Codelivery of VEGF sirna and Gemcitabine Monophosphate in a Single Nanoparticle Formulation for Effective Treatment of NSCLC

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1 The Americn Society of Gene & Cell Therpy originl rticle Codelivery of VEGF sirna nd Gemcitine Monophosphte in Single Nnoprticle Formultion for Effective Tretment of NSCLC Yun Zhng 1, Nicole MJ Schwerrock 2, Arlin B Rogers 3, Willim Y Kim 4 nd Lef Hung 1 1 Division of Moleculr hrmceutics nd Center for Nnotechnology in Drug Delivery, Eshelmn School of hrmcy, University of North Crolin t Chpel Hill, Chpel Hill, North Crolin, USA; 2 Qulier Inc., Chpel Hill, North Crolin, USA; 3 Deprtment of thology nd Lortory Medicine, School of Medicine, University of North Crolin t Chpel Hill, Chpel Hill, North Crolin, USA; 4 Lineerger Comprehensive Cncer Center, University of North Crolin t Chpel Hill, Chpel Hill, North Crolin, USA There is n urgent need for new therpeutics for the tretment of ggressive nd metsttic refrctory humn non-smll cell lung cncer (NSCLC). Antingiogenesis therpy nd chemotherpy re the two mjor tretment options. Unfortuntely, oth types of therpies when used individully hve their disdvntges. Integrting ntingiogenesis therpy with chemotherpy is expected to trget the tumor s vsculr endothelil cells nd the tumor cells simultneously. In this study, we coformulted Vsculr endothelil growth fctor (VEGF) sirna trgeting VEGFs nd gemcitine monophosphte (G) into single cell-specific, trgeted lipid/ clcium/phosphte (L) nnoprticle formultion. Antitumor effect of the comintion therpy using L loded with oth VEGF sirna nd G ws evluted in oth sucutneous nd orthotopic xenogrft models of NSCLC with systemic dministrtion. The improved therpeutic response, s compred with either VEGF sirna or G therpy lone, ws supported y the oservtion of 3 4% induction of tumor cell poptosis, eightfold reduction of tumor cell prolifertion nd significnt decrese of tumor microvessel density (MVD). The comintion therpy led to drmtic inhiition of tumor growth, with little in vivo toxicity. In ddition, the current studies demonstrted the possiility of incorporting multiple nucleic cid molecules nd phosphorylted smll-molecule drugs, trgeting to different pthwys, into single nnoprticle formultion for profound therpeutic effect. Received 11 Mrch 213; ccepted 3 My 213; dvnce online puliction 18 June 213. doi:1.138/mt INTRODUCTION The comintion of chemotherpy nd gene therpy could gretly increse their therpeutic efficcy in the tretment of mny humn diseses. The drwcks ssocited with stndrd chemotherpy regimens cn e llevited y the ddition of gene therpy to the tretment pln. 1 Furthermore, the efficcy of gene therpy cn e olstered y chemo-gents whose effects re often more potent nd widespred. This increse in efficcy could e prticulrly importnt in the tretment of ggressive humn cncers whose progression nd invsion involves vriety of physiologicl or pthologicl fctors, such s non-smll cell lung cncer (NSCLC). Antingiogenesis therpy nd chemotherpy re importnt tretment regimens for NSCLC. Vsculr endothelil growth fctor (VEGF) is overexpressed in mlignnt tumors nd is mjor driver of tumor ngiogenesis. Blocking the VEGF signling pthwy cn reduce tumor-ssocited ngiogenesis nd lood vessel dependent metstsis. 2,3 VEGF-receptor (VEGFR) inhiitors, such s smll-molecule inhiitors, nti-vegf monoclonl ntiodies, nd ptmers tht strongly ntgonize the VEGF-VEGFR inding with high specificity, hve een developed. 4 6 However, the efficcy of these inhiitors is often limited y unfvorle phrmcokinetics, low tumor ccumultion, nd undesired interction with the immune system. Additionl dverse effects lso compromise the therpeutic response in ptients 7,8 sirna specific to VEGF, if properly delivered to the tumor cells, my overcome some shortcomings of the trditionl drugs, especilly when it is codelivered with n efficient chemodrug. Gemcitine (2,2 -difluoro 2 -deoxycytidine) (Gem) is nucleoside nlogue widely used s the first-line chemotherpy of dvnced NSCLC. Gem relies on nucleoside trnsporters to enter into cells, sequentilly phosphorylted y deoxycytidine kinse tht forms mono-, di-, nd triphosphte derivtives. The ddition of the first phosphte group to ecome gemcitine monophosphte (G) is the rte-limiting step Triphosphte derivtive of Gem is then incorported into the DNA strnd where it inhiits repliction y terminting the DNA chin elongtion. 9 To comine the therpeutic dvntges of VEGF sirna nd Gem, while lso voiding their delivery rodlocks, we entrpped oth VEGF sirna nd G into single lipid/clcium/phosphte (L) nnoprticle formultion. Our im ws to pply multiple tumor-killing steps to progrmmticlly inhiit tumor growth nd eventully erdicte tumor progression. The smll molecule lignd, nismide (AA), ws modified to the L surfce to specificlly trget the sigm receptors tht re overexpressed in mny humn cncer cells. The rtionl design of L nnopltform lies Correspondence: Lef Hung, 1315 Kerr Hll CB# 7571, Chpel Hill, NC , USA. E-mil: lefh@emil.unc.edu Moleculr Therpy vol. 21 no. 8, ug

