Targeted Gene Silencing Using RGD-Labeled Chitosan Nanoparticles

This article is featured in Highlights of This Issue, p. 3809

RNA interference (RNAi)-based approaches hold great potential for cancer therapy (1–3). Short interfering RNA (siRNA)-based therapy may allow development of a broad armamentarium of targeted drugs against genes that are difficult to target with other traditional approaches such as small molecules or monoclonal antibodies. However, one of the key challenges to the use of siRNA for therapy is the need for efficient intracellular delivery because unprotected siRNA is rapidly cleared or degraded by nucleases. Delivery of siRNA across plasma membranes in vivo has been achieved using delivery systems such as liposomes (4–6), nanoparticles (7–9), and chemically modified siRNA (1). Although these delivery approaches have been shown to be effective in preclinical models, many cannot be used in clinical settings due to nonspecific delivery, which may lead to unwanted or unexpected side effects. Therefore, to overcome these limitations, novel delivery systems are needed. A desirable delivery system should lead to enhanced concentrations of therapeutic payloads at disease sites, minimize concerns about off-target effects (3), and ultimately raise the therapeutic index. Chitosan (CH) is particularly attractive for clinical and biological applications due to its low immunogenicity, low toxicity, and biocompatibility (10, 11). In addition to its advantages such as a protonated amine group, chitosan can increase binding efficiency with cells because of electrostatic interactions (12).

For a targeted delivery system (3, 8, 13), various receptors on the tumor cell surface have been established as a target binding site to achieve selective delivery. One such protein receptor of interest is the ανβ3 integrin, which has been considered for selective delivery (14–17). The ανβ3 integrin is overexpressed in a wide range of tumors, and is largely absent in normal tissues, which is a desirable feature for selective delivery. Here, we developed a cyclic Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle (RGD-CH-NP) for tumor targeted delivery of siRNA. The cyclic RGD has one or two ring structures, and provides conformation stability and improved binding selectivity for the ανβ3 integrin. Moreover, cyclic peptides are less susceptible to biodegradation than linear RGD peptides (18, 19). In the current study, we show highly selective delivery of targeted nanoparticles to ανβ3 integrin–expressing cells and the therapeutic efficacy of this approach in multiple ovarian cancer models.

Conjugation of RGD (c[RGDfK (Ac-SCH₂CO)], MW 719.82 Da) and chitosan (MW 50-190 KDa) is shown in Fig.1A. The RGD and chitosan were conjugated by thiolation reaction using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). Briefly, 10.5 mL of 2 mg/mL chitosan solution (1% acetate buffer) were added to 700 μg of SPDP to react the NH₂ group of the chitosan for 4 hours at room temperature. After that, 500 μg of RGD were added to SPDP-activated chitosan solution for 24 hours at room temperature. After this reaction, dialysis was done for 48 hours to isolate conjugates. The conjugates were confirmed by proton-NMR (H-NMR) (CH and CH-RGD: 1% acetic acid included D₂O, RGD: DMSOd6, 500 MHz, HRMAS-FT-NMR, Bruker, Germany). In addition, to determine the RGD concentration in RGD-CH-NPs, RGD peptide was labeled with FITC as shown in Supplementary Fig. S1 (20).

RGD-CH-NP was prepared based on ionic gelation of anionic tripolyphosphate and siRNA. Briefly, predetermined tripolyphosphate (0.25% w/v) and siRNA (1 μg/μL) were added in RGD-CH solution, and the siRNA/RGD-CH-NP were spontaneously formed under constant stirring at room temperature. After incubation at 4°C for 40 minutes, siRNA/RGD-CH-NP was collected by centrifugation (Thermo Biofuge, Germany) at 13,000 rpm for 40 minutes at 4°C. The pellet was washed by sterile water three times to isolate siRNA/RGD-CH-NP, which was stored at 4°C until used.

The RGD concentration in the RGD-CH-NPs was calculated by measuring FITC intensity based on a calibration curve of standard concentration of FITC-labeled with RGD by a fluorescence spectrophotometer (20). The size and ζ potential of RGD-CH-NP were measured by light scattering with a particle size analyzer and Zeta Plus (Brookhaven Instrument Co.), respectively. Coincorporation of FITC-labeled RGD and Alexa555 siRNA into siRNA/RGD-CH-NP was observed by fluorescence microscopy, and the physical morphology of siRNA/RGD-CH-NP was observed by scanning electron microscopy.

