more than 10 million patients with new cases of cancer are diagnosed every year , and about 27 million new cases of cancer will have been recorded by 2030.1,2 some traditional cancer therapies , such as radiotherapy and chemotherapy , have enhanced the 5-year survival rates of cancer patients . for improving the therapeutic efficiency against cancer , increasing amounts
have been used to develop more new approaches , with the aims of fewer side effects , enhanced safety , and decreased invasiveness .
hyperthermia is known to induce apoptotic cell death in many tissues , in which the local temperature is raised more than 40c .
the heat generation sources , radiofrequency waves , microwaves , or ultrasound , have been used to produce moderate heating in a specific target region.3 heat energy can cause irreversible cell damage by denaturing proteins and the local cells or tissues are selectively destroyed .
however , a lack of specificity for tumor tissues would induce unavoidable cell damage in the surrounding healthy tissues , which has limited use in cancer treatment.3 while still in a relatively immature stage , gold nanoparticle - mediated photothermal therapy has contributed to great advances in cancer therapy .
gold nanostructures , as highly biocompatible materials , are widely used for biological application and medical purposes including imaging , drug delivery , and hyperthermia therapy.46 gold nanostructures provide precise control of sizes , shapes , and flexible surface chemistry for bioconjugation of biological molecules , which can offer molecular - level specificity for particular biocoupling in cancer cells . due to
unique and highly tunable optical properties , when gold nanostructures are exposed to light at their resonance wavelength , the conduction band electrons at the gold surface generate a collective coherent oscillation , resulting in strong light absorption or light scattering of gold . the absorbed light can be converted into localized heat , which can be readily employed for therapy based on photothermal destruction of cancer cells.710 pitsillides et al first reported the photothermal therapy in lymphocytes with a short pulsed laser in the presence of gold nanoparticle immunoconjugates in 2003.11 zharov et al reported gold - induced thermal destruction of cancer cells using a nanosecond laser.12,13 research on the use of gold in cancer treatment has also been carried out by el - sayed et al.10,14 several studies have reported on the feasibility and efficiency of tumor - specific targeting of gold nanostructures for photothermal cancer therapy , such as gold nanorods,15 nanoshells,5,16 and nanocages.17 in this study , on the basis of successfully synthesizing gold nanoparticle - antibody conjugates , l-428 hodgkin s cell - killing experiments induced by the photothermal effect of gold nanoparticles were implemented . under laser irradiation , through specific targeting of ligands to receptors , light strongly absorbed by gold is transferred to the antibody molecules and the cell environment , so that the very high killing efficiency of cancer cells can be achieved .
the irradiation laser was a frequency doubled q - switched neodymium ( nd):yag laser ( surelite i ; continuum , santa clara , ca ) , with nonlinear crystals to enable conversion of the fundamental wavelength frequency from 1064 nm to 532 nm ( 2.5 mm spot size , 6 ns pulse width , 10 hz repetition rate ) , which was used for matching the gold surface plasmon resonance peak for photothermal cancer treatment .
the output laser power , which is measured with a power meter , was adjusted by using an attenuator placed between the laser and the first mirror .
then , the laser was irradiated on a sample micro - cuvette with 18 wells with a diameter of 2 mm , which was custom - made in a 25  75 mm optical glass slide .
the two antibodies , anti - cd30 monoclonal antibody ( mab ) berh2 and anti - cd25 mab act1 , were provided by the research center borstel ( borstel , germany ) .
gold nanoparticles 15 nm in size were purchased from british biocell international ltd ( cardiff , uk ) . the stable gold 
antibody conjugates were prepared by passive absorption of proteins to the surface of the gold . for steady conjugation of antibodies and gold , the ph of the gold solution
must be adjusted to be just above ( ~0.5 ) the isoelectric point ( pi ) of the antibody.18 an important parameter to consider is the amount of antibody bound to the gold . to find the amount of antibody needed to saturate and stabilize the gold , a titration procedure was used to fix the antibody concentration using a 96-well microtiter plate .
first , 50 l solution with the same concentration of gold per well was placed into ten plates .
the pi of berh2 and act1 antibodies was about 7.5 , the ph of the gold in every plate was adjusted by 1% k2co3 solution to 8.0 to match the pi of the protein .
