Patent Application: US-60191408-A

Abstract:
a method of developing degradable linear poly peg with multiple functioning capacities , which can be used as drug carriers for cancer cell delivery . a dpeg may be effective in targeting cancerous tumors through an enhanced permeation and retention effect . the dpeg will then degrade in the acidic extracellular fluid of solid tumors leading to fast cellular internalizations , finally degrading in the lysosome for efficient renal clearance . these may be used in conjunction with drugs and / or targeting groups . furthermore , dpegs are thermoresponsive , on an as needed basis , making them useful for in vivo application .

Description:
poly ( peg - diacrylate - dithiothreitol ) s ( pegda - dtt ), poly ( peg - diacrylate - ethanedithiol ) s ( pegda - det ), poly ( peg - diacrylate - propanedithiol ) s ( pegda - dpt ), poly ( peg - diacrylate - butanedithiol ) s ( pegda - dbt ), poly ( peg - dimethacrylate - dithiothreitols ( pegdma - dtt ), poly ( peg - dimethacrylate - ethanedithiol ) s ( pegdma - det ) were prepared by michael - type polyaddition of poly ( ethylene glycol ) diacrylate ( pegda ), or dimethacrylate ( pegdma ) with d , l - dithiothreitol ( dtt ), 1 , 2 - ethanedithiol ( det ), 1 , 3 - propanedithiol ( dpt ), or 1 , 4 - butanedithiol ( dbt ), respectively ( schemes 1 and 2 ). the synthesized dpegs are listed in table 1 . a typical procedure is as follows . dtt ( 0 . 4830 g , 3 . 1313 mmol ) was dissolved in 3 ml of dimethyl sulfoxide ( dmso ) at room temperature . pegda575 ( 1 . 8000 g , 3 . 1310 mmol ) was added to the dmso solution . triethylamine ( tea ) ( 0 . 05 ml , 0 . 3587 mmol ) was added dropwise to the above mixture and the polymerization was continued at room temperature for 72 h . the polymer was precipitated in ether and purified by repeated precipitations . the precipitant was dried in vacuum at 70 ° c . overnight . the polymer pegda575 - dtt was characterized by gpc ( table 1 ) and nmr . a typical procedure is as follows : cbda ( 0 . 4365 g , 2 . 2258 mmol ) and peg200 ( 0 . 4452 g , 2 . 2258 mmol ) were dissolved in 3 ml of anhydrous dmso at 60 ° c . for 72 h . the polymer was precipitated in ether and purified by repeated precipitation . the polymer was dried in vacuum oven at 70 ° c . overnight . yield = 92 %. synthesis of dpeg with terminal dithiols ( scheme 3 b ) or diacrylates ( scheme 3 c ) an example is shown as follows : pegda258 ( 0 . 8207 g , 3 . 1810 mmol ) and det ( 0 . 2996 g , 3 . 1811 mmol ) were dissolved in 3 ml of dmso and stirred at room temperature . tea ( 0 . 05 ml , 0 . 3587 mmol ) was added dropwise to the above mixture as catalyst . after 72 h , additional det ( 0 . 27 g , 2 . 87 mmol ) or pegda258 ( 0 . 53 g , 2 . 05 mmol ) was added to the polymerization solution and stirred at room temperature for another 30 h . the polymer was precipitated in ether for three times and dried under vacuum overnight ( mn : 36 , 900 , pdi : 1 . 58 ). the polymers with terminal diacrylates ( pegda258 - det - diacrylates ) or dithiols ( pegda258 - det - dithiols ) were analyzed by 1 h - nmr . to confirm the terminal dithiol groups , deuterium oxide ( d 2 o ) exchange experiment was carried out by the following procedure : d 2 o ( 0 . 2 ml ) was added into 0 . 6 ml of pegda258 - det - dithiols cdcl 3 solution and mixed well . after centrifuged at 2 , 500 rpm , pegda258 - det - dithiols cdcl 3 solution was collected for 1 h - nmr measurement . the dpeg with terminal dipyridyl disulfide groups ( pegda258 - det - dipyridyl disulfides ) was synthesized as follows : pegda258 - det - dithiols ( mn : 36 , 900 , pdi : 1 . 58 , 2 . 47 g , 0 . 067 mmol ) and 2 , 2 - dipyridyl disulfide ( 0 . 059 g , 0 . 27 mmol ) were dissolved in 20 ml of methanol and stirred at room temperature for 12 h . the mixture solution was precipitated in ether for three times . the precipitate was dried under a vacuum overnight . introducing folic acid targeting groups to the dpeg is as follows : folic acid ( 2 . 45 g , 5 . 55 mmol ), nhs ( 1 . 28 g , 11 . 12 mmol ), dcc ( 1 . 37 g , 6 . 64 mmol ) and tea ( 1 . 86 ml , 13 . 