Neeraj Chauhan

Curcumin-loaded magnetic nanoparticles for breast cancer therapeutics and imaging applications

Background The next generation magnetic nanoparticles (MNPs) with theranostic applications have attracted significant attention and will greatly improve nanomedicine in cancer therapeutics. Such novel MNP formulations must have ultra-low particle size, high inherent magnetic properties, effective imaging, drug targeting, and drug delivery properties. To achieve these characteristic properties, a curcumin-loaded MNP (MNP-CUR) formulation was developed. Methods MNPs were prepared by chemical precipitation method and loaded with curcumin (CUR) using diffusion method. The physicochemical properties of MNP-CUR were characterized using dynamic light scattering, transmission electron microscopy, and spectroscopy. The internalization of MNP-CUR was achieved after 6 hours incubation with MDA-MB-231 breast cancer cells. The anticancer potential was evaluated by a tetrazolium-based dye and colony formation assays. Further, to prove MNP-CUR results in superior therapeutic effects over CUR, the mitochondrial membrane potential integrity and reactive oxygen species generation were determined. Magnetic resonance imaging capability and magnetic targeting property were also evaluated. Results MNP-CUR exhibited individual particle grain size of ~9 nm and hydrodynamic average aggregative particle size of ~123 nm. Internalized MNP-CUR showed a preferential uptake in MDA-MB-231 cells in a concentration-dependent manner and demonstrated accumulation throughout the cell, which indicates that particles are not attached on the cell surface but internalized through endocytosis. MNP-CUR displayed strong anticancer properties compared to free CUR. MNP-CUR also amplified loss of potential integrity and generation of reactive oxygen species upon treatment compared to free CUR. Furthermore, MNP-CUR exhibited superior magnetic resonance imaging characteristics and significantly increased the targeting capability of CUR. Conclusion MNP-CUR exhibits potent anticancer activity along with imaging and magnetic targeting capabilities. This approach can be extended to preclinical and clinical use and may have importance in cancer treatment and cancer imaging in the future. Further, if these nanoparticles can functionalize with antibody/ligands, they will serve as novel platforms for multiple biomedical applications.

Anti-cancer activity of curcumin loaded nanoparticles in prostate cancer.

Prostate cancer is the most commonly diagnosed cancer disease in men in the Unites States and its management remains a challenge in everyday oncology practice. Thus, advanced therapeutic strategies are required to treat prostate cancer patients. Curcumin (CUR) is a promising anticancer agent for various cancer types. The objective of this study was to evaluate therapeutic potential of novel poly(lactic-co-glycolic acid)- CUR nanoparticles (PLGA-CUR NPs) for prostate cancer treatment. Our results indicate that PLGA-CUR NPs efficiently internalize in prostate cancer cells and release biologically active CUR in cytosolic compartment of cells for effective therapeutic activity. Cell proliferation (MTS), clonogenic, and Western blot analyses reveal that PLGA-CUR NPs can effectively inhibit proliferation and colony formation ability of prostate cancer cells than free CUR. PLGA-CUR NPs showed superior tumor regression compared to CUR in xenograft mice. Further investigations reveal that PLGA-CUR NPs inhibit nuclear β-catenin and AR expression in cells and in tumor xenograft tissues. It also suppresses STAT3 and AKT phosphorylation and leads to apoptosis via inhibition of key anti-apoptotic proteins, Mcl-1, Bcl-xL and caused induction of PARP cleavage. Additionally, significant downregulation of oncogenic miR21 and up-regulation of miR-205 was observed with PLGA-CUR NPs treatment as determined by RT-PCR and in situ hybridization analyses. A superior anti-cancer potential was attained with PSMA antibody conjugated PLGA-CUR NPs in prostate cancer cells and a significant tumor targeting of (131)I labeled PSMA antibody was achieved with PLGA-CUR NPs in prostate cancer xenograft mice model. In conclusion, PLGA-CUR NPs can significantly accumulate and exhibit superior anticancer activity in prostate cancer.