then , various amounts of antibodies with the same concentration were added . after stewing for 5 minutes
, 10 l of nacl solution ( 10% ) per well was added and mixed homogenously , while observing the solution color change .
the amount that maintained the red color before the blue envelope was used for conjugation . for gold - berh2 and gold - act1 conjugates ,
70 l 1% k2co3 solution was added into 5 ml gold to make the ph = 8.0 , and the best conjugation amount of berh2 ( 9.5 mg / ml ) was 7.5 l , while the best conjugation amount of act1 ( 7.4 mg / ml ) was 12.5 l .
to take the preparation of gold - berh2 conjugates as an example , 5 ml gold nanoparticles with 15 nm were transferred into a 15 ml tube , and 70 l 1% k2co3 solution was added into the tube to adjust the ph .
then , 7.5 l berh2 mabs ( 9.5 mg / ml ) were mixed into the gold solution . after incubating for 30 minutes at room temperature while stirring ,
the bottom scarlet solution was extracted and centrifuged for 45 minutes at 40,000 rpm to wash the unbound antibodies .
finally , the pellet was resuspended in phosphate - buffered saline ( pbs ) supplemented with 0.1% bovine serum albumin ( bsa ) , and stored at 4c until use .
cells were maintained in rpmi1640 medium supplemented with 10% heat - inactivated fetal calf serum , 2 mm l - glutamine , and antibacterial antibiotic solution .
the cell line was maintained at 37c in an incubator in 5% co2 and 95% air and passaged twice a week .
for the experiments , l-428 cells at the logarithmic growth phase were centrifuged at 1400 rpm for 5 minutes at 20c and then resuspended in pbs with cell densities of 10 ml . for testing the stability of gold 
antibody conjugates and the staining ability of different conjugates to the l-428 cells , either gold - berh2 or gold - act1 conjugates were added to the cell suspension at certain ratios between the conjugates and the cells .
after incubation for 20 minutes at 37c , the cells were centrifuged and washed twice and resuspended in pbs . then , a secondary antibody , goat anti - mouse alexa 488 ( am - a488 ) was added into the cell suspension to couple with the berh2 or act1 antibody .
after incubation for 20 minutes and centrifugation at 1400 rpm for 5 minutes , the cell samples were resuspended in pbs and analyzed using a flow cytometer ( facscan ; becton dickinson , franklin lakes , nj ) . equipped with a 488 nm air - cooled argon - ion laser .
data analysis was based on the collection of 10,00050,000 events . to implement the gold nanoparticle - induced l-428 cell - killing experiments , the gold 
antibody conjugates , gold - berh2 and gold - act1 , and unbound gold with the same optical density , were added into the cell suspension , respectively .
after incubation for 20 minutes at 37c , the cells were centrifuged at 1400 rpm for 5 minutes , and resuspended in pbs for the next photothermal treatment . in the l428 cell
killing experiments , there were four experimental groups for comparing the killing efficiency with or without gold , the control cell group , cells with unbound gold , cells with gold - berh2 conjugates , and cells with gold - act1 conjugates .
four groups of prepared cell samples was seeded into sample micro - cuvettes per well .
the cells were treated in the scanning mode of laser irradiation with 050 mw , 5 pulses .
after photothermal treatment , all of the cells were washed , resuspended in pbs , and incubated for 15 minutes , followed by the cell viability analysis .
cell viability was determined using calcein - am / propidium iodide ( pi ) double - staining solution . briefly , after photothermal treatment , 2 g / ml of calcein - am solution was added into the irradiated resuspended cell sample and incubated at 37c for 20 minutes .
the samples were analyzed using flow cytometry . to determine the percentage of necrotic cells relative to the total number of cells , 10,00050,000 events were acquired per sample using cellquest software ( bd biosciences ) .
all the data were processed with winmdi 2.8 software ( microsoft corporation , redmond , wa ) .
the results were confirmed by fluorescence microscopy ( bh2-rfl - t2 ; olympus corporation , tokyo , japan ) , and the fluorescent images were recorded using a digital camera .
the irradiation laser was a frequency doubled q - switched neodymium ( nd):yag laser ( surelite i ; continuum , santa clara , ca ) , with nonlinear crystals to enable conversion of the fundamental wavelength frequency from 1064 nm to 532 nm ( 2.5 mm spot size , 6 ns pulse width , 10 hz repetition rate ) , which was used for matching the gold surface plasmon resonance peak for photothermal cancer treatment .
