSI) and anti-biofilm agent to attenuate P. aeruginosa pathogenicity. In addition, they point to the promising role of metabolomics study in investigating the altered biochemical pathways in P. aeruginosa.

Effects of cyclic acute and chronic hypoxia on the expression levels of metabolism related genes in a pancreatic cancer cell line.

The aim of this study was to characterize cycling hypoxia-induced changes in the expression of metabolism-related genes in the pancreatic cancer cell line PANC1. PANC1 cells were exposed to either 7 h cycles of hypoxia every other day for 20 cycles (cyclic acute hypoxia), or for 72 h cycles of hypoxia once a week for 5 cycles (cyclic chronic hypoxia). Changes in gene expression were profiled using reverse transcription-quantitative PCR and compared to cells cultured under normoxic conditions. Western blotting analysis confirmed upregulation of HIF1-α, glucose-6-phosphate isomerase, and ribokinase at the mRNA level. Upregulation in genes encoding enzymes involved in glycolysis was greater in cells cultured under cyclic acute hypoxia compared with cells cultured under chronic hypoxia including hexokinase2 and phosphoglycerate kinase 1. Genes encoding the pentose phosphate pathway (PPP) enzymes (transketolase and transaldolase) were upregulated to a similar degree. The expression of genes encoding pyruvate dehydrogenases that block pyruvate flow to the TCA cycle was significantly upregulated. Thus, exposure of PANC1 cells to acute hypoxia resulted in the upregulation of genes that shift the metabolism of cells towards glycolysis and the pentose phosphate pathway (PPP) in adaptation to hypoxic stress.

Acetic Acid Mediated for One-Pot Synthesis of Novel Pyrazolyl s-Triazine Derivatives for the Targeted Therapy of Triple-Negative Breast Tumor Cells (MDA-MB-231) via EGFR/PI3K/AKT/mTOR Signaling Cascades.

Here, we described the synthesis of novel pyrazole-s-triazine derivatives via an easy one-pot procedure for the reaction of ß-dicarbonyl compounds (ethylacetoacetate, 5,5-dimethyl-1,3-cyclohexadione or 1,3-cyclohexadionone) with N,N-dimethylformamide dimethylacetal, followed by addition of 2-hydrazinyl-4,6-disubstituted-s-triazine either in ethanol-acetic acid or neat acetic acid to afford a novel pyrazole and pyrazole-fused cycloalkanone systems. The synthetic protocol proved to be efficient, with a shorter reaction time and high chemical yield with broad substrates. The new pyrazolyl-s-triazine derivatives were tested against the following cell lines: MCF-7 (breast cancer); MDA-MB-231 (triple-negative breast cancer); U-87 MG (glioblastoma); A549 (non-small cell lung cancer); PANC-1 (pancreatic cancer); and human dermal fibroblasts (HDFs). The cell viability assay revealed that most of the s-triazine compounds induced cytotoxicity in all the cell lines tested. However, compounds 7d, 7f and 7c, which all have a piperidine or morpholine moiety with one aniline ring or two aniline rings in their structures, were the most effective. Compounds 7f and 7d showed potent EGFR inhibitory activity with IC50 values of 59.24 and 70.3 nM, respectively, compared to Tamoxifen (IC50 value of 69.1 nM). Compound 7c exhibited moderate activity, with IC50 values of 81.6 nM. Interestingly, hybrids 7d and 7f exerted remarkable PI3K/AKT/mTOR inhibitory activity with 0.66/0.82/0.80 and 0.35/0.56/0.66-fold, respectively, by inhibiting their concentrations to 4.39, 37.3, and 69.3 ng/mL in the 7d-treatment, and to 2.39, 25.34 and 57.6 ng/mL in the 7f-treatment compared to the untreated control.

Synthesis of Mono-Amino Substituted γ-CD: Host-Guest Complexation and In Vitro Cytotoxicity Investigation.

Cyclodextrins (CDs) are cyclic oligosaccharides which can trap hydrophobic molecules and improve their chemical, physical, and biological properties. γ-CD showed the highest aqueous solubility with the largest cavity diameter among other CD types. The current study describes a direct and easy method for nucleophilic mono-aminos to be substituted with γ-CD and tested for their ability to host the guest curcumin (CUR) as a hydrophobic drug model. The mass spectrometry and NMR analyses showed the successful synthesis of three amino-modified γ-CDs: mono-6-amino-6-deoxy-cyclodextrine (γ-CD-NH2), mono-6-deoxy-6-ethanolamine-γ-cyclodextrine (γ-CD-NHCH2CH2OH), and mono-6-deoxy-6-aminoethylamino)-γ-cyclodextrin (γ-CD-NHCH2CH2NH2). These three amino-modified γ-CDs were proven to be able to host CUR as native γ-CDs with formation constants equal to 6.70 ± 1.02, 5.85 ± 0.80, and 8.98 ± 0.90 mM-1, respectively. Moreover, these amino-modified γ-CDs showed no significant toxicity against human dermal fibroblast cells. In conclusion, the current work describes a mono-substitution of amino-modified γ-CDs that can still host guests and showed low toxicity in human dermal fibroblasts cells. Therefore, the amino-modified γ-CDs can be used as a carrier host and be conjugated with a wide range of molecules for different biomedical applications, especially for active loading methods.