34 mmol ) were dissolved in 30 ml of anhydrous dmso and stirred at room temperature overnight . the mixture was precipitated in ether for three times to isolate folate - nhs ester . folate - nhs ester ( 1 . 21 g , 2 . 25 mmol ) and cysteamine ( 0 . 21 g , 2 . 70 mmol ) were dissolved in 10 ml of anhydrous dmso . tea ( 0 . 32 ml , 2 . 29 mmol ) was added to the reaction mixture . the reaction was stirred for 12 h and the solution was precipitated in water for three times to obtain folate - cysteamide . pegda258 - det - dipyridyl disulfides ( mn : 36 , 900 , pdi : 1 . 58 , 2 . 03 g , 0 . 055 mmol ) and folate - cysteamide ( 0 . 11 g , 0 . 22 mmol ) were dissolved in 20 ml of dmso and stirred at room temperature for 48 h . the polymer was repeatedly precipitated in ether until tlc ( solvent system : n - propanol : water : ammonium hydroxide = 8 : 1 : 2 , by volume ) showed no sign of free folate cysteamide . pegda258 - det - difolates was obtained in a yield of 81 %. conjugation of cpt to the dpeg with pendant hydroxyl groups is shown in scheme 4 . a typical procedure is as follows : the pegda700 - dtt ( mn = 10 , 900 , 5 . 24 g , 0 . 48 mmol ) and succinic anhydride ( 2 . 50 g , 24 . 98 mmol ) were dissolved in 50 ml of dry dmf and stirred at 60 ° c . for 72 h until all hydroxyl groups were reacted . the resulting pegda700 - dtt functionalized with pendant carboxylic acid groups ( pegda700 - dtt - acid ) was isolated in ether and dried under high vacuum at 60 ° c . overnight . the pegda700 - dtt - acid reacted with cpt catalyzed by dcc / dmap at different carboxylic acid / cpt molar ratios produced dpeg carrying different amounts of cpt molecules per chain . a typical example is as follows : the pegda700 - dtt - acid ( 1 . 42 g , 0 . 11 mmol ), dcc ( 0 . 35 g , 1 . 65 mmol ), dmap ( 17 . 1 mg , 0 . 14 mmol ) and cpt ( 0 . 50 g , 1 . 43 mmol ) were dissolved in 20 ml of dmso and stirred at room temperature for 48 h . the polymer was repeatedly precipitated in ether until the thin layer chromatograph ( tlc ) ( solvent system : chloroform : acetone = 2 : 1 , by volume ) showed no sign of free cpt in the pegda700 - dtt - cpt conjugate . yield = 86 %. 1 h - nmr analysis showed that the dpeg - cpt conjugate had 12 cpt molecules per chain ( pegda700 - dtt - 12cpt ). the conjugate with one cpt molecule per chain ( pegda700 - dtt - 1cpt ) was also prepared by the same procedure by adding less cpt . generally , all tested dithiols ( dtt , det , dpt , and dbt ) could react with pegda to produce high molecular weight dpegs in high yields . only dtt and det , however , reacted with pegdma to produce high molecular weight dpegs ( schemes 1 and 2 ). the molecular weights of the obtained dpegs ranged from 10 , 000 to 90 , 000 , depending on the dithiols and the di ( meth ) acrylates ( table 1 ). the polydispersity was about 1 . 7 to 2 , which is typical for polymers obtained from condensation polymerization . low polydispersed polymers could be easily obtained by repeated fractionation . for example , with chcl 3 as solvent and ether as precipitant , 3 - step fractionations of pegdma750 - det with mn of 49 , 000 and pdi of 1 . 83 produced fractions with lower pdis ( table 2 ). dpeg &# 39 ; s are degradable because they are hydrolysable . specifically , dpeg backbone contains β - thioester (— sch 2 ch 2 coo — or — sch 2 ch ( ch 3 ) coo —) groups that promote hydrolysis . the hydrolysis of dpegs was tested at ph 7 . 4 , 6 . 0 and 5 . 0 and monitored by measuring the decrease of the ester bonds using 1 h - nmr . fig1 shows the hydrolysis of pegda258 - det at different phs . at ph 7 . 4 , pegda258 - det was relatively stable . only 8 . 7 % of the ester groups hydrolyzed after 42 h . while at ph 5 . 0 , pegda258 - det hydrolyzed quickly . about 25 . 3 % in 15 min and 55 . 48 % in 9 h of the ester groups were hydrolyzed . at ph 6 . 0 , pegda258 - det hydrolyzed faster than at ph 7 . 4 , but more slowly than at ph 5 . 0 . about 34 . 8 % of the ester groups hydrolyzed in 9 h . correspondingly , the mn of pegda258 - det only decreased slightly at ph7 . 