Patent Application: US-201213465150-A

Abstract:
the present invention provides a method for screening the size of carrier for a subject in need , comprising : providing a series of labeled carriers which have different sizes ; administering one of the series of carriers to a subject who suffers from an organ dysfunction ; monitoring biodistribution of the carrier of step in said subject ; repeating steps and until all the series of carriers are administered and all the biodistribution of the series of carriers are monitored ; and determining the size of carrier for said subject in accordance with the retention time of the series of carriers in the dysfunctional organ of said subject . the method can be used as a screening platform for drug carrier , in which the optimal size of carrier can be screened for the dysfunctional organ of the subject .

Description:
different nanoparticle properties , such as shape and surface charge , have been investigated to understand how to enhance the efficacy of nanoparticles in biomedical applications . however , there has not been a comprehensive study characterizing the size - dependency of nanoparticle biodistribution under different pathophysiologic conditions . our study with fluorescent polystyrene nanoparticles revealed a size - dependent biodistribution of the nanopartieles that had been intravenously injected into normal mice . further investigation showed that systemic inflammation induced by lipopolysaccharide changed the retention of the nanoparticles and led to redistribution in vital organs . interestingly , we also observed a time - dependent distribution profile of the nanoparticles in a localized inflammatory hindlimb ischemia model . this model was validated by intravenous injection of polylactic - co - glycolic acid ) ( plga ) nanoparticles that circulated into the ischemic areas . these unprecedented results show the importance of considering size when designing nanoparticles for use in nanoscale therapeutics and diagnostics . cells ( a549 cells , a2058 cells , afscs and hmscs ) were seeded on to 12 - well culture plates at a density of 2 . 6 × 104 / cm2 in 1 ml total medium per well and allowed to adhere . 100 ul of 3 -( 4 , 5 - dimethyl - 2 - thiazolyl )- 2 , 5 - diphenyl - 2h - tetrazolium bromide ( mtt ) reagent ( 5 mg / ml 1xpbs ) were added into each well and incubated for 2 hours at 37 ° c . after medium removal , 0 . 6 ml of dmso was used to lyse cells and dissolve the formazan . the supernatant was collected and distributed into a 96 - well plate at 0 . 2 ml each well for analysis . the absorbance was measured using elisa reader ( spectramax 340pc384 , molecular devices , usa ) at 570 nm . the national cheng kung university animal care and use committee and the national laboratory animal center approved all animal research procedures . fvb and nude mice of either sex ( 6 to 8 weeks , weight 22 ± 0 . 6 g ) were purchased from the national laboratory animal center . fluorescent carboxylated polystyrene latex bead nanoparticles with uniform diameters of 20 , 50 , 100 , 200 , and 500 nm ( invitrogen or polyscience ) were used to investigate the biodistribution and retention of nanoparticles after intravenous injection into mice . these nanoparticles were non - degradable , thus excluding resorption as a variable . nanoparticles were quantified by high - performance liquid chromatography ( hplc , jasco , essex , uk ). fluorescence microscopy and an in vivo fluorescence imaging system ( ivis 200 , caliper life sciences , massachusetts , usa ) were used to observe the nanoparticle biodistribution in the tissues and organs . to quantify the nanoparticle retention , normal , healthy , mice were anesthetized with zoletil ( 50 mg / kg ; virbac , france ) and rompun ( 0 . 2 ml / kg ; bayer healthcare , germany ), and injected with one of the five sizes of nanoparticles through the jugular vein ( 150 μl / mouse ). mice were returned to their cages and received a normal diet and water for 4 hours . major organs and tissues , including the brain , heart , lungs , liver , spleen , kidneys , skin , fat and blood , and urine , were harvested . these harvested tissues , organs and urine were digested in 0 . 5 or 3 ml of 1 m potassium hydroxide ( koh ) solutions at 60 ° c . overnight , depending on the sample . the total volumes of the brain , heart , lung , liver , spleen , kidney , blood , skin and fat were digested in a 0 . 5 ml volume . due to the size of the liver , 3 ml of a koh solution was required for complete digestion . all of the samples were then mixed with 0 . 5 ml of o - xylene for fluorescein extraction by sonication for 1 min and placed into a 60 ° c . oven for 15 minutes . the samples were vortexed and incubated at 60 ° c . for 5 min ; this step was repeated twice . for the urine sample , 0 . 5 ml of xylene was added directly without koh digestion . the preparation of these samples then followed the procedures described previously . finally , all of the samples were centrifuged for 30 minutes at 14 , 000 rpm , and the supernatants were analyzed by hplc . hplc standards were measured by sampling 10 , 40 , 80 , 160 , and 200 μg of 20 , 100 , 200 , and 500 nm nanoparticle solutions and 12 . 