the output laser power , which is measured with a power meter , was adjusted by using an attenuator placed between the laser and the first mirror .
then , the laser was irradiated on a sample micro - cuvette with 18 wells with a diameter of 2 mm , which was custom - made in a 25  75 mm optical glass slide .
the two antibodies , anti - cd30 monoclonal antibody ( mab ) berh2 and anti - cd25 mab act1 , were provided by the research center borstel ( borstel , germany ) .
gold nanoparticles 15 nm in size were purchased from british biocell international ltd ( cardiff , uk ) . the stable gold 
antibody conjugates were prepared by passive absorption of proteins to the surface of the gold . for steady conjugation of antibodies and gold , the ph of the gold solution
must be adjusted to be just above ( ~0.5 ) the isoelectric point ( pi ) of the antibody.18 an important parameter to consider is the amount of antibody bound to the gold . to find the amount of antibody needed to saturate and stabilize the gold , a titration procedure was used to fix the antibody concentration using a 96-well microtiter plate .
first , 50 l solution with the same concentration of gold per well was placed into ten plates .
the pi of berh2 and act1 antibodies was about 7.5 , the ph of the gold in every plate was adjusted by 1% k2co3 solution to 8.0 to match the pi of the protein .
then , various amounts of antibodies with the same concentration were added . after stewing for 5 minutes
, 10 l of nacl solution ( 10% ) per well was added and mixed homogenously , while observing the solution color change .
the amount that maintained the red color before the blue envelope was used for conjugation . for gold - berh2 and gold - act1 conjugates ,
70 l 1% k2co3 solution was added into 5 ml gold to make the ph = 8.0 , and the best conjugation amount of berh2 ( 9.5 mg / ml ) was 7.5 l , while the best conjugation amount of act1 ( 7.4 mg / ml ) was 12.5 l .
to take the preparation of gold - berh2 conjugates as an example , 5 ml gold nanoparticles with 15 nm were transferred into a 15 ml tube , and 70 l 1% k2co3 solution was added into the tube to adjust the ph .
then , 7.5 l berh2 mabs ( 9.5 mg / ml ) were mixed into the gold solution . after incubating for 30 minutes at room temperature while stirring ,
the bottom scarlet solution was extracted and centrifuged for 45 minutes at 40,000 rpm to wash the unbound antibodies .
finally , the pellet was resuspended in phosphate - buffered saline ( pbs ) supplemented with 0.1% bovine serum albumin ( bsa ) , and stored at 4c until use .
cells were maintained in rpmi1640 medium supplemented with 10% heat - inactivated fetal calf serum , 2 mm l - glutamine , and antibacterial antibiotic solution .
the cell line was maintained at 37c in an incubator in 5% co2 and 95% air and passaged twice a week .
for the experiments , l-428 cells at the logarithmic growth phase were centrifuged at 1400 rpm for 5 minutes at 20c and then resuspended in pbs with cell densities of 10 ml . for testing the stability of gold 
antibody conjugates and the staining ability of different conjugates to the l-428 cells , either gold - berh2 or gold - act1 conjugates were added to the cell suspension at certain ratios between the conjugates and the cells .
after incubation for 20 minutes at 37c , the cells were centrifuged and washed twice and resuspended in pbs . then
, a secondary antibody , goat anti - mouse alexa 488 ( am - a488 ) was added into the cell suspension to couple with the berh2 or act1 antibody .
after incubation for 20 minutes and centrifugation at 1400 rpm for 5 minutes , the cell samples were resuspended in pbs and analyzed using a flow cytometer ( facscan ; becton dickinson , franklin lakes , nj ) . equipped with a 488 nm air - cooled argon - ion laser .
data analysis was based on the collection of 10,00050,000 events . to implement the gold nanoparticle - induced l-428 cell - killing experiments , the gold  antibody conjugates , gold - berh2 and gold - act1 , and unbound gold with the same optical density , were added into the cell suspension , respectively .
after incubation for 20 minutes at 37c , the cells were centrifuged at 1400 rpm for 5 minutes , and resuspended in pbs for the next photothermal treatment .
in the l428 cell killing experiments , there were four experimental groups for comparing the killing efficiency with or without gold , the control cell group , cells with unbound gold , cells with gold - berh2 conjugates , and cells with gold - act1 conjugates .
