since its approval in 1971 , cisplatin has been used to treat a variety of human cancers . whereas the drug has a nearly 100% cure rate for testicular cancer , its use in treating ovarian , bladder , lung , head , and neck cancers is limited by acquired resistance .
cisplatin and its analogues carboplatin and oxaliplatin act by binding to dna , thereby preventing its transcription and replication and through that inducing cellular apoptosis .
resistance can arise from reduced drug uptake / enhanced efflux , increased repair / tolerance of drugdna adducts , and increased intracellular levels of the tripeptide glutathione that can readily degrade and deactivate cisplatin .
recently , elevated intracellular chloride concentrations have been shown to be another possible mechanism of resistance .
the use of cisplatin is also limited because of its severe dose - limiting side effects that include nephrotoxicity , ototoxicity , and neurotoxicity . in the past decade , only one new platinum drug , oxaliplatin , has been approved worldwide , and although it has shown potential for treating a range of cancers , it is only currently used for colorectal tumors . like cisplatin ,
many of the side effects of cisplatin and oxaliplatin are due to their nonspecific attack of all rapidly dividing cells ; therefore , platinum - based chemotherapy can be greatly improved through enhanced drug delivery .
platinum drugs can be passively targeted to solid tumors through the enhanced permeability and retention effect .
alternatively , platinum drugs can be actively targeted to both solid tumors and leukemias through the use of aptamers , peptides , antibodies , or cancer - related substrates ( such as folate ) . over the past decade
, gold nanoparticles have been developed that demonstrate a wide variety of applications , including catalysis , bioanalysis , and imaging .
gold nanoparticles are also an ideal drug - delivery scaffold because they are known to be nontoxic and nonimmunogenic .
gold nanoparticles can also be readily functionalized with multiple targeting molecules and have so far shown excellent potential for the delivery of other nonplatinum - based drugs and a platinum(iv ) complex .
several gold nanoparticle - based drugs are currently under development by cytimmune , with their lead drug , aurimune , in clinical trials .
typically , gold nanoparticles ranging from 13 to 60 nm can be easily made by a simple reduction of the gold salts in water or by starting with seed particles of 13 to 20 nm followed by a second reduction step involving more gold salt .
as such , gold nanoparticles with very high surface areas suitable for the attachment of a large number of platinum drug molecules are readily available . in this article , we report the synthesis of a platinum - tethered gold nanoparticle for improved anticancer drug delivery , where the platinum molecule is the active component of oxaliplatin : [ pt(1r,2r - diaminocyclohexane ] ( [ pt(dach ] ) .
this was characterized using dynamic light scattering , scanning electron microscopy , electron probe microanalysis , and inductively coupled plasma mass spectrometry ( icp - ms ) .
its in vitro uptake , localization , and cytotoxicity in a549 lung and hct116 , hct15 , ht29 , and rko colon cancer cells was also examined using growth inhibition assays , icp - ms , and transmission electron microscopy .
nanoparticles ( 900 l , 26.7 nm based on the extinction coefficient of gold ) were digested using nacn solution ( 100 l , 0.067 m ) for 24 h and then analyzed on a thermo electron corp .
the instrument used a concentric nebulizer with a peltier - cooled conical single - pass spray chamber with impact bead and an integral peristaltic pump for sample uptake from a cetac asx-520 autosampler .
a hexapole for cct ed ( collision cell technology with energy discrimination ) mode was used to remove polyatomic interference .
the instrumental operating conditions were 1400 w rf forward power , 13 l min plasma flow , 1.0 l min nebulizer flow , and 0.8 l min auxiliary flow .
a sample flush time of 60 s , a wash time of 60 s , and a peak jump mode were used with a dwell time per isotope of 10 ms .
calibration solutions were prepared from a spex  certiprep  certified standard diluted as required with 2% fisher  primar plus  nitric acid .
samples of naked nanoparticle , pegylated nanoparticles , and platinum - tethered nanoparticle ( 1 l ) were dried on a silicon substrate and placed under vacuum .
sem images were collected using a fei sirion 200 ultrahigh - resolution schottky field - emission scanning electron microscope running fei software .
an accelerating voltage of 5 kv was applied to each sample , and a spot size of 3 was used . a sample of platinum - tethered nanoparticle ( 1 l ) was dried on a silicon substrate and placed under vacuum .
the wavelength - dispersive x - ray spectrum was acquired in a cameca sx100 electron probe microanalyzer using a pentaerythritol crystal with a lattice spacing of 2d = 8.75  at 20 kev , and a 40 na electron beam was used .
