Toxicological evaluation of therapeutically active zinc oxide nanoflowers in pre-clinical mouse model.

Our earlier reports established that zinc oxide nanoflowers (ZONF) show significant pro-angiogenic properties, where reactive oxygen species, nitric oxide and MAPK-AKT-eNOS cell signaling axis play an essential task. Considering the significance of angiogenesis in healthcare, our research group has recently demonstrated the in vivo therapeutic application of ZONF (10 mg/kg b.w.) for treating peripheral artery disease. Moreover, based on the angio-neural crosstalk between vascular and neuronal systems, we have further demonstrated the neuritogenic and neuroprotective characteristics of pro-angiogenic nanoflowers (10 mg/kg b.w.) for the treatment of cerebral ischemia. However, it is crucial for a therapeutic material to be non-toxic for its practical clinical applications and therefore assessment of its in vivo toxicity and adverse effect is highly important. Herein, for the first time, we investigate a detailed nanotoxicology of therapeutically active ZONF in Swiss albino mice to evaluate their safety profile and comprehend their aspects for future clinical applications. The maximum tolerated dose (MTD) of ZONF was found to be 512.5 mg/kg b.w. which was employed for acute exposure (2 weeks), showing slight toxicity. However, sub-chronic (4 weeks) and long term chronic (8-12 weeks) studies of nanoflowers exhibited their non-toxic nature particularly at lower therapeutic doses (1-10 mg/kg b.w.). Additionally, in depth genotoxicity study revealed that lower therapeutic dose of ZONF (10 mg/kg b.w.) did not exhibit significant toxicity even in genetic level. Overall, the present nanotoxicology of ZONF suggests their high biocompatible nature at therapeutic dose, offering the basis of their future clinical applications in ischemic and other vascular diseases.

Therapeutic angiogenesis using zinc oxide nanoflowers for the treatment of hind limb ischemia in a rat model.

Critical limb ischemia (CLI) is a severe type of peripheral artery disease (PAD) which occurs due to an inadequate supply of blood to the limb extremities. Patients with CLI often suffer from extreme cramping pain, impaired wound healing, immobility, cardiovascular complications, amputation of the affected limb and even death. The conventional therapy for treating CLI includes surgical revascularization as well as restoration of angiogenesis using growth factor therapy. However, surgical revascularization is only suitable for a small percentage of CLI patients and is associated with a high perioperative mortality rate. The use of growth factors is also limited in terms of their poor therapeutic angiogenic potential, as observed in earlier clinical studies which could be attributed to their poor bio-availability and non-specificity issues. Therefore, to overcome the aforesaid disadvantages of conventional strategies there is an urgent need for the advancement of new alternative therapeutic biomaterials to treat CLI. In the past few decades, various research groups, including ours, have been involved in developing different pro-angiogenic nanomaterials. Among these, zinc oxide nanoflowers (ZONFs), established in our laboratory, are considered one of the more potent nanoparticles for inducing therapeutic angiogenesis. In our earlier studies we showed that ZONFs promote angiogenesis by inducing the formation of reactive oxygen species and nitric oxide (NO) as well as activating Akt/MAPK/eNOS cell signaling pathways in endothelial cells. Recently, we have also reported the therapeutic potential of ZONFs to treat cerebral ischemia through their neuritogenic and neuroprotective properties, exploiting angio-neural cross-talk. Considering the excellent pro-angiogenic properties of ZONFs and the importance of revascularization for the treatment of CLI, in the present study we comprehensively explore the therapeutic potential of ZONFs in a rat hind limb ischemia model (established by ligating the hind limb femoral artery), an animal model that mimics CLI in humans. The behavioral studies, laser Doppler perfusion imaging, histopathology and immunofluorescence as well as estimation of serum NO level showed that the administration of ZONFs could ameliorate ischemia in rats at a faster rate by promoting therapeutic angiogenesis to the ischemic sites. Altogether, the present study offers an alternative nanomedicine approach employing ZONFs for the treatment of PADs.

Design of DNA-intercalators based copper(II) complexes, investigation of their potential anti-cancer activity and sub-chronic toxicity.

