in recent years , the morbidity and mortality of cancer still reach a high plateau and is a major public health problem world wide ( 1,2 ) . cancer is the major cause of human s death because of high incidence and mortality . the identification of new cytotoxic drug with low side effects on immune system has developed as important area in new studies of pharmacology ( 3 ) . many studies report that a heavy diet in fruits and vegetables lowers the incidence of cancer ( 4,5 ) . some fruits and vegetables are considered as the main anticancer foods , because of their abundant antioxidants such as phenols , vitamin c , vitamin e , beta - carotene and lipotene ( 6 ) . it has been reported that various fruit and vegetable extracts are capable of inhibiting the protoasome activity and this inhibition is associated with tumor cell apoptosis ( 7 ) . thymoquinone ( tq ) is the bioactive compound derived from black seed ( nigella sativa ) oil . it is a spice that grows in the mediterranean region and in western asian countries . in folklore medicine , the seed is reportedly associated with diverse therapeutic benefits to bronchial asthma , dysentery , headache , gastrointestinal problems , eczema , hypertension , and obesity . tq has been reported to exhibit antiproliferative effects on cell lines derived from breast , colon , ovary , larynx , lung , myeloblastic leukemia , and osteosarcoma . chemosensitization by tq is mostly limited to in - vitro studies , and it has potential in therapeutic strategy for cancer . the results favor efficacy and enhancement of therapeutic benefit against tumor cells resistant to therapy based on cellular targets that are molecular determinants for cancer cell survival and progression ( 9 ) . drug overdose remains a common clinical problem across the globe , occurring both inadvertently and intentionally . consequently , a broad range of non - specific therapies have been developed to approach this problem , ranging from cathartics to more invasive and sophisticated approaches . nanogels , sub - micron hydrogel nanoparticles , have highly desirable properties that may make them particularly suitable for such applications . like hydrogels , nanogels have a three dimensional , internally cross - linked microstructure , swell in aqueous solvents to provide free volume for non - specific sorption , and can shrink and swell according to changes in the gel environment . like nanoparticles , nanogels are injectable , have extremely high specific surface areas available for interaction with chemicals in the gel environment , and respond much faster to environmental stimuli . based on these properties , nanogels have already attracted significant interest as sensors , rheological modifiers , optical devices , mechanical actuators , diagnostics supports , and drugdelivery vehicles , among other applications ( 11 ) . the aims of the current study were : 1 ) the preparation of ma - chitosan nanogels loaded with thymoquinone and 2 ) comparison the effects of thymoquinone and nanothymoquinone on the viability of human breast adenocarcinoma cell line . cell cytotoxicity and viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) and dye exclusion assay . thymoquinone ( 2-isopropyl-5-methyl-1,4-benzoquinone ) , myristic acid ( sigma aldrich  99% , mw = 228.37 g / mol ) , dimethyl sulfoxide ( dmso , sigma aldrich ,  99.9% , mw = 78.13 g / mol ) , chitosan(sigma aldrich , medium molecular weight of 340 g / mol ) and edc ( 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide sigma aldrich , mw = 155.24 g / mol , d = 0.814 g / ml ) were purchased from sigma chemical co. ( st . louis , mo . preparation of the nanogels 