4 even after 42 h , but it hydrolyzed to oligomers ( mn , ˜ 2000 ) after 42 h at ph5 ( fig2 ). similar results were also obtained from pegda575 - det ( mn , 82 , 700 ) ( fig3 ). these results indicate that the dpegs are relatively stable at ph 7 . 4 while degrade rapidly at ph 5 . 0 and ph 6 . thus , high molecular weight dpegs can be used as drug carriers for effective cancer targeting while they can degrade into oligomers in lysosomes for effective renal clearance . in addition to the functional groups introduced by using dithiols having functional groups , such as dtt , the dpegs could easily be functionalized with terminal ( meth ) acrylate or thiol groups ( scheme 3 ). the ratio of di ( meth ) acrylate monomer to dithiol monomer was first kept at 1 / 1 molar ratio to make a high molecular weight polymer . after a desirable molecular weight was reached ( e . g ., pegda258 - det , mn : 36 , 900 , pdi : 1 . 58 ), an excess of dithiol or di ( meth ) acrylate monomer was added to the reaction solution to cap the polymer ends with either thiol or ( meth ) acrylate . typical 1 h - nmr spectra are shown in fig4 . the peaks at about 5 . 8 ppm , 6 . 1 - 6 . 2 ppm , and 6 . 4 ppm were present in the nmr spectrum of pegda258 - det - diacrylates , indicating the existence of terminal acrylate groups . the molecular weight calculated from the integrations of the acrylate peaks and the ester peak was about half of the values measured by gpc ( mn : 36 , 900 , pdi : 1 . 58 ), suggesting that the polymer chains indeed have acrylates at the both ends . 1 h - nmr spectrum of pegda258 - det - dithiols had a peak at 1 . 7 ppm , which disappeared after d 2 o exchange , indicating the existence of terminal dithiol groups . the terminal thiol groups were further continued by the reaction with pyridyl disulfide ( scheme 3 ). the presence of 2 - pyridyldithio group in the polymer again confirmed the presence of the terminal thiol groups . the calculation from the integrations also showed that there were pyridyl disulfide groups at the both ends . the use of terminal thiol groups for conjugation was demonstrated by introducing folic acid targeting groups ( scheme 3 d and e ). the pegda258 - det with terminal 2 - pyridyldisulfides reacted with folic acid - cysteamide prepared from folic acid and cysteamine and formed the disulfide bonds , anchoring the folic acid moieties to the dpeg ( pegda258 - det - difolates ). the presence of folic acid moieties was confirmed by nmr ( experimental section ) and was about 2 folic acid groups per chain . the dpeg hydrogels were synthesized by an in - situ copolymerization method or an end - capping method using crosslinking agents of pentaerythritol tetraacrylate ( teac ) or trimethylolpropane ethoxylate triacrylate ( triac ) with molecular weight of 428 ( triac428 ), 604 ( triac604 ), 912 ( triac912 ) ( scheme 5 ). in the in - situ copolymerization method , pegda700 , dtt and a crosslinking agent were copolymerized to form gels . triac428 at 5 , 10 or 15 wt -% of pegda700 were used . the corresponding molar ratios of the acrylate from pegda700 to that from triac428 were 8 , 4 , and 3 respectively . the amounts of other crosslinking agents in the copolymerization were calculated according to the molar ratios . a typical procedure is as follows . pegda700 ( 2 . 1479 g , 3 . 0685 mmol ), triac912 ( 0 . 2289 g , 0 . 2510 mmol ) and dtt ( 0 . 5314 g , 3 . 445 mmol ) were dissolved in 3 ml of dmso at room temperature . tea ( 0 . 05 ml , 0 . 3587 mmol ) was added dropwise to the above mixture and the crosslinking polymerization was continued at room temperature for 72 h . the solids were extracted for 24 h with 250 ml of acetone using a soxhlet extractor . the insoluble solid in a yield of 92 % was dried in vacuum at 70 ° c . overnight . in the end - capping method , the dpeg with terminal thiol groups at the both ends ( dpeg - dithiols ) was first synthesized as the precursor . a crosslinking agent and tea were then added to form the gels ( scheme 6 ). the molar ratio of the acrylate in the crosslinking agent to the thiol group in the dpeg - dithiols was kept close to 1 / 1 . a typical procedure is as follows . det ( 0 . 3353 g , 3 . 