The derivation and source of the human epithelial ovarian cancer cell lines SKOV3ip1, HeyA8, A2780, and A2780ip2, and murine ovarian endothelial cells (MOEC) have been previously described (4, 5, 21). The POSTN siRNA (target sequence: 5′-GGAUCUUGUGGCCCAAUUA-3′), FAK siRNA (target sequence: 5′-CCACCUGGGCCAGUAUUAU-3′), PLXDC1 siRNA (target sequence: 5′GACACCUGCGUCCUCGA-3′), and control siRNA (target sequence: 5′-UUCUCCGAACGUGUCACGU-3′) were purchased from Sigma (22).

To confirm the in vitro binding efficiency of RGD-CH-NP against ανβ3 integrin on the cell surface, we conducted both flow cytometry analysis and fluorescence microscopy. To measure the binding efficiency of Alexa555 siRNA/RGD-CH-NP, cells were incubated for 20 minutes at 4°C after nanoparticles were added, and then cells were collected by centrifugation (1,500 rpm, 3 minutes). The binding efficiency was measured by flow cytometry (23, 24). To observe cell binding of RGD-CH-NP, cells were fixed in a chamber slide using 4% paraformaldehyde and then the cells were stained with Hoechst 33258 for 10 minutes at 4°C (to stain nuclei blue) and observed under fluorescence microscopy (magnification, ×200; refs. 23, 24). In addition, we confirmed intracellular delivery of chitosan nanoparticles (CH-NP) or RGD-CH-NP by confocal microscopy. Briefly, we added CH-NP or RGD-CH-NP in cells and then incubated for 20 minutes at room temperature. The cells were then fixed using 4% paraformaldehyde, after which the cells were stained with sytox green (to stain nuclei green) for 10 minutes at room temperature and observed under confocal microscopy. To confirm binding of RGD-CH-NP by tumor cells, morphology of the cells was observed by transmission electron microscopy (23, 24).

Detection of uptake of Alexa555 siRNA/RGD-CH-NP was done as described previously (25, 26). Relevant tissues were harvested after single injection of either control siRNA/CH-NP, Alexa555 siRNA/CH-NP, or Alexa555 siRNA/RGD-CH-NP into SKOV3ip1-bearing mice. Uptake efficiency was determined by the percentage of Alexa555 siRNA-labeled nanoparticles location into tissue in five random fields at ×200 magnification for each tumor and organ. In addition, to confirm ανβ3 integrin–mediated delivery of RGD-CH-NP, we carried out ανβ3 integrin staining in tumor tissues as described above.

The preparation of cultured cell lysates and tumor tissue lysates has been previously described (5, 27). Protein concentrations were determined using a BCA Protein Assay Reagent Kit (Pierce Biotech.), and aliquots of 20 μg protein were subjected to gel electrophoresis on 7.5% or 10% SDS-PAGE gels. Transfer to membranes and immunoblotting were carried out as described previously (28).

Female athymic nude mice (NCr-nu) were purchased from the National Cancer Institute-Frederick Cancer Research and Development Center and maintained as previously described (29). All mouse studies were approved by the M.D. Anderson Cancer Center Institutional Animal Care and Use Committee. The mice used for in vivo experiments were 8 to 12 weeks old. To produce tumors, SKOV3ip1, HeyA8, and A2780 cells (1 × 10⁶ cells per 0.2 mL HBSS) were injected into the peritoneal cavity (i.p.) of mice. Mice (n = 10 per group) were monitored daily for adverse effects of therapy and were sacrificed when any of the mice seemed moribund.

To assess tumor growth, treatment began 1 week after i.p. injection of tumor cells into the mice. Each siRNA-incorporated CH-NP or RGD-CH-NP was given twice weekly at a dose of 150 μg/kg body weight through i.v injection. Docetaxel was diluted in PBS and injected i.p once a week, at a dose of 100 μg in 200 μL. Treatment continued until mice became moribund (typically 4 to 5 weeks depending on tumor-cell). Mouse weight, tumor weight, number of nodules, and distribution of tumors in the mice were recorded at the time of sacrifice. The individuals who did the necropsies, tumor collections, and tissue processing were blinded to the treatment group assignments. Tissue specimens were fixed either with formalin or optimum cutting temperature (Miles, Inc.) or were snap frozen.

