targeteddrugandgenedeliverysystemsforlungcancertherapy ·...

Cancer Therapy: Preclinical

Targeted Drug and Gene Delivery Systems for Lung Cancer Therapy

Sneha Sundaram,1,2 Ruchit Trivedi,2 Chandrasekar Durairaj,2 Rajagopal Ramesh,3

Balamurali K. Ambati,4 and Uday B. Kompella1,2

Abstract Purpose: To evaluate the efficacy of a novel docetaxel derivative of deslorelin, a lutei-

nizing hormone–releasing hormone (LHRH) agonist, and its combination in vivo withRGD peptide conjugated nanoparticles encapsulating an antiangiogenic, anti–vascular

endothelial growth factor (VEGF) intraceptor (Flt23k; RGD-Flt23k-NP) in H1299 lung can-

cer cells and/or xenografts in athymic nude BALB/c mice.

Experimental Design: The in vitro and in vivo efficacy of the deslorelin-docetaxel con-jugate was evaluated in H1299 cells and xenografts in athymic nude mice. Coadminis-

tration of deslorelin-docetaxel conjugate and RGD-Flt23k-NP was tested in vivo in mice.Tumor inhibition, apoptosis, and VEGF inhibition were estimated in each of the treat-

ment groups.

Results: The conjugate enhanced in vitro docetaxel efficacy by 13-fold in H1299 cellscompared with docetaxel at 24 hours, and this effect was inhibited following reduction

of LHRH receptor expression by an antisense oligonucleotide. Combination of the con-

jugate with the RGD-Flt23k-NP in vivo resulted in an 82- and 15-fold tumor growthinhibition on day 39 following repeated weekly i.v. injections and a single intratumoral

(i.t.) injection, respectively. These effects were significantly greater than individual tar-

geted therapies or docetaxel alone. Similarly, apoptotic indices for the combination

therapy were 14% and 10% in the i.v. and i.t. groups, respectively, and higher than

the individual therapies. Combination therapy groups exhibited greater VEGF inhibition

in both the i.v. and i.t. groups.

Conclusions: Docetaxel efficacy was enhanced by LHRH receptor–targeted deslorelin

conjugate and further improved by combination with targeted antiangiogenic nanopar-

ticle gene therapy. Combination of novel targeted therapeutic approaches described

here provides an attractive alternative to the current treatment options for lung cancer

therapy. (Clin Cancer Res 2009;15(23):7299–308)

Lung cancer is the leading cause of cancer related deaths world-wide (1). In 2009, an estimated 219,440 new lung cancer casesand 159,390 deaths are expected in the United States alone (2).Non–small cell lung cancer (NSCLC) accounts for 87% ofall cases (2, 3), with a dismal 5-year survival rate of 15% (1).Almost two thirds (70%) of patients present locally advanced

disease or metastatic disease at the time of diagnosis (1, 4),the primary treatment of choice for which is chemotherapy(5). In 2002, based on phase III clinical trial results, the U.S.Food and Drug Administration approved the use of docetaxelin combination with cisplatin in chemotherapy-naive NSCLCpatients with unresectable locally advanced or metastatic dis-ease (6). However, such as other chemotherapeutics, docetaxeldosing and efficacy are limited by its undesirable side effects,including febrile neutropenia and myelosuppression (7), neces-sitating the development of more efficacious derivatives ofdocetaxel. One potential solution to increase potency and min-imize nontarget effects is targeting docetaxel delivery to tumors.

Conjugating ligands targeting receptors specifically expressedor overexpressed on cancer cells is an attractive technique toachieve targeting of delivery systems. Deslorelin, a nonapeptideluteinizing hormone–releasing hormone (LHRH) superagonisthas a molecular weight of 1,285 Da and exerts its effectsthrough the LHRH-receptor (LHRH-R; refs. 8, 9). The LHRH-R,a member of the GPCR super family, is expressed in varioustissues, including normal lung (10), and overexpressed onlung cancers cells (11, 12). We have established that deslor-elin can be conjugated to docetaxel at the hydroxyl (-OH)group in the serine moiety (amino acid 4) through a gluta-rate linker (13). One objective of this study was to determine

Authors' Affiliations: 1Department of Pharmaceutical Sciences, University

of Colorado Denver, Aurora, Colorado; 2Department of Pharmaceutical

Sciences, University of Nebraska Medical Center, Omaha, Nebraska;3Department of Thoracic and Cardiovascular Surgery, M.D. Anderson

Cancer Center, University of Texas, Houston, Texas; and 4Moran Eye

Center, University of Utah, Salt Lake City VA Health Care System, Salt

Lake City, Utah

Received 7/6/09; revised 9/3/09; accepted 9/8/09; published OnlineFirst

11/17/09.

