Method of treating cancer by using siRNA nanocomplexes

Abstract

The present invention is related to a method for treating cancer by using siRNA nanocomplex consisting of a nucleic acid molecule, a monocationic drug and a biocompatible polymer surfactant. The present nanocomplex can deliver an active ingredient (for example, a nucleic acid molecule and monocationic drug) into a cell/tissue of interest in a stable manner, and may be effectively applied for treating or detecting diverse disorders (practically, cancers).

Claims

1. A method for treating cancer comprising the steps of: administrating to a subject in need thereof a nanocomplex comprising (i) a hydrophobically associated multiple monocomplex (HMplex) formed through self-assembly of a nucleic acid molecule selected from siRNAs with 19 to 100 nucleotides and benzethonium chloride (BZT) which is a monocationic drug and (ii) polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer which encapsulates the HMplex, wherein the siRNAs inhibit the expression of a gene selected from the group consisting of Bcl-2, Bcl-3, Bcl-4, Bcl-5, Bcl-6, HER2/Neu, HER3, HER4, raf, c-fos, c-jun, c-kit, c-met, c-ret, hTERT, and erbB, wherein the nanocomplex has a hydrodynamic size of 5 nm or more and 10 nm or less.

2. The method of claim 1, wherein the HMplex, the self-assembly of the nucleic acid molecule and the BZT, is formed through electrostatic interaction.

3. The method of claim 2, wherein a charge ratio of the BZT to the nucleic acid molecule is 2 or more.

4. The method of claim 1, wherein the polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer is a pluronic poloxamer selected from the group of pluronic F-68, F-38, F-77, F-98, F108 and F-127.

5. The method of claim 1, wherein the cancer is any one cancer selected from the group consisting of brain cancer, neuroendocrine cancer, stomach cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, Laryngeal cancer, pancreatic cancer, bladder cancer, adrenal cancer, colon cancer, colon cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer or ureteral cancer.

6. The method of claim 1, wherein the administration is parenteral administration.

7. The method of claim 6, wherein the parenteral administration is any one selected from peritumoral administration, intravenous administration and intraperitoneal administration.

8. The method of claim 7, wherein the subject is a mammal.

9. A method for delivering benzethonium chloride (BZT) comprising the steps of: administrating to a subject in need thereof a nanocomplex comprising (i) a hydrophobically associated multiple monocomplex (HMplex) formed through self-assembly of a nucleic acid molecule selected from siRNAs with 19 to 100 nucleotides and benzethonium chloride (BZT) which is a monocationic drug and (ii) polyoxyethylene-polyoxypropylene-polyoxyethylene block copolymer which encapsulates the HMplex, wherein the siRNAs inhibit the expression of a gene selected from the group consisting of Bcl-2, Bcl-3, Bcl-4, Bcl-5, Bcl-6, HER2/Neu, HER3, HER4, raf, c-fos, c-jun, c-kit, c-met, c-ret, hTERT, and erbB, wherein the nanocomplex has a hydrodynamic size of 5 nm or more and 10 nm or less.

10. The method of claim 9, wherein the administration is parenteral administration.

11. The method of claim 10, wherein the parenteral administration is peritumoral administration, intravenous administration, or intraperitoneal administration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1a, 1b, 1c and 1d are schematic illustrations of ternary HMplexation (1 a) and colloidal characteristics of the resulting ternary HMplexes (1 b-1 d): (1 b) TEM image; (1 c) Hydrodynamic size (open circle) and zeta potential (solid square) measured by DLS; and (1 d) Agarose gel electrophoresis of HMplexes prepared at various charge ratios indicated (N/P=BZT ammonium/siRNA phosphate) in PBS (pH 7.4).

(2) FIG. 2 represents hydrodynamic sizes of siRNA/BZT binary complexes (without F-68) at charge ratios of 1, 2 and 5 (N/P=BZT ammonium/siRNA phosphate), measured by DLS.