Relative expression of POSTN and FAK mRNA in mice after treatment was determined by real-time quantitative reverse transcriptase-PCR (qRT-PCR) using 50 ng total RNA isolated from treated tumor tissue using the RNeasy Mini Kit (Qiagen). Relative expression values were obtained using the 2^−ΔΔCT method, and normalized to control for percent fold changes (30).

Immunohistochemical analysis was done on tumor tissue from mice that were treated by i.v. injection of siRNA/CH-NP or siRNA/RGD-CH-NP. Procedures for immunohistochemical analysis of cell proliferation (Ki67), microvessel density (MVD; CD31), and POSTN expression (POSTN antibody) were done as described previously (27, 31). All of these analyses were recorded in five random fields for each slide at ×100 magnification. In addition, terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was done as described previously to determine cell apoptosis (32). The quantification of apoptotic cells was calculated by the number of apoptotic cells in five random fields at ×200 magnification. All staining was quantified by two investigators in a blinded fashion.

Differences in continuous variables were analyzed using Student's t test for comparing two groups, and ANOVA was used to compare differences for multiple group comparisons. For values that were not normally distributed, the Mann-Whitney rank sum test was used. The statistical package for the Social Sciences (SPSS, Inc.) was used for all statistical analyses. A P <0.05 was considered statistically significant.

In this study, we selected the ανβ3 integrin as a target receptor because it is selectively expressed in a large proportion of ovarian cancer cells and associated tumor vasculature. Therefore, we utilized a well-characterized targeting peptide, RGD, which can bind specifically to the ανβ3 integrin (33, 34). We first conjugated the RGD peptide with chitosan by thiolation reaction of SPDP (Fig. 1A) and the conjugates were confirmed by H-NMR analysis (Supplementary Fig. S2). As shown in Supplementary Fig. S2C, the peaks for the chitosan of benzene group in RGD (1) and CH₂ of methylene in RGD (1.5 ppm; ref. 2) were observed at 7 to 8 ppm and 1 to 2 ppm, respectively. Additionally, conjugation of RGD with chitosan and CH-NP was measured using FITC-labeled RGD by fluorescence intensity (Supplementary Fig. S1). Conjugation yield of RGD with chitosan was up to 60% (data not shown), and RGD concentration on RGD-CH-NP was determined by measuring FITC intensity based on a calibration curve of standard concentration of FITC-labeled with RGD.

Based on conjugation of RGD-CH, RGD-CH-NPs were prepared. We prepared five different siRNA incorporated RGD-CH-NPs (siRNA/RGD-CH-NP) with varying amounts of RGD (Fig. 1B). The size and ζ potential of siRNA/RGD-CH-NPs were around 200 nm and 40 mV, respectively (Fig. 1B). The histogram of RGD-CH-NP 5 is shown in Supplementary Fig. S3. These experiments indicate that RGD conjugation with CH-NP does not affect the formation and physicochemical properties of siRNA/RGD-CH-NPs. Additionally, the incorporation of RGD and siRNA into RGD-CH-NP 5 was confirmed by fluorescence microscopy using FITC-labeled RGD (green) and Alexa555-labeled siRNA (red; Fig. 1C, top). The morphology of siRNA/RGD-CH-NP 5 was determined by scanning electron microscopy. The particles were spherical in shape and the size was around 200 nm (Fig. 1C, bottom).