The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Uday B. Kompella, Department of Pharmaceutical

Sciences, University of Colorado Denver, 12700 East 19th Avenue,

Aurora, CO 80045. Phone: 303-724-4028; Fax: 303-724-4666; E-mail: uday.

[email protected].

F 2009 American Association for Cancer Research.

doi:10.1158/1078-0432.CCR-09-1745

7299 Clin Cancer Res 2009;15(23) December 1, 2009www.aacrjournals.org

Research. on April 6, 2020. © 2009 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst November 17, 2009; DOI: 10.1158/1078-0432.CCR-09-1745

http://clincancerres.aacrjournals.org/

whether the in vitro and in vivo efficacy of docetaxel can be en-hanced by its conjugation to deslorelin in a lung cancer model.

Cancer therapy benefits from a multipronged approach. Inaddition to targeted cytotoxic therapy, antiangiogenic therapiesare expected to be beneficial in lung cancer therapy (14). Thevascular endothelial growth factor (VEGF), a potent inducerof angiogenesis, is overexpressed in 61% to 92% of NSCLCpatients (15). Studies have also shown that VEGF in the tumormicroenvironment is capable of overcoming the effects of doc-etaxel, resulting in docetaxel resistance (16). Furthermore,VEGF causes impaired delivery of therapeutic agents to tumorcells (17), indicating that VEGF inhibition could improve theefficacy of anticancer agents. Indeed, the combination of beva-cizumab (an anti-VEGF antibody) with chemotherapy hasbeen shown to prolong progression-free survival, and overallsurvival in NSCLC patients (18). However, treatment with bev-acizumab is associated with serious side effects, includinghypertension, neutropenia, bleeding, thrombocytopenia, andrashes, compared with control groups (19). Hence, new tar-geted strategies are required for the inhibition of VEGF expres-sion and angiogenesis.

In this study, we investigated a targeted anti-VEGF gene ther-apy with an anti-VEGF intraceptor (Flt23k) based on nanopar-ticles (NP) with surface ligands targeting the integrin receptors.Flt23k is a recombinant construct of domains 2 and 3 of high-affinity VEGF-R1 fused with endoplasmic reticulum retentionsignal sequence [Lysine-Aspartic acid-Glutamic acid-Leucine(KDEL)] constructed in a pTracer plasmid (20). This constructsequesters VEGF by the VEGF receptor in the endoplasmic retic-ulum, inhibiting VEGF secretion and the VEGF autocrine loop(20), thereby inhibiting angiogenesis (21). Thus, gene therapywith targeted Flt23k NPs provides a potential alternative toexisting and currently tested anti-VEGF therapies. The secondobjective of this study was to determine whether the efficacyof the deslorelin-docetaxel conjugate (D-D) can be enhanced

by a combination therapy with novel integrin receptor–targetedantiangiogenic NPs encapsulating the anti-VEGF intraceptorplasmid (Flt23k-NP). A seven amino acid RGD peptide ofsequence, Gly-Arg-Gly-Asp-Ser-Pro-Lys, which binds integrinreceptors αvβ3 with high affinity and αvβ5 with low affinity(22, 23) was used as neovasculature targeting ligand on the sur-face of these Flt23k-NPs (RGD-Flt23k-NP).

This is the first report of the efficacy of D-D and a novel tar-geted anti-VEGF intraceptor plasmid–encapsulated NPs in vivoin subcutaneous H1299 cell xenograft–bearing mouse model.Further, it shows the advantage of combining chemotherapiesand gene therapies in treating cancer.

Materials and Methods

Chemicals. Deslorelin was a gift from Balance Pharmaceuticals, Inc.Docetaxel was obtained from Chempacific. H1299 cells were kindlyprovided by Dr. Pi-Wan Cheng (University of Nebraska Medical Center,Omaha, NE). Cell culture materials, reagents, and Lipofectin wereobtained from Invitrogen. Chemicals for conjugation and buffers, poly(vinyl alcohol), 3-(N-Morpholino) propanesulfonic acid, and RGDpeptide, were obtained from Sigma-Aldrich. Flt23k plasmid was a giftfrom Dr. Balamurali Ambati (Moran Eye Center, University of Utah,Salt Lake City, UT).

