Patent Application: US-12518605-A

Abstract:
the present invention relates to targeted cancer therapy , and concerns specifically the use of small matrix metalloproteinase inhibitors in improving targeting of liposomes to cancer cells , and in enhancing the uptake thereof to such cells . the invention thus provides a method for treating cancer , as well as a method for improving targeting of liposomes to tumor cells , a method for enhancing the uptake of liposomes by tumor cells , and a method for selected liposomal delivery of chemotherapeutic agents into tumor cells .

Description:
clp clpghwgfpsc ( seq id no : 7 ) ctt ctthwgftlc ( seq id no : 6 ) cwl cwltfthgtc ( seq id no : 9 ) dmem dulbecco &# 39 ; s modified eagle medium dmso dimethylsulphoxide dppe dipalmitoylphosphatidylethanolamine dpprho 1 , 2 - dihexadecanoyl - sn - glycero - 3 - phosphoethanolamino - thiocarbamoyl - n - 6 - tetramethylrhodamine ecm extracellular matrix egf epidermal growth factor eggpc egg yolk phosphatidylcholine luv large unilamellar vesicle mlv multilamellar vesicle mmp matrix metalloproteinase nbd - pe 1 - acyl - 2 -(( 7 - nitro - 2 - 1 , 3 - benzoxadiazol - 4 - yl ) amino ) dodecanoyl - 1 - sn - glycero - 3 - phospho - ethanolamine nhs n - hydroxysulfosuccinimide pa phosphatidyl acid pc phosphatidylcholine pe phosphatidyl ethanolamine peg polyethylene glycol popc 1 - palmitoyl - 2 - oleoyl - sn - glycero - 3 - phosphocholine pope 1 - palmitoyl - 2 - oleoyl - sn - glycero - 3 - phosphoethanolamine rfi relative fluorescence intensity sds sodium dodecyl sulphate stt stthwgftls ( seq id no : 8 ) timp - 2 tissue inhibitor of metalloproteinases - 2 gelatinase inhibiting peptides . three peptides were selected for this study ( table 1 ). ctt is a recently described cyclic collagenase inhibitor , which has been shown to target to tumors ( koivunen et al ., 1999 ). stt is the corresponding linear sequence of ctt in which the terminal cysteines were replaced by serines . the third peptide was ctt homolog clp , found by sequence homology search from swiss - prot and embl databases by blast 1 . 4 . 11 program . since our main interest was the conserved part of ctt , our query sequence was cxxhwgftxc ( seq id no : 2 )( koivunen et al ., 1999 ). computer search revealed nine homologs , among them the sequence of clp , which is a part of egf - 7 domain in human laminin β - 1 chain ( lmb1 ). three of the other eight homologs were laminins from other species , one was from immunoglobulin heavy chain , and four were thiazide - sensitive sodium - chloride cotransporters from human and other species . because this latter group had less apparent similarity to ctt , we focused instead on clp , the ctt - resembling sequence of the laminin egf - 7 domain . notably , all three peptides ( ctt , clp and stt ) inhibited the activity of mmp - 2 as assessed by casein zymography ( fig1 ). extent of inhibition was approx . 70 % at 85 μm ctt . also clp was a potent inhibitor of mmp - 2 , causing a 50 % inhibition at 85 μm peptide . the linear ctt analog stt ( 85 μm ) reduced the activity of mmp - 2 by approx . 30 %. similar results were also obtained with mmp - 9 . free ctt and ctt complexed with liposomes inhibited mmp - 9 also when studied by the gelatinase assay employing the fluorogenic peptide as a substrate ( fig1 b ) in keeping with the results obtained by casein zymography . the ic50 values for ctt and ctt - liposome were 8 μm in both assays . these results additionally demonstrate the mmp - 9 interacting an epitope of ctt to remain available for interaction with the enzyme when bound to the lipid membrane . complete inhibition of mmp - 9 ( 5 nm ) in this assay was obtained by 100 μm edta chelating the zn 2 + required for catalysis . interaction with phospholipid monolayers . the amino acid composition of ctt readily suggests this peptide to be hydrophobic . accordingly , it was of interest to study if ctt binds to lipids . for comparison we also investigated clp and stt . for this purpose we used phospholipid monolayers residing on an air / buffer interface , a model biomembrane which has been widely utilized to study lipid - protein interactions and the effects of proteins on the lateral organization of lipid monolayers ( söderlund et al ., 1999 ). penetration of the peptide injected underneath a lipid monolayer increases the surface pressure π , and peptides which do not insert into lipid monolayers cause no changes in surface pressure . initial surface pressure ( π 0 ) of eggpc monolayers was varied between 10 and 40 mn / m and increments in surface pressure ( δπ ) due to the addition of peptides ( final concentration 200 μm ) into the subphase were measured ( fig2 ). all three peptides readily penetrated into the monolayer films and the slopes of δπ vs . π 0 were qualitatively similar . at initial monolayer packing pressures exceeding 38 , 31 , and 33 mn / m for ctt , stt , and clp , respectively , the membrane penetration of these peptides was abolished . interactions with liposomes . the above experiments using eggpc lipid monolayers revealed ctt , clp , and stt to bind to membranes . this was confirmed by measuring trp fluorescence emission anisotropy for ctt in the presence of increasing concentrations of liposomes ( fig3 a ). accordingly , in the absence of liposomes the value for r was 0 . 065 , reflecting rapid brownian rotational diffusion of the peptide in solution . however , increasing concentrations of liposomes caused progressively increasing r , up to 0 . 349 measured at 230 μm phospholipid . approximately half - maximal effect was evident at ctt / phospholipid molar ratio close to 5 : 90 . intrinsic tryptophan fluorescence allows to estimate possible changes in the micro - environment of this fluorophore upon the association of the peptide with liposomes . the value for i 350 / i 330 measured in pbs for the trp residue of ctt is 1 . 4 , whereas in the presence of luvs ( 0 . 5 mm total phospholipid ) this ratio decreased to 1 . 00 . trp thus resides in a more hydrophobic environment in the presence of liposomes , in keeping with partitioning of ctt into lipid membrane . in addition , the quenching of trp by i − was reduced in the presence of liposomes , and reveals that trp in ctt is only partially exposed to the water soluble collisional quencher i —. the stern - volmer constants were 0 . 0087 and 0 . 0034 in absence and presence of liposomes , respectively . some lipid - binding peptides and proteins can induce fusion of lipid vesicles . in order to explore this possibility , labelled and non - labelled luvs were mixed in the absence and presence of peptides , and lipid mixing was measured as described earlier . however , neither ctt , stt , nor clp caused measurable changes in the fluorescence emission intensity of nbd - pe within 15 min , revealing lack of lipid mixing and thus also vesicle hemifusion and fusion ( data not shown ). effects on cellular uptake of liposomes . the above data show that ctt binds to phospholipids and that ctt does not cause liposome fusion . accordingly , it was of interest to investigate the possibility that ctt could be used in liposome targeting . we first approached this by including a fluorescent lipid marker dpprho , into liposomes as described in materials and methods . subsequently , ctt was added to these liposomes , whereafter they were added to u937 leukemia cells , serving here as a model of gelatinase - expressing cells . after 5 min the cells were washed and the fluorescence in cells measured by microplate reader . ctt promoted the association of the liposomes with u937 cells and approx . 3 . 