We first assessed the expression of ανβ3 integrin in ovarian cancer cell lines by flow cytometry. Although A2780ip2 cells were negative, the SKOV3ip1 cells showed positive expression on the cell membrane against the ανβ3 integrin (Fig. 2A). We next studied the binding efficiency of siRNA/RGD-CH-NPs in both cell lines with different concentrations of RGD (Fig. 1B). As expected, little binding was observed in A2780ip2 cells at any of the five different formulations tested (Fig. 2B). In the ανβ3-positive SKOV3ip1 cells, however, binding increased in a RGD concentration-dependent manner. Among the five formulations, siRNA/RGD-CH-NP 5 (1.45 μg RGD/mg chitosan) showed the highest binding (Fig. 2B). Therefore, we selected siRNA/RGD-CH-NP 5 for subsequent experiments. We next confirmed binding efficiency of Alexa555-labeled (Alexa555) siRNA/RGD-CH-NP by fluorescence microscopy against tumor cells. Alexa555 siRNA/RGD-CH-NP showed higher binding efficiency (94.25% induction versus CH-NP) in the SKOV3ip1 cells compared with non–RGD-labeled CH-NP. In contrast, similar binding efficiency was observed in the A2780ip2 cells between RGD-CH-NP and CH-NP (Fig. 2C). To observe binding of RGD-CH-NP in tumor cells, we utilized transmission electron microscopy. In the SKOV3ip1 cells, RGD-CH-NP showed higher binding compared with the ανβ3-negative A2780ip2 cells (Fig. 2D). In addition, we observed intracellular delivery of CH-NP or RGD-CH-NP using confocal microscopy. Alexa555 siRNA/RGD-CH-NP resulted in higher intracellular efficiency in the SKOV3ip1 cells compared with non–RGD-labeled CH-NP (Supplementary Fig. S4).

Prior to conducting proof-of-concept in vivo efficacy studies, we tested the extent of in vivo delivery following a single i.v. injection of Alexa555 siRNA/RGD-CH-NP into SKOV3ip1-bearing mice after 48 hours. The siRNA was observed in >80% of fields examined, and showed up to 3-fold higher localization into tumor tissues compared with CH-NP (Fig. 3A). Additionally, we stained harvested tumors for the ανβ3 integrin to evaluate colocalization. The Alexa555 siRNA/RGD-CH-NP (red) consistently showed colocalization (yellow) with the ανβ3 integrin (green) in tumor tissues (Fig. 3B). In contrast, delivery with siRNA/CH-NP showed Alexa555-positive siRNA in both ανβ3-positive and -negative cells. These findings indicate that siRNA/RGD-CH-NPs indeed result in selective delivery into ανβ3-positive cells. We also examined other organs, including liver, kidney, spleen, lung, heart, and brain, for delivery of siRNA using either CH-NP or RGD-CH-NP. However, minimal siRNA RGD-CH-NP was observed in these organs as compared with CH-NP due to the greater binding specificity for RGD-CH-NP in tumor tissues (Fig. 3C).

To determine the effectiveness of gene silencing and potential therapeutic efficacy, we focused on targeting periostin (POSTN), which plays a significant role in cell invasion, survival, and angiogenesis, leading to increased metastasis of cancer cells (35). The SKOV3ip1 and A2780 models were selected for these experiments as they have increased POSTN levels (Supplementary Fig. S5). Following a single i.v. injection of POSTN siRNA/RGD-CH-NP (150 μg siRNA/kg body weight) into SKOV3ip1-bearing mice, tumors were harvested. POSTN expression was reduced by >51% in RGD-CH-NP–treated tumors compared with control siRNA/CH-NP and by >20% compared with CH-NP at 24 hours (Fig. 4A). On the basis of this result, we evaluated POSTN expression by immunohistochemistry analysis. Delivery of POSTN siRNA with RGD-CH-NP resulted in significantly greater inhibition of POSTN expression in tumor tissues as compared with POSTN siRNA/CH-NP or control siRNA/CH-NP at 24 hours (Fig. 4B).