5 , 25 , 75 , 100 , and 150 μg of 50 nm nanoparticle solutions . the extraction procedures for nanoparticle standards were identical to the protocol described above . the relative amount of nanoparticle retention in each sample was calculated using the calibration standard curves . mice were anesthetized by injecting zoletil ( 50 mg / kg ; virbac , france ) and rompun ( 0 . 2 ml / kg ; bayer healthcare , germany ) before surgery was performed . for the systemic inflammation model , lipopolysaccharide ( lps , 5 mg / kg ; sigma , usa ) was injected into the mice through the tail vein , followed with intravenous injection of nanoparticles after 24 hours . after four hours , mouse tissues and organs were harvested for sample preparation , as described above . the hindlimb ischemia - reperfusion model was produced by ligating the right femoral artery of the unilateral right leg for 1 hour using a surgical suture . the sutures were then released for reperfusion for 6 hours , 1 day , or 3 days . hindlimb blood flows were measured by laser doppler ( o2c flow meter , lea medizintechnik , giessen , germany ) before and after surgery to confirm vessel occlusion . after 6 hours , 1 day and 3 days after reperfusion , the blood flow rates of both the injured leg ( ischemic , right side ) and the normal leg ( nonischemic , left side ) were measured , and different - sized nanoparticles were administered in the same procedure as outlined above . the muscles of both legs were harvested 4 hours after the nanoparticle injection . samples were prepared and analyzed in the same procedure outlined above . poly ( lactic - co - glycolic acid ) ( plga ) was dissolved in 5 ml of acetone at a final concentration of 10 mg / ml . ethanol / h 2 o ( 50 / 50 , % v / v ) solution was added dropwise ( 1 ml / min ) to the plga solution using a tubing pump and stirred at 400 rpm until turbid . after 5 minutes of additional stirring , the suspension was transferred into a glass beaker containing 20 ml of 1 mm polyethylenimine ( pei , sigma ) solution and homogenized at low speed for 20 minutes as previously described25 . the solution was filtered through a 0 . 22 μm membrane . the produced nanoparticles were washed three times with deionized water . the functional group of qd - cooh was linked to the nh 2 - terminated groups of plga nps by adding 1 - ethyl - 3 -( 3 - dimethylaminopropyl )- carbodiimide ( edc ). the surface morphology of plga nps and plga - qd nps , as shown in fig5 c , was examined using transmission electron microscopy ( tem ). next , we obtained real - time images and tracked the model nanodrug carrier , plga - qd nps , in the living animal . after injection of plga - qd nps into the hindlimb of ischemia - reperfusion nude mice after a 1 day reperfusion , whole - body fluorescence images of the mice were analyzed . plga - qd nps were excited at 605 nm and emitted at 660 nm . results are presented as the mean ± sem . statistical comparisons were performed with student &# 39 ; s t test . a probability value of p & lt ; 0 . 05 was considered statistically significant . there were at least 6 animals in each group , unless specified . to characterize the size - dependent effects of nanoparticles , commercially available 20 , 50 , 100 , 200 and 500 nm fluorescent polystyrene nanoparticles were acquired . the nanoparticle sizes and shapes were confirmed by transmission electron microscopy , which showed uniform size distribution and consistent spherical morphology ( fig1 a ). we detected minimal toxicity , which was similar for the nanoparticles of various sizes , using the ( 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide ) ( mtt ) assay for 4 different human cell lines , including a549 carcinoma cells , a2058 melanoma cells , cultured amniotic fluid - derived stem cells ( afscs ), and primary bone marrow mesenchymal stem cells ( hmscs ) ( fig6 ). therefore , we focused our studies on the in vivo characterization of different - sized fluorescent polystyrene nanoparticles following systemic injection . as depicted in fig1 b , nanoparticles were injected into the jugular vein of mice to investigate the biodistribution of the nanoparticles . organs and tissues were collected and digested in 1 m koh overnight at 60 ° c . the samples were then mixed with o - xylene to dissolve the nanoparticles for fluorescein extraction after centrifugation . supernatants were analyzed with high - performance liquid chromatography ( hplc ) for nanoparticle quantification by measuring fluorescence intensity . to ensure that the protocol did not compromise the nanoparticle fluorescence signal , stock nanoparticles were treated with koh and o - xylene at 60 ° c . overnight . hplc analysis revealed an excellent alignment of the differentially treated nanoparticle samples , indicating that neither koh nor o - xylene interfered with the fluorescence signal ( fig1 c ). standard calibration curves were also established by extracting different amounts of a nanoparticle stock solution with o - xylene before hplc analysis ( fig1 d ). nanoparticles are well - known for their short half - lives within the circulation and their rapid accumulation ( within hours ) in target tissues or organs 9 . to verify this biodistribution 3 mg ( 150 μl stock volume ) of nanoparticles was injected into the jugular vein of healthy fvb mice and allowed to circulate for 4 hours . at this point , organs were collected for imaging with an in vivo imaging system ( ivis ) or for sample preparation as outlined above for the hplc analysis . ivis images showed that the nanoparticles , regardless of size , were present in all of the vital organs , including the heart , lungs , liver , spleen , and kidneys ( fig2 a ). however , the fluorescence levels were extremely low in the brain when mice were treated with nanoparticles larger than 100 nm , suggesting that nanoparticles larger than 100 nm do not easily cross the blood - brain barrier ( bbb ). hplc quantification confirmed the ivis results , revealing that most of the nanoparticles were retained in the lungs , liver , and spleen in a size - dependent manner ( fig2 b ). a very steep cut - off size of 50 nm was determined for nanoparticle retention in the liver . when nanoparticle sizes changed from 50 nm to 200 nm , retention in the liver increased from approximately 5 % to more than 60 %. when designing nanoparticles for drug delivery , our results show that 200 nm is the optimal size for drug nanocarriers when targeting the liver . anything larger is unlikely to increase retention in the liver . when the biodistribution of the nanoparticles was analyzed by a weight - to - weight ratio of nanoparticles to organs , nanoparticles were revealed to be more evenly distributed by nanoparticle density among the heart , lungs , liver , spleen , and kidneys ( fig7 a ). the heart and lungs retained the nanoparticles in a manner that was linearly proportional to the nanoparticle size , suggesting that the larger nanoparticles were blocked from exiting the capillaries to a greater extent . the spleen demonstrated a retention similar to the liver . nanoparticle density increased dramatically from 0 . 1 mg / g to more than 2 . 7 mg / g when the nanoparticle size increased from 50 nm to 200 nm . again , this result suggests that a 200 nm diameter is the optimum size when designing a nanoparticle to be retained by the spleen . detailed inspection of the hplc quantification of brain nanoparticle retention revealed contradictory results . the ivis images only showed the presence of nanoparticles smaller than 100 nm ( fig2 a ). however , hplc results showed an increased retention in the brain proportional to the size of the nanoparticles ( fig2 b ). therefore , we suspect that most of the nanoparticles greater than 100 nm were located in the center of the tissue sections that were sliced in the coronal plane . in contrast , nanoparticles smaller than 100 nm were retained primarily in the cerebral cortex and white matter . ivis cannot image beyond a certain depth , depending on the tissue and the source of the fluorescence 15 . therefore , in the present study ivis was used only for preliminary biodistribution analysis , and . hplc was used for precise biodistribution analysis and quantification . the ivis images showed that nanoparticles accumulated in the center of the brain and were only preferentially distributed into the cortex when the size decreased . most of the larger nanoparticles ( 100 nm or greater ) were retained in the vital organs and the blood , and less than 20 % of the smaller nanoparticles ( below 100 nm ) were recovered from our samples ( fig2 c ). to assess the fate of the remainder of the smaller nanoparticles , additional tissues and samples were analyzed , including skin , muscle , adipose tissue ( fat ), and urine . the ivis images revealed a large amount of small nanoparticles present in the skin and muscle ( fig2 d ). hplc quantification also indicated that the retention of nanoparticles in the peripheral tissues , including skin , muscle and fat , was inversely proportional to the size of the nanoparticles ( fig8 a - d ). urine samples also contained more small nanoparticles than large nanoparticles , as one would expect . nanoparticles distribute differently among organs and are excreted according to their size . larger nanoparticles are more likely to be retained in the vital organs , either because of the size restriction of the renal system or of the organ itself . smaller nanoparticles have the ability to permeate more easily throughout the vasculature , pass through the renal system into the urine , and distribute into the peripheral tissues . components within the renal system , such as the glomerular endothelium or the glomerular basal membrane , filter small substances through a defined pore size 16 . as such , most nanoparticles