Synthesis of deslorelin-docetaxel conjugate. D-D was synthesized,purified, and characterized as previously described (13).

Cell lines and cell culture. NSCLC cell line, H1299, was used forin vitro and in vivo experiments. H1299 cells were maintained underincubation in a humidified incubator under 5% carbon dioxide inRPMI 1640 supplemented with 10% fetal bovine serum, 5% glutamine,and 10 mmol/L penicillin-streptomycin.

Antiproliferative effects of D-D in H1299 lung cancer cells. Antipro-liferative effects in H1299 cells were determined following a previouslydescribed method (13). H1299 cells in 96-well tissue culture plateswere treated with 1 to 250 μg/mL of docetaxel (1.25-300 nmol/L),1 to 250 μg/mL of deslorelin (0.7-200 nmol/L), and 1 to 250 μg/mLof D-D (0.19-46 nmol/L docetaxel equivalent) in serum-free mediumover 24, 48, or 72 h. Medium alone, untreated cells, and cells treatedwith approximate amounts of ethanol present in the 250 μg/mL doce-taxel group were used as controls. The plates were analyzed at 540 nmto determine efficacy.

LHRH-R expression on H1299 cells and the influence of decreasedLHRH-R expression on the efficacy of D-D. The LHRH-R expressionon H1299 cells was determined following a Western blot analysis aspreviously described (24), following pretreatment with antisense oligo-nucleotides (AON) and sense oligonucleotides (SON) against LHRH-R.Pretreatment was carried out using a previously reported (25) and val-idated (11) SON and AON against LHRH-R as previously described(11). Untreated cells were used as controls. Efficacy of D-D under de-creased receptor conditions following pretreatment with AON wascarried out following a previously described method (13).

Preparation and characterization of RGD peptide–conjugated, Flt23kplasmid–loaded PLGA NPs (RGD-Flt23k-NP). NPs were formulatedas per a previously described method using a water/oil/water doubleemulsion solvent evaporation technique (26). RGD peptide was conju-gated to NPs using a process as described previously (26). NPs werecharacterized for particle size using dynamic light scattering. Particleswere visualized using a scanning electron microscope following goldcoating.

In vivo tumor xenograft studies using H1299 cells. These studieswere done with approval and in accordance with guidelines of the In-stitutional Animal Care and Use Committee at the University of Ne-braska Medical Center. Tumor inoculation in male athymic nudeBALB/c mice (Harlan Sprague-Dawley) and tumor volume measure-ment were carried out as previously described (13).

Translational Relevance

Cytotoxic anticancer agents such as docetaxel suf-

fer from dose-limiting toxicities in treating lung can-

cer. This study advances deslorelin-docetaxel as a

novel luteinizing hormone–releasing hormone re-

ceptor–targeted cytotoxic agent for treating lung tu-

mors. Treatment of complex diseases such as lung

cancer requires multiple therapeutic modalities. In

this study, an integrin receptor–targeting nanoparti-

culate system encapsulating an anti–vascular endo-

thelial growth factor intraceptor (Flt23k) plasmid

has also been developed. Both targeted therapies

exerted superior antitumor effects compared with

their nontargeted counterparts in athymic nude mice

bearing lung tumor xenografts. Further, combining

these two targeted approaches resulted in greater

lung tumor growth inhibition. Thus, targeting lutei-

nizing hormone–releasing hormone receptors and

integrin receptors is a viable approach in enhancing

lung tumor efficacy of a cytotoxic drug and an anti-

angiogenic plasmid, respectively.

7300Clin Cancer Res 2009;15(23) December 1, 2009 www.aacrjournals.org





At 100 mm3 tumor volume, treatments with docetaxel (2.5 mg/kg),D-D (2.5 mg/kg of docetaxel equivalent), Flt23k-NP (100 μg of plasmidequivalent NPs), RGD-Flt23k-NP (100 μg of plasmid equivalent), andD-D (2.5 mg/kg of docetaxel equivalent) plus RGD-Flt23k-NP (100 μgof plasmid equivalent; as a single injection mixture) were administered.Treatments were administered as a single intratumoral (i.t.) dose(50 μL) using two 25-μL injections into two halves (halved along thelength) of the tumor xenografts or as repeated weekly i.v. doses(100 μL) through the tail vein. PBS (vehicle) was administered i.v. ascontrol. All animals were sacrificed and xenografts were isolated oncetumor volumes in vehicle-treated mice measured 1,500 mm3. Immuno-histochemical analyses were done on these xenografts.