5606 mmol ) and pegda700 ( 2 . 4924 g , 3 . 5606 mmol ) were dissolved in 3 ml of dmso and stirred at room temperature . tea ( 0 . 05 ml , 0 . 3587 mmol ) was added dropwise to the mixture as catalyst . after 72 h , additional det ( 0 . 31 g , 3 . 29 mmol ) was added to the polymerization solution and stirred at room temperature for another 30 h . the polymer was precipitated in ether for three times and dried under vacuum overnight . pegda700 - det with terminal thiol groups at both ends ( pegda700 - det - dithiols , mn : 22 , 900 , pdi : 1 . 62 ) was obtained . pegda700 - det - dithiols ( 1 . 6450 g , 0 . 0718 mmol ) and teac ( 12 . 6490 mg , 0 . 0359 mmol ) were dissolved in 10 ml of dmso at room temperature . tea ( 2 . 84 , 0 . 02 mmol ) was added dropwise to the above mixture and the polymerization was continued at 35 ° c . for 96 h . the resulting soft solid was extracted for 24 h with 250 ml of acetone using a soxhlet extractor . the insoluble solid in a yield of 83 % was dried in vacuum at 70 ° c . overnight . the dpeg gels resulted from the both methods are summarized in tables 3 and 4 . ionic strength and ph effects on the phase transition temperature were tested by measuring the cloud points of the dpeg ( 1 wt %) in nacl solutions at 0 . 15 , 0 . 5 , 1 . 0 , and 2 . 0 mol / l or at ph 6 . 0 , 7 . 0 , and 8 . 0 . the swelling ratio (%) of the hydrogels was measured in terms of the percent of absorbed water by the dry gels . the gel particles were equilibrated in water for 24 h . the hydrated particles were carefully taken out from the solution , wiped with a filter paper to remove the free water on the surface and then weighted . swelling ratio (%) of a sample was calculated by : where w is the hydrogel weight at equilibrium and w 0 is the weight of the dry gel . all tested dithiols ( dtt , det , dpt , and dbt ) could react with pegda to produce high molecular weight dpegs in high yields . only dtt and det , however , reacted with pegdma to produce high molecular weight dpegs ( schemes 1 and 2 ). the molecular weights of the obtained dpegs ranged from 10 , 000 to 90 , 000 , depending on the dithiols and the di ( meth ) acrylates ( table 1 ). the polydispersity was about 1 . 7 to 2 , which is typical for polymers obtained from condensation polymerization . low polydispersed polymers could be easily obtained by repeated fractionation . for example , with chcl 3 as solvent and ether as precipitant , 3 - step fractionations of pegdma750 - det with mn of 49 , 000 and pdi of 1 . 83 produced fractions with lower pdis ( table 2 ). their degradation was confirmed by the hydrolysis experiments . dpeg is stable at the neutral ph but hydrolyzes quickly at acidic phs . dpegs can be made to carry multifunctional groups . in addition to the functional groups introduced by using dithiols having functional groups , such as hydroxyl groups ( dtt ) and carboxylic acid groups ( dmsa ), the dpegs could easily be functionalized with terminal ( meth ) acrylate or thiol groups ( scheme 3 ). the ratio of di ( meth ) acrylate monomer to dithiol monomer was first kept at 1 / 1 molar ratio to make a high molecular weight polymer . after a desirable molecular weight was reached ( e . g ., pegda258 - det , mn : 36 , 900 , pdi : 1 . 58 ), an excess of dithiol or di ( meth ) acrylate monomer was added to the reaction solution to cap the polymer ends with either thiol or ( meth ) acrylate . typical 1 h - nmr spectra are shown in fig4 . the peaks at about 5 . 8 ppm , 6 . 1 - 6 . 2 ppm , and 6 . 4 ppm were present in the nmr spectrum of pegda258 - det - diacrylates , indicating the existence of terminal acrylate groups . the molecular weight calculated from the integrations of the acrylate peaks and the ester peak was about half of the values measured by gpc ( mn : 36 , 900 , pdi : 1 . 58 ), suggesting that the polymer chains indeed have acrylates at the both ends . 1 h - nmr spectrum of pegda258 - det - dithiols had a peak at 1 . 