2 Codelivery of sirna nd Gemcitine Monophosphte The Americn Society of Gene & Cell Therpy in the fct tht clcium ions cn precipitte oth sirna nd G. Thus, n ntingiogenic nd chemo-gents cn e simultneously delivered to the tumor cells to lock different mechnisms of tumor cell prolifertion (Figure 1). 1 Ls entrpping only VEGF sirna () nd Ls entrpping only G () were prepred nd tested seprtely to compre with the comintion therpy. Cytidine monophosphte (C), hving chemicl structure similr to G, ut without ny cytotoxic effect, serves s the surrogte for G. Ls entrpping oth C nd control sirna () were used s control nnoprticles. RESULTS Chrcteriztion of drug-loded Ls L is memrne/core type nnoprticle. It is composed of solid clcium phosphte precipitte core coted with single lipid VEGFR Tumor sites Norml tissues Vsculr endothelil cell memrne ER Anti-ngiogenesis Receptor-medited endocytosis Cytoplsm Endosome relese Gem sirna RISC Guide strnd mrna Trnsltion VEGF dck Gem mrna Nucleus Gem Incorportion Apoptosis Anti-prolifertion O 4 3 (ph = 9), G, DOA C 2+, sirna Wter-in-oil Microemulsion L core Entrpped G nd sirna Ethnol wsh Collect L core L core suspend in chloroform Ctionic lipid Evporte chloroform Outer lipids coting: DOTA, cholesterol, DSE-EG () DSE-EG Hydrtion L core: G + sirna + clcium phosphte Figure 1 Schemtic illustrtion of () the in vivo codelivery mechnism nd () the preprtion procedure of G- nd/or VEGF sirna loded L formultions. AA, nismide; ER, enhnced permeility nd retention; G, gemcitine monophosphte; L, lipid/clcium/phosphte vol. 21 no. 8 ug. 213

3 The Americn Society of Gene & Cell Therpy Codelivery of sirna nd Gemcitine Monophosphte c 1 nm 1 nm 1 nm Figure 2 TEM pictures of () VEGF-Ls, () G-Ls, nd (c) (G+VEGF)-Ls. Scle r = 1 nm. G, gemcitine monophosphte; L, lipid/clcium/phosphte; VEGF, Vsculr endothelil growth fctor. ilyer. The lipid memrne wrpping round the core is modified y grfting high density of polyethylene glycol (EG) chins, with tethered trgeting lignd AA. The preprtion scheme of L is illustrted in Figure 1. The trnsmission electron microscope (TEM) photogrphs showed tht ll drug-loded Ls hd sphericl shpe nd were monodispersed, with prticle size of round 2 nm (Figure 2). The reltively smll size of Ls renders them etter tumor penetrtion cpility over lrge size nnoprticles. 11 The zet potentils of,, nd were 3.2 ± 14.1 mv, 3.4 ± 3.1 mv, nd 1. ± 4. mv, respectively. The zet potentil of (C+Con)- L ws identicl to the zet potentil of (G+VEGF)- L. The encpsultion efficiency (EE%) of VEGF sirna nd G in L ws round 55 nd 75%, respectively. The EE% of VEGF sirna nd G in coformulted L ws lmost the sme s tht of the or single formultion, which indictes tht VEGF sirna nd G did not interfere with ech other in the process of coprecipittion with clcium ions within the L core. Drug-loded Ls induced VEGF downregultion nd poptosis in vivo Humn NSCLC H46 tumor ering mice were given three dily IV injections of different L formultions with dose of 5.4 μmol/kg G (19.5 mg/kg G, or 13.2 mg/kg in terms of Gem) nd/or.2 mg/kg VEGF sirna. Twenty-four hours fter the third injection, mice were killed nd tumor lystes were prepred for western lot, nd the RNAs in tumors were extrcted for the RT-CR mesurement of VEGF mrna levels. VEGF-VEGFR signling in the endothelil cells of tumor lood vessels cn e prevented y silencing VEGF. AR is nucler protein tht performs centrl roles in the repir of dmged DNA. Clevge of AR y cspses is considered to e hllmrk of poptosis. 12 As shown in Figure 3, showed significnt knockdown of VEGF, nd stimulted the overexpression of cleved AR. ctivted the cleved AR overexpression nd hd prtil effect on the VEGF expression level. clerly reduced the VEGF expression, ut hd limited effect on AR clevge. Compred with the control, hd no mesurle effect on the protein expression level of VEGF, nd ws not le to cleve AR. Tumor Reltive VEGF mrna level (%) VEGF: Cleved AR: β-ctin: Figure 3 Western lot nlysis nd VEGF mrna level fter systemic tretments. H46 tumor ering mice were given three dily IV injections, nd mice were killed 24 hours fter the finl injection. () Tumor lystes were prepred for western lot nlysis. () Tumor VEGF mrna levels in different tretment groups (n = 5) were mesured y RT-CR. Dt re shown s men ± SD. <.5 versus control. AA, nismide; G, gemcitine monophosphte; L, lipid/clcium/phosphte; VEGF, Vsculr endothelil growth fctor. VEGF mrna levels in different tretment groups (Figure 3) coincided with the VEGF protein expression levels in the western lot nlysis (Figure 3). Drug-loded Ls triggered cspse ctivtion nd tumor cell poptosis in vivo Cspses re proteolytic enzymes nd ply n importnt role in poptosis s effector molecules. Among the cspse enzymes, cspse-3 nd cspse-7 re especilly importnt, nd they re responsile for the proteolytic clevge of lrge numer of sustrtes during poptosis. 13 Twenty-four hours fter three dily Moleculr Therpy vol. 21 no. 8 ug