7 - fold more of the fluorescent marker dpprho was bound to the cells when ctt was present ( data not shown ). we then proceeded to study the ability of ctt to enhance the uptake by u937 , cho , nrk52e , and ht 1080 cells of the water - soluble fluorescent marker , rhodamine b encapsulated into liposomes . same liposomes but without ctt were used as a control . five minutes after the liposomes with ctt were added to cells , the soluble rhodamine label could be detected in cells by fluorescence microscopy . ctt enhanced rhodamine intake by u 937 and ht 1080 cells but not by cho cells ( fig4 ). all u 937 and ht 1080 cells have fluorescence but it is distributed in an uneven manner between the cells , so some cells emit more fluorescence than others . expression of mmp - 2 as mmp - 9 is essential for liposome uptake . accordingly , chinese hamster ovary ( cho ) cells which do not express gelatinases according to our zymographic assay , showed no uptake of ctt - liposomes under conditions where an enhanced liposome uptake was evident for ht1080 and u937 cells . uptake of liposome - encapsulated rhodamine was quantitated by fluorescence plate - reader . the uptake of liposome - encapsulated rhodamine into u937 cells was enhanced 3 . 6 - fold by ctt , compared to the uptake of liposomes lacking the peptide ( fig5 a ). only minor effect was observed for stt and clp , and the signal was too weak to be statistically significant in comparison with liposomes lacking the peptides . the scrambled cyclic peptide cwl was also inefficient in promoting the liposome uptake . similar effect of ctt on liposome uptake was evident for human ht1080 fibrosarcoma and nrk52e rat kidney epithelial - like cells ( data not shown ). ctt did not promote the uptake of free rhodamine b . enhanced liposome uptake by ctt was evident only at 37 ° c . but not at 4 ° c ., thus indicating active receptor - mediated endocytosis to be involved ( fig5 b ). the effect of tpa on the uptake of ctt - liposomes is biphasic and after a short exposure time ( 15 min ) liposome intake is increased , whereas after prolonged incubation ( 75 min ) uptake of liposomes is inhibited . this could be explained by gelatinase at low concentrations remaining bound mainly to the cell surface whereas at high concentrations the excess gelatinase locates in the extracellular space ( toth et al ., 1997 ). gelatinases as targets of ctt - liposome complexes . we further investigated if cell surface - bound gelatinases were the receptors for the ctt - containing liposomes . cells were washed to remove soluble forms of gelatinases , and then preincubated for 30 min with mmp inhibitors or specific antibodies before the addition of liposomes . these experiments showed that the internalization of ctt - containing liposomes by ht1080 cells could be particularly efficiently prevented by antibodies to mmp - 9 . in particular two such mmp - 9 antibodies , when used together , completely blocked the internalization of the liposome encapsulated fluorescent dye ( fig6 ). inhibition by an antibody to mmp - 2 was only partial . an antibody against cytosolic part of integrin β1 had no effect . these results indicate a specific blockade of the liposome uptake by the anti - mmp - 9 and anti - mmp - 2 antibodies . studies with a panel of proteinase inhibitors revealed that mmp inhibitors but not serine proteinase inhibitors prevented the liposome uptake . mmp inhibitors timp - 2 and marimastat and the cation - chelator edta each had a similar effect , causing an approx . 50 % inhibition of liposome transfer into cells ( fig7 ). serum trypsin inhibitors or aprotinin had no significant inhibitory effect in cells cultured in 10 % fetal calf serum which internalized the liposomes . adriamycin , a widely used anticancer drug , has been encapsulated into liposomes with high efficiency ( gokhale et al ., 1996 ). the efficiency of ctt to promote liposome uptake and to target this therapeutic agent to tumor cells was studied in u937 culture . ctt was added to the liposome solution to a 200 μm concentration , corresponding to a ctt / phospholipid molar ratio of ˜ 1 : 2 . this solution was then added to u937 cells to yield final ctt and adriamycin concentrations of 85 μm and 0 . 4 μm , respectively . up to the concentrations used in this study the synthetic peptides and liposomes as such were not toxic to the cells ( data not shown ). liposomes without added ctt were employed as a control and cell killing was assessed by the ethd - 1 assay . after 24 h , a 4 . 1 - fold increase in killing of the cells ( p & lt ; 0 . 