| BackgroundPancreatic cancer,which is characteristic by high malignant tumor,tumor process rapidly and poor prognosis,is one of gastrointestinal tumor having constitutional symptoms rather than local symptoms.Among the pancreatic cancer,the most regular pathological classification is pancreatic duct adenocarcinoma which is also known as PD AC and the incidence of PD AC occupy eighty percent of total incidence of pancreatic cancer.With the development of country,not only has the daily lifestyle changed,but also the eating patterns.People in nowadays tend to eat food with high cholesterol and unhealthy lifestyle such as go to work without eating breakfast leading to gallstone pancreatitis.Recently,the incidence of PDAC is increasing year after year.Besides,the early symptoms of PD AC are too invisible to be found by surgeons and unfortunately there are not economical and efficient methods to diagnose PDAC.Therefore,a lot of patients usually did not fell any uncomfortable until daily examination or have related symptoms,however,which is diagnosed as advanced pancreatic cancer.Account for this currency,the vital therapy tragedy of pancreatic cancer should not be placed emphasis on early diagnose but tumor totally resection during the operation or thoroughly adjuvant therapy after operation.Molecular imaging is a newly rising imaging method which comb:ines the biology subject with imageology,and can be used to observe changes in biological tissue.Molecular imaging can help people observe contrast agents' metabolism,distribution and function from the molecular level after injected into body.Then,according to the acquired images,surgeons can locate where the tumor is and the line between tumor and normal tissues which is beneficial to subsequent useful treatment.Compared to the clinical traditional imaging method,the molecular imaging use different probe or contrast agents primarily.Nowadays,X-ray,CT and MRI which have be utilized widely in clinic medical medicine usually take advantage of ordinary contrast agents to make difference between target part and normal tissues which maybe because the diverse density or water content and then highlight the image of tumor or lesion disease.The probe such as molecular probe or nanoprobe could combine to the specific target on the surface of tumor cells while using the molecular imaging technology to acquire the images needed.Owning to the molecular or nanometer probe could be refined to the level of cell imaging and perceive slight changes,so when compared to traditional imaging methods,the molecular imaging has advantages of high solution,high accuracy,high signal-background ratio and taking less time to image the body examined leading to most obviously advantages in distinguish the tumor and normal tissue.Near-infrared imaging could utilize the near-infrared imaging system to help doctors to find where the tumor is even to discover the tiny space-occupying lesions.Additionally,it is a safe and no radioactive risk for animal maybe for people in the future and can save exam time which is preferred by researchers in bio-medical field.Thus,many more scientific studies devote themselves to attain the aim to integrate the diagnosis and therapy through induce the nanometer probe accumulate in tumor site by using the principle of antigen antibody combination based on the near-infrared imaging ability of nanometer probe.Photothermal therapy,which is also known as PTT,is a newly method to kill tumor cells.Using 808 nm near-infrared light irradiate the tumor when the probe has accumulated there,the nanometer probe injected could convert luminous energy into thermal energy which is the mainly cause leading the cells' dead.Photothermal therapy has apparent feature compared to traditional adjuvant therapy account for its small trauma,few side effects and the characteristics of local applications.Among the numerous photothermal therapy material,single walled carbon nanotubes which are one kind of the carbon materials are widely applied to bio-medical area.Single walled carbon nanotubes can maximize the absorption of near-infrared light and convert them into thermal energy which lead to a local high temperature to kill the tumor cells.However,the toxicity of single walled carbon nanotubes is too great before functionalization,besides,it is insoluble in water neither insoluble in organic solvents.Therefore,many researchers tend to make surface modification to the single walled carbon nanotubes previous being used in bio-medical area which not only decrease the toxicity but also increase the bio-compatibility and then improve the circulating time in the blood which would assist nanotubes penetrating the tumor tissue.In terms of biological near-infrared light imaging,single-walled carbon nanotubes could accumulate in target tumor site through the blood circulation and enhanced permeability and retention effect(EPR).When single walled nanotubes conjugated to near-infrared dye,the tumor tissue could appear under the excitation and achieve the purpose of thermal therapy under the near-infrared wavelength of 808 irradiation which could cause local high temperature because light-thermal conversion.Cy5.5 is one kind of photoluminescence fluorescence dye and soluble in water.In addition,this kind of fluorescence dye also be used to detect and label some bio-protein or bio-molecule.When excited by 678-680 nm near-infrared wavelength,cy5.5 could release about 695-700 nm wavelength of near-infrared light which could be detected by near-infrared imaging system and expose the tumor site which could help surgeons to remove the tumors.On the basis of previous studies,we take advantage of the near infrared fluorescence molecular imaging techniques,using Cy5.5 fluorescent dyes