Novel Curcumin-Loaded Magnetic Nanoparticles for Pancreatic Cancer Treatment

Curcumin (CUR), a naturally occurring polyphenol derived from the root of Curcuma longa, has showed potent anticancer and cancer prevention activity in a variety of cancers. However, the clinical translation of CUR has been significantly hampered due to its extensive degradation, suboptimal pharmacokinetics, and poor bioavailability. To address these clinically relevant issues, we have developed a novel CUR-loaded magnetic nanoparticle (MNP-CUR) formulation. Herein, we have evaluated the in vitro and in vivo therapeutic efficacy of this novel MNP-CUR formulation in pancreatic cancer. Human pancreatic cancer cells (HPAF-II and Panc-1) exhibited efficient internalization of the MNP-CUR formulation in a dose-dependent manner. As a result, the MNP-CUR formulation effectively inhibited growth of HPAF-II and Panc-1 cells in cell proliferation and colony formation assays. The MNP-CUR formulation suppressed pancreatic tumor growth in an HPAF-II xenograft mouse model and improved the survival of mice by delaying tumor growth. The growth-inhibitory effect of MNP-CUR formulation correlated with the suppression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-extra large (Bcl-xL), induced myeloid leukemia cell differentiation protein (Mcl-1), cell surface–associated Mucin 1 (MUC1), collagen I, and enhanced membrane β-catenin expression. MNP-CUR formulation did not show any sign of hemotoxicity and was stable after incubation with human serum proteins. In addition, the MNP-CUR formulation improved serum bioavailability of CUR in mice up to 2.5-fold as compared with free CUR. Biodistribution studies show that a significant amount of MNP-CUR formulation was able to reach the pancreatic xenograft tumor(s), which suggests its clinical translational potential. In conclusion, this study suggests that our novel MNP-CUR formulation can be valuable for the treatment of pancreatic cancer. Mol Cancer Ther; 12(8); 1471–80. ©2013 AACR.

Interaction of curcumin nanoformulations with human plasma proteins and erythrocytes.

Background Recent studies report curcumin nanoformulation(s) based on polylactic-co-glycolic acid (PLGA), β-cyclodextrin, cellulose, nanogel, and dendrimers to have anticancer potential. However, no comparative data are currently available for the interaction of curcumin nanoformulations with blood proteins and erythrocytes. The objective of this study was to examine the interaction of curcumin nanoformulations with cancer cells, serum proteins, and human red blood cells, and to assess their potential application for in vivo preclinical and clinical studies. Methods The cellular uptake of curcumin nanoformulations was assessed by measuring curcumin levels in cancer cells using ultraviolet-visible spectrophotometry. Protein interaction studies were conducted using particle size analysis, zeta potential, and Western blot techniques. Curcumin nanoformulations were incubated with human red blood cells to evaluate their acute toxicity and hemocompatibility. Results Cellular uptake of curcumin nanoformulations by cancer cells demonstrated preferential uptake versus free curcumin. Particle sizes and zeta potentials of curucumin nanoformulations were varied after human serum albumin adsorption. A remarkable capacity of the dendrimer curcumin nanoformulation to bind to plasma protein was observed, while the other formulations showed minimal binding capacity. Dendrimer curcumin nanoformulations also showed higher toxicity to red blood cells compared with the other curcumin nanoformulations. Conclusion PLGA and nanogel curcumin nanoformulations appear to be very compatible with erythrocytes and have low serum protein binding characteristics, which suggests that they may be suitable for application in the treatment of malignancy. These findings advance our understanding of the characteristics of curcumin nanoformulations, a necessary component in harnessing and implementing improved in vivo effects of curcumin.