001 ) was observed in comparison to liposomes without ctt ( fig8 ). also gelatinase inhibiting peptides stt and clp , but not the scrambled cwl peptide , enhanced cell killing by liposome - encapsulated adriamycin , yet to a lesser degree than ctt . accordingly , stt and clp increased the number of dead cells 1 . 7 - and 1 . 3 - fold ( p & lt ; 0 . 01 ), respectively . peg - liposomes . we also made peg - liposomes , in which a peg - lipid derivative is attached to the carboxy terminal of the ctthwgftlc peptide via 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide hydrochloride ( grabarek and gergely , 1990 ). dppe , which has a carbamate linkage to peg ( 2000 ), has an amine group in one of its ends . the use of peg - lipid derivatives prolongs the in vivo circulation time of liposomes . it also anchors the peptide to the liposome surface , preventing the association of the peptide from the liposome in blood circulation . it can be used for targeting of tumor cells , tumor blood vessels , rheumatic arthritis lesions and other diseased tissues , where gelatinases are abundantly expressed . the molecular weights of the most commonly used pegs are 2 , 000 and 5 , 000 , but pegs ranging from 600 to 12 , 000 are also used . the lipid part of the conjugate can vary from saturated or unsaturated pes to cholesterol and ceramides with a short chain ( c8 ), intermediate chain ( c14 ) and long chain ( c20 ) fatty acids . on the lipid side an infinite variety of lipid anchors from pes to pas , cardiolipin , cholesterol or ceramides can be used . as a functionalized group amine is used here , but all other reactive groups , which can bind peptides or modified peptides to eg - derivatives , can also be used , such as biotin , maleimide , or carboxy - nhs - ester . ctt peptide / liposome is taken by the cells in a way that responds with adding of phorbol ester , known to stimulate gelatinase expression ( fig9 ). this shows that the gelatinase expression levels correlates with ctt - liposome intake . in our studies we have used chinese hamster ovary cells , which do not express in significant level gelatinases by our gelatin zymography assays . in our results liposome targeting did not happen in the case of cho cells . this brings us to suggest that ctt - liposome intake is cell type - dependent and correlates with gelatinase expression level of the cell type . liposome targeting experiments , which utilised liposomes having ctt peptide on the surface of the liposome and rhodamine inside of the liposome , revealed that liposome targeting is successful at 37 ° c . but not in 4 ° c . this implements that active receptor - mediated endocytosis has to take place when liposomes are internalized . ctt - liposome intake correlates with addition of phorbol ester in short induction times , which is known to stimulate gelatinase expression , and that way gelatinases , on the cell surface . when induction time prolongs , gelatinases are located also in the medium . the amount of free gelatinase in the medium rises , whereby liposome intake is inhibited ( fig1 ). in vivo experiments of liposome targeting were also carried out . fig1 a shows a gamma image of a nude mouse treated with radiolabeled ctt - peptidoliposome . ctt was labeled with technetium - 99m , which chelates between the two cysteine residues . gamma imaging was done 30 min following the injection via the tail vein . the site of the tumor ( implanted ks1767 kaposi &# 39 ; s sarcoma ) is indicated by an arrow . fig1 b shows a gamma image using ctt - peptide without liposomes , and fig1 c using liposomes without ctt - peptide , labeled with radioactive iodine - 125 - labeled bsa , encapsylated inside of the liposome . materials . egg yolk phosphatidylcholine ( eggpc ), 1 - palmitoyl - 2 - oleoyl - sn - glycero - 3 - phosphoethanolamine , ( pope ), adriamycin ( doxorubicin ), rhodamine b , and 0 . 01 m phosphate - buffered saline with 2 . 7 mm kcl and 0 . 137 m nacl , ph 7 . 