4 Codelivery of sirna nd Gemcitine Monophosphte The Americn Society of Gene & Cell Therpy Cspse-3/7 ctivity (compred with the control) Free VEGF sirna c % Apoptotic cells Figure 4 The cspse ctivtion nd induction of poptosis fter the systemic dministrtion of different Ls in H46 xenogrfts. () In vivo cspse-3/7 ctivity in tumors. <.1, versus control, versus control, versus VEGF- L, nd versus. () In vivo tumor poptosis y TUNEL ssy. (c) The percentge (%) of poptotic cells in the TUNEL ssy. <.5, versus control, versus control, versus control; <.1, versus, versus (n = 5 per group). AA, nismide; G, gemcitine monophosphte; L, lipid/ clcium/phosphte; VEGF, Vsculr endothelil growth fctor. IV injections, cspse-3/7 ctivity in H46 sucutneous tumors ws incresed threefold in mice injected with (G+VEGF)- L nd nd 1.5-fold in mice injected with, s compred with the control (Figure 4)., free VEGF sirna, nd free G displyed little cspses elevtion (Figure 4). The results indicted tht the Ls gretly improved the in vivo delivery efficiency of VEGF sirna nd G, nd the cspse ctivtion ws triggered minly y G rther thn VEGF sirna. We lso mesured the poptotic induction in H46 sucutneous tumor tissues using the TdT-medited dut Nick-End Leling (TUNEL) ssy. Twenty-four hours fter three dily IV injections, triggered drmtic killing effect, inducing 4% poptotic cells in tumors. G- L nd led to 22 nd 5% poptotic tumor cells, respectively. did not elicit tumor cell poptosis (Figure 4). The tumor cell poptotic induction of ws significntly higher thn G- L nd, indicting profound therpeutic effect of the comined L. G-loded Ls were more potent thn VEGF-loded L in terms of tumor cell killing effect. Drug-loded Ls inhiited the formtion of tumor vsculture Next, we evluted the effect of different L formultions on the formtion of tumor vsculture on H46 sucutneous tumor ering mice. We compred two tretment regimens: frequent tretment schedule nd n infrequent tretment schedule. The frequent tretment ws chrcterized y dily IV injections over 3 consecutive dys; the mice were killed 24 hours fter the finl injection. The infrequent tretment ws chrcterized y IV injections given every other dy over 8 dys, totling four injections. Mice treted with this regimen were killed 2 dys fter the finl injection. Figure 5 shows the results of stining CD31 ntigen, n endothelil cell-specific surfce mrker. tumor showed thick, elongted, nd disorgnized lyers of the vsculr endothelium. cused significnt reduction in tumor vsculture in oth frequent nd infrequent tretments, indicting n immedite nd lsting effect of tumor ngiogenesis inhiition. drmticlly shut down the tumor vsculture fter infrequent tretment, ut only displyed prtil ntingiogenesis effects with sprsely dispersed microvessels fter frequent tretments, indicting tht needs more time to effectively impir the tumor vsculture. It seems tht G codelivered in the comined Ls sensitized the tumor vsculture for ntingiogenesis therpy. Indeed, Gem cn induce poptosis in tumor-ssocited endothelil cells, leding to decrese in microvessel density (MVD). 14,15 cused prtil reduction of tumor vessel formtion fter multiple doses. nd free G hd little effect on the ltertion of the endothelil cells compred with the control. The quntittive MVD results were shown in Figure 5,d. Drug-loded Ls triggered tumor cell poptosis nd inhiited tumor cell prolifertion in vivo The H46 tumor ering mice were given IV injections of different L formultions every other dy for totl of four injections. Two dys fter the finl injection, the mice were killed nd the tumors were sectioned for TUNEL ssy, proliferting cell nucler ntigen (CNA) immunohistochemistry nd H&E stin. In the vol. 21 no. 8 ug. 213

5 The Americn Society of Gene & Cell Therpy Codelivery of sirna nd Gemcitine Monophosphte MVD c d 6 5 MVD Figure 5 (,) CD31 immunohistochemistry stining of H46 xenogrft tumors fter frequent tretment nd (c,d) infrequent tretment of different L formultions. () Sttistics for frequent tretment: <.5, <.5 versus control, <.5 versus control. (d) Sttistics for infrequent tretment: <.1, <.5 versus control, <.1 versus control. (n = 5 per group). G, gemcitine monophosphte; L, lipid/clcium/phosphte; MVD, microvessel density; VEGF, Vsculr endothelil growth fctor. TUNEL ssy (Figure 6), elicited the most effective killing effects nd triggered significnt mount (~3%) of poptotic cells in the tumor, more potent thn G- L which induced 12% poptotic cells. The results indictes ntingiogenesis-induced tumor cell strvtion my ugment the intrinsic cytotoxicity nd durtion of ntitumor effects of the coformulted G. 16 Tretment with ws less efficient thn with, only 3% of tumor cells underwent poptosis. nd free G hd limited ility to induce poptosis in tumor cells. We lso evluted the ntiprolifertion effect of different L formultions on H46 tumor ering mice. CNA is expressed in the cell nuclei during DNA synthesis nd cn e used s mrker for cell prolifertion. As shown in Figure 6c, significntly decresed the numer of CNA positive cells in H46 xenogrft tumors. nd lso cused reductions in the prolifertion of tumor cells. The ntiprolifertion effect of indictes tht VEGF not only cts s n endothelil-specific growth fctor, it cn lso promote prolifertion of tumor cells. 