four groups of prepared cell samples was seeded into sample micro - cuvettes per well .
the cells were treated in the scanning mode of laser irradiation with 050 mw , 5 pulses .
after photothermal treatment , all of the cells were washed , resuspended in pbs , and incubated for 15 minutes , followed by the cell viability analysis .
cell viability was determined using calcein - am / propidium iodide ( pi ) double - staining solution . briefly , after photothermal treatment , 2 g / ml of calcein - am solution was added into the irradiated resuspended cell sample and incubated at 37c for 20 minutes .
the samples were analyzed using flow cytometry . to determine the percentage of necrotic cells relative to the total number of cells , 10,00050,000 events were acquired per sample using cellquest software ( bd biosciences ) .
all the data were processed with winmdi 2.8 software ( microsoft corporation , redmond , wa ) .
the results were confirmed by fluorescence microscopy ( bh2-rfl - t2 ; olympus corporation , tokyo , japan ) , and the fluorescent images were recorded using a digital camera .
to evaluate the conjugation stability of gold and antibodies and the binding specificity of different conjugates to l-428 cells , the two conjugates , gold - berh2 ( anti - cd30 receptor ) and gold - act1(anti - cd25 receptor ) , and the goat anti - mouse alexa 488 were adopted in this experiment . both cd30 and cd25 are cell membrane proteins of the tumor necrosis factor receptors , but only cd30 has a high overexpression on the surface of l-428 . through the specific coupling of goat anti - mouse immunoglobulin g antibody to monoclonal antibody , stable coupling was achieved between am - a488 and berh2/act1 . whether or not am - a488 attaches to l-428 depends on the expression of protein on l-428 .
the results indicate that only for the cells in the gold - berh2 group , the detection of the fluorescence signal was positive .
the fluorescence signal on the cells in the gold - act1 group was the same as that for the control cell group ( negative ) .
the gold - berh2 conjugates bound specifically to cd30 receptors on the surface of l428 cells , which were also stained with am - a488 . a conceptual diagram of this binding process is shown in the inset box in figure 2 .
the conjugation stability was confirmed on one hand , but the different binding abilities of different conjugate - bound cells would bring a different performance in the cell - killing experiment .
figure 3 shows cell viability of l428 cells after some hours of exposure to a certain concentration of gold , or gold - berh2 conjugates .
the ratio of pure gold to cells was 10:1 , and the ratios of gold conjugates to cells were 10:1 and 10:1 , respectively .
after incubation , the number of viable cells were stained with calcein - am ( 2 g / ml ) and then measured using flow cytometry . for all samples , 20,000 events were acquired .
for the groups of unbounded gold and gold - berh2 conjugates incubated with l-428 cells at a ratio of 10 , the cell viability was greater than 90% after 24 hours of incubation , which had no significant difference compared with the control cell group . at the ratio of 10:1 ,
a bigger decrease in cell viability was observed after incubation for 24 hours and l-428 cell viability dropped to 84% .
therefore , for a short incubation period ( less than 12 hours ) , there was no significant increase in the number of dead cells incubated with gold at a relatively low concentration . to avoid the cytotoxicity induced by gold with high doses and a long incubation period , the highest ratio of gold to cells used for the photothermal killing experiment was not more than 10:1 , and the incubation period was less than 6 hours . to monitor the necrotic effect of laser irradiation on living cells in the presence of gold , we performed cell damage experiments on l-428 cells , which were incubated with gold - berh2 conjugates at a 10:1 ratio for 20 minutes , and then irradiated with a laser at 50 mw , 5 pulses .
after irradiation , cell viability was assessed using calcein - am ( 2 g / ml ) and pi ( 1 g / ml ) staining , and tested using flow cytometry and optical fluorescence microscopy . the events for facs were 20,000 events .
the cells positively stained with pi presumably represented the later stages of cell death , when membrane integrity was lost .
the percentage of death was calculated as the number of pi positive cells divided by the total number of cells .
figure 4 shows the photothermal treatment results , where severe destruction of l-428 cells was observed when the cells were exposed to laser irradiation ( 50 mw , 5 pulses ) with gold - berh2 conjugates .
the flow cytometry results of l-428 cells incubated with gold - berh2 conjugates treated with or without laser irradiation are shown on the left side .
