the technical contents and characteristics of the present invention will be apparent with the detailed description of a preferred embodiment accompanied with related drawings as follows . it is noteworthy that the drawings are provided for the purpose of illustrating the present invention , but not intended for limiting the scope of the invention . ly294002 is encapsulated on the surface of plga without being modified by a modifier to form a nano - scale composition . with reference to fig1 for a schematic view of ly294002 encapsulated on the surface of poly ( lactic - co - glycolic acid ) ( plga ) powder ( 101 ) without being modified by a modifier , and the plga powder ( 101 ) includes a plga hydrophilic region ( 102 ) and a plga hydrophobic region ( 103 ), and ly294002 ( 104 ) with the molecular structure as shown below is added , so that the plga hydrophobic region ( 103 ) of the plga powder ( 101 ) is encapsulated on the ly294002 ( 104 ), and the semi - finished good ( 105 ) of the composition formed by ly294002 and plga is used for forming nanoparticles in a molding process . in other words , a finished good ( 106 ) of the composition formed by ly294002 and plga is produced . the detailed experiment procedure of the aforementioned manufacturing process is described as follows : plga polymers ( sf ) and ly294002 ( ly ) of different quantities are dissolved in 5 ml of acetone , and then a peristaltic pump is used to drop an ethanol / water ( 50 / 50 %, v / v ) solution into the plga solution slowly at a speed of 1 ml / min . the solution is blended at 240 rpm until the mixture becomes cloudy , and then the suspension is moved to 20 ml of deionized water and blended at 300 rpm for 15 minutes . the solution is placed in a suction flask for 30 minutes to remove the organic solvents . to prevent possible aggregations of the plga , a 90 - mm filter is used to filter the solution to obtain nanoparticles . to measure the encapsulation rate of sf - ly nps , a centrifuging method is used in this preferred embodiment to collect plga nanoparticles , and the plga nanoparticles are dissolved into acetonitrile completely . an uv - vis spectrometer with a wavelength of 300 nm is used to measure the absorption rate . the change of encapsulation rate is observed after the ratio of ly294002 to plga is changed . based on the preparation method as described in example 1 , the ratio of ly294002 to plga is changed , and then the change of encapsulation rate is observed , and the encapsulation rate is calculated by the following formula : the results of the encapsulation rates are listed in table 1 . from the results obtained by using a constant quantity of plga to be encapsulated on ly294002 in different ratios , we found that the more the ly294002 , the smaller is the encapsulation rate . however , the drop of the encapsulation rate will reach a saturation point below a certain ratio of plga : ly294002 . for example , 20 mg of plga encapsulated on ly294002 in different ratios , we found that although the encapsulation rate drops for the 0 . 5 mg ˜ 3 mg of plga , yet the difference of the encapsulated contents is not large . in this preferred embodiment , parameters with the most appropriate ratio can be found for biological experiments . although a larger quantity of plga can be used to achieve a better encapsulation rate , excessive plga may cause an insufficient dispersity of the solution and result in a sticky solution , or too - large plga nanoparticles that will be precipitate easily and cannot be dispersed uniformly . the size of nanoparticles is approximately equal to 200 ˜ 800 nm when more than 100 mg of plga is encapsulated on ly294002 in different ratios . in the following experiments , approximately 50 mg of plga and 1 or 3 mg of ly294002 are used for conducting the experiments . to measure the properties of plga nanoparticles ( sf - ly nps ) encapsulated on ly294002 , the following experiment is performed to analyze the particle diameter of sf - ly nps in this preferred embodiment . firstly , 50 mg of plga powder and 3 mg of ly294002 are mixed into 5 ml of acetone , and then a peristaltic pump is used to drop an ethanol / water ( 50 / 50 %, v / v ) solution into the plga solution slowly at a speed of 1 ml / min . the solution is blended at 240 rpm until the mixture becomes cloudy , and then the suspension is moved to 20 ml of deionized water and blended at room temperature at 300 rpm for 15 minutes . the solution is placed in a suction flask for 30 minutes to remove the organic solutes . to prevent possible aggregations of the plga , a 90 - mm filter is used to filter the solution to obtain nanoparticles . the average particle diameter is measured by a dynamic light scattering ( dls ) measuring method . the results of this experiment are shown in fig2 a . the nanoparticles are dropped into a copper net and vacuumed to remove moisture . the nanoparticles are stained with an 1 % sodium phosphotungstate solution ( ph 7 . 0 ) and developed before viewing under an electronic microscope . the results of this experiment are shown in fig2 b . the nanoparticles are contained in a 1 . 5 ml - eppendorf tube containing a pbs buffer solution ( 10 mm , ph 7 . 4 ). a suspension containing ionized ly2940025 is connected after a fixed time , and an uv - vis spectrometer with a wavelength of 300 nm is used to measure the released ly294002 . the results of this experiment are shown in fig2 c . in fig2 a , the particle diameter is measured to be 96 . 