4 at 25 ° c . ( pbs ) were purchased from sigrna and 1 - acyl - 2 -(( 7 - nitro - 2 - 1 , 3 - benzoxadiazol - 4 - yl ) amino )- dodecanoyl - 1 - sn - glycero - 3 - phospho - ethanolamine ( nbd - pe ) and 1 - palmitoyl - 2 - oleoyl - sn - glycero - 3 - phosphocholine ( popc ) from avanti ( birmingham , ala .). the other fluorescent lipid 1 , 2 - dihexadecanoyl - sn - glycero - 3 - phosphoethanolamino - thiocarbamoyl - n - 6 - tetramethylrhodamine ( dpprho ), and the fluorescent probe phenanthridinium , 5 , 5 ′-[ 1 , 2 - ethanediylbis ( imino - 3 , 1 - propanediyl )] bis ( 3 , 8 - diamino - 6 - phenyl )-, dichloride , dihydrochloride ( ethd - 1 ) were from molecular probes ( leiden , netherlands ). mmp - 2 was purchased from boehringer mannheim gmbh ( germany ). dulbecco &# 39 ; s modified eagle medium ( dmem ) and rpmi 1640 cell culture medium with glutamax - 1 were from gibco life technologies ( paisley , scotland ). phospholipid stock solutions were made in chloroform . the purity of lipids was checked by thin - layer chromatography on silicic acid coated plates ( merck , darmstadt , germany ) using chloroform / methanol / water ( 65 : 25 : 4 , v / v ) as a solvent . examination of the plates after iodine staining , or when appropriate , upon fluorescence illumination revealed no impurities . concentrations of the nonfluorescent phospholipids were determined gravimetrically using a high precision electrobalance ( cahn instruments , inc ., cerritos , calif ., usa ) and those of the fluorescent phospholipid analogs spectrophotometrically using the molar extinction coefficients ε = 93 , 000 at 540 nm for dpprho and ε = 21 , 000 at 463 nm for nbd - pe , with methanol as a solvent . anti - human mmp - 9 and mmp - 2 were kindly provided by dr . timo sorsa ( department of medical chemistry and periodontology , university of helsinki , finland ). marimastat was obtained from british biotech . timp - 2 and the fluorogenic substrate for mmp - 2 , mca - pro - leu - ala - nva - dpa - ala - arg from calbiochem ( la jolla , calif ., usa ). sequence homology search . ctt ( ctthwgftlc )( seq id no : 6 ) homologs were searched with blastp 1 . 4 . 11 mp using strategy ( identity matrix , word size one , and expectation value as 1000 ) recommended for small peptides by national center of bioinformation . the other parameters were kept at their default values . our query sequence was cxxhwgftxc ( seq id no : 2 ). nine analogs were found , among them was a part of the egf - 7 domain in human laminin β - 1 chain precursor clpghwgfpsc ( denoted here as clp , seq id no : 7 ). homologs to clp were also searched for and were compared with the swiss - prot sim program . synthetic peptides . peptides were synthesized on an applied biosystems 433 a ( foster city , calif ., usa ) automatic synthesizer using fmoc - chemistry . disulfide bridges were formed in 5 % acetic acid ( ph 6 . 0 ) containing 20 % dimethyl sulphoxide ( dmso ) by incubation overnight at room temperature with continuous stirring ( domingo et al ., 1995 ). after 1 : 2 dilution with 0 . 1 % trifluoroacetic acid , peptides were loaded onto a preparative reversed phase hplc column and eluted by an acetonitrile gradient . the identity of the peptides was verified by mass spectrometry . the peptides used in this study , with their amino acid sequences and respective abbreviations , are compiled in table 1 . assay for gelatinases . inhibition of mmp - 9 and mmp - 2 by the different synthetic peptides was measured using casein zymography ( halinen et al ., 1996 ). mmp - 9 was purified as described ( sorsa et al ., 1997 ). subsequently , mmp - 2 ( 2 . 5 μg ) or mmp - 9 ( 2 . 5 μg ) was run on a 10 % sds - page containing two mg / ml casein . the gel was first washed in triton x - 100 containing buffer to remove sds , and it was cut into five slices which were immersed into peptide ( ctt , clp , cwl or stt ) containing solutions ( 85 μm ). after incubation for 48 hrs at 37 ° c ., the gels were stained with coomassie blue , scanned , and the digested areas quantitated using image analysis ( global lab image 3 . 