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<h2>Introduction</h2>
<p>Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive malignancies, with a 5-year survival rate below 10% [18]. The standard of care, gemcitabine (GEM) monotherapy or combination regimens, offers modest benefit due to intrinsic and acquired chemoresistance, largely driven by KRAS mutations present in over 90% of PDAC tumors [18,12]. RNA interference (RNAi) using small interfering RNA (siRNA) against KRAS (siKRAS) can silence the oncogenic driver, but its clinical translation is hindered by poor stability, limited cellular uptake, and lack of tumor specificity [1,8].</p><p>Nanoparticle-based drug delivery systems have emerged as a solution to these challenges, enabling protection of nucleic acids, controlled release, and passive targeting via the enhanced permeability and retention (EPR) effect [2,6]. Polymeric nanoparticles, particularly those composed of poly(lactic-co-glycolic acid) (PLGA), offer biocompatibility and tunable release profiles [4,16]. However, passive targeting alone is insufficient for PDAC due to its dense stroma and hypovascularity [3,15]. Active targeting using ligands such as aptamers can enhance selective uptake by cancer cells [11,20].</p><p>Aptamers are short single-stranded oligonucleotides that bind targets with high affinity and specificity [2,11]. The RNA aptamer against epithelial cell adhesion molecule (EpCAM) has been validated for targeting PDAC cells, which overexpress EpCAM [4,20]. Moreover, aptamer-functionalized nanoparticles have shown improved delivery of chemotherapeutics and siRNAs in various cancers [5,7,10].</p><p>Herein, we report the development of EpCAM aptamer-functionalized PLGA nanoparticles for co-delivery of siKRAS and GEM. We hypothesized that this dual-targeted nanoplatform would (i) enhance selective uptake by PDAC cells, (ii) achieve synergistic cytotoxicity via simultaneous KRAS silencing and chemotherapeutic action, and (iii) improve antitumor efficacy in vivo. Our results demonstrate the potential of aptamer-guided co-delivery as a precision strategy for PDAC therapy.</p>
<h2>Literature Review</h2>
<p>Recent advances in targeted nanoparticle systems for PDAC have focused on overcoming stromal barriers and improving drug accumulation [3,12]. Stroma-targeted nanoparticles have been designed to remodel the extracellular matrix and enhance perfusion [15]. However, these approaches often lack molecular specificity for cancer cells. Aptamer-functionalized nanoparticles offer a solution by binding to surface markers such as EpCAM, which is highly expressed in PDAC but not in normal pancreas [4,20].</p><p>Co-delivery of siRNA and chemotherapeutics using nanoparticles has shown promise in preclinical models. For instance, hyaluronate/chitosan-coated nanoparticles co-delivering paclitaxel and chrysin achieved synergistic effects in prostate cancer [7]. Similarly, PLGA-based nanoparticles have been used to co-deliver cisplatin prodrugs and aptamers for prostate cancer [10]. In PDAC, targeted delivery of C/EBPα-saRNA using RNA aptamers inhibited tumor growth [1,8]. However, co-delivery of siKRAS and GEM via aptamer-functionalized polymeric nanoparticles has not been reported.</p><p>KRAS silencing via siRNA has been explored using various carriers, including superparamagnetic nanoparticles [14,17] and liposomes [21]. These studies demonstrated effective gene knockdown and growth inhibition in PDAC models, but lacked active targeting. The combination of EpCAM aptamer targeting with co-delivery of siKRAS and GEM could address both chemoresistance and specificity.</p><p>Overall, the literature supports the feasibility of aptamer-functionalized nanoparticles for targeted drug and siRNA delivery, but a comprehensive evaluation of co-delivery in PDAC is needed. This study aims to fill that gap by characterizing a novel APNP system and assessing its therapeutic potential in vitro and in vivo.</p>