33 nm by a dynamic light scattering ( dls ) measuring method . in fig2 b , a photo taken under an electronic microscope shows that the nanoparticles are in a spherical shape , and the average particle diameter of sf - ly nps is equal to 80 nm . in fig2 c , the time of releasing ly in this preferred embodiment is measured , and the curve as shown in fig2 c indicates that the releasing speed increases rapidly in the first 9 hours , and continues increasing for 48 hours before reaching a saturation . this phenomenon shows a continuously releasing effect . the test results also show that the nano - scale composition has a size falling within a range from 80 nm to 120 nm and capable of preventing the immune system of living organisms from being attacked . if the size of the nanoparticles is too large , the nanoparticles may be accumulated in the living organisms easily , and thus there is a possibility of having vacular occulations or the immune system may recognize the nanoparticles as external foreign matters and engulf the nanoparticles by phagocytosis , and thus failing to achieve the medical effect in the living organisms . on the other hand , if the size of the nanoparticles is too small , the metabolism will be too quick , so that the nanoparticles will be discharged with excrements quickly . for the too - large nanoparticles , peg or another equivalent surface modifier is required to modify the nanoparticles in order to prevent attacks to the immune system . however , the plga nanoparticles of the present invention do not require any surface modification by using a modifier . the invention simply controls the nano size to prevent attacks to the immune system . to measure the ionized ly , plag nanoparticles ( sf - nps ), and sf - ly nps , plga nps are applied to four selected types of lung cancer cell strains including as2 ( pten null ), h157 ( pi3kca , pten null ), h460 ( pi3kca ) and h1650 ( pten null ) in this preferred embodiment , and different concentrations are used to process the ionized ly or sf - ly nps and a mtt testing method is used to measure the cell activity , and the experiment procedure is described in details as follows : ( 1 ) for the electrophoresis , cells are placed in ice for 30 minutes , and a cell lysis ( containing a mixture of tris 50mm ph 7 . 2 ˜ 7 . 8 , np - 40 1 %, edta 2 mm , nacl 100 mm , 0 . 1 % sds supplementary liquid and protease prohibitor ) ( roche applied sciences , indianapolis , ind ., usa ) for cell lysis . the lysate is collected by a centrifuge at 14000 rpm for 10 minutes , and bradford testing method ( bio - rad , richmond , calif ., usa ) is used to measure the protein concentration . before the protein extract is separated from sds - page , 20 ˜ 50 mg of each protein is prepared and boiled for 5 minutes . the samples are processed by gel electrophoresis for 90 minutes , and then a blotter ( amersham pharmacia biotech inc ., piscataway , n . j ., usa ) is used to blot the samples to a pvdf film ( millipore , billerica , mass ., usa ) by 400 ma of current . the pvdf film is shaken by using skim milk ( 5 % in tbst ) at room temperature for 6 . 0 minutes , and then the milk is washed away , and a tbst buffer solution containing antigens ( such as pakt - s473 , akt , perk , erk , p - 4ebp1 , 4ebp 1 and actin ) is shaken uniformly at 4 □ till the next day , and horseradish peroxidase - conjugated secondary antibodies are shaken at room temperature for 60 minutes . after the secondary antibodies are washed away , an ecl kit ( amersham ) is used to perform a luminescence test according to the instructions given by the manufacturer &# 39 ; s manual . the results of this test are shown in fig3 . the cells are inoculated in 96 - hole culture boards ( each hole has 5 × 10 3 cells ) and cultured by 5 % of co 2 at 37 □ till the next day . in four types of different cells , different doses of sf nps , ly or sf - ly nps are added and processed for 48 hours , and then a stock solution ( with a content of 5 mg / ml in pbs ) of a mtt agent at 37 ° c . is added to the 96 - hole culture boards containing different processing drugs and wait for 4 fours before centrifuging the culture boards at 1200 rpm for 5 minutes , and after adding dmso ( capable of dissolving and precipitating products produced by the reaction of mtt and cells ) for 5 minutes , the suspension is moved to a new elisa board , and an elisa reader ( varioskan , thermo electron ) is used to measure the light absorption by 490 nm . the results of this test are shown in fig3 . ( 3 ) results : in 48 hours after the culture takes place , the lung cancer cells have no significant toxicity under the treatment of sf nps . the ionized ly group shows a slight toxicity of the cells . for a higher dosage , a slight toxicity to h157 cells is shown . on the contrary , observations show that sf - ly nps has significant cytotoxic effect on the three types of cell strains ( h460 , h157 and h1650 ) with a concentration falling within a range of 0 . 5 ˜ 1 mm . in fig3 , the as2 cells have a significant cytotoxic effect of the sf - ly nps at a higher concentration . in this preferred embodiment , a western blotting can be used for monitoring and measuring the phosphorylated akt content in 473 serine residue and quantifying the activated pakt / akt content . undoubtedly , the sf - ly nps group as shown in fig3 shows a significant drop of the overall activated akt content . with the same concentration , the data of this preferred embodiment show that sf - ly nps can increase the cytotoxicity more than the ionized ly of different concentrations . this result shows that sf - ly nps among the four types of cell strains has a stronger suppressing effect , and only a concentration above 0 . 