We next examined the therapeutic efficacy of POSTN silencing with POSTN siRNA/RGD-CH-NP in mice bearing orthotopic SKOV3ip1 (ανβ3-positive) or A2780 (ανβ3-negative) tumors. Seven days following injection of tumor cells into the peritoneal cavity, the mice were randomly allocated to the following groups (n = 10 mice/group): (a) control siRNA/CH-NP + PBS; (b) POSTN siRNA/CH-NP + PBS; (c) POSTN siRNA/RGD-CH-NP + PBS; (d) control siRNA/CH-NP + docetaxel; (e) POSTN siRNA/CH-NP + docetaxel; and (f) POSTN siRNA/RGD-CH-NP + docetaxel. All mice were sacrificed when animals in any group appeared moribund (4 to 5 weeks after cell injection depending on the cell line). In the SKOV3ip1 model, POSTN siRNA/RGD-CH-NP + PBS resulted in significant inhibition of tumor growth compared with POSTN siRNA/CH-NP + PBS (24% reduction, P < 0.04) and control siRNA/CH-NP + PBS (71% reduction, P < 0.001). Notably, combination of POSTN siRNA/RGD-CH-NP + docetaxel showed the greatest inhibition of tumor growth compared with control siRNA/CH-NP + docetaxel (32% reduction, P < 0.006) and CH-NP + docetaxel (22% reduction, P < 0.01; Fig. 5A). After treatment, the decrease in POSTN mRNA level was confirmed by qRT-PCR (Fig. 5A). In the A2780 model, POSTN siRNA/RGD-CH-NP + PBS showed significant inhibition of tumor growth compared with control siRNA/CH-NP + PBS (73% reduction, P < 0.01), however, POSTN siRNA/RGD-CH-NP + PBS showed no additional benefit compared with POSTN siRNA/CH-NP + PBS (P < 0.22; Fig. 5B). As above, decrease in POSTN mRNA level was confirmed by qRT-PCR (Fig. 5B). There were no differences in total body weight, feeding habits, or behavior between the groups, suggesting that there were no overt toxicities related to therapy.

To determine potential mechanisms underlying the efficacy of siRNA/RGD-CH-NP therapy in tumor tissues, we examined tumors for markers of cell proliferation (Ki67), MVD (CD31), and apoptosis (TUNEL). In the SKOV3ip1 model, POSTN siRNA/RGD-CH-NP + PBS showed significant inhibition of cell proliferation (P < 0.001 versus control siRNA/CH-NP + PBS; P < 0.05 versus POSTN siRNA/CH-NP + PBS), MVD (P < 0.001 versus control siRNA/CH-NP + PBS; P < 0.01 versus POSTN siRNA/CH-NP + PBS), and increased apoptosis (P < 0.001 versus control siRNA/CH-NP + PBS; P < 0.002 versus POSTN siRNA/CH-NP + PBS). The combination group of POSTN siRNA/RGD-CH-NP + docetaxel had significantly reduced cell proliferation (P < 0.05) and MVD (P < 0.001), and increased apoptosis as compared with the single-treatment groups (P < 0.001; Fig. 5C). In the A2780 model, POSTN siRNA/RGD-CH-NP + docetaxel showed significant inhibition of cell proliferation (P < 0.003), MVD (P < 0.001), and increased cell apoptosis compared with control siRNA/CH-NP + PBS (P < 0.004). As expected, neither POSTN siRNA/RGD-CH-NP nor POSTN siRNA/CH-NP showed any significant effects on cell proliferation (P < 0.10) and apoptosis (P < 0.33; Supplementary Fig. S6A).

To establish that the effects of RGD-CH-NP are not unique to just one target, we also did in vivo experiments with siRNA against additional targets. We targeted FAK due to its prominent role in ovarian cancer growth and progression (36). The HeyA8 cells were both ανβ3 integrin and FAK positive (37). Mice were randomly allocated to one of following six groups (n = 10 mice/group): (a) control siRNA/CH-NP + PBS; (b) FAK siRNA/CH-NP + PBS; (c) FAK siRNA/RGD-CH-NP + PBS; (d) control siRNA/CH-NP + docetaxel; (e) FAK siRNA/CH-NP + docetaxel; and (f) FAK siRNA/RGD-CH-NP + docetaxel. Treatment with FAK siRNA/RGD-CH-NP + PBS resulted in significant inhibition of tumor growth as compared with FAK siRNA/CH-NP + PBS (P < 0.04) and control siRNA/CH-NP + PBS (P < 0.001; Fig. 5D). Combination of FAK siRNA/RGD-CH-NP + docetaxel resulted in the greatest effect on tumor growth compared with the other single-treatment groups (P < 0.02) and FAK siRNA/CH-NP + PBS (P < 0.01; Fig. 5D). After treatment, FAK mRNA levels (by qRT-PCR) were found to be significantly lower in the FAK siRNA–treated groups (Fig. 5D). Treatment with FAK siRNA/RGD-CH-NP + docetaxel resulted in significant inhibition of cell proliferation (P < 0.05), MVD (P < 0.01), and increased cell apoptosis (P < 0.01). The combination group of FAK siRNA/RGD-CH-NP + docetaxel showed even further decreases in cell proliferation (P < 0.001) and MVD (P < 0.03), and increased apoptosis compared with the single-treatment group (P < 0.04; Supplementary Fig. S6B).