dynamic light scattering and zeta potential experiments were conducted on a malvern zetasizer nano zs . the machine was calibrated using a 60 nm polystyrene standard .
each 5 nm , 1 ml sample was loaded into a cell , and the particle size and zeta potential were measured simultaneously three times and in triplicate .
a549 lung epithelial cancer cells were grown in dulbecco s modified eagle s medium ( dmem ) containing 10% fetal calf serum at 37 c and in a 5% co2 atmosphere .
the cells were trypsinised , counte , and adjusted to 100  000 cells ml and 2 ml added per plate .
stock solutions of platinum - tethered nanoparticles were diluted using dmem and added to the plate in 800 l quantities .
after incubation for 24 h , cells were thoroughly washed with phosphate - buffered saline ( pbs ) in order to eliminate nanoparticles that were not internalized .
cell processing for tem analysis was carried out in situ , without displacement from the culture dish .
cells were fixed in a 0.1 m pbs solution containing 2.5% gluteraldehyde and 4% paraformaldehyde for 1 h. they were then rinsed with 0.1 m pbs and postfixed in 1% osmium tetroxide solution ( extremely toxic ; use caution ) for 1 h , rinsed with distilled water , stained with 0.5% uranyl acetate for 1 h , dehydrated in a graded series of ethanol ( 30 , 60 , 70 , 90 , and 100% ) , and embedded in epoxy resin .
the resin was polymerized at 60 c for 48 h. ultrathin sections ( 5075 nm ) obtained with an lkb ultramicrotome were stained with 2% aqueous uranyl acetate and 2% aqueous lead citrate and imaged under a 120 kv fei tecnai spirit tem . for the human lung cancer cell line a549 and
the human colon cancer cell lines hct116 , hct15 , ht29 , and rko , cytotoxicity was determined by a tetrazolium dye - based microtitration assay .
cells were plated out in 96-well plates at a density of 5001000 cells per well and were allowed to attach and grow for 2 days .
cells were exposed to oxaliplatin , pegylated gold nanoparticles , or platinum - tethered gold nanoparticles over a range of concentration for 72 h , and then the medium was replaced with a drug - free medium for another 24 h. on the final day , mtt ( 50 l of a 5 mg / ml solution ) was added to the 200 l of medium in each well , and the plates were incubated at 37 c for 4 h in the dark . the medium and mtt
were then removed , and the mtt - formazan crystals were dissolved in 200 l of dmso .
glycine buffer ( 25 l per well , 0.1 m , ph 10.5 ) was added , and the absorbance was measured at 570 nm in a multiwell plate reader .
a typical doseresponse curve consisted of eight drug concentrations , and two wells were used per drug concentration .
a549 cells were plated out at a density of 10 cells per well in six well plates and incubated overnight at 37 c in a humidified atmosphere of 10% co2 in air .
the medium was then replaced with fresh medium alone or with medium containing oxaliplatin or platinum - tethered gold nanoparticles for 4 h. the medium was removed , and the cells were then washed with ice - cold pbs and lysed by the addition of 215 l of nitric acid ( optima 68% ) to each well .
the cells were then collected in an eppendorf tube and incubated overnight at 65 c .
they were then diluted with water/0.1% triton - x100 to a final concentration of 1% acid before the platinum content of the samples was determined by icp - ms .
naaucl42h2o ( 50 mg , 0.14 mmol ) was dissolved in distilled water ( 500 ml ) and heated to 100 c with continuous stirring . upon boiling , sodium citrate ( 1% m / v , 7.5 ml )
was added , and the solution boiled for 15 min before cooling to room temperature .
uvvisible spectra ( max = 519523 nm ) indicated the successful formation of the colloid .
wang resin ( 0.5 g , 1 mmol / g loading ) was suspended in dcm ( 50 ml ) . to this , succinic anhydride ( 0.25 g , 5 equiv ) and dimethylaminopyridine ( 0.3 g , 5 equiv ) were added .
this was then refluxed at 60 c for 6 h. the resin was separated from the mixture by filtration and washed several times with dcm and meoh .
jeffamine ed-2003 ( 4.25 g , 5 equiv ) and n , n - diisopropylcarbodiimide ( 387 l , 5 equiv ) were added , and the mixture was shaken overnight .
the resin was then separated from the mixture as before , using filtration , and was washed several times with copious amounts of dcm and meoh .
the resin was once again suspended in dcm ( 50 ml ) , and to this n , n - diisopropylcarbodiimide ( 387 l , 5 equiv ) and thioctic acid ( 0.516 g , 5 equiv ) were added .