  nanogels were prepared by the technique of self - assembly with the method of chen , et al . myristic acid ( ma ) solution was prepared by dissolving 228 mg ma in 23 ml dimethyl sulfoxide . chitosan ( 0.5 g ) was dissolved in 1% acetic acid solution ( 100 ml ) . edc was dissolved in methanol ( 126 l/15 ml ) ; then added to the ma solution . subsequently , chitosan solution was added drop wisely to the ma solution while stirring under the hood at room temperature . ma was coupled to chitosan by the formation of amid linkages through the edc - mediated reaction . the molecule oriented itself to exposed hydrophilic regions to the water and the hydrophobic regions assembled in the core ; by this process micelle like nanogels are prepared . dried nanogels were dissolved in 1% acetic acid solution and kept in refrigerator at -4 c until required for the tests . . a variable ratio of thymoquinone ( 0 , 5 , 50 , 100 , 200 and 300 g / ml ) was added to the nanogels while sonicating in water bath ( powersonic s10 ) with high - power ultrasound at room temperature for 30 min . thymoquinone -loaded nanogels ( tlns ) were characterized by malvern zetasizer ( malvern instruments ltd . , united kingdom ) to determine median size of nanogels and size distribution when loaded with different amount of the thymoquinone . scanning and transmission electron microscopy ( sem and tem ) was preformed to observe tlns morphology , shape , size and core and shell structures . for preparing sem photographs , one drop of tln was dropped onto studs and gold sputtered using an auto sputter coater ( biorad polaron division ) , then analyzed with hitachi field emission scanning electron microscope ( fe - sem ) s-4160 ( japan ) at 15 kv acceleration voltage . ( 13 ) . by using transmission electron microscope ( zeiss em 10c tem - gmbh ) at an accelerating voltage of 80 kv . the increasing amount of the thymoquinone led to an increase in the particles sizes with irregular shape ( figure 1 ) . cell culture and thymoquinone treatment 
  mcf7 cell lines were obtained from the national cell bank of iran ( ncbi ) . the cells were cultured in rpmi-1640 containing 10% fbs , 2 mm glutamine , antibiotics ( penicillin g , 60 mg / l ; streptomycin , 100 mg / l ; amphotericin b , 50 l / l ) under a humid atmosphere ( 37 c , 5% co2 , 95% air ) .   upon reaching appropriate confluence , the cells were passaged and incubated with 0(control group ) , 5 , 50 , 100 , 200 and 300 g / ml concentrations of thymoquinone and nanothymoquinone for defined times . the cells were treated with ma - chitosan nanogels at the same dose of that used in the maximum dose ( 300 microgram / ml ) group too . determination of cell viability 
  the assay detects the reduction of mtt [ 3-(4,5-dimethylthiazolyl)-2,5-diphenyl - tetrazolium bromide ] ( sigma ) ( a colorimetric technique ) by mitochondrial dehydrogenase to blue formazan product , which reflects the normal function of mitochondria and hence the measurement of cytotoxicity and cell viability . briefly , l0 cells / well were treated with various concentrations of thymoquinone and nanothymoquinone . after 24 hour incubation the cells were washed twice with phosphate buffered saline ( pbs ) and mtt ( 0.5 mg / ml pbs ) was added to each well and incubated at 37 c for 3 h. the formazan crystals that formed were dissolved by adding dmso ( 100 l / well ) , and the absorbance was read at 570 nm using a microplate scanning spectrophotometer ( elisa reader , organon teknika , netherlands ) . toxicity level was calculated by the following formula : 
 cytotoxicity%=1-mean absorbance of toxicantmean absorbance of negative control100 
  viability % = 100 - cytotoxicity % to diminish test error level , mtt strain was added to some wells without cells and along with other wells , absorbance level was read and ultimately subtracted from whole the absorbance . statistical analysis 
  statistical significances of difference throughout this study were calculated using a students t - test and by one - way variance analysis . p - values ( statistically significant if<0.05 ) were determined using the graph pad prism v.5 . surface morphology 
  the shape and surface texture of the nanoparticles could be detected using a number of sophisticated techniques such as tem or sem , respectively . the morphology of tq - loaded ma - chitosan nanogels as prepared is shown in  figure 1(a and b ) . transmission electron ( a ) and scanning electron ( b ) microscopy study of optimized nanoparticles 
 the e 
  
 ects of thymoquinone and nanothymoquinone on inhibition and proliferation of mcf7 cell line 
