breast cancer is the second most common cancer worldwide , the fifth most common cause of cancer death , and the leading cause of cancer death in women .
the global burden of breast cancer exceeds all other cancers and the incidence rates of breast cancer are increasing . despite significant advances that have improved breast cancer detection and treatment ,
women continue to die of the disease even when disease is detected at an early stage .
while locoregional treatment has improved , there has been little progress to improve the diagnosis and treatment of patients with significantly advanced disease . at present
, there are limited curative options for patients with disease that has advanced beyond regional nodes .
novel treatment strategies , such as nanoparticle drug delivery systems , offer the opportunity to enhance the delivery of anticancer therapeutics into breast cancer cells and may eventually improve patient outcomes .
nanoscale drug delivery systems formulated from biocompatible and biodegradable polymers constitute an evolving approach to improve drug delivery and tumor targeting .
alg particles offer promise as drug delivery nanoparticles because of the biocompatibility , water solubility , and bioadhesive properties that improve drug - binding .
advantages of alg also include specific uptake by tumor cells , nontoxic effect on healthy tissue , and accumulation in tumor tissue with demonstrable antitumor efficacy .
ptx is a microtubule - stabilizing agent that promotes tubulin polymerization causing cell death by disrupting the dynamics needed for proper cell division .
ptx is poorly soluble in aqueous solutions but soluble in organic solvents , for example , alcohols , which lends itself to advanced formulation strategies .
the currently available formulation of ptx includes cremophor el ( polyethoxylated castor oil ) and ethanol for solubilization . however , cremophor el generates unwanted toxic effects as well as unwanted side effects , for example , hypersensitivity reactions , nephrotoxicity , and neurotoxicity .
biodegradable nanoparticle formulations using poly(lactic - co - glycolic acid ) ( plga ) have shown comparable activity to traditional formulations with much faster rates of administration .
these same nanoparticles ranged in diameter from 117 to 160  nm and in vitro released over 50% of loaded drug over the first 24  h with a much slower release rate of release over the next 96  h. cellular studies showed up to a 70% loss of viability in nci - h69 human small cell lung cancer cells at levels as low as 0.025  g / ml .
another group has added vitamin e tpgs ( d--tocopheryl polyethylene glycol 1000 succinate ) as an emulsifier and matrix component to plga nanoparticle for ptx release .
psa - peg nanoparticle containing ptx as a model drug has also been prepared and evaluated by nanoprecipitation methods [ 58 ] .
cancer nanotechnology is emerging as a new field of interdisciplinary research expected to advance cancer detection , diagnosis , and treatment .
nk105 is a ptx - containing polymeric micelle that consists of poly(ethylene glycol ) chemically linked to poly(aspartate ) [ 1012 ] .
genexol - pm ( samyang pharmaceuticals , daejeon city , korea ) is another polymer - based micelle that contains ptx . in a phase
i study , genexol - pm was used to deliver ptx to tumors without additional adverse toxicity and had an increased response rate in metastatic breast cancer patients compared to treatment with taxol alone [ 1315 ] .
poly(l - glutamic acid)-ptx conjugate ( ct-2103 ) and poly(l--glutamyl - glutamine)-ptx nanoconjugate ( pgg - ptx ) are also ptx - containing polymers being investigated for anticancer activity [ 1622 ] .
pgg - ptx has shown antitumor activity in vivo that was superior to abraxane ( abraxis bioscience , summit , nj , usa ) in some mouse models .
the pharmacokinetic and tissue distribution of pgg - ptx was improved over ptx alone and the level of active free ptx in the plasma and tumor was increased compared to cremophor - ethanol mixtures of ptx in mice bearing lung cancer xenografts .
we hypothesized that a ptx - micelle conjugate could create a novel class of antitumor nanoparticles with improved therapeutic index compared to that of ptx alone .
here , we generated alg - ptx nanoparticles and demonstrate their ability to mediate efficient drug delivery and killing of breast cancer cells . to achieve this goal ,
alg - ptx was synthesized and prepared in nanoparticles using a simple emulsion method ( figure 1 ) .
the resulting nanoparticles were then compared using uv / vis methods , hplc - fluorescence detection , and tem with unmodified ptx .
our results clearly demonstrate the potential of alg - ptx nanoparticles to efficiently target and eradicate breast cancer cells .
louis , mo ) , sodium alginate from cica - reagent , tokyo , japan , and all other reagents were from sigma - aldrich or merck .
water was purified using the milli - q reverse osmosis system ( emd millipore , billerica , ma ) .
briefly , 25  l of the drug ( 30  mg/5  ml ) , in 10  ml of chloroform , is emulsified under sonication in 30  ml of 0.1%  m  ( w / v ) aqueous solution of sodium alginate as emulsifier for 30  min and glycerol ( 15  ml ) as stabilizer , and also emulsifier was added into the mixture .
nanoparticles were separated by ultracentrifugation ( cooling centrifuge , sigma , usa ) at 10,000  rpm , 0c for 10  min . to remove the nonincorporated drug ,
the obtained nanosuspension was filtered ( s&s  filter paper circles ,  pore size 1  mm ) .