the corresponding fluorescent images are shown on the right side . without laser irradiation , about 93% of the cell population was alive , which exhibited bright green fluorescence after calcein - am staining .
more than 95% of the cell population was dead after laser irradiation at 50 mw , 5 pulses , and the dead targets exhibited red fluorescence after pi staining .
furthermore , the effect of laser influence on laser - induced cell damage has been considered in this section . to test whether the increased cell death rate is linked to laser irradiated power , we treated cell samples with different laser power settings .
l-428 cells incubated with gold - berh2 conjugates and the control cell group were seeded into the sample micro - cuvette respectively .
the cell concentration was 4  10/ml , and the gold - berh2 conjugate to cell ratio was 10:1 .
the cell samples were irradiated with 5 pulses and different laser power settings , and then tested using the flow cytometry by calcein - am / pi double staining .
figure 5a and b show a representative flow cytometry dot plot illustrating the change in cell viability induced by different power laser irradiation .
right lower quadrants expressed the calcein - am positive cells as a percentage of the total cell population . left upper quadrants expressed the pi positive cells as a percentage of the total cell population .
as shown in figure 5a , for the control cell group , there was no remarkable change in cell viability when increasing the laser - irradiated power from 0 mw to 50 mw .
but for the cells in the gold - berh2 conjugates group as shown in figure 5b , cell damage efficiency was affected significantly by the laser - irradiated power .
most of the cells died when the laser power was increased to 50 mw . in the presence of gold 
for the control cell group incubated without gold , the cell viability curve did not change much with the variation of laser - irradiated power ; however , the curve of the cells with gold - berh2 conjugates showed a rapid decrease in cell viability as the laser power increased .
excessive irradiated laser power caused devastating damage or complete necrosis of l-428 cells incubated with gold 
antibody conjugates . due to the specific aggregation of the cd30 receptor and berh2 antibodies ,
efficient conversion of light strongly absorbed by the gold to heat energy induced a significant increase in cell death .
the effect of the photothermal treatment of l-428 cells was evaluated in the above experiments by combining laser irradiation and gold nanoparticles . in this section ,
we compared the efficiency of different binding modes of gold to cells and cell viability .
there were four experimental groups for comparing the killing efficiency with or without gold nanoparticles : the control cell group , cells with gold , cells with gold - berh2 conjugates , and cells with gold - act1 conjugates .
from the cell - binding specificity tests , it was known that berh2 antibodies could bind specifically on the surface of l-428 cells , but act1 antibodies could not .
thus , the above four experimental groups represented four combination conditions between gold and cells : pure cells , cells with unbound gold , cells targeted by few nonspecific gold  antibody conjugates , and cells targeted by many specific gold  antibody conjugates .
the cell samples were treated by laser irradiation with 40 mw , 5 pulses , which would be compared with the nonirradiated part .
then , 10,000 events were tested using flow cytometry by calcein - am / pi double - staining .
the experimental analysis results are shown in figure 6 , and were divided into two parts : irradiated and non - irradiated . in the non - irradiated part , there was no difference of cell viability between the four cell samples without laser irradiation .
in the irradiated part , there was no significant difference from the control group in the cell viability .
for the cells in the unbound gold group and in the gold - act1 conjugates group , a slight increase in the death rate can be found . due to the small volume of the sample micro - cuvettes , gold or gold - act1 conjugates in the sample volume
might distribute in the surrounding cells , which could induce some heat energy shift to the cells .
compared with the other three groups , due to the specific coupling of cd30 antigen and berh2 antibody , gold - berh2 conjugates have a tight binding on the surface of l428 cells . under laser irradiation with 40 mw ,
5 pulses , a large damage rate of about 96% was observed in the l-428 cells .
to evaluate the conjugation stability of gold and antibodies and the binding specificity of different conjugates to l-428 cells , the two conjugates , gold - berh2 ( anti - cd30 receptor ) and gold - act1(anti - cd25 receptor ) , and the goat anti - mouse alexa 488 were adopted in this experiment . both cd30 and cd25 are cell membrane proteins of the tumor necrosis factor receptors , but only cd30 has a high overexpression on the surface of l-428 . through the specific coupling of goat anti - mouse immunoglobulin g antibody to monoclonal antibody , stable coupling was achieved between am - a488 and berh2/act1 . whether or not am - a488 attaches to l-428 depends on the expression of protein on l-428 .