17 However, nd free G showed little ntiprolifertive effect. From this infrequent dosing tretment, we hd oserved dditive tumor cell killing nd ntiprolifertion effects of compred with the monotherpy of or. Drug-loded Ls inhiited mitotic figures in tumors As shown in the H&E stin (Figure 6e), fter infrequent tretment in H46 tumor ering mice, the control tumor hd mny mitotic figures, showing high mitotic ctivity of tumor cells. Some typicl mitotic figures were shown y rrows in lue. The chromosomes of the mitotic figures re visile s tngled, drk-stining threds or spots. Counting mitotic figures serves s tool for differentiting enign tumors from mlignnt ones. 18 The control tumor displyed vrious cell morphologies with drk clumped chromtin, indicting uncontrollle tumor growth nd poor differentition, otherwise known s mlignncy. Cellulr nd nucler fetures lso correlted with prolifertive ctivity, with untreted tumors exhiiting mrked nisocytosis nd nisokryosis (vrition in cell nd nucler size), polyploidy, nd open vesiculr nuclei with dispersed chromtin nd prominent nucleoli. Tumors tht were treted with (G+VEGF)- L experienced drmtic decrese in mitotic figures nd exhiited more sophilic nd uniform nuclei. However, some chromosome condenstion remined due to the cytotoxicity induced y the comined therpy. The decrese in mitotic figures supported the forementioned CNA stining, indicting the ntiprolifertion effects of prtilly results from the inhiition of cell mitosis in tumors. nd lso triggered decrese in mitotic figures compred with the control. The mitotic figures of tumors in ech group re shown quntittively in Figure 6f. Moleculr Therpy vol. 21 no. 8 ug

6 The Americn Society of Gene & Cell Therpy Codelivery of sirna nd Gemcitine Monophosphte 4 % Apoptotic cells ol A ntr Co -A -LC GM V A -A -LC F EG )-L F EG + +V e Fre GM (C (G c )-L n Co d 12 % CNA positive cells ol ntr Co VE L F)- EG G +V f -A LC )on +C e Fre GM (C (G e A -L -L GF 25 MF counts ol -A -LC V F EG A A ntr Co -A -LC GM +V (G )-L F EG )-L + n Co e Fre GM (C Figure 6,,, nd free G were dministered intrvenously every other dy for totl four injections. Two dys fter the lst injection, H46 tumor ering mice were killed nd tumor tissues were sectioned for (,) TUNEL ssy nd (c,d) CNA immunohistochemistry. (e) Mitotic figures (MF) in tumors were evluted y H&E stin. () Sttistics of the TUNEL ssy in H46 xenogrfts: <.5, versus control, versus control, versus, versus, versus ; <.1, versus control. (d) Sttistics of the CNA immunohistochemistry in H46 xenogrfts: <.5, versus control, versus control, versus VEGFL, versus ; <.1, versus control. (f) Sttistics of mitotic figures in tumors: <.1, versus control, versus control; <.1, versus control. (n = 5 per group). AA, nismide; C, Cytidine monophosphte; G, gemcitine monophosphte; L, lipid/clcium/phosphte; VEGF, Vsculr endothelil growth fctor. Tumor growth inhiition The tumor growth inhiition ws evluted in nude mice ering H46 sucutneous tumors. Mice were treted every other dy for totl of four IV injections with dose of 5.4 μmol/kg G nd/or.2 mg/kg VEGF sirna. As shown in Figure 7, 1564 exhiited the most effective tumor growth inhiition; growth ws lmost completely rrested during the comined therpeutic regimen. lso suppressed tumor growth effectively, ut not s potently s the comined nnoprticle tretment. Antingiogenic monotherpy vol. 21 no. 8 ug. 213

7 The Americn Society of Gene & Cell Therpy Codelivery of sirna nd Gemcitine Monophosphte 3, 2,5 Tumor volume (mm 3 ) 2, 1,5 1, ost injection time (dys) c d % Tumor nodules in the lung Figure 7 Tumor growth inhiition in sucutneous nd orthotopic tumor models of NSCLC. () Tumor growth inhiition effects of different L formultions on H46 tumor ering mice.,,,, nd free G were dministered intrvenously every other dy for totl four injections. Tumor volumes were mesured every other dy. Dt re men ± S.D. (n = 6 7 per group) Sttistics re s follows: <.1, versus control; φ <.1, versus control, versus control, versus ; <.5, versus. <.1, versus (C+Con)- L. () Visul oservtions of the H46 tumor sizes in ech tretment group t the end time point. (c) Representtive histopthology exmintion (H&E stining) of orthotopic lung tumors fter L tretments. (d) ercentges (%) tumor nodules in the lung in the orthotopic tumor model. <.5, <.5 versus control. (n = 6 per group). AA, nismide; C, Cytidine monophosphte; G, gemcitine monophosphte; L, lipid/clcium/phosphte; VEGF, Vsculr endothelil growth fctor. with ws not sufficient to otin long-term tumor suppression. stilized the tumor growth t erly stges of tretment, ut unfortuntely, ws ssocited with insufficient nticncer ctivity nd tumor progression t lter stges. The nd free G hd little effect on tumor growth inhiition compred with the control. At the tretment end point, representtive tumors in ech tretment group were hrvested (Figure 7) for visul comprison. Tumors treted with were smller thn tumors in other tretment groups. Thus, the comined tretment with (G+VEGF)- L ws significntly more effective thn tretment with nd individully. We further tested the comintion therpy in murine orthotopic models of humn NSCLC tht closely recpitulte the clinicl pttern nd progression of lung cncer in humns using A549 NSCLC cell lines. Incresed visile lung surfce nodules were indictive of incresed tumor urden in orthotropic tumor model. As shown in the H&E histopthology nlysis (Figure 7c), the lung of untreted mice reveled some neoplstic nodules scttering in the prenchym of the lung loe nd some tumor mss dhering to the periphery of the lung or ttched in etween the irwy. Some orthotopic tumor nodules displyed mssive nd contiguous invsion into the surrounding lveolr wll nd lung lveoli, nd necrotic zones cn e oserved in the centrl region of some tumors. Compring with the untreted mice, mice treted with nd displyed 5% reduction in size of orthotopiclly implnted lung tumors; mice treted with displyed 7% reduction in size of the orthotopic lung tumors. reduced the in vivo toxicity To test whether G nd VEGF sirna loded Ls would induce in vivo toxicity, especilly heptic nd renl dysfunction fter frequent multiple dosing, CD-1 mice were given three dily IV injections. Twenty-four hours fter the finl injection, lood smples were otined for hemtologicl nlysis nd histopthology of different orgns were evluted y H&E stin. As shown in Tle 1, within the norml rnge, induced reltively high level of lood urine nitrogen. led to higher sprtte minotrnsferse nd lnine minotrnsferse compred with the Moleculr Therpy vol. 21 no. 8 ug