conjugated with single walled carbon nanotubes to expose the tumor location and its outline.The nanotubes can utilize the ability of converting the light energy to thermal energy under the irradiation of 808nm and then generate local high temperature to kill cancer cells which not only remove residual tumor but also minimize the damage in normal tissues.During our experiment we continue to explore the appropriate concentration,irradiating time and irradiating power and make a comparison among different situations in order to acquire the optimal and efficient conditions.Animals' weight,tumor volume and survival time were closely observed which also reflect the toxicity and the potential application of thermal therapy.Objective1.To optimize the biological properties of single-walled carbon nanotubes in order to improve the biological dispersibility and compatibility.2.To explore the distribution and metabolic characteristics of single-walled carbon nanotubes after conjugated with cy5.5 in vivo.3.To analysis comprehensively the thermal therapy effects of single walled nanotubes conjugated to cy5.5.Methods1.Agents:PEG-WM3500,nitric acid,sulfuric acid,deionized water,single walled carbon nanotubes,0.9%NaCl solution,Cy5.5,95%ethyl alcohol.2.Biological material:Pancreatic cancer cell line Panc-1,breast cancer cell line MCF-7,cell culture fluid,Balb/c nude mice,10%FBS,1%of penicillin and streptomycin(double resistance),Tripsin,DMEM medium,RMPI 1640 medium,75%ethyl alcohol.3.Equipment:(1)oil bath pot,liquid receiver,(2)electronic balance,(3)spatulas,(4)weighing paper,(5)magnetic stirrer,(6)stirrer,(7)blow-dry device,(8)100 nm aperture membrane filter,(9)vacuum suction filter device,(10)ultra-centrifuge(1000 r/min).(11)Syringes,(12)alarm clock,(13)surgical scissors,(14)tweezers,(15)ice boxes,(16)insulin injection needle.4.Apparatus:(1)ultraviolet visible light absorption spectrometer,(2)Living Imaging System(IVIS,Caliper Sciences IVIS Spectrum 3D),(3)inverted fluorescence microscope,(4)thermogravimetric analyzer,(5)animal blood analyzer,(6)transmission electron microscopy(TEM),(7)thermal imaging device,(8)808nm laser device,(9)animals anesthesia device.5.Cell passage and preparation of tumor-bearing mice.Pancreatic cancer cell line Panc-1 and breast cancer cell line MCF-7 were cultivated under the standard conditions.Balb/c nude mice were bought from Beijing Vital River Laboratory Animal Technology Co.,Ltd,and raised according to the animal center.Subcutaneously implanted tumor model in nude mice with human breast cell carcinoma were used in the experiments.Human breast cell carcinoma,MCF-7 cancer cell line and human pancreatic cancer(Panc-1 cell line)were cultured in DMEM,supplemented with 10%FBS,1%penicillin and streptomycin under 5%CO2 at 37?.The MCF-7 and Panc-1 cells(1×106 cells)were suspended respectively in 150 mL PBS and subcutaneously injected into the right armpit of female BALB/c mice.6.The distribution of single walled carbon nanotubes conjugated with Cy5.5 in vivo.The biodistribution of probe was detected by IVIS according to the fluoresce of cy5.5.single walled carbon nanotubes conjugated with Cy5.5 was injected into tumor-bearing mice through the tail vein.The probe biodistribution was observed using IVIS contentiously in order to find the time when the probe accumulated most in tumor and the time would be treated as the best time for photothermal therapy.The quality of life,such as weight,tumor volume and survival time,of the treated mice was continuously observed after therapy.7.Photothermal therapy.The time for photothermal therapy was evaluated from the biodistribution.We used 808nm wavelength irradiate the tumor site in order to generate the local high temperature which can kill the cancer cells.The tumor volume,monitored with a digital caliper,was calculated by formula V=axbxb/2(a,longest dimension;b,shortest dimension).ResultsThe tumors of mice which were intravenously injected with single-walled carbon nanotubes coupling united Cy5.5 meanwhile being treated by photothermal therapy were disappear and developed scar after two days therapy.The scars fall off without tumor recurrence.The tumors of mice which were intravenously injected with single-walled carbon nanotubes coupling united Cy5.5 without photothermal therapy increased gradually.The increase rate of tumor in this group was slower than control group,however,these two group did not show any therapeutic efficacy.The result indicated that no matter using near-infrared irradiation only or just probe injection,it cannot reach the purpose of killing cancer cells.ConclusionWhen we tried to remove tumor with photothermal therapy,low irradiated power almost kill all the cancer cells.Meanwhile,using near-infrared light irradiating the tumor site also can prevent damaging the normal tissue.Therefore,we suggested that we would acquire much better outcome if under the circumstances of not damaging normal tissue but with advanced power properly.We mainly explored the optimize situation and surface modification of single walled carbon nanotubes in this article.In addition,we will acquire much better outcome if carry out the experiments from next aspects.(1)Combine photothermal therapy with other therapy method,the tumor would be removed thoroughly.(2)Explore the detail about how the single walled carbon nanotubes act during the photothermal therapy.Such as the proper temperature and the irradiating time.(3)How to eliminate the probe from the live body much more rapidly though surface modification.(4)The key factor of this article is the concentration for injection and the quantity of probe accumulated in tumor tissue which would improve the photothermal therapy effect. |
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