within the kidneys were found to accumulate in the glomerulus ( fig1 ). together with the finding that smaller nanoparticles were 2 to 3 times more likely to be found in the urine , the glomerulus was confirmed as a filter of 100 nm particles , which has been established by previous studies 16 . drug nanocarriers have been designed to target tissues under specific disease conditions 2 , 4 , 10 , 11 . a system under a disease condition responds differently to foreign bodies than a normal , healthy , system . the diseased system responds differently by altering microenvironmental conditions , varying cell behavior , and using signal transduction pathways that result in specific responses against the foreign bodies 17 - 19 . thus , it is crucial that the uptake and distribution of nanoparticles be fully characterized in the diseased state of the model for the drug delivery system . to investigate whether a change in pathophysiologic conditions affects the biodistribution of nanoparticles , the same procedure was repeated as outlined above ( fig1 b ) for mice pretreated with the bacterial endotoxin lipopolysaccharide ( lps ) to induce a systemic inflammatory response . ivis imaging revealed a preliminary distribution profile of the nanoparticles that indicated the accumulation of the nanoparticles in all of the vital organs during systemic inflammation ( fig3 a ). surprisingly , a distinctive signal was clearly detected for the 200 and 500 nm nanoparticle - injected brains after lps treatment , and this signal was previously only slightly detectable . furthermore , signals in the brain from all of the sizes of nanoparticles were more pronounced . hplc analysis revealed that while the distribution of nanoparticles in healthy mice was size dependent , lps - treated mice no longer exhibited the distinct size - dependent biodistribution ( fig3 b and fig7 b ). these results will be useful in drug delivery design for therapy targeted to the cerebral cortex . the diseased state of the body may allow more large nanoparticles to reach the cerebral cortex , but this effect is less pronounced for particles with a diameter of more than 200 nm . beyond that size limit , the retention efficiency did not increase ( fig1 a ). in contrast , although smaller nanoparticles were capable of reaching the cerebral cortex , they were more likely to perfuse into the skin and muscles , similar to the case for normal tissues and organs . additionally , small nanoparticles showed very high clearance rates and short half - lives ( fig2 d , 3 d and fig8 d ). in the heart , lungs , liver , spleen , and kidney , larger nanoparticles were evenly distributed ( fig3 b and fig7 b ). lps - treated livers did not retain many nanoparticles compared to a normal liver . in fact , the spleen , which plays a vital role in the immune system , retained most of the nanoparticles when the mice experienced systemic inflammation . in the spleen , b cell proliferation is heavily induced by lps treatment 20 . a previous report also demonstrated the mechanisms of marginal zone antigen capture by b cells 21 . besides b cells , dendritic cells may also be involved in antigen retrieval processes during early time points 22 . in our case , we suspect that the b cells may have facilitated nanoparticle transport through migration to follicular dendritic cells or through the retrieval capabilities of dendritic cells that migrated to the spleen . tissue sections , ivis imaging , and hplc analysis also confirmed that more nanoparticles were retained in the brain and spleen but that fewer were retained in the lungs and liver of lps - treated mice ( fig4 a - d and fig9 and 10 a - d ). a summary of the nanoparticle retention in the vital organs revealed that in lps - treated mice , there was a decrease in the nanoparticles in the vital organs compared to healthy mice ( fig3 d ). interestingly , fewer nanoparticles accumulated in the heart during inflammation ( fig1 ). the increased blood flow and vasodilation , along with the high circulatory effect of the heart , may eject nanoparticles from this organ . as mentioned above , additional tissues were analyzed . the ivis images showed that larger nanoparticles were detected in the skin and muscle ( fig3 e ), which was confirmed by tissue sections ( fig1 and 14 ). these larger nanoparticles were previously undetectable under the normal physiologic conditions in the mice . these results indicate that during a systemic inflammatory response , the mice experienced vasodilation and increased blood flow , which allowed larger nanoparticles to more readily permeate the peripheral tissues . the systemic inflammation induced by lps changed the pathophysiologic conditions of the mice , causing a different fate for the nanoparticles . in contrast , local injuries may be not accompanied by the same heightened systemic response by the body as with systemic inflammation . to investigate whether changes in the microenvironmental conditions surrounding the diseased tissue altered the nanoparticle