the results indicate that only for the cells in the gold - berh2 group , the detection of the fluorescence signal was positive .
the fluorescence signal on the cells in the gold - act1 group was the same as that for the control cell group ( negative ) .
the gold - berh2 conjugates bound specifically to cd30 receptors on the surface of l428 cells , which were also stained with am - a488 . a conceptual diagram of this binding process is shown in the inset box in figure 2 .
the conjugation stability was confirmed on one hand , but the different binding abilities of different conjugate - bound cells would bring a different performance in the cell - killing experiment .
figure 3 shows cell viability of l428 cells after some hours of exposure to a certain concentration of gold , or gold - berh2 conjugates .
the ratio of pure gold to cells was 10:1 , and the ratios of gold conjugates to cells were 10:1 and 10:1 , respectively .
after incubation , the number of viable cells were stained with calcein - am ( 2 g / ml ) and then measured using flow cytometry . for all samples , 20,000 events were acquired .
for the groups of unbounded gold and gold - berh2 conjugates incubated with l-428 cells at a ratio of 10 , the cell viability was greater than 90% after 24 hours of incubation , which had no significant difference compared with the control cell group . at the ratio of 10:1 ,
a bigger decrease in cell viability was observed after incubation for 24 hours and l-428 cell viability dropped to 84% .
therefore , for a short incubation period ( less than 12 hours ) , there was no significant increase in the number of dead cells incubated with gold at a relatively low concentration . to avoid the cytotoxicity induced by gold with high doses and a long incubation period ,
the highest ratio of gold to cells used for the photothermal killing experiment was not more than 10:1 , and the incubation period was less than 6 hours .
to monitor the necrotic effect of laser irradiation on living cells in the presence of gold , we performed cell damage experiments on l-428 cells , which were incubated with gold - berh2 conjugates at a 10:1 ratio for 20 minutes , and then irradiated with a laser at 50 mw , 5 pulses .
after irradiation , cell viability was assessed using calcein - am ( 2 g / ml ) and pi ( 1 g / ml ) staining , and tested using flow cytometry and optical fluorescence microscopy .
the cells positively stained with pi presumably represented the later stages of cell death , when membrane integrity was lost .
the percentage of death was calculated as the number of pi positive cells divided by the total number of cells .
figure 4 shows the photothermal treatment results , where severe destruction of l-428 cells was observed when the cells were exposed to laser irradiation ( 50 mw , 5 pulses ) with gold - berh2 conjugates .
the flow cytometry results of l-428 cells incubated with gold - berh2 conjugates treated with or without laser irradiation are shown on the left side .
about 93% of the cell population was alive , which exhibited bright green fluorescence after calcein - am staining .
more than 95% of the cell population was dead after laser irradiation at 50 mw , 5 pulses , and the dead targets exhibited red fluorescence after pi staining .
furthermore , the effect of laser influence on laser - induced cell damage has been considered in this section . to test whether the increased cell death rate is linked to laser irradiated power , we treated cell samples with different laser power settings .
l-428 cells incubated with gold - berh2 conjugates and the control cell group were seeded into the sample micro - cuvette respectively .
the cell samples were irradiated with 5 pulses and different laser power settings , and then tested using the flow cytometry by calcein - am / pi double staining .
figure 5a and b show a representative flow cytometry dot plot illustrating the change in cell viability induced by different power laser irradiation .
right lower quadrants expressed the calcein - am positive cells as a percentage of the total cell population .
left upper quadrants expressed the pi positive cells as a percentage of the total cell population .
as shown in figure 5a , for the control cell group , there was no remarkable change in cell viability when increasing the laser - irradiated power from 0 mw to 50 mw .
but for the cells in the gold - berh2 conjugates group as shown in figure 5b , cell damage efficiency was affected significantly by the laser - irradiated power .
the cell death rate was about 40% when using 15 mw laser irradiation ; most of the cells died when the laser power was increased to 50 mw . in the presence of gold 
antibody conjugates , the cell death rate mostly depends on the laser irradiated power . the statistical analysis of the repeated experiments is shown in figure 5c .
for the control cell group incubated without gold , the cell viability curve did not change much with the variation of laser - irradiated power ; however , the curve of the cells with gold - berh2 conjugates showed a rapid decrease in cell viability as the laser power increased .