the mixture was shaken overnight in the absence of light and then washed as before with dcm and meoh .
the resin was suspended in 10% trifluoroacetic acid in dcm at room temperature for 3 h and shaken to cleave the desired compound .
the resin was removed by filtration , and the filtrate was concentrated under reduced pressure . the remaining liquid was then washed with diethylether , which was subsequently decanted , and the product was left to dry under high vacuum overnight .
yield : 386 mg , 18% . to an eppendorf tube containing gold nanoparticles ( 1 ml , 17 nm ) was added peg linker ( 100 l , 1 mm ) , and the solution was allowed to form a monolayer on the nanoparticles by shaking for 4 h. to wash away unbound peg linker , the nanoparticles were centrifuged at 7000 rpm for 20 min , the supernatant was removed , and the remaining pellet was dissolved in milli - q water ( 1 ml ) .
n , n - diisopropylethylamine ( 100 l , 0.1 mm ) was added to the nanoparticles .
[ pt(r , r - dach)(h2o)2]2no3 ( 10 mg ) was dissolved in 1,3-dimethyl-3,4,5,6-tetrahydro-2(1h)-pyrimidone ( 1 ml ) , added to the nanoparticles in 10100 l aliquots , and left overnight .
finally , the gold nanoparticles were centrifuged , the supernatant was removed , the remaining pellet was resuspended in milli - q water ( 1 ml ) , and the wash was repeated once more .
the production of the platinum - tethered nanoparticles began with the synthesis of citrate - reduced gold nanoparticles from sodium tetrachloroaurate(iii ) dihydrate ( figure 1 ) .
the attachment of [ pt(r , r - dach ) ] then required the design , synthesis , and attachment of a suitable linker .
alkyl thiolgold chemistry has been well established in terms of functionalizing gold surfaces and nanoparticles to form stable monolayers ; however , these are normally hydrophobic in nature and , as such , are not ideal for biological applications . in addition , the thiolgold bond is susceptible to displacement by strong nucleophiles commonly found in vivo . to overcome this
, we used a cyclic disulfide to bind the linker to the nanoparticles . in the past , this has increased the stability to such an extent that displacement by millimolar concentrations of dithiothreitol ( an unusually strong reducing agent ) was not possible .
additionally , our monolayer also contains a stabilizing linkage that is hydrophobic on the inner core of the nanoparticle but then converts to being hydrophilic on the outer part of the sphere to make it compatible with a biological environment ( e.g. , use of polyethylene glycol ( peg ) spacers ) .
the peg linker used on our nanoparticles in this study was made using solid - phase synthesis beginning with refluxing succinic anhydride with wang resin to yield a carboxylic acid group available for amide formation with a bis - amino peg copolymer ( figure 1 ) .
one amine group of the copolymer was coupled to the carboxylic acid on the resin , and the other was reacted with thioctic acid .
washing away unbound reagents from the resin in between each step and cleavage with trifluoroacetic acid yielded a peg linker with an average molecular weight of 2080 g mol .
the formation of a peg monolayer on the surfaces of the gold nanoparticles was achieved through shaking in water , with excess linker removed by centrifugation .
the active component of oxaliplatin , [ pt(r , r - dach ) ] , was then added to the surface of the pegylated nanoparticles ( figure 1 ) .
the platinum - tethered gold nanoparticles were characterized by electron probe microanalysis ( epma ) to confirm that both gold and platinum were present through their elemental peaks at 2.12 and 2.05 kev , respectively ( figure 2 ) . although the amounts of gold and platinum in the sample could not be determined quantitatively because of scattering effects from the rough surfaces of the nanoparticles , the spectra clearly indicated that each nanoparticle contained a much larger percentage of gold compared to platinum .
the number of [ pt(r , r - dach ) ] molecules per gold nanoparticle , after digestion with sodium cyanide , was determined to be 280 ( 20 ) by icp - ms .
this represents a significant improvement in the number of drug molecules that can be carried on other delivery scaffolds , such as carbon nanotubes ( 82 drug molecules per nt ) , hyperbranched polymers ( 36 drug molecules ) , or dendrimers ( 30104 depending on the dendrimer generation / size ) .
it is possible that our gold nanoparticles could be made to deliver even more drug molecules if other linker terminal groups , such as bidentate carboxylates , are used to couple the platinum molecules more strongly to the nanoparticle .