nanoparticles were washed with milli - q reverse osmosis water and then dried at room temperature .
tem samples were prepared by drop coating the stock suspension on carbon - coated copper grids .
tem photograph shows nanoparticles ( scale bar representing 500  nm , image taken at 80,000x magnification ) .
breast cancer tumor tissue specimens were obtained at the time of surgery after informed written consent in accordance with south egypt cancer institute ethical committee guidelines .
tumor tissues were immediately disaggregated mechanically using a 16-gauge stainless steel mesh . for analysis of apoptosis by flow cytometry , propidium iodide
( pi ) and fitc conjugated annexin - v ( iq products , netherlands ) were used with the appropriate mouse isotype controls ( bd pharmingen , san diego , ca ) .
all antibodies were used at concentrations titrated for optimal staining and carried out using standardized protocols .
after treatment with anticancer drugs as indicated , breast cancer cells were fixed and permeabilized with 70% ice - cold ethanol for at least 1  h and then washed twice in phosphate - buffered saline ( pbs ) .
dna was stained by incubating cells at 37c for 1  h in 40  g / ml pi and 100  g / ml dnase - free rnase in pbs .
as the fluorescence area ( fl2-a ) is the main parameter in the cell cycle analysis , histogram plots of fl2-a served as a cell cycle graph using modfit lt 3.0 software ( bd , san jose , ca , usa ) for analysis .
cells were analyzed for expression of annexin - v and both annexin - v and pi to identify early and late apoptosis , respectively .
flow cytometry was performed with fluorescence - activated flow cytometry ( facs ) calibur system ( bd , san jose , ca ) .
fluorocytometric results were subsequently analyzed and displayed with cell quest software ( bd , san jose , ca ) .
the tungsten - filament 100  kv advanced high performance jeol jem-100cxii system ( jeol , tokyo , japan ) was used to obtain high - resolution tem images .
absorption spectra of solutions were recorded using a spectro uv - vis double beam pc scanning spectrophotometer uvd-2950 ( labomed inc .
hplc analysis was carried out using agilent hplc 1200 ( santa clara , ca ) system consisting of degasser , quaternary pump , and a fluorescence detector .
hplc chemstation software ( agilent ) was used for instrument control , data acquisition , and data analysis .
ptx , alg , and alg - ptx nanoparticles were eluted using a zorbax extend c18 column ( 150  4.6  mm i d  5  m particle size ) and detected at 232  nm excitation and 353  nm emission with a flow rate of 1.5  ml  min .
column temperature was maintained at 25c . the mobile phase consisted of 40% of water , 35% acetonitrile ( acn ) , and 25% methanol .
nanoparticles were prepared and the zeta potential (  ) was measured using an agilent 1600 ce system ( agilent technologies , germany ) , equipped with a diode - array uv / vis detection system ( 190 to 600  nm ) .
ce chemstation software was used for instrument control , data acquisition , and data analysis .
bare fused - silica capillaries ( agilent ) are with 64.5 cm total length , 56.0  cm effective length , and 50.0  m internal diameter
. the applied voltage was maintained at 20  kv with a controlled temperature at 25.0c to give the current value of 15  a .
a small amount of pbs was added to obtain charges in the solution and to mimic the ionic concentration in the human body . a total h2o  :  pbs ratio equal to 20  : 
is then added to the cell to measure . a new capillary was conditioned by rinsing with 1.0  mol  l naoh for 40  min at 40.0c and water for 20  min at 25.0c .
the flushing procedure was optimized to give precise analysis by flushing with 0.1  mol  l naoh for 2  min , followed by water for 2  min , and then equilibrated with running buffer for 5  min .
immediately after sonication and stabilization , figure 2(a ) shows the transmission electron microphotographs of the alg nanoparticles and figures 2(b ) and 2(c ) show the ptx - loaded alg nanoparticles .
minimal drug degradation ( 0.1% ) was seen after storage at 4c for 8 months to indicate that the ptx - loaded alg nanoparticles remained stable in polymer - generated matrices .  
figure 3 indicates the spectrophotometric analysis of ptx , alg nanoparticles , and ptx - alg nanoparticles .
a shift is observed for ptx - loaded alg nanoparticles ( red ) from that of ptx alone ( black ) .
also it is shifted from the alg nanoparticles alone ( blue ) to indicate the formation of a new compound that is detected between alg nanoparticles and ptx .
fluorescence spectroscopy is a type of electromagnetic spectroscopy , which then measures fluorescence within a given sample .
the interest of monitoring the fluorescence spectral changes is to study the interactions of ptx with the biodegradable molecules , for example , alg . in these studies , we used liquid chromatography combined with fluorescence ( figure 4 ) .
zeta potential (  ) is defined as the measure of magnitude for the electrostatic charge repulsion or attraction determined between two particles .