excessive irradiated laser power caused devastating damage or complete necrosis of l-428 cells incubated with gold 
antibody conjugates . due to the specific aggregation of the cd30 receptor and berh2 antibodies ,
efficient conversion of light strongly absorbed by the gold to heat energy induced a significant increase in cell death .
the effect of the photothermal treatment of l-428 cells was evaluated in the above experiments by combining laser irradiation and gold nanoparticles . in this section ,
we compared the efficiency of different binding modes of gold to cells and cell viability .
there were four experimental groups for comparing the killing efficiency with or without gold nanoparticles : the control cell group , cells with gold , cells with gold - berh2 conjugates , and cells with gold - act1 conjugates .
from the cell - binding specificity tests , it was known that berh2 antibodies could bind specifically on the surface of l-428 cells , but act1 antibodies could not .
thus , the above four experimental groups represented four combination conditions between gold and cells : pure cells , cells with unbound gold , cells targeted by few nonspecific gold  antibody conjugates , and cells targeted by many specific gold  antibody conjugates .
the cell samples were treated by laser irradiation with 40 mw , 5 pulses , which would be compared with the nonirradiated part .
then , 10,000 events were tested using flow cytometry by calcein - am / pi double - staining .
the experimental analysis results are shown in figure 6 , and were divided into two parts : irradiated and non - irradiated . in the non - irradiated part , there was no difference of cell viability between the four cell samples without laser irradiation .
, there was no significant difference from the control group in the cell viability . for the cells in the unbound gold group and in the gold - act1 conjugates group , a slight increase in the death rate can be found . due to the small volume of the sample micro - cuvettes , gold or gold - act1 conjugates in the sample volume
might distribute in the surrounding cells , which could induce some heat energy shift to the cells .
compared with the other three groups , due to the specific coupling of cd30 antigen and berh2 antibody , gold - berh2 conjugates have a tight binding on the surface of l428 cells . under laser irradiation with 40 mw ,
5 pulses , a large damage rate of about 96% was observed in the l-428 cells .
with the preliminary successes of photothermal therapy in cancer studies , gold nanoparticle - mediated thermal therapy became a new and minimally invasive tool for cancer treatment.19,20 in the present study , to evaluate the effectiveness of gold in the destruction of cancer cells or tumor tissue , three main aspects were considered : the photothermal therapy system , specific gold nanoparticle 
we set up a photothermal therapy system ( figure 1 ) . a pulsed light with 532 nm wavelength was chosen to match the surface plasmon resonance absorption peak of 15 nm gold nanoparticles used in the experiment for photothermal cancer treatment . specificity of targeting to achieve high efficiency of photothermal conversion of gold nanoparticles was a key problem .
specific targeting should produce a high photothermal effect on the targeted cancer , and only minimal heat should be applied to the surrounding normal cells and tissues . after synthesizing two different gold nanoparticle 
antibody conjugates , gold - berh2 and gold - act1 conjugates , their conjugation stability and binding specificity for l-428 cells were evaluated ( figure 2 ) .
l-428 cells are positive for cd30 , but negative for cd25 . through flow cytometry measurements using alexa 488 ,
the results indicate that the gold - berh2 conjugates bound specifically to the surface of l-428 cells , but that gold - act1 conjugates did not , which seems to agree with our predictions . prior to considering gold nanoparticles for photothermal therapy application ,
it is important to understand their characterization of biocompatibility.21 the toxicity and side effects of nanoparticles are determined by the shape , dose , surface chemistry , incubation duration , etc.2224 we compared the toxicity of pure gold nanoparticles and gold  antibody conjugates to l-428 cells under different incubation time periods and different concentrations .
after incubation for 24 hours , pure gold and gold  antibody conjugates incubated with l-428 cells did not induce a decrease in cell viability compared with control cells under a relatively low concentration ( the ratio of gold to cells was not more than 10:1 ) ; but , with an increase in the gold concentration , a decrease in cell viability was observed .