  the eect of thymoquinone and nanothymoquinone were studied as a dose - response experiment . proliferation of mcf7 cells was significantly inhibited by thymoquinone and nanothymoquinone in a concentration - dependent manner in 24 h. ( p<0.05 ) different concentrations of thymoquinone and nanothymoquinone at 24 h had different cytotoxicity effects on mcf7 cell line ( ) . the 50% inhibition concentration ( ic50 ) values of thymoquinone and nanothymoquinone on mcf7 cells were determined ( figure 3 ) . ic50 was determined by probit analysis using the pharm pcs ( pharmacologic calculation system ) statistical package ( springer- verlag , usa ) . preclinical studies reveal the potential of tq in improving the therapeutic effect of anticancer drugs and also protection of nontumor tissues against chemotherapy - induced damages . for example , tq ameliorated nephrotoxicity and cardiotoxicity by cisplatin , ifosfamide , and doxorubicin ( 14 - 16 ) . however , no phase i pilot study has so far been reported evaluating its promising anticancer effect in human subjects . of interest , gali - muhtasib et al . ( 17 ) noted that tq induces a sharp increase in p16 protein levels within 2 h of treatment ; this observation is of interest , since as mentioned by gali - muhtasib et al . ( 18 ) and hochhauser ( 19 ) , modulation of p16 protein expression increases tumor sensitivity to chemotherapeutic drugs . bioavailability of a drug to the cells , whether in - vitro or in - vivo , is critical for its optimal efficacy . physical entrapment of bioactive molecules such as drugs , proteins , carbohydrates , and dna in the polymeric network , as well as their in - vitro release behavior , have been extensively investigated as targeted drug delivery carriers for biomedical applications ( 20 - 23 ) . to enhance the solubility of drugs in aqueous solvents , increase their bioavailability , enhance serum half - life , for tumor cell targeting and bio imaging , conjugations of drugs with lipids , carbohydrates , and proteins have been used to make nanoparticles . recent studies from our laboratory have shown that enhanced uptake of cur cumin nanoparticles correlates with enhanced antitumor effects ( 20 ) . tq is the bioactive compound , but its poor solubility  in aqueous medium and relative toxicity affect its bio - availability and administration in human .   of particular relevance to pathophysiology of tumor growth is proliferation of tumor cells . thus far , tq has been shown to inhibit cell proliferation in cultured cells derived from human breast and ovarian adenocarcinoma , myeloblastic leukemia cells , hl-60 , squamous carcinoma [ sccvii ; ] , fibro sarcoma [ fssar ; ) ] , laryngeal neoplastic cells - hep-2 , and prostate and pancreatic cancer ( pc ) cell lines . interestingly , human pancreatic ductal epithelial cells ( hpde ) cells , noncancerous ( bph-1 ) prostate epithelial cells , normal kidney cells , and normal human osteoblasts are relatively resistant to inhibitory effect of tq as reported by us and others ( 9 ) . in the present investigation , tq - encapsulated chitosan nanoparticles were prepared successfully . we  prepared  thymoquinone -loaded nanogels to  enhance  solubility and investigate their ability to inhibit the proliferation of breast cancer cells in - vitro . when examined for its ability to suppress the growth of cancer cells , we found that thymoquinone -loaded nanogels was at least twice as potent as free tq , possibly due to enhanced uptake . the initial stage involved study on breast cancer cell proliferation and exposure to wide range of thymoquinone and nanothymoquinone concentrations . the long - term treatment with thymoquinone was studied for 72 h in breast cancer cell line mcf-7 . further analysis on cell cycle was previously carried out for prolonged treatment of 72 h ( 21 ) . thymoquinone was found to inhibit molecules which are involved in the s phase with 25 m thymoquinone concentration . furthermore , sub - g1  phase arrest occurred with either 25 m when treated for 12 h. it was previously reported more than 60 m thymoquinone led to sub - g1  arrest within 1248 h. this indicated cell growth inhibition could be sustained with minimal dose 25 m thymoquinone in an extended duration of treatment ( 22 ) . cells were found to be more sensitized following 72 h. the proliferation of mcf-7 cell line was regulated by dose of thymoquinone and in overall 50% cell death occured with 186.89 g / ml