8 Codelivery of sirna nd Gemcitine Monophosphte The Americn Society of Gene & Cell Therpy Tle 1 Serum levels of BUN, cretinine, AST, nd ALT fter three dily IV injections in H46 xenogrft model BUN mg/dl Cretinine mg/dl AST U/l ALT U/l 15. ± 3..4 ± ± ± ± ± ± ± 1..4 ± ± ± 15. (G+VEGF)-L- AA 15. ± ± ± 1. Reference rnge Dt re men ± SD (n = 5 per group). AA, nismide; ALT, lnine minotrnsferse; AST, sprtte minotrnsferse; BUN, lood urine nitrogen; G, gemcitine monophosphte; L, lipid/ clcium/phosphte; VEGF, Vsculr endothelil growth fctor. control. However, tretment with llevited the elevtions of lood urine nitrogen, sprtte minotrnsferse, nd lnine minotrnsferse nd kept the prmeters of kidney nd liver functions within the norml rnge. The dministrtion of chemotherpy to ptients with liver impirment my result in complicted sfety issues, tretment regimen using the nnoprticles formulted with oth G nd VEGF sirna cn help llevite the potentil liver toxicity s well s enhnce the therpeutic response, compred with tretment with single gent. From the H&E-stined tissue sections of hert, liver, spleen, lung, nd kidney (Supplementry Figure S1), there were no noticele histologicl chnges etween the control nd (G+VEGF)- L tretment group, which showed no evidence of orgn toxicity. In ddition, t the therpeutic dose, no significnt immune response ws elicited fter olus IV dministrtion, s evidenced y the production of serum cytokines, including interleukin (IL)- 6, IL-12, tumor necrosis fctor (TNF)-α, nd interferon (IFN)-γ (Figure 8). Tken together, L formultions encpsulting chemotherpeutic gents nd therpeutic sirnas show sfety nd low immunogenicity in vivo. DISCUSSION In this study, we report novel systemic delivery pltform consisting of the coformultion of nucleic cid molecules nd phosphorylted, smll-molecule drugs in single L nnoprticle. G nd VEGF sirna were coformulted using L nnotechnology to trget two therpeutic pthwys of n ggressive humn cncer mlignncy (i.e., NSCLC). Specificlly, our delivery system imed to induce poptosis though G chemotherpy nd to shut down the tumor neovsculture y locking the VEGF-VEGFR signling cscde with VEGF RNAi. hosphte groups on the molecules cn interct with clcium in microemulsion, enling the encpsultion of G nd VEGF sirna in the nnoprticle system contining clcium phosphte precipitte () core (i.e., L). A lipid ilyer surrounds the core to llow high density of DSE-EG to e grfted onto the surfce. The surfce modifiction with EG helps shield the ctionic chrge of the lipid ilyer nd my minimize the interction with circulting lood components to prolong circultion time of the prticle. The prolonged circultion hlf-life is prerequisite for enhnced tumor trgeting nd increses the proility tht the Ls will encounter the leky tumor vsculture nd pg/ml pg/ml IL-6 IL-12 TNF-α IFN-γ IL-6 IL-12 TNF-α IFN-γ Untreted Untreted cmyc-l (G+cMyc)-L Figure 8 Immunotoxicity nlysis on norml mice. Mouse serum ws collected () 4 hours nd () 24 hours fter olus IV dministrtion of different Ls t the therpeutic dose. Interleukin (IL)-6, IL-12, tumor necrosis fctor (TNF)-α, nd interferon (IFN)-γ were mesured y ELISA. Dt = men ± SD (n = 5 per group). AA, nismide; C, Cytidine monophosphte; G, gemcitine monophosphte; L, lipid/ clcium/phosphte; VEGF, Vsculr endothelil growth fctor. e exposed to the enhnced permeility nd retention effect. 19 The L s high density EG nd smll size llow it to evde the reticuloendothelil system surveillnce, which promote tumor ccumultion (Supplementry Figure S2) nd decrese toxicity in the liver nd kidney. With AA s tumor-specific trgeting lignd, Ls cn e more effectively tken up into tumor cells vi sigm-receptor medited endocytosis. In ddition, the design of the core lso promotes endosoml relese of the crgo while preventing lysosoml degrdtion of the entrpped VEGF sirna nd G. When Ls re delivered into cidic endosomes, the core of the Ls rpidly dissolves to increse the osmotic pressure in the endosome, eventully ursting the endosomes nd enling the entrpped G nd VEGF sirna to escpe (Figure 1). 2 The ctionic lipid, DOTA, surrounding the L core my lso promote the relese of the entrpped crgo y destilizing the nionic endosome memrne. 21 Loding Gem derivtives in Ls cn potentilly void drug efflux proteins (e.g., MR, BCR) nd the deficiency of cellulr uptke cused y the muttions of nucleoside trnsporters (e.g., ENT1, ENT2, CNT1, CNT3) in the cell memrne. In our previous study, we hve vlidted tht the nucleotide nlogue Gem triphosphte (GT) loded L nnoprticle showed significnt ntitumor efficcy on lung nd pncretic xenogrft models. 22 We lter found tht compring with GT which ws synthesized in n ig orgnic slt form, G (disodium slt form) gve us higher vol. 21 no. 8 ug. 213