electron probe microanalysis spectrum of platinum - tethered gold nanoparticles showing the presence of gold ( 2.12 ev ) and platinum ( 2.05 ev ) in the sample .
the size and charge of the naked gold nanoparticles , the pegylated nanoparticles , and the platinum - tethered nanoparticles were determined by dynamic light scattering ( table 1 ) .
upon addition of the peg linker , the nanoparticles increase in size slightly but maintain a similar zeta potential because of the negative charge of the terminal carboxylate groups .
upon addition of [ pt(r , r - dach ) ] , the nanoparticles increase significantly in size ( 4.5-fold ) and change from a net negative to a positive charge .
this increase was unexpected and is thought to represent either aggregation of the nanoparticles through hydrophobic interactions between the cyclohexane components of [ pt(r , r - dach ) ] or the cross linking of multiple nanoparticles using [ pt(r , r - dach ) ] as a bridging ligand ( figure 1 ) .
this increase in size might be a potential problem when using these nanoparticles in vivo , and methods to prevent the aggregation of platinum - tethered gold nanoparticles may therefore need to be developed .
, however , has shown that the vasculature of solid cancers leaves them susceptible to nanoscale - sized particles .
recent research has shown that nanoparticles with diameters of less than 200 nm are ideal for targeting tumors , which means that the aggregation seen for our platinum - tethered gold nanoparticles , rather than being a problem , may even be beneficial and assist them in being more selective for solid cancers . the size and aggregation of the platinum - tethered nanoparticles
the naked nanoparticles are approximately 2129 nm in diameter , and the pegylated nanoparticles are roughly 3040 nm , with some evidence of aggregation .
the platinum - tethered nanoparticles appear to be approximately the same diameter as the pegylated nanoparticles but are now more aggregated . because of the drying nature of the technique , it is impossible to calculate the degree of aggregation or whether this aggregation is due to [ pt(r , r - dach ) ] molecules bridging multiple nanoparticles or simply is due to hydrophobic interactions . scanning electron microscope pictures of ( a ) naked gold nanoparticles , ( b ) gold nanoparticles with a monolayer of peg linker , and ( c ) platinum - tethered gold nanoparticles , showing their increase in size with functionalization and the high propensity of the platinum - tethered nanoparticles to aggregate in both the solution and solid state .
lung and colorectal cancers are the first and third most common cancer types in the world , respectively . as such , the ability of the nanoparticles to induce cellular apoptosis was determined using growth - inhibition assays with the a549 human lung epithelial and the hct116 , hct15 , ht29 , and rko human colon cancer cell lines .
the pegylated nanoparticles demonstrated no cytotoxicity at concentrations up to 10 nm in all five cancer cell lines ( table 2 ) .
in contrast , the platinum - tethered gold nanoparticles had an ic50 of 0.495 nm in the a549 cell line ( based on the gold nanoparticle concentration ; 0.135 m based on the number of [ pt(r , r - dach ) ] molecules per nanoparticle ) , which is almost 6-fold more active than oxaliplatin ( ic50 = 0.775 m ) . in the colon cancer cell lines , the platinum - tethered gold nanoparticles are up to 5.6-fold more cytotoxic or at least as active as oxaliplatin ( table 2 ) .
the cytotoxicity of the platinum - tethered gold nanoparticles is given in terms of both the concentration of gold nanoparticles and as the platinum drug concentration .
the fold increase is the cytotoxicity of the platinum - tethered nanoparticles ( with respect to the concentration of platinum ) in relation to oxaliplatin itself .
it was originally thought that the benefit of the platinum - tethered gold nanoparticles would be from their enhanced plasma retention and circular times and better uptake into solid tumors , in vivo , compared with cisplatin .
therefore , the enhanced in vitro cytotoxicity was unexpected and suggests that gold nanoparticle - based delivery of platinum drugs may significantly improve their potency , possibly through enhanced cellular uptake .
importantly , our nanoparticles do not rely on near - infrared radiation to induce their cytotoxic effect , as many gold nanoparticle drug - delivery systems do , thus greatly simplifying their application in treating cancer .
given the excellent cytotoxicity , the uptake and localization of the platinum - tethered nanoparticles were then examined using icp - ms and transmission electron microscopy .
a549 lung cancer cells were incubated with oxaliplatin and platinum - tethered gold nanoparticles at equal [ pt(r , r - dach ) ] concentrations . after 4 h ,
the results demonstrate a significantly higher uptake of [ pt(r , r - dach ) ] by the gold nanoparticles inside the cell ( 21.34  0.89 ng pt/10 cells ) compared with that of oxaliplatin ( 10.19  0.95 ng pt/10 cells ) and also demonstrate that the gold nanoparticles are capable of delivering [ pt(r , r - dach ) ] to the cell and then releasing it .