(3) FIG. 3a shows an agarose gel electrophoresis of free siRNA and ternary HMplexes, both of which were incubated in 50% of serum at 37° C. for the indicated times. After 24 h serum incubation, HMplexes were decomplexed with 200 μg heparin sodium. The rightmost panel shows the released intact siRNA that was protected by ternary HMplexation against nucleases in serum. FIG. 3b is a heparin polyanion competition assay for siRNA/BZT nanocomplexes with and without micellar encapsulation by F-68. Samples were treated with various amounts of heparin sodium for 30 min at room temperature. Control (C) is free siRNA. FIG. 3c is fluorescence spectra of ternary HMplexes prepared with Cy5.5-labeled siRNA (λ.sub.ex=670 nm), with and without heparin treatment for 30 min.

(4) FIG. 4 is optical and NIRF images of MDA-MB-231 cells treated with ternary HMplexes prepared with Cy5.5-labeled siRNA (left column) or Cy5.5-labeled F-68 (right column) for 1 h and 4 h at 37° C.

(5) FIG. 5 shows fluorescence microscopic images of MDA-MB-231 cells treated for 4 h with ternary HMplexes composed of TAMRA-labeled siRNA, BZT and Cy5.5-labeled F-68.

(6) FIG. 6a represents a down-regulation of Bcl-2 gene expression in MDA-MB-231 cells analyzed by semi-quantitive RT-PCR. The Bcl-2 mRNA level was plotted after normalizing with the gene expression intensity of 1-actin. FIG. 6b is a flow cytometric analysis on apoptosis induced in MDA-MB-231 cells treated with ternary HMplexes of siRNA or scRNA. Apoptotic cells were stained with FITC-labeled annexin V. FIG. 6c is a cytotoxicity of ternary HMplexes (siRNA) against normal cells (NIH-3T3 and MRC-5) and cancer cells (MDA-MB-231), evaluated by the colorimetric MTT assay.

(7) FIG. 7a is in vivo NIRF images showing tumor accumulation of ternary HMplexes prepared with Cy5.5-labeled siRNA that were injected peritumorally to MDA-MB-231 xenograft mice (λ.sub.ex=675 nm, λ.sub.em=720 nm). FIG. 7b shows low (×200) and high (×1000) magnification histological micrographs of tumor sections resected at 4 d after peritumoral injection of free Cy5.5-labeled siRNA or HMplexes made of Cy5.5-siRNA. The fluorescence signals of Cy5.5-siRNA and DAPI-stained nuclei are presented in red and blue, respectively.

(8) FIG. 8 is in vivo NIRF images showing tumor accumulation of free Cy5.5-labeled siRNA that was injected peritumorally to MDA-MB-231 xenograft mice (λ.sub.ex=675 nm, λ.sub.em=720 nm).

(9) FIGS. 9a and 9b are results representing tumor growth suppression (9a) and body weight changes (9b) for 30 d in MDA-MB-231 xenografts mice peritumorally injected with Bcl-2 targeting or nontargeting HMplexes (HMplex/siRNA or HMplex/scRNA) or untreated (control).

DETAILED DESCRIPTION OF THIS INVENTION

(10) The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.

(11) Examples Experimental Materials and Methods Experimental Materials

(12) All chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA), and used without further purification. Lipofectamine 2000 and SYBR® Gold were purchased from Invitrogen (Carlsbad, Calif., USA) and used according to the manufacturer's instructions. Custom Bcl-2 siRNA (sense 5′-GUACAUCCAUUAUAAGCUG (SEQ ID NO: 1)-dTdT; antisense CAGCUUAUAAUGGAUGUAC (SEQ ID NO: 2)-dTdT), scrambled siRNA (scRNA, AccuTarget™ Negative Control siRNA), Cy5.5- or TAMRA-labeled siRNA end PCR premix (AccuPower® PCR PreMix) were available from Bioneer (Daejeon, Korea). For near-infrared fluorescence (NIRF) labeling of F-68, vinylsulfone-functionalized Cy5.5 (Cy5.5-VS, BioActs Co. Ltd., Korea) was conjugated to the hydroxyl group of F-68 by mixing F-68 (100 mg) and Cy5.5-VS (0.5 mg) in PBS (pH 8.0) for 2 h at room temperature. The Cy5.5-labeled F-68 was dialyzed against Milli-Q water in a cellulose ester membrane (Spectra/Por Membrane®, MWCO: 3.5 kDa) for 1 d and then lyophilized.