kinetics , a hindlimb ischemia - reperfusion model was performed as described previously 23 with some modifications . after femoral arterial ligation for 1 hour , the artery was allowed to reperfuse for 6 hours , 1 day , or 3 days ( fig1 a ). ligation and reperfusion of the femoral artery was confirmed by measuring blood flow ( fig1 b ). at different time points during reperfusion , nanoparticles were injected , and the muscles were collected , prepared , and analyzed in the same manner as outlined above . after 6 hours of reperfusion , large nanoparticles were retained in the ischemic muscle to a greater extent than in the non - ischemic muscle ( fig5 a and fig1 and 17 ). after 1 day , there was no difference in the distribution of nanoparticles larger than 100 nm between ischemic and nonischemic muscles . however , nanoparticles smaller than 100 nm were present in the ischemic leg , and this was also confirmed in tissue sections ( fig5 b ). after 3 days of reperfusion , there were no differences in distribution among the different sizes of nanoparticles ( fig1 ). taking blood flow into consideration with the inflammation response , these factors may have caused the changes in the size - dependent biodistribution . during the inflammation stages of ischemia and reperfusion , the biodistribution of the nanoparticles differed . these findings suggest that the intervention time may be crucial , depending on the disease and the size of the drug nanocarriers . drug nanocarriers larger than 100 nm have a retention time window of several hours after reperfusion , but smaller drug nanocarriers may require a 1 day period after reperfusion to achieve optimal effects . we used poly ( lactic - co - glycolic acid ) nanoparticles ( plga nps ) to confirm our results in a relevant clinical setting . plga nps have been well - established and well - characterized in several drug delivery systems 3 , 24 , 25 . previous studies have demonstrated success in therapeutic treatments , and drug - containing plga nps are also approved by the us food and drug administration for clinical use . using plga nps , the drug nanocarrier design considerations were tested with more relevant biomaterial than fluorescent polystyrene nps , allowing us to generalize design principles for all drug nanoparticles , regardless of the material . we theorized that a plga np drug delivery carrier smaller than 100 nm in diameter would result in greater retention in the muscle of a hindlimb ischemia - reperfusion model . we synthesized 80 and 300 nm plga nps conjugated with quantum dots ( plga - qd nps ; fig5 c ), injected them into hindlimb ischemic mice subjected to a 1 day reperfusion , and analyzed the retention using ivis imaging ( fig5 d ). consistent with our initial size - dependent data , the in viva imaging of the mice in a supine position showed the presence of 80 nm plga - qd nps on the surface near the skin at the ischemic region , but the 300 nm plga - qd nps were absent ( fig5 d ). ex vivo images of the muscle and subsequent quantification revealed that ischemia and reperfusion increased the retention of 80 nm plga - qd nps compared with the 300 nm nanoparticles ( fig5 d , e ). this result was also consistent with our 1 day reperfusion data . hindlimb ischemia and reperfusion induced local inflammatory responses , including vasodilation and increased blood flow . the 80 nm plga nps were small enough to reperfuse into the muscle region of the inflamed hindlimb and escape renal reabsorption . in summary , we have validated the size - dependent biodistribution pattern of nanoparticles injected intravenously into mice and have confirmed that the alteration in the distribution pattern is caused by a physiologic change . in the present study , the size - dependent biodistribution of nanoparticles ranging from 20 to 500 nm was systematically characterized in a mouse model . our results indicate that most of the vital organs retained the nanoparticles in a size - dependent manner . larger nanoparticles , particularly those with a diameter greater than 100 nm , were more likely to be distributed in the vital organs . small nanoparticles , with a diameter of less than 100 nm , were mostly retained in the peripheral tissues or were excreted via the urine . additionally , systemic inflammation and local hindlimb ischemia altered the biodistribution pattern to allow large nanoparticles to be retained in the vital organs and in the peripheral tissues . our results were validated by the injection of a nanoparticles produced from an fda approved material , plga . we consider that the comprehensive characterization of nanoparticle behavior in vivo presented in this study is important for nanomedicine design considerations . the conclusions drawn from our results should be taken into account when designing nanoparticles for intravenous drug delivery . 1 . anderson , d . g , burdick , j . a . & amp ; langer , r . smart biomaterials . science 305 , 1923 - 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