In vivo terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling assay. Apoptotic cells in tumor tissues were detected bya terminal deoxynucleotidyl transferase-mediated dUTP nick end label-ing (TUNEL) method using the DeadEnd Colorimetric kit (Promega)according to the manufacturer's instructions. Following incubation with

the rTdT enzyme, the sections were developed with 3,3′-diaminobenzi-dine, and images were obtained using Hitachi HV-D25 digital cameraand EPIX-XCAPLite software V2.2 for windows at ×10 magnification.Apoptotic indices were calculated by counting number of TUNEL-positive cells/total number of cells in a field ×100. A total of 10 fieldswere counted in a random and blinded manner.

Immunohistochemistry for VEGF expression and CD31 plus TUNELdual staining in tumor xenografts. Immunohistochemistry for VEGFwas done using a diaminobenzidine method in formaldehyde-fixed/paraffin-embedded sections wherein antigen retrieval was carried outwith 10 mmol/L citrate buffer (pH 6.0). VEGF staining was carriedout by overnight incubation (4°C) of primary goat anti-VEGF anti-body (1:200 dilution; R&D Systems) and secondary antibody rabbitanti- goat IgG (1:200 dilution). The sections were developed with3,3′-diaminobenzidine and images were obtained using HitachiHV-D25 digital camera and EPIX-XCAPLite software V2.2 for windowsat ×10 magnification.

Fig. 1. In vitro assessment of antiproliferative effects of D-Ds in H1299 lung cancer cells. A, time course of antiproliferative effects over 24, 48, or 72 h.B, expression of LHRH-R—a Western blot analysis. Lane 2, AON-treated H1299; lane 3, SON-treated H1299; lane 4 to 6, control H1299, LNCaP, andPC-3 cells respectively. The bands at 64 kDa represent the LHRH-R, whereas those at 43 kDa represent internal protein standard, β-actin. The densitometricanalysis of the band intensities of LHRH receptor versus β-actin (internal standard) are presented as mean ± SD for three experiments. *, P < 0.05compared with all other groups. C, cells pretreated with SON and AON followed by treatment with various concentrations of deslorelin, docetaxel, and D-Dsfor 24 h. Points, mean (n = 6); bars, SD. The IC50 values were calculated using the inhibitory sigmoid Emax model. The lines were fit using this model.


Nanosystems for Lung Cancer Therapy




Dual staining for CD31 and TUNEL was carried out using an immu-nofluorescence method. CD31 staining was carried out by overnightincubation (4°C) of primary rabbit anti-CD31 antibody (1:200 dilu-tion; Abcam), followed by Texas red–labeled goat anti-rabbit IgGsecondary antibody for 2 h (1:200 dilution). TUNEL staining was car-ried out according to manufacturer's protocol (Promega). Followingincubation with the rTdT enzyme, the sections were developed withStreptavidin-FITC and confocal images were obtained with a ×100 oilimmersion lens on a Zeiss Axiovert 135 microscope equipped with aCARV optical module (“spinning disc confocal”) and a HamamatsuOrca (C4742-95) digital camera (6.7 × 6.7 μm of physical pixels).

Statistical analysis. The experiments were carried out with ann = 8 wells for MTT assays and n = 6 mice for in vivo studies. Data inall cases are expressed as mean ± SD. Comparison of mean valuesbetween different treatments was carried out using two-way ANOVAfollowed by Tukey's post hoc analysis with the SPSS (version 8) software.The level of significance was set at P < 0.05.