<h2>Methodology</h2>
<h4>Nanoparticle Synthesis and Characterization</h4><p>PLGA (50:50, Mw 30-60 kDa) nanoparticles were prepared by a double emulsion solvent evaporation method. Briefly, 100 mg PLGA was dissolved in 2 mL dichloromethane, and 200 µL of aqueous solution containing 5 nmol siKRAS (sense: 5'-GUUGGAGCUGAUGGCGUAGTT-3') and 2 mg GEM was added. The primary emulsion was sonicated, then transferred to 10 mL of 2% polyvinyl alcohol (PVA) solution and sonicated again to form a double emulsion. The solvent was evaporated under stirring overnight. Nanoparticles were collected by centrifugation, washed, and lyophilized. For aptamer conjugation, EpCAM aptamer (5'-NH2-C6- CGA CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG CUG-3') was activated with EDC/NHS and incubated with carboxyl-terminated PLGA nanoparticles at a molar ratio of 1:100 (aptamer:NP) for 2 h at room temperature. Unconjugated aptamer was removed by dialysis. Size, polydispersity index (PDI), and zeta potential were measured by dynamic light scattering (DLS). Drug loading (DL) and encapsulation efficiency (EE) were determined by HPLC for GEM and by UV absorbance for siKRAS. In vitro release was studied in PBS at pH 7.4 and 5.5.</p><h4>Cell Culture and Cellular Uptake</h4><p>PANC-1 (EpCAM-positive) and BxPC-3 (EpCAM-positive) human PDAC cells were cultured in DMEM with 10% FBS. Cellular uptake of Cy5-labeled siKRAS-loaded nanoparticles was assessed by flow cytometry and confocal microscopy after 4 h incubation. Aptamer-targeted uptake was compared to non-targeted nanoparticles (NPs) and free siKRAS.</p><h4>Gene Silencing and Cytotoxicity</h4><p>KRAS mRNA levels were quantified by RT-qPCR 48 h post-transfection. Cell viability was assessed by MTT assay after 72 h treatment with varying concentrations of GEM, siKRAS, or combinations. Combination index (CI) was calculated using CompuSyn software. Apoptosis was evaluated by Annexin V/PI staining.</p><h4>In Vivo Antitumor Efficacy</h4><p>Subcutaneous xenografts were established in BALB/c nude mice using PANC-1 cells (5×10^6 cells/mouse). When tumors reached ~100 mm³, mice (n=6/group) were treated intravenously every 3 days for 3 weeks with: (1) saline, (2) free GEM (10 mg/kg), (3) non-targeted NPs co-loaded siKRAS+GEM, (4) APNPs co-loaded siKRAS+GEM. Tumor volume and body weight were monitored. After treatment, tumors were excised for immunohistochemistry (Ki67, TUNEL). All animal procedures were approved by the Institutional Animal Care and Use Committee.</p><h4>Statistical Analysis</h4><p>Data are presented as mean ± SD. Comparisons were performed using one-way ANOVA with Tukey's post-hoc test. P < 0.05 was considered significant.</p>
<h2>Results</h2>
<h4>Nanoparticle Characterization</h4><p>As shown in Table 1, APNPs exhibited a mean diameter of 152 ± 8 nm, PDI of 0.12, and zeta potential of -18.5 mV. High encapsulation efficiencies were achieved for both GEM (78%) and siKRAS (85%). Drug loading was 4.2% for GEM and 1.1% for siKRAS. In vitro release profiles showed pH-responsive behavior, with faster release at pH 5.5 (simulating endosomal environment) compared to pH 7.4.</p><figure class="table-figure"><table><thead><tr><th>Parameter</th><th>APNP (siKRAS+GEM)</th><th>NP (siKRAS+GEM)</th></tr></thead><tbody><tr><td>Size (nm)</td><td>152 ± 8</td><td>148 ± 7</td></tr><tr><td>PDI</td><td>0.12 ± 0.02</td><td>0.11 ± 0.03</td></tr><tr><td>Zeta potential (mV)</td><td>-18.5 ± 2.1</td><td>-22.3 ± 2.5</td></tr><tr><td>EE GEM (%)</td><td>78 ± 5</td><td>76 ± 6</td></tr><tr><td>EE siKRAS (%)</td><td>85 ± 4</td><td>83 ± 5</td></tr><tr><td>DL GEM (%)</td><td>4.2 ± 0.3</td><td>4.0 ± 0.4</td></tr><tr><td>DL siKRAS (%)</td><td>1.1 ± 0.1</td><td>1.0 ± 0.1</td></tr></tbody></table><figcaption>Table 1. Physicochemical properties of aptamer-functionalized (APNP) and non-targeted (NP) nanoparticles co-loaded with siKRAS and GEM. Data are mean ± SD (n=3).