(13) Preparation and Characterization of HMplex

(14) HMplexes were prepared by mixing 9.3 μg of siRNA (or scRNA) and varying amounts of BZT (23.8-476.8 μg) in the presence or absence of Pluronic F-68 (2.5 mg) in 500 μL PBS (pH 7.4) for 3 h at room temperature, which resulted in various charge ratios (N/P=BZT ammonium/siRNA phosphate=1-20). For fluorescence labelling of the HMplexes, Cy5.5-labeled F-68 or Cy5.5-labeled siRNA was used for complexation. Transmission electron microscopic (TEM) images were recorded with a CM30 electron microscope (FEI/Philips) operated at 200 kV. For the TEM sample preparation, a drop of sample dispersion was dried on a 200 mesh copper grid coated with carbon and negatively stained with a 2 wt % uranyl acetate solution. The hydrodynamic size and zeta potential of HMplexes were determined using a zeta-sizer (Nano-ZS, Malvern, UK). For gel retardation assay, HMplexes were loaded onto 2% agarose gel and electrophoresis was performed in TBE buffer solution at 100 V. After electrophoresis, the SYBR Gold-stained siRNA bands were visualized using a gel documentation system (MiniBis Pro, DNR Bio-Imaging Systems, Israel). All the in vitro and in vivo experiments were performed with HMplexes at a fixed charge ratio (NIP=4).

(15) Cell Culture

(16) Cell culture media, antibiotics, fetal bovine serum (FBS) and bovine calf serum (BCS) were purchased from Welgene Inc. (Korea). NIH3T3 (a mouse embryonic fibroblast cell line), MRC-5 (a human fetal lung fibroblast cells) and human TNBC cells (MDA-MB-231) were cultured according to the manufacturer's specifications. MRC-5 was maintained in DMEM (Dulbecco's modified eagle medium) with 10% FBS, L-glutamine (5×10.sup.−3 M) and gentamicin (5 μg/mL), in a humidified 5% CO.sub.2 incubator at 37° C. MDA-MB-231 and NIH-3T3 were maintained in RPMI 1640 medium supplemented with 10% serum (FBS for MDA-MB-231 or BCS for NIH-3T3), L-glutamine (5×10.sup.−3 M) and gentamicin (5 μg/mL) in a humidified 5% CO.sub.2 incubator at 37° C.

(17) Evaluation of In Vitro Cytotoxicity and Stability

(18) In vitro cytotoxicity studies of HMplexes were performed against normal cells (MRC-5 and NIH-3T3) and cancer cells (MDA-MB-231) using the colorimetric MTT assay. Cells were treated with HMplexes at various concentrations for 4 h and then incubated in a serum-free medium for another 44 h after washing twice with cold PBS (pH 7.4). After washing with PBS, the cells were treated with MTT reagents and the absorbance at 540 nm was measured with a microplate reader (Spectra Max 340, Molecular Devices, Sunnyvale, Calif., USA). The complex stability of HMplexes was evaluated by a heparin polyanion competition assay and a serum stability assay. For the heparin polyanion competition assay, HMplexes containing 1 μg siRNA were incubated with varying amounts of heparin (0-200 μg) for 30 min at room temperature and then subjected to 2% agarose gel electrophoresis. The decomplexed siRNA was detected with SYBR Gold staining. For serum stability studies, HMplexes or naked siRNA were incubated in 50% FBS/PBS (pH 7.4) at 37° C. for predetermined time periods (0-24 h). Aliquots from each sample were analyzed by 2% agarose gel electrophoresis and visualized with SYBR Gold.