Aptamer-Aptamer Chimera for Targeted Delivery and ATP-Responsive Release of Doxorubicin into Cancer Cells.

Aptamers offer a great opportunity to develop innovative drug delivery systems that can deliver cargos specifically into targeted cells. In this study, a chimera consisting of two aptamers was developed to deliver doxorubicin into cancer cells and release the drug in cytoplasm in response to adenosine-5'-triphosphate (ATP) binding. The chimera was composed of the AS1411 anti-nucleolin aptamer for cancer cell targeting and the ATP aptamer for loading and triggering the release of doxorubicin in cells. The chimera was first produced by hybridizing the ATP aptamer with its complementary DNA sequence, which is linked with the AS1411 aptamer via a poly-thymine linker. Doxorubicin was then loaded inside the hybridized DNA region of the chimera. Our results show that the AS1411-ATP aptamer chimera was able to release loaded doxorubicin in cells in response to ATP. In addition, selective uptake of the chimera into cancer cells was demonstrated using flow cytometry. Furthermore, confocal laser scanning microscopy showed the successful delivery of the doxorubicin loaded in chimeras to the nuclei of targeted cells. Moreover, the doxorubicin-loaded chimeras effectively inhibited the growth of cancer cell lines and reduced the cytotoxic effect on the normal cells. Overall, the results of this study show that the AS1411-ATP aptamer chimera could be used as an innovative approach for the selective delivery of doxorubicin to cancer cells, which may improve the therapeutic potency and decrease the off-target cytotoxicity of doxorubicin.

Curcumin-tannic acid-poloxamer nanoassemblies enhance curcumin's uptake and bioactivity against cancer cells in vitro.

Curcumin (CUR) is a bioactive natural compound with potent antioxidant and anticancer properties. However, its poor water solubility has been a major limitation against its widespread clinical use. The aim of this study was to develop a nanoscale formulation for CUR to improve its solubility and potentially enhance its bioactivity, by leveraging the self-assembly behavior of tannic acid (TA) and amphiphilic poloxamers to form CUR-entrapped nanoassemblies. To optimize drug loading, formulation variables included the CUR: TA ratio and the type of amphiphilic polymer (Pluronic® F-127 or Pluronic® P-123). The optimal CUR nanoparticles (NPs) were around 200 nm in size with a high degree of monodispersity and 56% entrapment efficiency. Infrared spectroscopy confirmed the presence of intermolecular interactions between CUR and the NP formulation components. X-ray diffraction revealed that CUR was entrapped in the NPs in an amorphous state. The NPs maintained excellent colloidal stability under various conditions. In vitro release of CUR from the NPs showed a biphasic controlled release pattern up to 72 h. Antioxidant and antiproliferative assays against a panel of human cancer cell lines revealed significantly higher activity for CUR NPs compared to free CUR, particularly in MCF-7 and MDA-MB-231 breast cancer cells. This was attributed to greater cellular uptake of the NPs compared to the free drug as verified by confocal microscopy imaging and flow cytometry measurements. Our findings present a highly promising NP delivery platform for CUR prepared via a simple self-assembly process with the ability to potentiate its bioactivity in cancer and other diseases where oxidative stress is implicated.

A Potential MRI Agent and an Anticancer Drug Encapsulated within CPMV Virus-Like Particles.

BACKGROUND: Virus nanoparticles have been extensively studied over the past decades for theranostics applications. Viruses are well-characterized, naturally occurring nanoparticles that can be produced in high quantity with a high degree of similarity in both structure and composition. OBJECTIVES: The plant virus Cowpea Mosaic Virus (CPMV) has been innovatively used as a nanoscaffold. Utilization of the internal cavity of empty Virus-Like Particles (VLPs) for the inclusion of therapeutics within the capsid has opened many opportunities in drug delivery and imaging applications. METHODS: The encapsidation of magnetic materials and anticancer drugs was achieved. SuperscriptCPMV denotes molecules attached to the external surface of CPMV and CPMVSubscript denotes molecules within the interior of the capsid. RESULTS: Here, the generation of novel VLPs incorporating iron-platinum nanoparticles TCPMVFePt and cisplatin (Cis) (TCPMVCis) is reported. TCPMVCis exhibited a cytotoxic IC50 of TCPMVCis on both A549 and MDA-MB-231 cell lines of 1.8 µM and 3.9 µM, respectively after 72 hours of incubation. The TCPMVFePt were prepared as potential MRI contrast agents. CONCLUSION: Cisplatin loaded VLP (TCPMVCis) is shown to enhance cisplatin cytotoxicity in cancer cell lines with its potency increased by 2.3-folds.