Curcumin Nanoformulation for Cervical Cancer Treatment

Cervical cancer is one of the most common cancers among women worldwide. Current standards of care for cervical cancer includes surgery, radiation, and chemotherapy. Conventional chemotherapy fails to elicit therapeutic responses and causes severe systemic toxicity. Thus, developing a natural product based, safe treatment modality would be a highly viable option. Curcumin (CUR) is a well-known natural compound, which exhibits excellent anti-cancer potential by regulating many proliferative, oncogenic, and chemo-resistance associated genes/proteins. However, due to rapid degradation and poor bioavailability, its translational and clinical use has been limited. To improve these clinically relevant parameters, we report a poly(lactic-co-glycolic acid) based curcumin nanoparticle formulation (Nano-CUR). This study demonstrates that in comparison to free CUR, Nano-CUR effectively inhibits cell growth, induces apoptosis, and arrests the cell cycle in cervical cancer cell lines. Nano-CUR treatment modulated entities such as miRNAs, transcription factors, and proteins associated with carcinogenesis. Moreover, Nano-CUR effectively reduced the tumor burden in a pre-clinical orthotopic mouse model of cervical cancer by decreasing oncogenic miRNA-21, suppressing nuclear β-catenin, and abrogating expression of E6/E7 HPV oncoproteins including smoking compound benzo[a]pyrene (BaP) induced E6/E7 and IL-6 expression. These superior pre-clinical data suggest that Nano-CUR may be an effective therapeutic modality for cervical cancer.

Ormeloxifene Suppresses Desmoplasia and Enhances Sensitivity of Gemcitabine in Pancreatic Cancer

The management of pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to lack of an efficient therapy and development of chemoresistance to the current standard therapy, gemcitabine. Recent studies implicate the intimate reciprocal interactions between epithelia and underlying stroma due to paracrine Sonic hedgehog (SHH) signaling in producing desmoplasia and chemoresistance in PDAC. Herein, we report for the first time that a nonsteroidal drug, ormeloxifene, has potent anticancer properties and depletes tumor-associated stromal tissue by inhibiting the SHH signaling pathway in PDAC. We found that ormeloxifene inhibited cell proliferation and induced death in PDAC cells, which provoked us to investigate the combinatorial effects of ormeloxifene with gemcitabine at the molecular level. Ormeloxifene caused potent inhibition of the SHH signaling pathway via downregulation of SHH and its related important downstream targets such as Gli-1, SMO, PTCH1/2, NF-κB, p-AKT, and cyclin D1. Ormeloxifene potentiated the antitumorigenic effect of gemcitabine by 75% in PDAC xenograft mice. Furthermore, ormeloxifene depleted tumor-associated stroma in xenograft tumor tissues by inhibiting the SHH cellular signaling pathway and mouse/human collagen I expression. Xenograft tumors treated with ormeloxifene in combination with gemcitabine restored the tumor-suppressor miR-132 and inhibited stromal cell infiltration into the tumor tissues. In addition, invasiveness of tumor cells cocultivated with TGFβ-stimulated human pancreatic stromal cells was effectively inhibited by ormeloxifene treatment alone or in combination with gemcitabine. We propose that ormeloxifene has high therapeutic index and in a combination therapy with gemcitabine, it possesses great promise as a treatment of choice for PDAC/pancreatic cancer.

Ormeloxifene suppresses prostate tumor growth and metastatic phenotypes via inhibition of oncogenic β-catenin signaling and EMT progression