7 ppm , which disappeared after d 2 o exchange , indicating the existence of terminal dithiol groups . the terminal thiol groups were further confirmed by the reaction with pyridyl disulfide ( scheme 3 ). the presence of 2 - pyridyldithio group in the polymer again confirmed the presence of the terminal thiol groups . the calculation from the integrations also showed that there were pyridyl disulfide groups at the both ends . the use of terminal thiol groups for conjugation was demonstrated by introducing folic acid targeting groups ( scheme 3 d and e ). the pegda258 - det with terminal 2 - pyridyldisulfides reacted with folic acid - cysteamide prepared from folic acid and cysteamine and formed the disulfide bonds , anchoring the folic acid moieties to the dpeg ( pegda258 - det - difolates ). the presence of folic acid moieties was confirmed by nmr ( experimental section ) and was about 2 folic acid groups per chain . the use of the hydroxyl groups in dtt - based dpeg was demonstrated by conjugation of cpt ( scheme 4 ). pegda700 - dtt was first reacted with succinic anhydride to convert the hydroxyl groups into acid groups ( pegda700 - dtt ). it is important to ensure all the hydroxyl groups reacted ; otherwise , crosslinking would occur in the next step of cpt conjugation . cpt was anchored to the chains using conventional dcc / dmap catalyzed reaction . the amount of cpt per chain was controlled by cpt / carboxylic acid ratio . the resulting pegda700 - dtt - cpt was analyzed by nmr ( experimental section ). pegda700 - dtt - cpt samples having 12 cpt / chain and 1 cpt / chain were prepared . these dpegs were found to have thermosensitive properties . a clear solution of the dpeg became cloudy upon heating . a typical optical transmittance of the solution of pegda575 - dtt as a function of temperature is shown in fig5 . clearly , the pegda575 - dtt precipitated out from the solution at temperatures around 40 - 60 ° c ., depending on its concentration . below 1 wt %, the cloud point decreased as the polymer concentration increased . at 0 . 125 wt %, the cloud point was 55 ° c . the phase transition region was also broad . when the concentration was 1 . 0 wt % or higher , the cloud point of the dpeg was about 36 ° c . independent of the dpeg concentration . the phase transition was also sharp , within a 2 ° c . range . thus , pegda575 - dtt had a lcst at about 36 ° c . the influences of molecular weight and structure of the peg - di ( meth ) acrylate macromonomers on the dpeg &# 39 ; s lcst are shown in fig6 . with the same dithiol ( dtt ), the dpeg obtained from a pegda with molecular weight of 700 ( pegda700 - dtt ) had a lcst of 45 ° c ., 9 ° c . higher than that of dpeg obtained from pegda575 ( pegda575 - dtt ). similarly , the dpeg from pegdma with mn of 750 ( pegdma750 - dtt ) had a lcst of 39 ° c ., significantly higher than that of the dpeg from pegdma550 ( pegdma550 - dtt ). thus , the lcst of the dpeg is very sensitive to the number of the ethylene glycol units in the peg -( meth ) acrylate macromonomers ( table 1 ). a longer peg chain in the peg - di ( meth ) acrylate resulted in a higher lcst of the polymer . fig6 also shows that the dpeg made from pegdma had a lower lcst than that made from pegda with the same peg molecular weight ( or n in table 1 ) and dithiol . the cloud point of pegda700 - dtt was 6 ° c . higher than that of pegdma750 - dtt , and the cloud point of pegda575 - dtt was 22 ° c . higher than that of pegdma550 - dtt . the phase transitions of dpegs obtained from pegda700 with different dithiols are shown in fig7 . a long methylene - chain in the dithiol decreased the lcst of the resulting dpeg . the lcsts of pegda700 - det , pegda700 - dpt and pegda700 - dbt were 20 , 8 and 2 ° c ., respectively . introducing hydroxyl groups to the dithiol increased the lcst of the dpeg . pegdma750 - dtt had a lcst of 39 ° c ., but the lcst of pegdma750 - det was 14 ° c ., a 25 ° c . difference . a similar difference was found in pegda700 - dtt and pegda700 - det . the effects of ionic strength on the lcsts of dpegs were tested by measuring the lcsts in the presence of varied concentrations of nacl ( fig8 ). the presence of nacl significantly decreased the lcsts of dpegs . this is consistent with the “ salt out ” effect found in other thermoresponsive polymers . 