Aptamer-functionalized pH-sensitive liposomes for a selective delivery of echinomycin into cancer cells.

Echinomycin (quinomycin A) is a peptide antibiotic from the quinoxaline family, which has a DNA bifunctional intercalating activity and an inhibitor of hypoxia-inducible factor (HIF1α). Echinomycin was discovered in 1957 as a potent antitumor agent; however, it was not successful in clinical use due to its low water solubility and short half-life. To revitalize this potent drug, it is important to increase its aqueous solubility and bioavailability. In this study, echinomycin was loaded into PEGylated pH-sensitive liposomes (PEGLippH) and functionalized with anti-nucleolin aptamer (AptNCL) for selective targeting and pH-responsive release of echinomycin into cancer cells. Echinomycin was complexed with γ-cyclodextrin (ECγCD) to enhance its water solubility and then encapsulated into pH-sensitive liposomes (PEGLippH-ECγCD). Then, liposomes were functionalized with AptNCL (AptNCL-PEGLippH-ECγCD) and the successful functionalization was confirmed by dynamic light scattering (DLS) measurements and gel electrophoresis. Cellular uptake for AptNCL-PEGLippH was evaluated by flow cytometry analysis using MDA-MB-231, MCF7, A549 cancer cell lines with respect to the normal fibroblast cells. The results showed a higher uptake and selectivity for AptNCL-PEGLippH compared to PEGLippH. The anti-proliferative effects of AptNCL-PEGLippH-ECγCD were more potent than PEGLippH-ECγCD by 3.5, 4, and 5 folds for A549, MDA-MB-231, and MCF7, respectively. Selectivity indices (SI) for AptNCL-PEGLippH-ECγCD for the tumor cell lines compared to the normal cell line after 72 h were MDA-MB-231 (43.3), MCF7 (16.9), and A549 (8.5). Furthermore, SI after 3 h for the three cancer cell lines were 4.7, 2.5, 2.8, respectively.

Enhancing chemosensitivity of wild-type and drug-resistant MDA-MB-231 triple-negative breast cancer cell line to doxorubicin by silencing of STAT 3, Notch-1, and ß-catenin genes.

BACKGROUND/OBJECTIVE: The absence of receptors in triple-negative breast cancer limits therapeutic choices utilized in clinical management of the disease. Doxorubicin is an important member of therapeutic regimens that is hindered by emergence of resistance. The current work aim to investigate of therapeutic potential of single and combinations of siRNA molecules designed for silencing STAT 3, Notch-1, and ß-catenin genes in wild type and doxorubicin resistant MDA-MB-231 triple negative breast cancer cell line. METHODS: Doxorubicin resistant MDA-MB-231 cell line was developed and characterized for the expression of multidrug resistance-related genes, CD44/CD24 markers, inflammatory cytokines, and the expression of STAT 3, Notch-1, and ß-catenin targeted genes. Further, the effect of single and combinations of siRNA on cell viability and chemosensitivity of both wild type MDA-MB-231 cells (MDA-MB-231/WT) and doxorubicin resistant MDA-MB-231 cells (MDA-MB-231/DR250) were assessed by MTT assay. RESULTS: The IC50 of doxorubicin was 10-folds higher in MDA-MB-231/DR250 resistant cells compared to MDA-MB-231/WT control cells, 1.53 ± 0.24 µM compared to 0.16 ± 0.02 µM, respectively. The expression of targeted genes was higher in resistant cells compared to control cells, 3.6 ± 0.16 folds increase in ß-catenin, 2.7 ± 0.09 folds increase in Notch-1, and 1.8 ± 0.09 folds increase in STAT-3. Following treatment with siRNAs, there was a variable reduction in mRNA expression of each of the targeted genes compared to scrambled siRNA and a reduction in IC50 in both cell lines. The effect of a combination of three genes produced the largest reduction in IC50 in resistant cell line. CONCLUSION: Our study showed that the silencing of single and multiple genes involved in drug resistance and tumor progression by siRNA can enhance the chemosensitivity of cancer cells to conventional chemotherapy.