Ormeloxifene is a clinically approved selective estrogen receptor modulator, which has also shown excellent anticancer activity, thus it can be an ideal repurposing pharmacophore. Herein, we report therapeutic effects of ormeloxifene on prostate cancer and elucidate a novel molecular mechanism of its anticancer activity. Ormeloxifene treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), β-catenin/TCF-4 transcriptional activity, and induced the expression of pGSK3β. In molecular docking analysis, ormeloxifene showed proficient docking with β-catenin and GSK3β. In addition, ormeloxifene induced apoptosis, inhibited growth and metastatic potential of prostate cancer cells and arrested cell cycle in G0–G1 phase via modulation of cell-cycle regulatory proteins (inhibition of Mcl-1, cyclin D1, and CDK4 and induction of p21 and p27). In functional assays, ormeloxifene remarkably reduced tumorigenic, migratory, and invasive potential of prostate cancer cells. In addition, ormeloxifene treatment significantly (P < 0.01) regressed the prostate tumor growth in the xenograft mouse model while administered through intraperitoneal route (250 μg/mouse, three times a week). These molecular effects of ormeloxifene were also observed in excised tumor tissues as shown by immunohistochemistry analysis. Our results, for the first time, demonstrate repurposing potential of ormeloxifene as an anticancer drug for the treatment of advanced stage metastatic prostate cancer through a novel molecular mechanism involving β-catenin and EMT pathway. Mol Cancer Ther; 16(10); 2267–80. ©2017 AACR.

Abstract 2893: Curcumin loaded magnetic nanoparticles for breast cancer therapeutics and imaging applications

Background: Magnetic nanoparticles (MNPs) with theranostic features (diagnosis and treatment) have attracted significant attention and will greatly improve the scope of nanomedicine in cancer applications. The aim of this study is to develop a novel MNPs formulation composed of an iron oxide core coated with α-cyclodextrin and pluronic polymer (F68), to load an anti-cancer drug (curcumin, CUR) and prevent particle aggregation, respectively. The therapeutic efficacy and imaging capabilities of this novel formulation were evaluated in breast cancer cell line models. Methods: The physico-chemical analyses of curcumin loaded MNPs (MNP-CUR) were performed using dynamic light scattering (DLS), transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) analyses. MNP-CUR internalization was evaluated after 6 hrs incubation with MDA-MB-231 cancer cells by Prussian blue stain and TEM. Anti-cancer effects of the MNP-CUR formulation was determined by a tetrazolium based dye (MTT) and colony formation assays using “triple negative” MDA-MB-231 cancer cells. Tetramethylrhodamine, ethyl ester, perchlorate (TMRE), red mitochondrial superoxide indicator (Mitosox), and propidium iodide stains were used to evaluate the loss of mitochondrial membrane potential, reactive oxygen species (ROS) generation, and apoptosis/dead cells, respectively, to determine effects MNP-CUR on these cellular features. The imaging capability of this MNP-CUR formulation was analyzed using a magnetic resonance imaging (MRI) system. The magnetic targeting feature of the MNP-CUR formulation was evaluated using fluorescence microscopy. Results: We prepared MNP-CUR with an average aggregative particles size of ∼ 150 nm (individual particle grain size, ∼ 9-11 nm). Prussian blue stain data represent a preferential uptake of MNP-CUR in MDA-MB-231 cells in a dose dependant manner. TEM analysis demonstrates that accumulation of MNP-CUR nanoparticles in cancer cells indicate that particles are not attached on the surface of cells but internalized within the cells. The MNP-CUR formulation showed strong anti-cancer effects in breast cancer cells including “triple negative” MDA-MB-231 cancer cells compared to free curcumin. This formulation also enhanced loss of mitochondrial membrane potential, generation of ROS and apoptosis compared to free curcumin treatment. Additionally, the MNP-CUR formulation exhibits superior T2 imaging characteristics compared to T1. A significant increase of the targeting feature was observed with MNP-CUR in breast cancer cells. Conclusion: These data suggest that our novel MNP-CUR formulation exhibits potent anti-cancer activity along with imaging and magnetic targeting capabilities. This approach can be extended to pre-clinical and clinical use and may have importance in cancer treatment and cancer imaging in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2893. doi:1538-7445.AM2012-2893

Resilience and receptivity worked in tandem to sustain a geothermal mat community amidst erratic environmental conditions