a relatively low concentration of gold and a short incubation time were adopted in the photothermal experiment to exclude the influence of cytotoxicity of gold on cell viability . during the photothermal treatment ,
the temperature distribution on the surrounding cell surface was the dominant factor leading to cell death.2526 it is important to note that the temperature distribution is determined by the gold s shape and concentration and the wavelength and power of laser irradiation . at the molecular level
, hyperthermic effects can induce protein denaturation , membrane rupture , and cell shrinkage.27 cytotoxic effects have occurred in cells maintained for 1 hour at 42c , but this duration could be shortened to 34 minutes by using a higher temperature such as 80c.14,2729 numerical analysis found that cells with higher gold - loading required a lower laser - irradiated power to achieve the temperature rise for cell destruction.14 photothermal therapy tests on living l-428 cells were initially performed to confirm the gold nanoparticle - mediated photothermal effect on cell viability . when cells were treated with gold or laser alone ,
after laser irradiation in the presence of the gold , the cell death rate had a severe increase ( figure 4 ) , and the results were also confirmed by fluorescence microscopy . with the combination of gold nanoparticles and laser irradiation , few calcein - am positive cells were observed and most l428 cells were positive for pi staining .
the effect of laser irradiation on cell damage has been evaluated ( figure 5 ) . for the control cells without gold , there was no remarkable change in cell viability when increasing the power of laser irradiation .
but in the presence of gold - berh2 conjugates , cell damage efficiency was affected significantly by the laser - irradiated power , and the cell death rate mostly depended on the power of laser irradiation .
for successful cancer killing , the cells must be heated to a minimum temperature by proper laser irradiation to cause cancer cell death .
the different binding mode of gold to cells can also induce different therapy efficiency on cell viability ( figure 6 ) . under the same laser irradiation , the specific binding of gold - berh2 conjugates to l-428 induced extensive cell damage , but there was a slight increase in the cell death rate for the nonspecific binding groups , cells with the unbound gold group , and cells with the gold - act1 conjugates group .
the results again confirmed the advantage of specific targeting of gold to cells and high therapy efficiency for gold - targeted cancer cells and slight damage to untargeted cells . in vivo photothermal therapy of gold nanostructures in preclinical and , potentially , clinical use is a crucial direction for our future research . due to the low absorption by tissues in the 700850 nm near infrared ( nir ) region , gold nanostructure - mediated photothermal therapy
is predominantly designed to operate in this window of wavelengths to enhance light penetration capability , and to prevent undesirable damage to healthy tissue .
the absorption peak of gold nanorods , nanoshells , and nanocages are all in the nir region .
some recent studies have focused on in vitro and in vivo photothermal cancer treatments;3036 however , the optimization of gold nanoparticle - based therapy techniques to physiological environments needs further study , to determine the clinical success of gold nanostructure - based nanomedicine.19,37,38
many experimental parameters , including the absence of gold or laser irradiation , the laser - irradiated power , and the binding mode of gold to cells , were all tested for their influence on cell viability .
when conjugated with berh2 ( anti - cd30 receptor ) antibodies , these gold nanoparticles specifically bind l-428 cells that overexpress the cd30 receptor ; and , with laser irradiation at suitable wavelength and power , very high effectiveness in killing l-428 cells is achieved .
many optimization problems , including the compatibility of gold nanostructures , the stability of gold ligand conjugates , and laser irradiation modes , need further modification to make them more suitable for in vivo tumor model studies .
gold nanoparticle - mediated photothermal cancer therapy provides a relatively safe microsurgery system for cancer therapy development .
 purposedue to the efficient bioconjugation and highly photothermal effect , gold nanoparticles can stain receptor - overexpressing cancer cells through specific targeting of ligands to receptors , strongly absorb specific light and efficiently convert it into heat based on the property of surface plasmon resonance , and then induce the localized protein denaturation and cell death.methodstwo gold nanoparticle  antibody conjugates , gold - berh2 antibody ( anti - cd30 receptor ) and gold - act1 antibody ( anti - cd25-receptor ) , were synthesized . 
 gold - berh2 conjugates can specifically bind to the surface of l-428 hodgkin s cells , and gold - act1 conjugates were used for the control . 
 the gold nanoparticle - induced l-428 cell - killing experiments were implemented with different experimental parameters.resultsat a relatively low concentration of gold and short incubation time , the influence of cytotoxicity of gold on cell viability can be overlooked . under laser irradiation at suitable power , 
 the high killing efficiency of gold - targeted l-428 cells was achieved , but little damage was done to nontargeted cancer cells.conclusiongold nanoparticle - mediated photothermal therapy provides a relatively safe therapeutic technique for cancer treatment . 