zeta is one known to affect stability and allows insight into the causes of dispersion and aggregation . to get information about the electrostatic potential of the particles in solution , as well as their colloidal stability ,  of the prepared ptx - alg nanoparticles have to be measured .
a low  value is wanted in order for the particles to be as neutrally charged as possible and therefore less interference will occur in vivo . that will allow a better circulation all the way to the target site . at the same time
colloidal stability is obtained at a value higher than + 30  mv or lower than 30  mv .
also , a positive value is not desired because it is often a sign of toxicity .  is determined by measuring the electrophoretic mobility , which is the velocity of the particles when an electric field is applied to them . indeed ,
if a particle is charged , it will move with a constant velocity ( after equilibrium ) to the electrode of opposite charge , when near electrolytes .
the magnitude of the zeta potential ,  , is given by(1)=4eo,where  is the viscosity , eo the electroosmotic mobility , and  the dielectric constant .
ptx - loaded alg nanoparticles showed a significantly higher cytotoxicity than commercially available ptx ( taxol ) .
chemotherapeutics have been shown to inhibit cellular proliferation , block cell cycle progression , and induce apoptosis in cancer cells .
therefore , we evaluated the effect of ptx - alg nanoparticles on the cell cycle and apoptosis of breast cancer cells using pi and annexin - v staining . shown in figure 5 are the results of one such experiment .
the administration of ptx - encapsulated alg nanoparticles led to tumor cell apoptosis and block in the cell cycle , compared with that observed with taxol .
we determined that the percentage of apoptotic cells in untreated cells was 11% and was increased to 83% after treatment of the breast cancer cells with ptx nanoparticles .
treatment with alg - ptx nanoparticles further increased the percentage of apoptotic cells to 97% .
cell cycle analysis was performed to determine the effect of the nanoparticles on the cell cycle of patient primary breast cancer cells freshly isolated by tissue biopsy .
a high amount of debris was detected making the exact percentage of cells in each phase difficult to be calculated by modfit software .
after treatment with ptx - alg nanoparticles , cell cycle arrest in g2 phase was significantly increased ( p < 0.05 ) ( figure 6 ) .
we have shown that ptx - alg nanoparticles inhibit breast cancer cell viability and offer major advantages over ptx or ptx nanoparticles alone .
ptx - alg nanoparticles may reduce the unwanted side effects of ptx , increase drug solubility and dispersibility , offer biocompatibility , improve conjugation with other bioactive molecules , and increase the thermal and chemical stability of the drug .
since hydrophilicity of nanoparticles is very important to disperse nanoparticles in biological ( aqueous ) systems with biocompatibility a valuable alternative to conventional drug delivery systems , the ease of synthesis of the nanoparticles reported here also plays an important role in future bioapplications .
recently the microparticle properties on ptx release and in vitro and in vivo pharmacodynamics have been evaluated .
biodegradable polymeric nanoparticles , based upon in vivo stability and safety in human patients , may provide an improved delivery method for anticancer agents [ 17 , 18 , 24 ] .
the release of cancer drugs from poly(lactide - co - glycolide ) plga - containing particles is currently under study .
ptx delivery using plga - derived nanoparticles resulted in a reduction in ptx clearance from the peritoneal cavity as well as higher tumor exposure to ptx , compared to a cremophor solution of ptx .
future studies will address the precise molecular mechanisms by which alg - ptx nanoparticles inhibit cancer cell growth and induce apoptosis .
future studies will also investigate whether the findings reported here are applicable to other cancers .
an in - depth mechanistic understanding of the interactions between nanoparticles and tumors will lead to new strategies that advance clinical development of alg nanoparticles as an anticancer therapy .
 
 purpose . 
 the antitumor activity of a novel alginate ( alg ) polymer - based particle that contained paclitaxel ( ptx ) was evaluated using human primary breast cancer cells . 
 materials and methods . 
 ptx was combined with alg in a nanoparticle as a drug delivery system designed to improve breast cancer tumor cell killing . 
 ptx - alg nanoparticles were first synthesized by nanoemulsification polymer cross - linking methods that improved the aqueous solubility . 
 structural and biophysical properties of the ptx - alg nanoparticles were then determined by transmission electron microscopy ( tem ) and high performance liquid chromatography ( hplc ) fluorescence . 
 the effect on cell cycle progression and apoptosis was determined using flow cytometry . 
 results . 
 ptx - alg nanoparticles were prepared and characterized by ultraviolet ( uv)/visible ( vis ) , hplc fluorescence , and tem . 
 ptx - alg nanoparticles demonstrated increased hydrophobicity and solubility over ptx alone . 
 synthetically engineered ptx - alg nanoparticles promoted cell - cycle arrest , reduced viability , and induced apoptosis in human primary patient breast cancer cells superior to those of ptx alone . conclusion . taken together , 
 our results demonstrate that ptx - alg nanoparticles represent an innovative , nanoscale delivery system for the administration of anticancer agents that may avoid the adverse toxicities with enhanced antitumor effects to improve the treatment of breast cancer patients . 
1. Introduction
2. Methods 
3. Results
4. Discussion