Results

D-D is more effective when compared with docetaxel. Antipro-liferative effects and IC50 values of D-D on H1299 cells areshown in Fig. 1A. Over periods of 24-, 48-, and 72-hour expo-sure, it is observed that D-D is significantly more cytotoxic com-pared with docetaxel alone. Further, drugs/conjugates exhibitcytotoxicities in H1299 cells in the order: D-D > docetaxel >deslorelin at all three time points tested. The IC50 values ofthe drugs based on a fit to a sigmoidal inhibitory Emax phar-macodynamic model confirm this notion. Deslorelin did nothave any cytotoxic effects on H1299 cells even at doses as highas 100 nmol/L. IC50 values could not be accurately determinedfor deslorelin because there was no measurable antiproliferativeeffect.LHRH receptor expression in H12299 cells is decreased by

pretreatment with AON against LHRH-R. The expression ofLHRH-R in H1299 cells following pretreatment with AON

against LHRH-R is shown in Fig. 1B. H1299 cells (untreated)exhibit LHRH-R expression. Pretreatment with an AON againstLHRH-R in H1299 cells caused a decrease in LHRH-R expres-sion. The level of expression was determined by a densitometricanalysis of band intensities from three separate experiments.Based on the densitometric analysis, LHRH-R expressionfollowing pretreatment exhibits the order: untreated (control)∼ SON treated > AON treated.LHRH receptor contributes to the effects of D-D. The effects of

decreased expression of LHRH-R on antiproliferative efficacyand IC50 values of D-D are shown in Fig. 1C. Pretreatment withan AON against LHRH-R significantly decreased the cytotoxicityof D-D, which was not observed in SON-treated groups. Theseresults indicate that deslorelin retains its LHRH-R interactiveability when conjugated with docetaxel. Deslorelin-treatedgroups did not exhibit any cytotoxicity in any pretreatmentgroup even at doses as high as 100 nmol/L. The IC50 valuesfor deslorelin could not be determined because there was nomeasurable antiproliferative effect.Characterization of NPs. NPs were characterized by dynamic

light scattering for three batches each of Flt23k-NP and RGD-Flt23k-NP. Flt23k-NP exhibited mean diameters of 284.92 ±24.35 nm with a polydispersity index of 0.234 ± 0.028. RGD-Flt23k-NP exhibited a mean diameter of 291.48 ± 34.58 nmwith a polydispersity index of 0.132 ± 0.023. The surface mor-phology of the NPs using scanning electron microscopy isshown in Fig. 2.Combination therapy enhances tumor growth inhibition. A

schematic of tumor inoculation, treatments, and mechanismof action is presented in Fig. 3. Treatment efficacy followingi.t. and i.v. administration are shown in Fig. 4. Animals invehicle-treated group attained a tumor volume of ∼1,509 ±9 mm3 on day 39 (Fig. 4A), at which time all tumors were mea-sured and animals in various groups were sacrificed for isola-tion and further characterization of xenografts. Mice treated

Fig. 2. Surface morphology of NPs. A and B,Flt23k-NP; C and D, RGD-Flt23k-NP. A and C wereobtained at a magnification of ×50,000, whereasB and D were obtained at a magnification of×100,000.






both i.v. (Fig. 4B) and i.t. (Fig. 4C) exhibited tumor inhibitionin the following order: D-D + RGD-Flt23k-NP > D-D ≥ RGD-Flt23k-NP > docetaxel ≥ Flt23k-NP >> PBS. Tumor growth re-duction was observed in i.t. groups until day 16 from treatmentinitiation, followed by an increase toward baseline over thenext 23 days before sacrifice. No significant changes were ob-served in body weight and morbidity was not observed, indicat-ing that all treatments were well tolerated.

Combination therapy enhances apoptosis in tumor and endothe-lial cells. The treatment efficacy to induce apoptosis in the tu-mor cells (TUNEL assay) and endothelial cells (dual stainingwith CD31 and TUNEL) is shown in Fig. 5. TUNEL-positivecells were observed in all groups. As expected vehicle (PBS)-treated mice alone exhibited lesser TUNEL-positive cells andmore necrotic regions. TUNEL-positive cells observed in variousgroups were in the following order: D-D + RGD-Flt23k-NP >

Fig. 3. A, initiation of H1299 lungadenocarcinoma xenografts in athymicnude mice for assessing in vivopharmacologic efficacy of varioustreatments. B, novel targeted drugdelivery systems used for treatments.C, novel treatments in mice bearingH1299 lung adenocarcinomaxenografts. Various treatmentsincluding docetaxel, D-D, Flt23k-NP,RGD-Flt23k-NP, and a combination ofD-D + RGD-Flt23k-NP wereadministered either as repeated weeklyi.v. injections or a single i.t. injection.D, mechanism of action of D-D andRGD-Flt23k-NP in H1299 lungadenocarcinoma xenograft–bearingmice. The delivery systems are takenup by cells through receptor-mediatedmechanisms. The NPs release theplasmid and the VEGF intraceptor istranscribed, translated, and transportedto the endoplasmic reticulum. TheVEGF intraceptor sequesters andultimately inhibits VEGF secretion.D-D is endocytosed and expected tobreakdown to its parent componentswithin cells. In the tumor cells,docetaxel leads to stabilization oftubulins in spindle fibers leading to aG2-M phase arrest, ultimately causingapoptosis.