In the present paper, we synthesized and characterized four N-donor polypyridyl copper(II) complexes (C1-C4); [Cu(mono-CN-PIP)2]2+ (C1), [Cu(tri-OMe-PIP)2]2+ (C2), [Cu(di-CF3-PIP)2]2+ (C3) and [Cu(DPPZ)2]2+ (C4). The (Calf-Thymus) CT-DNA binding studies depicted that the complexes could interact with DNA via intercalative mode. All the complexes, particularly C3 and C4 attenuated the proliferation as well as migration of various cancer cells, indicating their anti-cancer and anti-metastatic activity. Additionally, chick embryo angiogenesis (CEA) assay exhibited the inhibition of vascular sprouting in presence of C3 and C4, suggesting their potential in inhibiting the blood vessel growth. Mechanistic studies revealed that the complexes induced the excessive production of cellular reactive oxygen species (ROS) leading to apoptosis through up regulation of p53 and downregulation of Bcl-xL, which might be the plausible mechanisms underlying their anti-cancer properties. To understand the feasibility of practical application of anti-cancer copper complexes C3 and C4, in vivo sub-chronic toxicity study (4 weeks) was performed in C57BL6 mice and the results exhibited almost non-toxic effects induced by these complexes in terms of haematology and serum biochemical analyses, suggesting their biocompatible nature. The current study provides the basis for future advancement of other novel biocompatible metal complexes that could be employed for the therapy of different cancers.

Pro-angiogenic Properties of Terbium Hydroxide Nanorods: Molecular Mechanisms and Therapeutic Applications in Wound Healing.

The process of angiogenesis, involving generation of new blood vessels from the existing ones, is vital for the supply of oxygen and nutrients to various tissues of body system. Angiogenesis is directly associated with several physiological and pathological processes. It is well-established that impairment in angiogenesis process results in various fatal conditions. Recently, few research groups including ours demonstrated therapeutic angiogenesis through nanomedicine approach using metal oxide/hydroxide nanoparticles. However, there is still a thorough necessity for the development of novel, eco-friendly, pro-angiogenic nanomaterials. Hence, in the present study we demonstrate the in vitro and in vivo pro-angiogenic properties of terbium hydroxide nanorods (THNRs) synthesized using an advanced microwave irradiation method, along with the detailed molecular signaling cascade underlying THNRs induced angiogenesis. The in vivo wound healing and nonimmunogenicity of the THNRs have been validated in the mouse models. We thus strongly believe that the present study establishing the pro-angiogenic properties of THNRs will aid in the development of alternative treatment strategies for wound healing along with cardiovascular and ischemic diseases, where angiogenesis is the chief target.

Synthesis and biological evaluation of novel 2-imino-4-thiazolidinone derivatives as potent anti-cancer agents.

A new series of 2-imino-4-thiazolidinone derivatives (7a-7t) has been synthesised and screened for their cytotoxicity against three cancer cell lines (B16F10, A549, PANC-1) and normal cell line (CHO). Among the compounds tested, compounds 7k, 7m, 7n showed potent cytotoxicity against B16F10 cell line with IC50 between 3.4 and 7µM. Interestingly these three compounds are non toxic to non cancerous CHO cells and induced apoptosis in B16F10 cells observed by DNA damage analysis through PI/Hoechst double staining method. Compounds 7k and 7n induced G0/G1 cell cycle arrest while compound 7m induced G2/M cell cycle arrest in B16F10 cells which confirms that these compounds have role in cancer cell cycle regulation. Additionally, compound 7m showed generation of intracellular reactive oxygen species (ROS) in B16F10 cells that may contribute in the cell cycle arrest whereas compounds 7k and 7n show anti-cancer activity through independent of ROS formation. Induction of apoptosis, cell cycle arrest in B16F10 cells are found to be the anti-cancer mechanism of these three compounds. The results all together demonstrate the potent cytotoxic nature of these compounds in cancer cells could be considered as new class of chemotherapeutic agents in near future.

Green Synthesis and Characterization of Monodispersed Gold Nanoparticles: Toxicity Study, Delivery of Doxorubicin and Its Bio-Distribution in Mouse Model.

In the present article, we report the in vitro and in vivo delivery of doxorubicin using biosynthesized gold nanoparticles (b-Au-PP). Gold nanoparticles were synthesized by a simple, fast, efficient, environmentally friendly and economical green chemistry approach using an extract of Peltophorum pterocarpum (PP) leaves. Because the biosynthesized b-Au-PP was highly stable in various physiological buffers for several weeks and biocompatible in both in vitro and in vivo systems, we designed and developed a biosynthesized gold nanoparticle (b-Au-PP)-based drug-delivery system (DDS) using doxorubicin (Dox) (b-Au-PP-Dox). Both b-Au-PP and b-Au-PP-Dox were thoroughly characterized using several analytical tools. Administration of doxorubicin-loaded DDS (b-Au-PP-Dox) resulted in a significant inhibition of the proliferation of cancer cells (A549, B16F10) in vitro and of tumor growth in an in vivo model compared to doxorubicin alone. Furthermore, we found that the cellular uptake and release of Dox in the nanoconjugated form (b-Au-PP-Dox) were faster than the uptake and release of unconjugated Dox. The in vivo toxicity study did not show any significant changes in the hematology, serum clinical biochemistry or histopathology in the C57BL6/J female mice after consecutive intraperitoneal (IP) injections over a period of seven days. To the best of our knowledge, our study is the first to report the application of a biosynthesized gold nanoparticle-based DDS for cancer therapy in an animal model, in addition to a detailed in vivo toxicity study. Together, the results demonstrate that a biosynthesized gold nanoparticle-based drug-delivery system (b-Au-PP-Dox) could be used in the near future as an alternative cost-effective treatment strategy for cancer therapy.