the a549 lung cells were also incubated with 225 nm platinum - tethered nanoparticles for 24 h , and the uptake was examined using transmission electron microscopy ( tem ) .
the resultant images show a number of important features , particularly intracellular accumulation of the gold nanoparticles ( figure 4 ) . at the nanoparticle concentration tested ,
cell growth was very significantly hindered and cell stress is seen by a large contraction of the cell wall toward the nucleus .
gold nanoparticles are observed both inside and in contact with the plasma membrane of the cell but more importantly also within the cell nucleus .
nonfunctionalized gold nanoparticles are known not to be able to penetrate cell nuclei , and neither the naked nanoparticles nor the pegylated nanoparticles when tested under the same conditions as the platinum - tethered nanoparticles are observed in the nucleus .
this result implies some sort of [ pt(r , r - dach)]-mediated transport mechanism into the nucleus , where presumably the drug is then released to bind with dna .
section of a whole cell transmission electron microscope image showing both the intracellular and intranuclear uptake of the platinum - tethered gold nanoparticles into a549 lung cancer cells : ( 1 ) extracellular nanoparticles , ( 2 ) intracellular nanoparticles , ( 3 ) intranuclear nanoparticles , ( 4 ) contracted cell wall , ( 5 ) double - envelope nuclear membrane , ( 6 ) endosome , and ( 7 ) lysosome .
whereas both endosomes and lysosomes are observed in the tem images , no platinum - tethered nanoparticles are located within them .
conversely , both the naked and pegylated nanoparticles demonstrated cellular uptake through endocytosis ( figure 5 ) .
previously , some cationic nanoparticles have been shown to enter cells by generating transient holes in the membrane , and it is possible that our platinum - tethered nanoparticles are taken up by the cancer cells in a similar manner .
interestingly , the previously observed aggregation of the nanoparticles was not observed and did not appear to inhibit uptake .
the reason for the lack of aggregation is unclear but may be due to the lower concentration of the nanoparticles or because [ pt(r , r - dach ) ] had already been released and thus was not acting as a bridging ligand between two individual nanoparticles .
tem images of a549 lung cancer cells showing ( a ) endocytosis of naked gold nanoparticles into the cell and ( b ) pegylated gold nanoparticles .
in this article , we have developed a drug - delivery strategy for platinum - based anticancer drugs based on gold nanoparticles .
the versatility of gold nanoparticles means that a large family of goldplatinum drug nanoparticles can now be produced to tune and improve the technology .
this includes the synthesis of different sized gold particles ( 20100 nm ) on which to attach the drugs ; linkers of different lengths and structure , including different terminal end groups such as dicarboxylates or even thiols ; the use of different drugs including cisplatin and multinuclear drugs such as bbr3464 ; and the attachment of active targeting groups such as folate and estrogen , prostate or leukemia targeting aptamers , lung cancer targeting peptides , or b - cell lymphoma targeting antibodies .
this genuinely nanomedicine - based strategy offers a different approach to cancer therapy and holds significant potential to improve disease management and the treatment of cancer in the future .
 the platinum - based anticancer drugs cisplatin , carboplatin , and oxaliplatin are an important component of chemotherapy but are limited by severe dose - limiting side effects and the ability of tumors to develop resistance rapidly . 
 these drugs can be improved through the use of drug - delivery vehicles that are able to target cancers passively or actively . in this study , we have tethered the active component of the anticancer drug oxaliplatin to a gold nanoparticle for improved drug delivery . 
 naked gold nanoparticles were functionalized with a thiolated poly(ethylene glycol ) ( peg ) monolayer capped with a carboxylate group . 
 [ pt(1r,2r - diaminocyclohexane)(h2o)2]2no3 was added to the peg surface to yield a supramolecular complex with 280 ( 20 ) drug molecules per nanoparticle . 
 the platinum - tethered nanoparticles were examined for cytotoxicity , drug uptake , and localization in the a549 lung epithelial cancer cell line and the colon cancer cell lines hct116 , hct15 , ht29 , and rko . 
 the platinum - tethered nanoparticles demonstrated as good as , or significantly better , cytotoxicity than oxaliplatin alone in all of the cell lines and an unusual ability to penetrate the nucleus in the lung cancer cells . 
Introduction
Experimental Section
Results and Discussion
Conclusions