(19) In Vitro Cellular Uptake and Evaluation of Apoptosis

(20) MDA-MB-231 cells were seeded onto 35 mm coverglass bottom dishes (2×10.sup.5 cells). At a confluence of 70-80%, cells were treated with free Cy5.5-siRNA or HMplexes of Cy5.5-siRNA for 1 h or 4 h in a serum-free medium. Afterward, cells were washed twice with cold PBS (pH 7.4) and fixed with 4% (v/v) paraformaldehyde. Fluorescence images were taken using a LEICA DMI3000B equipped with a Nuance FX multispectral imaging system (CRI, USA). For determination of apoptosis, MDA-MB-231 cells were seeded on 6-well culture plates (2×10.sup.5 cells) and incubated for 12 h. After treatment with HMplexes of siRNA or scRNA for 2 h in a serum-free medium, the cells were washed twice with cold PBS (pH 7.4) and incubated for another 44 h. Then the cells were washed twice with cold PBS (pH 7.4) and collected by centrifugation. Annexin V-FITC (BioVision, Inc., USA) was added into the cells for selectively staining apoptotic cells following a literature procedure [8]. Green fluorescence intensity of the stained apoptotic cells was analyzed on flow cytometry (Guava easyCyte™ Flow Cytometers, EMD Millipore, USA).

(21) Evaluation of Bcl-2 Down-Regulation

(22) The in vitro gene silencing by HMplexes was assessed using semi-quantitative reverse transcription-PCR (RT-PCR). MDA-MB-231 cells were seeded on 6-well culture plates (2×10.sup.5 cells) and incubated for 12 h. At a confluence of 70-80%, HMplexes of siRNA or scRNA were added into the cells at a final RNA concentration of 140 nM. After incubation of 2 h in a serum-free medium, the cells were washed twice with cold PBS (pH 7.4) and incubated for another 44 h. Then the cells were collected by centrifugation and total RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Down-regulation of Bcl-2 mRNA was determined by normalizing Bcl-2 expression levels to the endogenous actin levels under the following conditions of semi-quantitative RT-PCR in PCR premix by using Veriti® 96-Well Thermal Cycler (Applied Biosystems, Foster City, Calif.): a total of 25 cycles consisting of 94° C. for 60 s, 50° C. for 60 s, and 72° C. for 60 s, with a final extension step for 10 min at 72° C. Results were presented as average of at least 3 independent experiments. Custom primers for Bcl-2 and β-actin were as follows.

(23) TABLE-US-00001 1) Bc1-2, sense 5′-CGACGACTTCTCCCGCCGCTACCGC-3′, and antisense 5′-CCGCATGCTGGGGCCGTACAGTTCC-3′; and 2) β-actin, sense 5′-GCTCGTCGTCGACAACGGCTC-3′, and antisense 5′-CAAACATGATCTGGGTCATCTTCTC-3′.

(24) In Vivo Studies on Stability, Peritumoral Delivery and Tumor Growth Inhibition

(25) Animal experiments were performed according to the guidelines established by Korea Institute of Science and Technology (KIST). For animal experiments, Crlj nude mice (5-week-old female; Orient Bio Inc., Korea) were anaesthetized with intraperitoneal injection of 0.5% pentobarbital sodium (0.010 mug). For tumor xenograft model, MDA-MB-231 cells (1.0×10.sup.7 cells) were inoculated into the flank of mice by subcutaneous injection. For in vivo stability test, HMplexes (60 μL, 140 nM siRNA) were injected into a tumor xenograft mouse model by peritumoral route (n=3/group). In vivo fluorescence images were taken at predetermined time points by an IVIS Spectrum imaging system (Caliper, USA). To examine the intratumoral distribution of the peritumorally applied HMplexes, tumors were excised from the mice, fixed in neutral buffered formalin and embedded in paraffin. The tissue blocks were cut into 5 μm sections and fluorescence images were taken by a LEICA DMI3000B equipped with a Nuance FX multispectral imaging system (CRI, USA). Tumor growth inhibition was observed by measuring the tumor volume. When the tumor volume reached approximately 50 mm.sup.3, HMplexes (6 mg/kg bodyweight) were administered locally by peritumoral route (day 1) and the local administration was repeated on day 4 and 6. Tumor volume and weight were recorded over a period of 4 weeks (n=3/group).