D-D > docetaxel > RGD-Flt23k-NP > Flt23k-NP >> PBS in bothi.v. (Fig. 5A) and i.t. (Fig. 5B) groups. However, i.v. groups ex-hibited significantly higher TUNEL-positive cells comparedwith i.t. group at all times. The above trend was also observedin the apoptotic index calculated by counting the number ofTUNEL-positive cells (Fig. 5C). CD31 and TUNEL dual stainingwas assessed qualitatively in the xenograft sections (Fig. 5D).As evident, the endothelial cells exhibited apoptosis in thetreated groups, with a rank order similar to that observed inthe tumor cells.

Combination therapy enhances anti-VEGF efficacy. Anti-angiogenic effect by VEGF inhibition in various treatmentgroups are shown in Fig. 6. Immunohistochemical analysis ex-hibited VEGF inhibition in mice receiving treatments comparedwith vehicle-treated mice alone. However, groups treated withFlt23k-NP, RGD-Flt23k-NP, and D-D + RGD-Flt23k-NP exhib-ited greater VEGF inhibition compared with groups receivingdocetaxel or D-D. VEGF inhibition was in the order: D-D +RGD-Flt23k-NP > RGD-Flt23k-NP > Flt23k-NP > D-D > doce-taxel > PBS. The same trend was observed in mice treated bothi.v. (Fig. 6A) as well as i.t. (Fig. 6B). However, VEGF inhibitionwas higher in i.v. groups compared with i.t. groups.

Discussion

By using a novel D-D and RGD-conjugated Flt3k anti-VEGFintraceptor plasmid–loaded biodegradable NPs (RGD-Flt23k-NP) for lung cancer therapy (Fig. 3), we report the followingkey findings: (a) D-D, a targeted chemotherapeutic agent, exertsbetter in vitro and in vivo cytotoxic effects compared with doce-taxel alone, likely by targeting the receptors for LHRH. (b) RGD-Flt23k-NP, a targeted NP gene delivery system, improves in vivotherapeutic efficacy compared with nontargeted NPs of the anti-VEGF intraceptor plasmid. (c) Combination therapy of D-D, atargeted chemotherapeutic agent, with RGD-Flt23k-NP, a tar-geted NP antiangiogenic agent, provides enhanced antitumorefficacy compared with individual therapies or docetaxel alone.

Our antiproliferative studies showed that D-D is significantlymore cytotoxic compared with docetaxel alone after 24, 48,or 72 hours of exposure, with D-D being 10- to 13-fold morepotent compared with docetaxel. The IC50 of D-D was as low as0.8 nmol/L after 72-h exposure to H1299 cells. Deslorelinby itself did not exhibit any cytotoxic effects in H1299 cells,which is consistent with our previous studies in prostate cancercells (13).

Deslorelin, an LHRH agonist, exerts its pharmacologic effectsthrough the LHRH-R (12). Our Western blot analysis confirmsexpression of LHRH-R in H1299 similar to LNCaP and PC-3,prostate cancer cell lines, suggesting that D-D can exert its ef-fects through LHRH-R in H1299 cells. The AON againstLHRH-R significantly reduced LHRH-R expression by ∼2-foldand the potency of D-D by 3.5-fold at the end of 72-h drugexposure in H1299 cells. Thus, D-D likely exerted its effects inH1299 cells, partly through a LHRH-R–mediated uptake of theconjugate. Our prior studies indicated that deslorelin undergoescellular uptake and transport through LHRH receptors (11, 27,28). Thus, our in vitro studies suggest that lower doses of con-jugate can achieve the same effect as docetaxel alone. Alterna-tively, the conjugate at doses comparable with docetaxel clinicaldoses might achieve greater tumor reductions.