2 , data translation inc . and acuity imaging inc ., marlboro , mass ., usa ). the activity of mmp - 9 was measured also using the fluorogenic peptide substrate mca - pro - leu - ala - nva - dpa - ala - arg ( calbiochem , san diego , calif ., usa ). more specifically , 50 ng of mmp - 9 was preincubated in pbs for 30 min at rt in the absence or presence of ctt or its liposome complex . subsequently , the fluorogenic substrate was added to a final concentration of 0 . 1 mm and the incubation was continued at 37 ° c . for 15 min , whereafter fluorescence intensities were measured with exitation at 340 nm and emission at 390 nm in a microtiter plate reader . interaction of peptides with lipid monolayers . penetration of peptides into mono - molecular lipid films was measured using magnetically stirred circular wells ( subphase volume 400 μl ). surface pressure ( π ) was monitored with a wilhelmy wire attached to a microbalance ( μ troughs , kibron inc ., helsinki , finland ) connected to a pentium pc . lipids were spread on the air - buffer ( pbs , ph 7 ) interface in chloroform ( approx . one mg / ml ) to different initial surface pressures ( π 0 ) and were allowed to equilibrate for 15 min before the addition of the indicated peptides ( 4 μ1 , 10 mg / ml in h 2 o and cwl in dmso ) into the subphase . the increment in π from the initial surface pressures ( π 0 ) after the addition of peptide was complete in approximately 20 min and the difference between π 0 and the value observed after binding of the peptide into the film was taken as δπ . all measurements were performed at ambient temperature (˜+ 24 ° c .). the data are represented as δπ vs . 7π 0 ( brockman , 1999 ). preparation of liposomes . lipid stock solutions were mixed in chloroform to obtain the desired compositions . the solvent was removed under a gentle stream of nitrogen and the lipid residue was subsequently maintained under reduced pressure for at least 2 h . multilamellar liposomes were formed by hydrating the dry lipids at room temperature with one ml of pbs containing rhodamine b ( 10 μm ) or adriamycin ( 1 . 8 μm ) to encapsulate these compounds into liposomes , so as to yield a lipid concentration of one mm . multilamellar liposomes were freeze / thawed five times to enhance encapsulation ( clifford et al ., 1990 ). large unilamellar vesicles ( luvs ) were obtained by extruding the mlv dispersions 19 times through a 100 nm pore size polycarbonate membrane ( nucleapore , pleasanton , calif ., usa ) with a liposofast pneumatic gas pressure operated small - volume homogenizer ( avestin , ottawa , canada ). the pressure used for extrusion of vesicles through the filters was 25 psi (˜ 170 kpa ). when indicated , the peptides ( 2 mg / ml in pbs , except cwl in dmso ) were mixed with the luvs to yield final lipid and peptide concentrations of 1 mm and 0 . 5 mg / ml , respectively . preparation of peg - liposomes dppe , which has a carbamate linkage to peg ( 2000 ), has an amine group in one of its ends . peg - lipid derivative is attached to the carboxy terminal of the ctthwgftlc peptide via 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide hydrochloride ( grabarek and gergely , 1990 ). ctt - peg - pe is purified by gel filtration . 4 % of ctt - peg - pe is added to 16 % of pope and 80 % popc ( mol / mol ) and prepared as liposomes above . fluorescence spectroscopy . the environments of the tryptophan residues of ctt , stt , clp , and cwl in liposomes were studied by fluorescence spectroscopy . the center of trp fluorescence peak is at ˜ 350 nm when in water , whereas in a hydrophobic environment the emission is centered near 330 nm . accordingly , changes in the microenvironment of trp can be monitored by measuring i 350 / i 330 , the ratio of the emission at 350 nm to that at 330 nm ( lakowicz , 1999 ). tryptophan fluorescence was recorded with a perkin elmer ls 50 b spectrofluorometer equipped with a magnetically stirred and thermostated cuvette compartment . all measurements were done at 37 ° c . in 5 mm hepes , 0 . 1 mm edta , ph 7 . 