9 The Americn Society of Gene & Cell Therpy Codelivery of sirna nd Gemcitine Monophosphte EE% nd drug loding, such tht similr ntitumor efficcy ws ttined. So, G ws used in the current study. The monophosphte modifiction on G llows it to ypss the first rte-limiting phosphoryltion step, gretly incresing the conversion rte to iologiclly ctive Gem triphosphte derivtives. On the other hnd, the downregultion of VEGF expression in tumors leds to lockge of the sequentil survivl signling initited y dimeriztion nd utophosphoryltion of VEGFR molecules. Blocking VEGF/VEGFR signling y downregulting VEGF potently inhiits errnt ngiogenesis (Figure 1). In ddition, Ls cn potentilly protect VEGF sirna nd G from enzymtic degrdtion (i.e., nuclese nd deoxycytidine deminse, respectively) nd renl clernce in vivo. This study indictes tht comining ntingiogenesis tretment with chemotherpy through systemic dministrtions of L nnoprticles contining oth VEGF sirna nd phosphorylted Gem results in n dditive ntitumor effect. The codelivered VEGF sirna tht cn dmge existing lood vessels in tumors might influence response to the concurrent chemotherpy. Comintion therpies re most likely successful ecuse dmging the estlished tumor endothelium hs een shown to increse vessel permeility nd fcilitte the delivery of susequently dministered Ls nd the coformulted G. 23 The significnt knockdown of the prongiogenic protein, VEGF, cused y the systemic dministrtions of illustrtes tht this tretment regime cn led to decresed tumor ngiogenesis (Figure 3). Furthermore, MVD is reduced through the reduction of VEGF/ VEGFR interctions nd the resulting signling cscdes, s indicted y the expression of CD31 ntigen in vsculr endothelil cells (Figure 5). These effects of modify the tumor microenvironment to potentite the delivery of the chemodrug. G entrpped in the comined L susequently inhiits the prosurvivl progrm s evidenced y elevted AR clevge nd cspse ctivtion (Figures 3 nd 4), significnt induction of tumor cell poptosis nd reduction of tumor cell prolifertion (Figures 4 nd 6). The dvntges of nnoprticles contining multiple drugs re tht they cn offer the unique fetures of vehicle uniformity, rtiometric drug loding nd temporl drug relese, while mintining the ility to unify the phrmcokinetics of different drugs they encpsulte. These nnoprticles re therey le to simultneously deliver multiple gene- nd/or chemotherpeutic gents to the trget site. 24 We hve done some preliminry work on the comprison of nd the codministered mixture of nd on the ntitumor therpeutic response. The results indicted the comined hd more potency in inducing cell-killing effects compred with the dministrtion of seprte Ls loded with ech individul gent (dt not shown). The phrmcokinetic profiles nd the therpeutic effects of the drug loding rtio re now under investigtion. The strtegy of coformulting multiple gene therpeutics nd phosphorylted chemodrugs in single vector vi L nnotechnology is expected to led to superior therpeutic improvement in mny humn diseses, nd to modulte multiple therpeutic pthwys simultneously. MATERIALS AND METHODS Mterils. G disodium slt ws synthesized y HDH hrm (Reserch Tringle rk, NC). VEGF sirna (trget sequence: 5 -ACC UCA CCA AGG CCA GCA C-3 ) nd control sirna (trget sequence: 5 U UCU CCG AAC GUG UCA CGU-3 ) were synthesized y Sigm-Aldrich (St Louis, MO). 1,2-Dioleoyl-3-trimethylmmonium-propne chloride slt (DOTA), dioleoylphosphtydic cid (DOA), nd 1,2-disteroryl-snglycero-3-phosphoethnolmine-N-[methoxy(polyethylene glycol-2) mmonium slt (DSE-EG 2 ) were purchsed from Avnti olr Lipids (Alster, AL). DSE-EG ws synthesized in our l s descried previously. 25 DedEnd Fluorometric TUNEL ssy kits nd Apo-ONE Homogeneous Cspse-3/7 ssy sustrtes were otined from romeg (Mdison, WI). Other chemicls were otined from Sigm-Aldrich. Cell culture. H46 humn NSCLC cells, originlly otined from Americn Type Culture Collection (ATCC, Mnsss, VA), were cultured in RMI-164 medium (Invitrogen, Crlsd, CA) supplemented with 1% fetl ovine serum, 1 U/ml penicillin, nd 1 μg/ml streptomycin (Invitrogen). Cells were cultivted in humidified incutor t 37 C nd 5% CO 2. Cells were hrvested with.5% trypsin-edta efore suculture. Experimentl nimls. Femle nude mice 6 8 weeks of ge were used in ll studies. To estlish the xenogrft models, H46 cells in 1 μl of phosphte-uffered sline were injected sucutneously into the right flnk of mice. Experiments were performed 11 dys fter the tumor implnttion. For the orthotopic model, nude mice were nesthetized nd.5 cm incision ws mde fter skin disinfection A549 cells in 4 μl of Mtrigel-BS medium (v/v = 1:1) were injected vi 28-guge needle out 2 cm up from the ottom of the ricge vi the left dorsl side of nude mice. The incision ws closed with surgery clmp. All nimls were mintined t surgicl plne of nesthesi during the procedure. The mice in control group were untreted mice with no injections. All work performed on nimls ws pproved y the Institutionl Animl Cre nd Use Committee t University of North Crolin t Chpel Hill. reprtion of,, nd. L cores were prepred using wter-in-oil microemulsions, with the oil phse contining cyclohexne/igepl CO-52 solution (71/29, v/v). 