To elucidate how geothermal irregularities affect the sustainability of high-temperature microbiomes we studied the synecological dynamics of a geothermal microbial mat community (GMMC) vis-à-vis fluctuations in its environment. Spatiotemporally-discrete editions of a photosynthetic GMMC colonizing the travertine mound of a circum-neutral hot spring cluster served as the model-system. In 2010 a strong geyser atop the mound discharged mineral-rich hot water, which nourished a GMMC continuum from the proximal channels (PC) upto the slope environment (SE) along the mound’s western face. In 2011 that geyser extinguished and consequently the erstwhile mats disappeared. Nevertheless, two relatively-weaker vents erupted in the southern slope and their mineral-poor outflow supported a small GMMC patch in the SE. Comparative metagenomics showed that this mat was a relic of the 2010 community, conserved via population dispersal from erstwhile PC as well as SE niches. Subsequently in 2012, as hydrothermal activity augmented in the southern slope, ecological niches widened and the physiologically-heterogeneous components of the 2011 “seed-community” split into PC and SE meta-communities, thereby reclaiming either end of the thermal gradient. Resilience of incumbent populations, and the community’s receptiveness towards immigrants, were the key qualities that ensured the GMMC’s sustenance amidst habitat degradation and dispersal to discrete environments.

Abstract 4459: Characterization of a novel magnetic nanoparticles formulation for cancer therapeutic applications

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have successfully engineered a magnetic nanoparticles (MAG-NPs) formulation using a multi-layer approach which can be used for drug/gene/biomolecule delivery, hyperthermia, and magnetic resonance imaging (MRI) applications in cancer therapeutics. Overcoming nanoparticles clearance by the immune system remains a major challenge. This formulation is designed to provide an additional surface layer as molecular “authentication” that the body does not recognize as foreign material. The interaction between the surface of nanoparticles and plasma proteins leads to nanoparticle-protein complex which determines the rational design of clinically useful formulations for cancer therapeutics. This formulation was very effective for inhibiting tumorigenic features in in vitro and in vivo cancer models. Therefore, we further studied our MAG-NPs formulation for biologically and clinically relevant characteristic features for its clinical translation. For this, we have examined the change in particle size, zeta potential, human serum protein adsorption, hemotoxicity, uptake, targeting cancer cells, and magnetic resonance imaging of the MAG-NPs formulation. No significant change was observed in particle size and zeta potential of nanoformulation with the incubation of 0.5 to 50 wt/wt% human serum. With increase of serum concentration and particle concentration there was a clear indication that apart from serum albumin and transferrin; apolipoprotein E (Apo-E, a surrogate marker for improved circulation of nanoparticles and enhanced crossing of the blood-brain barrier) also adsorbed on the surface of nanoparticles (determined by Western blot analysis). No significant primary or secondary structural alterations were observed in serum proteins through circular dichroism study. Hemolysis assay demonstrated almost no hemolysis at the tested concentrations (up to 1 mg/mL) for nanoparticles compared to the positive control (sodium dodecyl sulphate). Particle internalization and targeting cancer cells were observed in dose dependent manner and were not influenced by protein architecture on surface of nanoparticles. Additionally, this formulation has exhibited identical T1 relaxation times (∼1.1-1.6 s) and T2 relaxation times (∼ 13-14 ms) which suggests that our nanoparticles retained MRI properties even after serum proteins adsorption and drug/biomolecular encapsulation. Based on all these superior clinical characteristics, this MAG-NPs formulation can effectively be used for the delivery of drug/siRNA/miRNAs for cancer therapy, diagnosis, and imaging. Citation Format: Murali M. Yallapu, Neeraj Chauhan, Shadi F. Othman, Vahid Khalilzad-Sharghi, Meena Jaggi, Subhash C. Chauhan. Characterization of a novel magnetic nanoparticles formulation for cancer therapeutic applications. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4459. doi:10.1158/1538-7445.AM2014-4459