(26) Results

(27) Design of siRNA/Drug HMplex Formulation

(28) The HMplex-based siRNA/drug co-delivery formulation has been designed as a self-assembled ternary complex composed of three active components (FIG. 1a). As a monocationic surfactant, we selected benzethonium chloride (BZT) that has been reported to induce cancer-specific apoptosis by activating caspase-2, caspase-8, caspase-9 and caspase-3. Herein, BZT plays dual functions, i.e., acts not only as a surfactant for the HMplex formation but also as an anticancer agent by itself. Hence, it can get rid of the necessity of using separate anticancer agents and cytotoxic monocationic surfactants, which simplifies the co-delivery constitution as well as minimizes its nonspecific cytotoxicity. For genetic chemosensitization, BZT was complexed with Bcl-2 targeting siRNA. Silencing the Bcl-2 gene has been recognized as a promising target for chemosensitization because the anti-apoptotic Bcl-2 protein is overexpressed in 50-70% of all human cancers and makes them resistant to the treatment by chemodrugs and radiation [9]. Therefore, the combination of BZT with Bcl-2 siRNA would present a synergistic effect of treatment by sensitizing resistant cancer cells to the anticancer action of BZT. Finally, the organic-soluble binary HMplex formed between Bcl-2 siRNA and BZT was further formulated into the ternary HMplex by encapsulating with micelles of a biocompatible polymeric surfactant, Pluronic F-68 (U.S. FDA approved as a local/i.v. injectable pharmaceutical ingredient), which would improve the complex stability as well as the efficiencies of cell penetration and in vivo delivery.

(29) The colloidal size is an important parameter that governs the tumor-targeting efficiency of nanotherapeutics. Tumor tissues have a special microenvironment composed of dense interstitial matrix where interstitial hypertension hinders passive diffusion of nanoparticles, restricting deep penetration into the tumor. Recent studies revealed that smaller nanoparticles show higher rates of tumor permeation. In this regard, further micellar formulation of the HMplex with F-68 allows us to miniaturize the ternary complex to increase the intratumoral diffusion and penetration efficiency.

(30) Formation and Characteristics of Ternary HMplexes

(31) When water-soluble siRNA and BZT were mixed in water, water-insoluble binary complexes formed spontaneously in a form of big nanoaggregates with a hydrodynamic size of 200-500 nm (FIG. 2). In the presence of pre-dissolved Pluronic F-68, however, the mixing of siRNA and BZT produced much smaller colloids with no big aggregates, as designed for the colloidal size minimization. As sketched in FIG. 1a, the HMplexation is composed of two sequential processes: 1) multiple monocomplexation-induced hydrophobic association between the oppositely charged counterparts into big nanoaggregates (binary complexation); and 2) micellar dissolution and encapsulation of the binary complexes into the surfactant-passivated tiny nanoparticles (ternary complexation). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) studies manifested that the resulting ternary HMplex was indeed greatly miniaturized by micellar encapsulation (smaller than 10 nm; FIGS. 1b and 1c), being suitable for facile penetration into the dense tumor tissue.

(32) With increasing charge ratio (N/P=BZT ammonium/siRNA phosphate; increasing BZT while keeping the siRNA amount constant) in the presence of F-68, the zeta potential of the ternary HMplex was gradually inverted from negative to positive values with a slight increase in the hydrodynamic size (FIG. 1c). The gradual changes in both the surface charge and size reached saturation at above a charge ratio of 5 or more, suggesting that the ternary HMplexation is efficient and can be completed at a fairly low N/P ratio. The efficient HMplex formation was confirmed by the gel retardation assay with varying charge ratio (FIG. 1d). At charge ratios of 2 or higher, uncomplexed free siRNA was completely absent, demonstrating that all the siRNA chains were HMplexed by excess BZT. Owing to the charge reversal and size increase by HMplexation, the migration of siRNA through the gel was greatly retarded under the applied electric field, with the HMplex bands remaining at the loading slots. Importantly, the retarded HMplex bands showed no staining with an intercalating dye (SYBR® Gold), as generally observed in strongly interacting DNA/surfactant complexes. This suggests that the cationic intercalating dye is not accessible to the HMplexed siRNA because siRNA chains are tightly packed and passivated within the HMplex structure through the strong collective interactions with BZT and F-68.