26 To prepre the VEGF-L cores, 48 μg VEGF sirna ws mixed with 6 μl 2.5 mol/l CCl 2 nd dded into 2 ml of oil phse, where the other emulsion contined 6 μl 12.5 mmol/l N 2 HO 4 (ph = 9.). The G-L core ws formulted using 18 μl of 6 mmol/l G mixed with 12.5 mmol/l N 2 HO 4 (ph = 9.) (finl concentrtion) to rech totl volume of 6 μl. This solution ws then dded into 2 ml of oil phse. A 6 μl 2.5 mol/l CCl 2 ws dded to seprte the 2 ml oil phse. To prepre the (G+VEGF)-L core, the phosphte phse met the sme specifictions outlined in the preprtion of the G-L core. The clcium phse contined 6 μl of 2.5 mol/l CCl 2 mixed with 48 μg of VEGF sirna. A 4 μl of 2 mmol/l DOA in chloroform ws dded to the phosphte phse of the G-L nd (G+VEGF)-L, wheres only 2 μl of 2 mmol/l DOA ws dded to the phosphte phse during the preprtion of the VEGF-L. The two seprte microemulsions were then mixed. After stirring for 5 minutes, nother 4 μl of 2 mmol/l DOA ws dded into the emulsion of G-L nd (G+VEGF)-L; for VEGF-L, 2 μl of 2 mmol/l DOA ws dded. The emulsion ws llowed to continully stir for nother 2 minutes efore 4 ml of solute ethnol ws dded. The ethnol emulsion mixture ws centrifuged t 1,g for 15 minutes to pellet the L core nd the superntnt ws then discrded. The L core ws wshed twice with solute ethnol nd dried under N 2. The L core pellets were suspended in 2 ml chloroform nd stored in glss vil t 2 C for further use. To prepre the finl,, nd with outer lipid outing, 33 μl L core in chloroform ws mixed with 38.7 μl of 1 mg/ml Cholesterol, 28 μl of Moleculr Therpy vol. 21 no. 8 ug

10 Codelivery of sirna nd Gemcitine Monophosphte The Americn Society of Gene & Cell Therpy 25 mg/ml DOTA, 76.8 μl of 25 mg/ml DSE-EG, nd 19.2 μl of 25 mg/ ml DSE-EG. After evporting the chloroform, the residul lipids were dissolved in 3 μl THF followed y 5 μl solute ethnol, nd then suspended in 16 μl wter. After rief soniction, the solution ws dilyzed in distilled wter to remove the THF nd ethnol. The preprtion procedure of ws identicl to tht of, except tht G nd VEGF sirna were replced y equl molr mount of C nd control sirna. Chrcteriztion of,, nd (G+VEGF)- L. The prticle size nd zet potentil of Ls were determined y dynmic light scttering using Mlvern ZetSizer Nno series (Westorough, MA). The EE% of G or sirna ws mesured fter lysing the Ls with THF/1 mol/l HCl (v/v = 7/3) solution. G EE% ws mesured using UV spectrophotometer (Beckmn Coulter, Bre, CA; DU 8 spectrophotometer) t wvelength of 275 nm. The EE% of sirna ws mesured y mixing smll mount of Texs-red leled sirna with VEGF sirna in L cores, nd the fluorescence intensity of Texs-red ws detected t the wvelength of Ex = 589 nm nd Em = 615 nm. TEM imges of L formultions were cquired through the use of JEOL 1CX II TEM (Tokyo, Jpn). Briefly, 4 μl of L solution ws dropped onto 3 mesh cron coted copper grid (Ted ell, Redding, CA) for 2 minutes. Excess fluid ws then removed with filter pper, nd the copper grid ws dried efore oservtion using the TEM. 27 Western lot nd RT-CR nlysis. Twenty-four hours fter the third injection, H46 tumor ering mice were killed nd tumor lystes were prepred with rdioimmunoprecipittion ssy uffer, supplemented with protese inhiitor cocktil (romeg). rotein concentrtions were determined using BCA ssy kit (ierce Biotechnology, Rockford, IL) following the mnufcturer s recommendtions. A 4 μg of protein per lne ws seprted y 4 12% SDS-AGE electrophoresis (Invitrogen) efore eing trnsferred into polyvinylidenedifluoride memrnes. The memrnes were locked for 1 hour with 5% silk milk t room temperture nd then incuted with rit polyclonl VEGF ntiody nd mouse monoclonl poly(ad-riose) polymerse-1 (AR-1) ntiody (1:5 dilution; Snt Cruz Biotechnology) overnight t 4 C. β-ctin ntiodies (1:4, dilution; Snt Cruz Biotechnology) served s the loding control. The memrnes were wshed three times nd then incuted with secondry ntiodies (1:4, dilution; Snt Cruz Biotechnology) t room temperture for 1 hour. Finlly, the memrnes were wshed four times nd developed y n enhnced chemiluminescence system ccording to the mnufcturer s instructions (Thermo Scientific, Milford, MA). RNAs in tumors were extrcted with n RNesy Mini Kit (Qigen, Vlenci, CA). The CR primers nd their fluorogenic proes for the trget genes were designed y using the computer progrm rimer Express (E Biosystems, Foster City, CA). The primer sequences for humn VEGF were: forwrd, 5 -TCC AAC ATC ACC ATG CAG ATT-3 ; reverse, 5 - GCA TTC ACA TTT GTT GTG CTG T-3. The primer sequences for humn β-ction were: forwrd, 5 -GGT CAT CAC CAT TGG CAA TG-3 ; reverse, 5 -TAG TTT CGT GGA TGC CAC AG-3. A reporter dye (FAM for the trget RNA nd TET for the β-ction control) of ech fluorescent proe ws covlently ttched t its 5 end nd quencher dye (TAMRA) ws ttched t its 3 end. The proes were purified in the olyk II crtridge (Glen Reserch, Sterling, VA) following the mnufcturer s instructions. RT-CR mplifictions were performed in 96-well plte in the ABI rism 77 sequence detector system in totl volume of 3 μl, including 1 μl RNA smple nd 2 μl of rection mixture mde y following the mnufcturer s instructions (E Biosystems). Ech RT-CR mplifiction ws performed in duplicte: 3 minutes t 48 C for the RT rection, then 1 minutes t 94 C, followed y totl of 4 temperture cycles (15 seconds t 94 C nd 1 minutes t 6 C). 28 During the mplifiction, the fluorescence of FAM (or TET), TAMRA, nd ROX ( pssive reference dye) ws mesured y the 77 sequence detector in ech well of the 96-well plte. The numers of copies of the CR templte in the strting smple were clculted y using the Sequence Detector Softwre incorported in the ABI rism 77 Sequence Detector System (Applied Biosystems, Foster City, CA). 