Curcumin-loaded silica-based mesoporous materials: Synthesis, characterization and cytotoxic properties against cancer cells.

Two different silica based (MSU-2 and MCM-41) curcumin loaded mesoporous materials V3 and V6 were synthesized and characterized by several physico-chemical techniques. Release kinetic study revealed the slow and sustained release of curcumin from those materials in blood simulated fluid (pH: 7.4). The materials V3 and V6 were found to be biocompatible in non-cancerous CHO cell line while exhibiting significant cytotoxicity in different cancer cells (human lung carcinoma cells: A549, human breast cancer cells: MCF-7, mouse melanoma cells: B16F10) compared to pristine curcumin indicating the efficacy of the mesoporous silica materials based drug delivery systems (DDSs). The generation of intracellular reactive oxygen species (ROS) and down regulation of anti-apoptotic protein leading to the induction of apoptosis were found to be the plausible mechanisms behind the anti-cancer activity of these DDSs. These results suggest that curcumin-loaded drug delivery system may be successfully employed as an alternative treatment strategy for cancer therapeutics through a nanomedicine approach in near future.

Evaluation of in vivo cytogenetic toxicity of europium hydroxide nanorods (EHNs) in male and female Swiss albino mice.

Our group already demonstrated that europium hydroxide nanorods (EHNs) show none or mild toxicity in C57BL/6 mice even at high dose and exhibited excellent pro-angiogenic activity towards in vitro and in vivo models. In the present study, we evaluated the in vivo cytogenetic toxicity of intraperitoneally administered EHNs (12.5-250 mg/kg/b.w.) in male and female Swiss albino mice by analyzing chromosomal aberrations (CAs), mitotic index (MI), micronucleus (MN) from bone marrow and peripheral blood. Furthermore, we performed the cytogenetic toxicity study of EHNs towards Chinese hamster ovary (CHO) cells, in order to compare with the in vivo results. The results of CA assay of mice treated with EHNs (12.5-125 mg/kg/b.w.) showed no significant change in the formation of aberrant metaphases compared to the control group. Also, there was no significant difference in the number of dividing cells between the control group and EHNs-treated groups observed by MI study, suggesting the non-cytotoxicity of EHNs. Additionally, FACS study revealed that EHNs do not arrest cells at any phase of cell cycle in the mouse model. Furthermore, MN test of both bone marrow and peripheral blood showed no significant differences in the induction of MNs when compared with the control group. In vitro results from CHO cells also support our in vivo observations. Considering the role of angiogenesis by EHNs and the absence of its genotoxicity in mouse model, we strongly believe the future application of EHNs in treating various diseases, where angiogenesis plays an important role such as cardiovascular diseases, ischemic diseases and wound healing.

A green chemistry approach for the synthesis of gold nanoconjugates that induce the inhibition of cancer cell proliferation through induction of oxidative stress and their in vivo toxicity study.

In this paper, we report the synthesis of gold nanoconjugates (b-Au-LM) using the aqueous leaf extract of Lantana montevidensis (LM), a naturally available medicinal plant. The biosynthesized b-Au-LM was biocompatible in both in vitro and in vivo systems. However, the LM extract as well as b-Au-LM exhibited significant inhibition of the proliferation of cancer cells. Interestingly, b-Au-LM showed enhanced anti-cancer activity compared to the pristine LM extract. Generation of reactive oxygen species (ROS) and oxidative stress, which triggered the upregulation of caspase-3, might be the plausible reason for anti-cancer activity. Cell cycle analysis demonstrated G2/M (in A549 cells) or Sub-G1 (in MCF-7) cell cycle arrest, which might lead to apoptosis. Together, the results support the future therapeutic application of an in situ biosynthesized gold nanoconjugates based drug delivery system (b-Au-LM) towards cancer therapy and other biomedical applications.