4 buffer . excitation and emission bandpasses were 10 nm and 10 nm , respectively . excitation wavelength was 295 nm , and emission spectra were recorded in the range 300 to 400 nm . trp emission spectra of ctt , stt , clp , and cwl were recorded both in absence of and presence of popc / pope ( 80 / 20 , mol / mol ) luvs , yielding final lipid concentrations of 10 μm and 100 μm , as indicated . fluorescence polarization of the trp residue of ctt as a function of increasing concentration of liposomes was measured using rotating polarizers in the excitation and emission beams , with a bandpass of 10 nm . the results are expressed as emission anisotropy ( lakowicz , 1999 ) calculated as follows : in this equation i ⊥ and i ∥ represent the emission intensity perpendicular and parallel , respectively , to the excitation polarizer . the extent of exposure of trp to the aqueous phase was studied using i — as a collisional quencher ( lakowicz , 1999 ). the extent of quenching was calculated by using the equation : where f 0 and f are trp fluorescence intensities at 345 nm in the absence and presence of the quencher , q , τ 0 is trp fluorescence lifetime in the absence of the quencher , k is the stern - volmer constant ( obtained from the slope of the linear fit of the data ). fluorescence of trp in ctt ( 5 μm ) and its quenching were measured in the absence and presence of popc / pope ( 80 : 20 , mol / mol ) luvs ( final lipid concentration of 0 . 5 mm ). the results were corrected for background caused by pbs and liposomes . assay for lipid mixing . the ability of the ctt , clp , stt , and cwl peptides to induce liposome fusion was assessed by measuring lipid mixing , as described earlier ( struck et al ., 1981 ). in brief , nbd - pe ( x = 0 . 01 ) and dpprho ( x = 0 . 01 ) were incorporated into liposomes ( popc / pope , 78 / 20 , mol / mol ). due to the spectral overlap of nbd - pe ( donor ) emission and dpprho ( acceptor ) absorption resonance energy transfer between these dyes is highly efficient . as liposomes containing nbd - pe and dpprho are mixed with non - labeled liposomes , fusion is observed as an increase in the emission intensity of nbd - pe due to the dilution of probes . excitation and emission wavelengths for nbd - pe at 430 nm and 530 nm were used . all fluorescence measurements were done at 37 ° c . assays for liposome uptake by cells . in order to study the effects of the different peptides on the cellular uptake of liposomes , rhodamine - labelled phospholipid analog ( dpprho ) was incorporated into liposomes as a tracer , to yield a composition popc / pope / dpprho ( 80 : 19 : 1 , mol / mol ). the indicated peptides were mixed with luvs and subsequently added to cells cultured in microtiter well plates . after an incubation for 5 min at 38 ° c . liposomes not bound to the cells were removed by rinsing the cells three times with cold pbs . the relative amount of dpprho associated with the cells was determined using tecan spectrafluor plus microplate reader ( tecan , hombrechtigon , switzerland ) with excitation at 535 nm and emission at 595 nm . in another series of experiments the water - soluble fluorescent marker rhodamine b was encapsulated into liposomes and its uptake by cells was measured . samples for rhodamine b uptake were prepared by mixing 60 μl of popc / pope ( 80 / 20 , mol / mol , one mm ) liposomes containing 10 μm rhodamine b to 300 μl of dmem medium supplemented with 10 % fetal bovine serum , glutamax i , penicillin 100 u / ml and streptomycin 0 . 1 mg / ml . peptides were then added to obtain a final concentration of 8 . 