32 fig9 shows that the solution ph had little effect on the lcst of pegdma750 - dtt . dpeg gels were first prepared by in - situ polymerization of pegda700 and dtt in the presence of a crosslinking agent triac or teac . the copolymerization with 5 % or more crosslinking agent produced gels in high yields ( table 1 ). the gels swelled at low temperatures but deswelled at elevated temperatures . the thermoresponsive property in terms of the swelling ratio as a function temperature of the hydrogels is shown in fig1 and 11 . in contrast to the sharp phase transition at 45 ° c . of noncrosslinked pdeda700 - dtt ( fig6 ), the pegda700 / dtt / triac428 hydrogels deswelled gradually as the temperature increased . the deswelling started even at around 0 ° c ., much lower than the lcst of pegda700 - dtt . this is because that triac428 contains only one ethylene glycol unit on average in each of its branch ( n = 1 in scheme 5 ) and thus it is a relatively hydrophobic molecule . the incorporation of triac428 into pegda700 - dtt chains lowers the lcst . triac428 units were randomly distributed in the pegda700 - dtt chains , causing the transition temperature to span from 0 to 45 ° c . as expected , the swelling ratio of the hydrogels decreased as the triac428 content in the gel increased from 5 % to 15 % ( fig1 ). at 5 ° c ., the swelling ratio of the hydrogels was 320 %, 225 % and 194 %, respectively when triac428 was 5 wt %, 10 wt %, and 15 wt % of pegda700 . the swellability of the hydrogels was also affected by the type of crosslinking agents ( fig7 ). as the triac molecular weight , i . e ., the ethylene glycol unit n shown in scheme 5 , increased , the swelling ratio of the resulting hydrogel increased . for example , at the molar ratio of the acrylate from pegda700 to that from the crosslink agent of 4 , the swelling ratios at 0 ° c . of the hydrogels with triac 912 , triac 604 , triac 428 and teac were 310 %, 292 %, 231 % and 218 %, respectively . the hydrogel from teac had even lower swelling ratios at the same temperature . the cocondensation polymerization in the presence of crosslinking agents produced thermoresponsive hydrogels , but their phase transitions were broad due to the random distribution of the crosslinking agents in the dpeg chains . thus , an end - capping method was used to synthesize the gels . in this method , the dpeg chains with terminal thiol groups at the both ends were first synthesized and then reacted with a crosslinker agent triac or teac ( scheme 6 ). the dpeg chains were thus minimally disturbed to retain their thermoresponsive properties . fig1 shows the swelling ratio as a function of temperature of hydrogels made from pegda700 - det with different crosslinking agents ( table 2 ). noncrosslinked linear pegda700 - det had a lcst of 20 ° c . ( fig7 ). the hydrogels made by the end - capping method had much higher swelling ratios than those made by the copolymerization method ( fig1 ). for example , pegda - det - triac912 and pegda - det - teac had a swelling ratio about 600 % at 10 ° c . in contrast to the gradual deswelling of the hydrogels made by the copolymerization method , the hydrogels made by the end - capping method deswelled in a certain range of temperature close to the lcst of pegda700 - det even though the transition temperature region was broad compared to that of linear pegda700 - det . thus , the end - capping crosslinking produced hydrogels with improved thermoresponsive properties . the foregoing description and drawings comprise illustrative embodiments of the present inventions . the foregoing embodiments and the methods described herein may vary based on the ability , experience , and preference of those skilled in the art . merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method . the foregoing description and drawings merely explain and illustrate the invention , and the invention is not limited thereto , except insofar as the claims are so limited . those skilled in the art that have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention . 2 . satchi - fainaro , r . ; 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