Because the ανβ3 integrin is known to be selectively expressed in the tumor vasculature, we also selected PLXDC1, a target we recently identified as being upregulated in ovarian cancer vasculature (38). We first confirmed that the mouse ovarian endothelial cells (in vitro) and mouse origin tumor vasculature in A2780 tumor tissue (in vivo) express the ανβ3 integrin although the A2780 cells were negative for ανβ3 integrin expression (Supplementary Fig. S7A and B). As expected, the A2780 tumors harvested from mice lacked ανβ3 expression on tumor cells; however, it was clearly present in tumor vasculature compared with the corpus luteum in the mouse ovary (Supplementary Fig. S7B). Prior to in vivo experiments, we confirmed delivery of Alexa555 siRNA/RGD-CH-NP into the tumor vasculature after injection into A2780-bearing mice. Alexa555 siRNA/RGD-CH-NP showed colocalization (yellow) with endothelial cells (CD31, green) in the tumor vasculature compared with CH-NP (Supplementary Fig. S7C). We next carried out experiments with siRNA targeting PLXDC1 (38), which was incorporated into RGD-CH-NP. For these experiments, A2780 tumor–bearing mice were randomly allocated to one of the following three groups (n = 10 mice/group): (a) control siRNA/CH-NP; (b) PLXDC1 siRNA/CH-NP; and (c) PLXDC1 siRNA/RGD-CH-NP. Treatment with PLXDC1 siRNA/RGD-CH-NP resulted in significant inhibition of tumor growth compared with control siRNA/CH-NP (87% reduction, P < 0.001; Fig. 6A). The targeted delivery of PLXDC1 siRNA with RGD-CH-NP resulted in even greater efficacy compared with PLXDC1 siRNA with CH-NP (P < 0.01; Fig. 6A). Because the A2780 cells lack ανβ3 integrin expression, these results suggest that the RGD-mediated CH-NP targeting is highly effective in targeting the tumor vasculature. We next examined the effects of PLXDC1 gene silencing on the tumor vasculature. PLXDC1 siRNA/RGD-CH-NP resulted in increased apoptosis in the tumor vasculature compared with PLXDC1 siRNA/CH-NP (Fig. 6B). Additionally, to confirm PLXDC1 silencing in the tumor vasculature following PLXDC1 siRNA/RGD-CH-NP injection into A2780-bearing mice, we carried out dual immunofluorescence staining for endothelial cells (CD31, red) and PLXDC1 (green). The PLXDC1 siRNA/RGD-CH-NP–treated group resulted in complete PLXDC1 silencing in the tumor vasculature compared with PLXDC1 siRNA/CH-NP (Fig. 6C).

We show here that a novel receptor-targeted delivery system (RGD-CH-NP) loaded with siRNA targeted to key ovarian cancer–associated genes leads to potent antitumor efficacy in ovarian carcinoma. This approach has broad utility for selectively targeting tumor cells as well as the associated endothelial cells. RGD-CH-NP was effective in silencing multiple targets of interest and in achieving therapeutic efficacy.

RNAi-based cancer therapy is a highly specific method of gene silencing, but hurdles related to systemic in vivo delivery of siRNA need to be overcome to realize its full potential in clinical settings. Moreover, delivery efficiency of free siRNA without the use of a nanoparticle is quite low, and most of the free siRNA is rapidly degraded following i.v. injection (5, 39). Therefore, to overcome this limitation, selective targeted delivery systems are needed. Although a number of nanoparticle systems have been utilized for therapeutic applications, most of these are widely distributed in the body, and could lead to undesirable toxicities in normal tissues. In addition, wide drug distribution may require higher doses for gene silencing in the target tissue of interest. Therefore, to overcome these limitations against conventional passive delivery, targeted delivery is highly desirable.