(33) Protection of siRNA by Ternary HMplexation

(34) The susceptibility of siRNA toward degradation in nuclease-rich serum was compared between the ternary HMplex (N/P=4) and free siRNA by incubating them in 50% serum media at 37° C. At each predetermined time point, an aliquot was sampled and analyzed by gel electrophoresis. As shown in FIG. 3a, free siRNA was gradually degraded with time in serum and completely disappeared at 24 h. In contrast, the HMplexed siRNA presented improved stability with no notable sign of degradation or decomplexation into free siRNA during serum incubation. Upon induced decomplexation by heparin treatment, intact free siRNA was released from the serum-treated HMplexes even after 24 h treatment, confirming the effective protection of siRNA by HMplexation.

(35) To evaluate the micellar shielding effect on the complex stability, HMplexes (NIP=4) with and without F-68 encapsulation were treated with varying concentrations of heparin sodium and the induced decomplexation by competition between siRNA and heparin was monitored by gel electrophoresis (FIG. 3b). It was shown that the binary siRNA/BZT HMplex without F-68 began to release decomplexed siRNA even at a low concentration of heparin (10 μg). In sharp contrast, the ternary HMplex with F-68 passivation disassembled only at much higher heparin concentrations, unambiguously attributed to the micellar shielding effect which blocks the competition between the complexed siRNA and external polyanions.

(36) The decomplexation feature was further examined by using Cy5.5-labeled siRNA in HMplexation. As shown in FIG. 3c, the intense near-infrared fluorescence (NIRF) of free Cy5.5-siRNA was totally quenched by ternary HMplexation. When heparin was added to the nonfluorescent HMplex, the original fluorescence signal of Cy5.5-siRNA (an indicative factor of siRNA release depending on the formation of HMplexation) was recovered depending on the heparin amount, indicative of the siRNA release by decomplexation of HMplexes. The initial formation of nonfluorescent HMplex is attributed to the self-quenching of fluorescence typical of common organic dyes in the aggregated state, clearly evidencing that the multiple monocomplexed Cy5.5-siRNA/BZT chains were closely aggregated by strong hydrophobic association with each other to construct the compact and stable HMplex nanostructure.

(37) Intracellular Uptake of HMplexes

(38) Cell internalization of HMplexes was examined with TNBC cells (MDA-MB-231) by using Cy5.5-labeled ternary complexes (N/P=4). Cy5.5 was labeled to either siRNA or F-68 to prepare Cy5.5-siRNA/BZT/F-68 or siRNA/BZT/Cy5.5-F-68 for NIRF tracking of siRNA or F-68, respectively (FIG. 4). At an early stage of cell treatment (1 h), the F-68 signals were mostly localized near the cell membrane whereas the siRNA fluorescence was not dearly observed due to the low intensity. As discussed in FIG. 3c, the quenched fluorescence of siRNA suggests that the densely complexed HMplex preserved its structural integrity at an early stage of cell internalization. After 4 h treatment, however, the siRNA fluorescence was recovered and diffusely seen in the cytoplasm. The distribution of F-68 signals was also shifted to the similar cytoplasmic region, being seemingly colocalized with siRNA (FIG. 5). It is highly probable that the transfected HMplex was decomplexed in the cytoplasmic environment and thereby free siRNA and BZT were released from the dense complex to regain the fluorescence intensity of free Cy5.5-siRNA.