5 μm . ten μl ( 105 cells ) of medium containing the indicated cells were combined with the luvs . after an incubation for 15 min at 37 ° c . or + 4 ° c . liposomes not bound to the cells were removed from 96 - well plates by rinsing three times with cold pbs . the + 4 ° c . temperature served as a control for non - endocytotic liposome uptake by cells ( oess and hildt , 2000 ). the relative amount of rhodamine b associated with the cells was determined using microplate reader with excitation at 535 nm and emission at 595 nm . we also preincubated cells with phorbol ester ( tpa ) ( 50 nm ). preincubation times varied from 15 to 75 min . treatment of cells by phorbol ester ( tpa ) increases both expression and secretion of mmp - 9 ( toth et al ., 1997 ). results are presented as rfi = i / i 0 , where rfi is relative fluorescence intensity , i 0 is the emission intensity for the control , 2 μm rhodamine b in luvs without the peptides , and i is the rhodamine emission for the peptide - liposome complexes taken up by the cells . effects of the indicated antibodies ( αmmp - 9 and αmmp - 2 ) and gelatinase inhibiting compounds ( timp - 2 , marimastat and edta ), on the uptake of rhodamine b containing liposomes ( pc / pe , 80 / 20 molar ratio ) by ht 1080 cells was performed as above . m - 17 inhibits the binding of mmp - 9 to the substrate and this inhibition is expected to be concentration dependent . it is not known if rabbit polyclonal antibody blocks human mmp - 2 ( murphy et al ., 1994 ). final concentrations of the indicated peptide , adriamycin , antibodies and liposomes were 85 μm , 0 . 2 mm , 20 μg / ml , and 0 . 4 μm , respectively . cell cultures and fluorescence microscopy . u937 ( ecacc 85011440 ), ht 1080 ( ecacc 85111505 ), and cho ( ecacc 85050302 ) cells were cultured in rpmi or dmem medium supplemented with 10 % fetal bovine serum , glutamax i , penicillin ( 100 u / ml ), and streptomycin ( 0 . 1 mg / ml ). uptake of rhodamine b containing liposomes by the cultured cells was verified by fluorescence microscopy . an inverted fluorescence microscope ( zeiss im 35 ) equipped with nikon extra long working distance objectives ( 20 ×) and ( 40 ×) were used . samples were prepared as described for the rhodamine uptake assay , with slight modifications . accordingly , instead of washing , u937 , ht 1080 , and cho cells with the rhodamine - containing liposomes and in cell culture medium were transferred into nunclon 48 plate wells . excitation and emission wavelengths were selected by suitable bandpass filters ( melles - griot ) transmitting in the range 535 nm and & gt ; 600 nm , respectively . fluorescence images were viewed with a peltier - cooled digital camera ( c4742 - 95 , hamamatsu , japan ) connected to a computer and operated by software ( hipic 5 . 0 ) provided by the instrument manufacturer . viability assays . peptide - liposome complexes with the encapsulated adriamycin were made as described above , yielding final concentrations of peptide , lipid , and adriamycin of 85 μm , 0 . 2 mm , and 0 . 4 μm , respectively , and were subsequently added to a ten μl ( 10 5 cells ) sample of u937 cells . for the detection of dead cells ethd - 1 was used . this fluorophore is membrane - impermeable and emits fluorescence only when bound to dna ( papadopoulos et al ., 1994 ). accordingly , ethd - 1 readily enters dead cells and binds to their dna , its fluorescence intensity correlating to the amount of dead cells . ethd - 1 emission was measured using microplate reader with excitation at 495 nm and emission at 635 nm . student &# 39 ; s t - test was used to assess statistical significance . in vivo liposome targeting experiment . iodination of bsa was performed by the iodogen 1 , 3 , 4 , 6 - tetrachloro - 3 , 6 - diphenylglycoluril method . briefly , 100 μl ( 10 mg / ml ) of bsa and 125 i ( 50 mbg ) were mixed in a iodogen - coated vial . to separate free iodogen pd - 10 gel column was used to filter the reaction products . the forming of liposomes popc / pope ( 80 : 20 mol / mol ) and encapsylation of iodinated bsa was made as described earlier . 99m tc labeled ctt - peptide was made by doc . sl karonen from helsinki university hospital . injected concentrations of ctt and lipids were 8 . 5 to 85 μm and 0 . 2 mm , respectively . injection was performed via the tail vein and the volume varied from 50 μl to 200 μl . the final plasma concentration of the peptide was 0 . 085 μm to 17 μm . gamma imaging was done 30 min following the injection . gamma imaging was performed with a picker prism 1500 × p single - 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