Targeted delivery systems have been designed to increase or facilitate uptake into target tissue (3), and to protect siRNA payloads and inhibit nonspecific delivery (3). Recent work comparing nontargeted and targeted nanoparticles has shown that the primary role of the targeting ligands is to enhance selective cellular uptake into cancer cells and to minimize accumulation in normal tissues (40). The addition of targeting ligands that provide specific nanoparticle-cell surface interactions can play a vital role in the ultimate location of nanoparticles. For example, nanoparticles can be targeted to cancer cells if their surfaces contain moieties such as peptides, proteins, or antibodies. These moieties can bind with cancer cell-surface receptor proteins such as transferrin (41) or folate (42) receptors, which are known to be increased in number on a wide range of cancer cells. These targeting ligands enable nanoparticles to bind to cell-surface receptors and penetrate cells by receptor-mediated endocytosis. However, a limited number of nanoparticle systems have reached clinical development (40).

Nanoparticles can carry a large payload of drugs as compared with antibody conjugates (40, 43). Furthermore, nanoparticle payloads are frequently located within the particles, and their type and number may not affect the pharmacokinetics and biodistribution of the nanoparticles. This is unlike molecular conjugates in which the type and number of therapeutic entities conjugated to the targeting ligand significantly modify the overall properties of the conjugate.

Several synthetic materials have been proposed for effective nonviral siRNA delivery systems such as lipid-based particles, oligofectamine, cyclodextrin, polyethyleneimine, and cholesterol (3). Although many types of compounds have potential utility as delivery agents, some have concerns regarding safety. For example, toxicity of cationic lipid particles has been reported both in vitro and in vivo (44, 45), and some synthetic agents have been found to induce a gene signature of their own that might increase the off-target effects of siRNA (46, 47). Therefore, development of siRNA therapeutics for cancer treatment requires clinically suitable, safe, and effective drug delivery systems.

Chitosan nanoparticles (CH-NP) have been recently developed for siRNA delivery (12, 48, 49). Chitosan is an attractive nanoparticle for siRNA delivery because its positive charge allows transport across cellular membranes and subsequent endocytosis. Moreover, chitosan is biodegradable, biocompatible, and has low immunogenicity (10, 12, 48). Chitosan nanoparticles without a therapeutic payload have no effect on tumor growth compared with untreated animals (50). The ανβ3 integrin is known to be overexpressed in most cancer cells and the tumor vasculature (33, 37, 51). The ανβ3 integrin is a family of cell surface receptors, which plays an important role in tumor biology and may serve as a useful target (13). In addition to its role in cell matrix recognition, the ανβ3 integrin has been a focus for drug delivery strategies because it assists with internalization and gene transfer (33). In the current study, we developed and characterized RGD-CH-NP incorporated with siRNA as an ανβ3 integrin–targeted delivery system because of its high affinity and highly specific binding. Indeed, our targeted delivery-mediated gene silencing significantly enhanced antitumor therapeutic efficacy compared with a nontargeted delivery system in preclinical ovarian cancer models. Although the RGD ligand can be useful for binding to integrin family members, additional targeting approaches may be useful. Nevertheless, the targeted delivery strategy presented here has broad potential as a delivery platform in human disease and could be adapted for other targeting ligands.

No potential conflicts of interest were disclosed.

We thank Nicholas B. Jennings and Donna Reynolds for their technical expertise, and Drs. Robert Langley and Michael Birrer for helpful discussions.

Grant Support: NIH grants (CA 110793, 109298, 128797, and RC2GM 092599), DOD (OC-073399, W81XWH-10-1-0158), the Ovarian Cancer Research Fund, Inc. (Program Project Development Grant), U. T. M. D. Anderson Cancer Center SPORE in ovarian cancer (P50CA083639), the Zarrow Foundation, the Medlin Foundation, and the Betty Anne Asche Murray Distinguished Professorship. A.M. Nick and R. Stone are supported by NCI-DHHS-NIH T32 Training Grant (T32 CA101642). M.M. Shahzad was supported by the Baylor WRHR grant (HD050128) and the GCF Molly-Cade ovarian cancer research grant.

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