(39) Bcl-2 Gene Silencing and Sensitization to Apoptosis

(40) Human TNBC cells (MDA-MB-231) are known to overexpress Bcl-2 proteins which inhibit the apoptosis pathway and develop resistance to treatment. As demonstrated in FIG. 4, ternary HMplexes are capable of transfection and release of Bcl-2 targeting siRNA into the cytoplasmic area of TNBC cells, which are key requirements for the successful RNA interference by siRNA delivery [1-4]. Moreover, it is reasonably speculated that the co-complexed BZT is concomitantly released intracellularly to trigger apoptosis. Hence, we explored potential of the ternary HMplex (N/P=4) for siRNA-mediated Bcl-2 gene silencing and thereby chemosensitization of TNBC cells to apoptosis induction by the co-delivered BZT. For this, TNBC cells were treated with siRNA complexes for 2 h and incubated for another 44 h after washing the samples. FIG. 6a displays the result of Bcl-2 down-regulation in TNBC cells depending on the siRNA formulation. HMplexes of non-targeting scrambled siRNA (scRNA) showed a minute silencing effect on Bcl-2 expression (about 80% with respect to the untreated control level), which is possibly due to the cytotoxicity of the co-delivered BZT against TNBC cells. With genetic targeting by HMplexes of Bcl-2 siRNA, TNBC cells showed a remarkably reduced level of Bcl-2 expression down to about 20%, which is even more efficient than a standard siRNA delivery system (Lipofectamine).

(41) The sensitized induction of apoptosis by siRNA/BZT co-delivery was evaluated with MDA-MB 231 cells by flow cytometry (FIG. 6b). HMplexes of non-targeting scRNA (HMplex/scRNA) presented a minute increase in the population of apoptotic cells, again reflecting the cancer-specific toxicity of BZT. Importantly, the level of apoptosis was greatly increased by treatment with HMplexes of Bcl-2 targeting siRNA. This result confirms that Bcl-2 gene targeting by the delivered siRNA remarkably sensitized the resistant cancer cells to anticancer effect of the co-delivered BZT, validating our HMplex-based design strategy for the synergistically enhanced combination therapy. It is noteworthy that the HMplex between Bcl-2 siRNA and BZT manifested a significant anticancer effect on TNBC cells (MDA-MB-231) with no notable nonspecific toxicity against normal cells (NIH-3T3 and MRC-5), which makes it a potential therapeutic formulation candidate with clinical utility (FIG. 6c).

(42) In Vivo Tumor Delivery by Peritumoral Injection

(43) In vivo therapeutic utility of the co-delivering HMplex was explored with human triple-negative breast tumor (MDA-MB-231) xenografts in mice. HMplexes were administered by peritumoral route to examine their intratumoral penetration and accumulation. Peritumoral administration route was chosen because it offers benefits for treating localized tumors by adjuvant chemotherapy prior to or after a local treatment such as surgery. In general, systemic administration of therapeutic nanoparticles often causes side effects and requires high dosage due to a variety of reasons such as filtration loss by the reticuloendothelial system (RES) or limited systemic blood flow directed to the tumor. Direct intratumoral injection is the most common route for local delivery of chemodrugs but has limitation such as drug effusion from the injection area of tumor. Peritumoral injection sometimes showed better anticancer effects than intratumoral route, and can offer an opportunity to treat undetected cancer cells hidden in the surgical margin. However, peritumoral application still demands better nanoparticles with higher tumor penetration that can overcome physical barriers at the periphery of solid tumors. In this respect, we applied our HMplex to peritumoral administration for locoregional chemotherapy because its small colloidal size and Pluronic-coated surface would facilitate deep penetration into the tumor by helping to cross the peripheral barriers.