Although i.v. injections are more common clinically, i.t. in-jections are expected to localize and retain the dose better thani.v. injections. Therefore, we assessed a single i.t. injection andrepeated weekly i.v. injections in this study. Weekly i.v. admin-istrations of D-D improved tumor inhibition by ∼40% at theend of the study, compared with equivalent docetaxel therapy.Single i.t. administration (equivalent to a single, weekly i.v.dose), on the other hand, resulted in a significant ∼20% greatertumor inhibition at the end of the study, compared with equiv-alent docetaxel therapy. Thus, D-D is significantly more effectivecompared with docetaxel. This enhanced tumor reduction,

Fig. 4. In vivo efficacy assessment of various treatments in H1299 lungcancer xenografts in mice. A, vehicle control (PBS); B,weekly i.v. treatment;and C, single i.t. treatment. The mice were given docetaxel, D-D, Flt23k-NP,RGD-Flt23k-NP, or D-D + RGD-Flt23k-NP either i.v. or i.t. Control groupsreceived PBS alone i.v. Solid arrows and days indicate the days whentreatments were administered. Points, mean (n = 6 mice); bars, SD.






although interesting, warrants the use of additional approachesfor further inhibiting lung tumor growth. Therefore, we assessedthe usefulness of an antiangiogenic therapy by itself or in com-bination with D-D cytotoxic therapy.

Targeted anti-VEGF gene therapies are expected to be signifi-cantly safer compared with currently available VEGF antibodytherapies. Polymeric NPs are effective carriers of macromole-cules, protecting against macromolecule degradation in bloodand/or tissues (29). Therefore, we designed integrin receptor–targeted polymeric NPs (Flt23k-NP) encapsulating the anti-VEGF intraceptor plasmid (Flt23k; ref. 20). The RGD-Flt23k-NP

exhibited approximately 30% and 20% greater, statistically sig-nificant, tumor inhibition compared with Flt23k-NP alone inthe i.v. and i.t. groups, respectively.

Because cytotoxic and antiangiogenic approaches treat differ-ent aspects of tumor progression, a combination of these twoapproaches might provide an additive effect. Therefore, wetested a combination of D-D and RGD-Flt23k-NP in vivo. It isnoteworthy that following weekly i.v. injections, D-D + RGD-Flt23k-NP achieved 82-fold tumor inhibition compared with38-, 36-, 50-, and 46-fold growth inhibition achieved by doce-taxel, Flt23k-NP, D-D, and RGD-Flt23k-NP, respectively,

Fig. 5. In vivo assessment of apoptotic efficacy of various treatments in H1299 lung cancer xenografts in mice. Representative photomicrographs ofapoptotic cells in (A) i.v. and (B) i.t. groups using TUNEL assay in one representative section of six xenografts. C, the apoptotic index following i.v. andi.t. treatments with docetaxel, D-D, Flt23k-NP, RGD-Flt23k-NP, and D-D + RGD-Flt23k-NP. Arrows, some TUNEL-positive cells in a field. Columns, mean (n = 6);bars, SD. D, representative photomicrographs of apoptosis in proliferative endothelial cells using a dual CD31 and TUNEL staining in one representativesection of six xenografts.






relative to vehicle-treated group on day 39 after dose initiation.At this time, all i.v. treated groups maintained the tumor sizebelow the baseline (∼100 mm3) by 60% or better. On the otherhand, following a single i.t. injection, the i.t. combinationtherapy with D-D and RGD-Flt23k-NP resulted in a -6% tumorvolume compared with the baseline. These effects were superiorto i.t. docetaxel and Flt23k-NP, the nontargeted counterparts.

The TUNEL assay suggested that D-D + RGD-Flt23k-NPachieved greater apoptosis compared with Fl23k-NP andRGD-Flt23k-NP in both the i.v. and i.t. groups. Further, theD-D + RGD-Flt23k-NP coadministration achieved greater apo-ptosis compared with docetaxel and D-D in both the groups. D-D enhanced apoptosis compared with docetaxel alone in boththe groups. Fl23k-NP-treated groups also exhibited greater apo-ptosis compared with the vehicle treated groups, which in-creased further when treated with RGD-Fl23k-NP. In additionto the above measures for apoptosis in tumor tissue at theend of the study, we also assessed apoptosis in endothelial cells

of the tumor vasculature. The qualitative rank order for apopto-sis in endothelial cells was similar to what was observed abovein tumor cells, with the combination therapy exhibiting thehighest apoptotic activity. Although the above observed apo-ptotic activity of docetaxel is well known, it is interesting tonote that Flt23k plasmid, capable of inhibiting VEGF secretion(20), also resulted in apoptotic activity. There is some evidenceindicating that inhibition of VEGF leads to apoptosis (14). Bev-acizumab administered i.p. inhibits tumor growth of lung ade-nocarcinoma xenografts by ∼20% and leads to an increase inproapoptotic gene expression (30). Inhibition of VEGF is pos-tulated to inhibit tumor growth and induce apoptosis throughloss of an endothelial cell–derived paracrine factor (31, 32) ordue to a direct interaction between VEGF and the tumor cellitself (14).