and 68.24 g / ml with 48 h treatment for thymoquinone and nanothymoquinone respectively . the design and preparation of nanogels have attracted a great deal of interest in biomedical engineering , pharmaceutical applications , and biomaterials science because of their tunable chemical and three - dimensional ( 3d ) physical structure , good mechanical properties , high water content , and biocompatibility . these unique properties offer great potential for the utilization of nanogels in tissue engineering , biomedical implants , drug delivery , and bionanotechnology ( 23 ) . a variety of polysaccharide  based nanogels have been subjected to intensive research in order to address the concerns related to the spontaneous self assembly behaviours of the nanogels in aqueous media . all the tested nanogels have clearly presented adequate cell viability and non - toxicity ( 23 ) . these nanogels have high content of functional group , compatibility , adjustable size , low immunogenicity and large surface area for multivalent bio - conjugation . furthermore , their abundance in nature , renewability , nontoxicity and low manufacturing cost make them unique candidates for biotechnology and pharmaceutics applications . chitosan myristic acid nanogel is used as a support for the organophosphorus hydrolase which degrade organophosphorus pesticides . acting as an inhibitor of acetylcholinestrase , organophosphorus compounds pose important hazards to the human and environment while they are used as effective pesticides and insecticides . growing usage of organophosphorus compounds in agriculture and manufacture of nerve agents has stimulated introduction of stabilization methods for degradative enzymes such as organophosphorus hydrolase ( 24 ) . recently reported chitosan polymers blended non - covalently to encapsulate curcumin using the capillary - microdot technique ( 25 ) . these curcumin nanoparticles showed higher efficacy against breast cancer cells and had the potential to treat breast tumors by local , sustained , and long - term therapeutic delivery as a biodegradable system . curcumin can block tnf - action and production in in - vitro models ( 26 ) . ( tnf)- stimulates the production of bcl-2 family member protein from the cytoplasm to the outer mitochondrial membrane ( 27,28 ) . this is the first study that describes the encapsulation of tq in chitosan myristic acid nanogel . it could be concluded that these nanogels are effective at low dose rates , with controlled and effective release and could be applied as an anticancer strategy .
cancer is one of the main causes of mortality in the world which is created by the effect of enviromental physico - chemical mutagen and carcinogen agents . 
 the identification of new cytotoxic drugs with low side effects on immune system has developed as important area in new studies of pharmacology . 
 thymoquinone ( tq ) , derived from the medicinal spice nigella sativa ( also calledt black cumin ) exhibit anti - inflammatory and anti - cancer activities . in this study we employed nanogel - based nanoparticle approach to improve upon its effectiveness . 
 myristic acid - chitosan ( ma - chitosan ) nanogels were prepared by the technique of self - assembly . 
 thymoquinone was loaded into the nanogels . 
 the surface morphology of the prepared nanoparticles was determined using sem and tem . 
 the other objective of this study was to examine the in - vitro cytotoxic activity of cell death of thymoquinone and nanothymoquinone on human breast adenocarcinoma cell line ( mcf7 ) . 
 cytotoxicity and viability of thymoquinone and nanothymoquinone were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) and dye exclusion assay.transmission electron microscopy confirmed the particle diameter was between 150 to 200 nm . 
 proliferation of mcf7 cells was significantly inhibited by thymoquinone and nanothymoquinone in a concentration - dependent manner in defined times . 
 there were significant differences in ic50 thymoquinone and nanothymoquinone . 
 tq - loaded nanoparticles proved more effective compared to tq solution . 
 the high drug - targeting potential and efficiency demonstrates the significant role of the anticancer properties of tq - loaded nanoparticles .