25 ˜ 5 μm is required . in this preferred embodiment , poly ( lactic - co - glycolic acid ) nanocapsules ( pva - ly nps ) can be synthesized and modified by a modifier pva to perform the aforementioned electrophoresis and mtt assays is used to test the activity of cytotoxicity of the four types of cell strains , wherein the experiment procedures of the electrophoresis and cell activity are the same as those described above , and thus will not be repeated . the method of preparing pva - ly nps is described as follows : 50 mg of plga powder and 3 mg of ly294002 are mixed uniformly in 2 . 5 ml of acetone , and added into 25 ml of 2 % polyacrylic acid solution , and a homogenizer is used for emulsification . such liquid is poured into 100 ml of a liquid containing 2 % polyacrylic acid solution to disperse the nanoparticles , and the liquid is stirred uniformly at room temperature for 4 hours to vaporize the organic solvents . finally , the nanoparticles pva - ly nps are collected by an ultracentrifuge . the results of this test are shown in fig4 , wherein pva - ly nps has a less effect on suppressing the growth of cancer cells , and the required concentration is as high as 25 ˜ 50 μm . balb / c female nude mice were obtained from national laboratory animal center , taiwan . mice of 6 ˜ 8 weeks old are used . the mice are randomly divided into four groups , and situated in the same environment with controlled tempeature , humidity and 12 h - light / dark cycle , and the controlled environmental conditions follows the environmental conditions for animal breeding set forth by national cheng kung university . the balb / c immunodeficient mice are innoculated with pc14pe6 / as2 cells ( 1 × 10 6 cells / 1004 of pbs ) by the subcutaneous inoculation method . if the tumor size is approximately equal to 50 - 60 cubic millimeters , the experiment starts taking place . according to the experiment requirements , a saline group ( n = 5 ), a plga nanoparticles ( sf nps ) group ( n = 5 ), a ly294002 ( ly ) group with the same dose ( 1 mg / kg ) ( n = 5 ) or a plga nanoparticles ( sf - ly nps ) group encapsulated with ly294002 ( n = 6 ) are provided , and the mice are injected three times a week ( monday , wednesday , and friday ) continuously for two weeks . the mice with the innoculations are observed in every other two days to check whether there is an abnormality . the tumor size is measured by a caliper and a standard tumor volume measurement method ( volume = long axis × short axis 2 × 0 . 5 ). based on human moral standards , euthanasia of the mice takes place when the tumor size reaches an average size of 4000 mm 3 . the results of this animal experiment are shown in fig5 . after the procedure as shown in fig5 takes place , the body weight of the mice is measured in every two other days to check whether there is a loss of weight . if there is no loss of weight , then it will be considered that there is no toxicity produced in the circulatory systems . the survival rate is determined by the number of days when a natural death of each group of mice occurs or the tumor reaches a size of 4000 mm 3 . the results of this experiment are shown in fig6 . the experiment results show that after the cells are transplanted , an as2 tumor mode of the bald / c mice is developed in 14 days . at the beginning , the average tumor size is 50 to 60 mm 3 , and injections are applied in the tumor three times a week for two weeks , and then the following treatments are given : ( i ) saline , ( ii ) ly ( 1 mg / kg ), ( iii ) sf nps ( 1 mg / kg ) and ( iv ) sf - ly nps ( 1 mg / kg ). the change of tumor size is monitored from the beginning until the average tumor size reaches 3000 ˜ 4000 mm 3 after the injections . this result shows that saline , sf nps and ionized ly injected into the mice will increase the tumor size steadily with time . after 12 ˜ 14 days , the tumor size will be approximately 40 times bigger , indicating that the sf nps treatments does not cause toxicity , and the ionized ly is insufficient to reduce the growth of tumors . on the other hand , the sf - ly nps ( 1 mg / kg ) group as shown in fig5 can effectively slow down the growth of tumors and inhibit the overall volume of the tumors from increasing to approximately 2 . 5 ˜ 3 times of their original size . in fig6 , the mice of each group do not have any significant loss of weight . in fig5 , the time for a tumor reaching a size of 4000 mm 3 is used to measure the survival rate , and the mice treated with sf - ly nps has a significantly higher survival rate than those treated with saline , sf nps and ionized ly . overall speaking , these results show that the injection of sf - ly nps into tumors induces a long - term sustainable effect of suppressing tumors . while the invention has been described by means of specific embodiments , numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope and spirit of the invention set forth in the claims .
the present invention is related to a composition of pi3k inhibitor , comprising : 0 . 01 ˜ 10 mg of pi3k inhibitor ; 10 ˜ 500 mg of poly which is encapsulated onto the surface of the pi3k inhibitor and the surface is non - modified by a modifier ; and the composition has a size of 10 ˜ 1000 nm . thereby , an excellent effect on suppressing the growth of tumor cells will be achieved by the encapsulation of pi3k inhibitor into plga nanomaterials without any modifier on its surface , the optimization of a ratio of pi3k inhibitor to plga , and the accordingly slow release of the composition .