In this study, all tumor xenografts exposed to Flt23k plasmidtreatments exhibited a significant VEGF inhibition at the endof the study. We also observed that VEGF inhibition was also

Fig. 6. In vivo assessment of efficacy of varioustreatment groups in inhibiting VEGF expressionin H1299 lung cancer xenograft models.Representative photomicrographs of VEGFexpression (brown stain) using a VEGFimmunohistochemistry assay in (A) i.v. and (B)i.t. groups following various treatments includingdocetaxel, D-D, Flt23k-NP, RGD-Flt23k-NP, andD-D + RGD-Flt23k-NP. These are representative ofsections in one of six xenografts.






increased in the D-D groups. The highest VEGF inhibition wasevident in the D-D + RGD-Flt23k-NP group, possibly due toVEGF inhibition by both docetaxel as well as the anti-VEGF in-traceptor. Previous studies have shown that docetaxel itself canlead to VEGF inhibition (33). Docetaxel is known to inhibitVEGF in colorectal carcinoma cells (34). However, docetaxelby itself did not inhibit VEGF in the current study, possiblydue to low drug levels at the end of the study. Docetaxel, asmall molecule, might have been cleared to a large extent bythe end of the study (day 39) after single i.t. injections. In i.v.injection groups, 5 days elapsed between last injection and thetime of sacrifice. Docetaxel concentrations present in tumor tis-sue at this time might not have been adequate to inhibit VEGF.Furthermore, docetaxel at 10 mg/kg achieves only a slight VEGFinhibition (35). We have used only 2.5 mg/kg dose, which isequivalent to the human dose, which likely is not adequate toobserve VEGF inhibition as observed with D-D. With D-D, great-er drug delivery or drug retention is anticipated. Possibly for thisreason, D-D induced apoptosis as well as VEGF inhibition.

The i.v. administration differentiated targeted systems muchbetter compared with i.t. administration. Overall, repeated i.v.administrations of treatments were able to sustain tumor vo-lumes well below baseline compared with single i.t. administra-tion of treatments. The higher efficacy following i.v. injectioncompared with i.t. injections could be attributed to the frequentdosing and 6-fold greater overall dose administered in the i.v.groups. Our studies suggest that targeted chemotherapeutic andantiangiogenic gene delivery systems are both more effectiveapproaches compared with nontargeted delivery systems for

lung cancer treatment. Further, a combination of targeted che-motherapeutic and antiangiogenic therapy is superior to indi-vidual therapies. The assessed targeting features likely improvelocalization of the treatments to the tumor compared with non-targeted systems. The relative advantage of targeted systemseven after i.t. administration suggests better uptake and/or re-tention of targeted systems at the tumor site compared withnontargeted systems.

In conclusion, conjugation with deslorelin enhances antican-cer activity of the clinically used chemotherapeutic agent doce-taxel in vitro and in vivo in lung cancer models. Further,combination of D-D with targeted anti-VEGF intraceptor genetherapy offers additive effects in inhibiting tumor growth andenhancing apoptotic index. Thus, this study advances novel tar-geted chemotherapeutic and antiangiogenic agents based onnanomaterials for cancer therapy.

Disclosure of Potential Conflicts of Interest

Patent application pending.

Acknowledgments

We thank Dr. Vidhya Rao for her critical input and assistance in prepa-

ration of this manuscript, the American Heart Association for predoctoral

fellowship award to S. Sundaram, Drs. Rakesh Singh and Seema Singh of

University of Nebraska Medical Center for their input and assistance in

immunohistochemical analyses, and Dr. Natalia Varaksa and the NCF

facility at the University of Colorado Boulder for assistance with electron

microscopy.

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2009;15:7299-7308. Published OnlineFirst November 17, 2009.Clin Cancer Res Sneha Sundaram, Ruchit Trivedi, Chandrasekar Durairaj, et al. TherapyTargeted Drug and Gene Delivery Systems for Lung Cancer

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