(44) FIG. 7a shows the tumor accumulation behavior of the peritumorally injected HMplex. For comparison, free Cy5.5-siRNA and HMplexes of Cy5.5-siRNA (N/P=4) were administered to MDA-MB-231 xenograft mice by peritumoral injection and the NIRF signals were monitored by an IVIS imaging system. Free Cy5.5-siRNA without HMplexation displayed weak fluorescence from the tumor at 1.5 h post-injection, which was further reduced with time (FIG. 8). This implies low in vivo stability of naked siRNA or its clearance due to low tumor accumulation. In sharp contrast, HMplexes containing the same amount of Bcl-2 siRNA exhibited recovery of the initially self-quenched Cy5.5-siRNA fluorescence from the tumor after peritumoral injection, elucidating that HMplexes were internalized into the tumor and decomplexed to release fluorescent siRNA chains as discussed in FIG. 2c. The recovered tumor signal reached a maximum intensity at 1-3 h after injection and then gradually decreased. Nonetheless, a notable signal was detected from the tumor even at 6 d post-injection, indicative of successful in vivo stabilization and peritumoral delivery of siRNA by ternary HMplexation. To confirm this argument, histological examination was done with tumor sections excised at 4 d after peritumoral injection of free Cy5.5-siRNA or HMplexes containing the same amount of Cy5.5-siRNA (FIG. 7b). In the case of HMplexes, clear Cy5.5-siRNA signals were observed in the cytoplasm of cancer cells (distinct from the DAPI-stained nuclear areas), strongly supporting that the decomplexation between siRNA and BZT occurred after cell internalization. This contrasts strikingly to the negligible cytoplasmic signal of the tumor sections from mice treated with naked Cy5.5-siRNA. From all these results, it is concluded that ternary HMplexes indeed co-deliver siRNA and BZT from the peritumoral exterior into the tumor tissue and further to the cytoplasm of cancer cells in vivo.

(45) Tumor Suppression by HMplexes in MDA-MB-231 Xenografts

(46) Finally, the in vivo tumor suppression effect was comparatively assessed with HMplexes (N/P=4) of Bcl-2 siRNA or scRNA. The HMplex samples were administered to MDA-MB-231 xenograft mice in triple doses by peritumoral injection on day 1, 4 and 6, and the tumor growth and body weights were monitored for 30 d (FIG. 9). As shown in FIG. 6a, the experimental group treated with HMplexes of Bcl-2 targeting siRNA (HMplex/siRNA) exhibited a significant tumor suppression effect which was sustained throughout the experimental period. It is noted that HMplexes of nontargeting scRNA (HMplex/scRNA) also presented tumor suppression to some extent, most probably due to the anticancer effect of the co-complexed BZT as found in vitro (FIG. 6). In spite of the apoptosis-inducing BZT effect, both HMplexes showed no toxic impact on the animal viability, as monitored with the minimal body weight changes during the experiment (FIG. 9b). Importantly, the suppressed tumor volume by HMplex/siRNA at the end of treatment (205 mm.sup.3) is less than half of the value reached by HMplex/scRNA (440 mm.sup.3), clearly evidencing that combining gene-targeting RNAi and chemotherapy by the co-delivering HMplex is indeed operative in vivo to synergistically enhance the treatment efficacy for locoregional chemotherapy of resistant TNBC.

(47) Advantages and Benefits of the Present Invention

(48) We have developed a simple and biocompatible formulation for the siRNA/drug co-delivery, based on multiple monocomplexation-induced hydrophobic associations between Bcl-2 targeting siRNA and a monocationic anticancer agent (BZT). It was found that the physical mixing of siRNA, BZT and Pluronic F-68 causes spontaneous formation of a tightly complexed nanostructure owing to the strong cooperative electrostatic/hydrophobic interactions between the ingredients. By virtue of the resulting compact complexation with micellar passivation as well as small colloidal size less than nm, the ternary HMplex was capable of suitable protection of siRNA and successful in vivo co-delivery of siRNA and BZT into the cytoplasm of cancer cells by peritumoral administration. It was shown in vitro and in vivo that cell-internalized HMplexes are decomplexed and release payloads in the cytoplasm to trigger a cooperative action, i.e., silencing anti-apoptotic Bcl-2 by siRNA and thereby sensitized induction of apoptosis by BZT. Thanks to this gene-targeted chemosensitization and cancer-specific toxicity of BZT, the co-delivering HMplex presented a synergistically enhanced therapeutic effect on the aggressive and resistant TNBC model in mice, demonstrating potential for locoregional cancer treatment by targeted combination therapy.

(49) Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.