28 Cspse ctivtion. Twenty-four hours fter three dily IV injections, 4 μg protein of ech tumor lyste ws used to detect cspse-3/7 ctivity in tumors ccording to the mnufcturer s instructions (Invitrogen, New York, NY). Briefly, 25 μl smple solution contining 4 μg of protein ws dded to 96-well plte, nd 25 μl cspse-3/7 regent ws dded to ech smple well. The contents of the wells were gently mixed t 4 rpm for t lest 1 hour t room temperture. Their fluorescence ws mesured using microplte reder t wvelength of Ex = 485 nm nd Em = 535 nm. The fluorescence intensity of tretment groups ws normlized to tht of the control group to indicte the extent of cspse ctivtion. TUNEL ssy. After predetermined dosing schedule, H46 tumor ering mice were killed nd tumors were fixed in 1% formlin for 24 hours efore emedded in prffin nd sectioned t thickness of 5 μm. The TUNEL stining ws performed s recommended y the mnufcturer (romeg). Then DAI mounting medium ws dropped on the sections for nucleus stining. Imges of TUNEL-stined tumor sections were cptured with fluorescence microscope (Nikon, Tokyo, Jpn). The percentge of poptotic cells ws otined y dividing the numer of poptotic cells (TUNEL positive cells shown s green dots) from the numer of totl cells (lue nuclei stined y DAI, dt not shown) in ech microscopic field, nd 1 representtive microscopic fields were rndomly selected in ech tretment group for this nlysis. Immunohistochemistry. rffin-emedded tumor sections were otined s mentioned ove. The CD31 (1:5 dilution, Acm, Cmridge, MA) nd CNA (1:2 dilution, Snt Cruz Biotechnology) immunohistochemistry ws performed using the HR/DAB detection IHC kit s recommended y the mnufcturer (Acm). Immunostining imges were oserved under light microscope (Nikon). The percentge of prolifertion cells ws otined y dividing the numer of CNA positive cells (shown s rown dots) from the numer of totl cells (lue nuclei stined y hemtoxylin) in ech microscopic field. MVD ws evluted y counting CD31 positive stining vessels in ech microscopic field. At lest 1 representtive microscopic fields were rndomly selected in ech tretment group for counting. Tumor growth inhiition. Tumor growth inhiition of the nnoprticles system ws evluted in n H46 sucutneous xenogrft mouse model. When the tumor volumes reched out 15 2 mm, 3 the mice were rndomly ssigned into six tretment groups (n = 6 7), nd intrvenously injected different Ls, including,,,, nd free G. The IV injections were performed every other dy for totl of four injections with dose of 5.4 μmol/kg G nd/or.2 mg/kg VEGF sirna. Tumor sizes were mesured every other dy with clipers cross their two perpendiculr dimeters, nd the tumor volume ws clculted using the following formul: V =.5 (W 2 L), where V = tumor volume, W = the smller perpendiculr dimeter nd L = the lrger perpendiculr dimeter. Two dys fter the finl injection, the mice were killed nd the tumors were stripped off. Some tumors were fixed in 1% formlin nd cut into prffin-emedded tissue sections for TUNEL ssy, immunohistochemistry nlysis nd n H&E stin. Other tumors were rrnged nd the photogrphs of tumors were tken s visul comprison of the representtive tumor sizes in ech tretment group. Four weeks fter the orthotopic lung tumor implnttion, mice were rndomly ssigned into 6 tretment groups (n = 6), nd intrvenously injected,,,, nd free G twice per week (once every 3 dys) over 3 weeks, with dose of 5.4 μmol/kg G nd/or.2 mg/kg VEGF vol. 21 no. 8 ug. 213

11 The Americn Society of Gene & Cell Therpy Codelivery of sirna nd Gemcitine Monophosphte sirna. Mice were killed 1 week fter the finl injection. Lung tissues were fixed in 1% formlin nd emedded in prffin for histologicl nlysis. In vivo toxicity. Twenty-four hours fter three dily IV injections, lood ws drwn from the venous plexus of the eyes of the mice. Blood smples were immeditely centrifuged t 3,g for 5 minutes t 4 C, nd the superntnt lood serums were collected for hemtologicl nlysis. Blood urine nitrogen, cretinine, sprtte minotrnsferse, nd lnine minotrnsferse vlues were recorded, s indictions of heptic nd renl functions. Orgns (hert, liver, spleen, lung, nd kidney) of mice in different tretment groups were fixed nd sectioned for H&E stining. Imges were collected using Nikon light microscope (Nikon). For immunotoxicity experiment, mice were given one IV injection of different L formultions, nd 24 hours lter, the cytokines in serum were determined y using enzyme-linked immunosorent ssy kits for IL-6, IL-12, TNF-α, nd IFN-γ (BD Biosciences, Sn Diego, CA). Sttisticl nlysis. Results were expressed s men ± SD. Student s t-tests were used to evlute sttisticl significnce. A result of <.5 ws considered to e sttisticlly significnt. SULEMENTARY MATERIAL Figure S1. Histopthology of different orgns evluted y H&E stin. Figure S2. Tumor uptke of NBD-leled in H46 tumor ering mice. ACKNOWLEDGMENTS Reserch ws supported y NIH grnts CA nd CA The uthors would like to thnk Chrlene M Sntos, Lei eng, nd rofessor Russell J Mumper for their kind help with the estlishment of A549 lung orthotopic mouse model. Kelly Rcette helped to edit the mnuscript. The technology of L hs een licensed to Qulier Inc. The uthors declred no conflict of interest. REFERENCES 1. 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