Downregulation of STAT3, ß-Catenin, and Notch-1 by Single and Combinations of siRNA Treatment Enhance Chemosensitivity of Wild Type and Doxorubicin Resistant MCF7 Breast Cancer Cells to Doxorubicin.

Combinatorial therapeutic strategies using siRNA and small molecules to eradicate tumors are emerging. Targeting multiple signaling pathways decreases the chances of cancer cells switching and adapting new signaling processes that may occur when using a single therapeutic modality. Aberrant functioning of Notch-1, Wnt/ß-catenin, and STAT3 proteins and their crosstalk signaling pathways have been found to be involved in tumor survival, drug resistance, and relapse. In the current study, we describe a therapeutic potential of single and combinations of siRNA designed for silencing Notch-1, Wnt/ß-catenin, and STAT3 in MCF7_DoxS (wild type) and MCF7_DoxR (doxorubicin resistant) breast cancer cells. The MCF7_DoxR cells were developed through treatment with a gradual increase in doxorubicin concentration, the expression of targeted genes was investigated, and the expression profiling of CD44/CD24 of the MCF7_DoxS and MCF7_DoxR cells were detected by flow cytometry. Both MCF7_DoxS and MCF7_DoxR breast cancer cells were treated with single and combinations of siRNA to investigate synergism and were analyzed for their effect on cell proliferation with and without doxorubicin treatment. The finding of this study showed the overexpression of targeted genes and the enrichment of the CD44-/CD24+ phenotype in MCF7_DoxR cells when compared to MCF7_DoxS cells. In both cell lines, the gene silencing efficacy showed a synergistic effect when combining STAT3/Notch-1 and STAT3/Notch-1/ß-catenin siRNA. Interestingly, the chemosensitivity of MCF7_DoxS and MCF7_DoxR cells to doxorubicin was increased when combined with siRNA treatment. Our study shows the possibility of using single and combinations of siRNA to enhance the chemosensitivity of cancer cells to conventional antitumor chemotherapy.

Encapsulation of echinomycin in cyclodextrin inclusion complexes into liposomes: in vitro anti-proliferative and anti-invasive activity in glioblastoma.

Echinomycin, a DNA bis-intercalator peptide, belongs to the family of quinoxaline antibiotics. Echinomycin exhibits potent antitumor and antimicrobial activity. However, it is highly water insoluble and suffers from low bioavailability and unwanted side effects. Therefore, developing new formulations and delivery systems that can enhance echinomycin solubility and therapeutic potency is needed for further clinical application. In this study, echinomycin has been complexed into the hydrophobic cavity of γ-cyclodextrin (γCD) then encapsulated into PEGylated liposomes. The anti-proliferative and anti-invasive effect has been evaluated against U-87 MG glioblastoma cells. Echinomycin-in-γCD inclusion complexes have been characterized by phase solubility assay, TLC, and 1H-NMR. The echinomycin-in-γCD inclusion complexes have been loaded into liposomes using a thin film hydration method to end up with echinomycin-in-γCD-in-liposomes. Drug-loaded liposomes were able to inhibit cell proliferation with IC50 of 1.0 nM. Moreover, echinomycin-in-γCD-in-liposomes were found to inhibit the invasion of U-87 MG cells using the spheroid gel invasion assay. In conclusion, the current work describes for the first time γCD-echinomycin complexes and their encapsulation into PEGylated liposomes.

Ligand-based modeling of diverse aryalkylamines yields new potent P-glycoprotein inhibitors.

The P-glycoprotein (P-gp) efflux pump has an important role as a natural detoxification system in many types of normal and cancer cells. P-gp is implicated in multiple drug resistance (MDR) exhibited by several types of cancer against a multitude of anticancer chemotherapeutic agents, and therefore, it is clinically validated target for cancer therapy. Accordingly, in this study we combined exhaustive pharmacophore modeling and quantitative structure-activity relationship (QSAR) analysis to explore the structural requirements for potent P-gp inhibitors employing 130 known P-gp ligands. Genetic function algorithm (GFA) coupled with k nearest neighbor (kNN) or multiple linear regression (MLR) analyses were employed to build self-consistent and predictive QSAR models based on optimal combinations of pharmacophores and physicochemical descriptors. Successful pharmacophores were complemented with exclusion spheres to optimize their receiver operating characteristic curve (ROC) profiles. Optimal QSAR models and their associated pharmacophore hypotheses were validated by identification and experimental evaluation of new promising P-gp inhibitory leads retrieved from the National Cancer Institute (NCI) structural database. Several potent hits were captured. The most potent hit decreased the IC50 of doxorubicin from 0.906 to 0.190 µM on doxorubicin resistant MCF7 cell-line.