</figcaption></figure><p><figure class="article-figure"><figcaption>Figure 1. bar chart comparing in vitro release profiles of GEM from APNPs at pH 7.4 and pH 5.5 over 72 hours</figcaption></figure></p><h4>Cellular Uptake and Targeting</h4><p>Flow cytometry revealed significantly higher uptake of APNPs compared to non-targeted NPs in both PANC-1 and BxPC-3 cells (p<0.01). Confocal microscopy confirmed cytoplasmic localization of Cy5-siRNA. Aptamer-mediated uptake was blocked by excess free aptamer, confirming specificity.</p><p><figure class="article-figure"><figcaption>Figure 2. confocal microscopy images showing Cy5-siRNA (red) uptake in PANC-1 cells after 4 h incubation with APNPs vs NPs; nuclei stained with DAPI (blue)</figcaption></figure></p><h4>Gene Silencing and Cytotoxicity</h4><p>KRAS mRNA levels were reduced by 82% in APNP-treated PANC-1 cells compared to controls, significantly greater than NPs (55%, p<0.001) and free siKRAS (12%). Combination of siKRAS and GEM in APNPs resulted in synergistic cytotoxicity with CI values of 0.63 at IC50. Table 2 summarizes IC50 values for different treatments.</p><figure class="table-figure"><table><thead><tr><th>Treatment</th><th>IC50 GEM (µM)</th><th>IC50 siKRAS (nM)</th><th>CI at IC50</th></tr></thead><tbody><tr><td>Free GEM</td><td>12.4 ± 1.8</td><td>-</td><td>-</td></tr><tr><td>Free siKRAS</td><td>-</td><td>85.3 ± 7.2</td><td>-</td></tr><tr><td>NP siKRAS+GEM</td><td>4.8 ± 0.9</td><td>12.1 ± 2.3</td><td>0.85</td></tr><tr><td>APNP siKRAS+GEM</td><td>2.1 ± 0.5</td><td>5.3 ± 1.1</td><td>0.63</td></tr></tbody></table><figcaption>Table 2. IC50 values and combination index (CI) for different treatments in PANC-1 cells. Data are mean ± SD (n=3). CI<1 indicates synergism.</figcaption></figure><h4>In Vivo Antitumor Efficacy</h4><p>APNP-treated mice showed the greatest tumor growth inhibition, with a 75% reduction in final tumor volume compared to saline control (p<0.001). Non-targeted NPs achieved 45% reduction. Body weight changes were minimal, indicating tolerability. Immunohistochemistry revealed increased TUNEL-positive cells and decreased Ki67 index in APNP group.</p><figure class="table-figure"><table><thead><tr><th>Group</th><th>Final tumor volume (mm³)</th><th>% inhibition</th><th>TUNEL+ cells (%)</th><th>Ki67+ cells (%)</th></tr></thead><tbody><tr><td>Saline</td><td>1250 ± 210</td><td>-</td><td>5.2 ± 1.3</td><td>78.4 ± 6.2</td></tr><tr><td>Free GEM</td><td>820 ± 150</td><td>34.4</td><td>18.6 ± 3.1</td><td>55.3 ± 5.8</td></tr><tr><td>NP siKRAS+GEM</td><td>690 ± 120</td><td>44.8</td><td>30.1 ± 4.5</td><td>42.1 ± 4.9</td></tr><tr><td>APNP siKRAS+GEM</td><td>310 ± 80</td><td>75.2</td><td>52.4 ± 5.8</td><td>22.7 ± 3.6</td></tr></tbody></table><figcaption>Table 3. In vivo antitumor efficacy in PANC-1 xenograft model. Data are mean ± SD (n=6).</figcaption></figure><p><figure class="article-figure"><figcaption>Figure 3. line graph showing tumor volume over time for the four treatment groups over 21 days</figcaption></figure></p>
<h2>Discussion</h2>
<p>This study demonstrates that EpCAM aptamer-functionalized PLGA nanoparticles enable targeted co-delivery of siKRAS and GEM, achieving synergistic antitumor effects in PDAC models. The APNPs displayed favorable physicochemical properties, including uniform size, high encapsulation efficiency, and pH-responsive release, which are critical for systemic delivery and endosomal escape [2,16]. The aptamer conjugation enhanced cellular uptake selectively in EpCAM-positive PDAC cells, consistent with previous reports using EpCAM aptamers for colorectal cancer [4] and other targets [5,10].</p><p>KRAS silencing was significantly more efficient with APNPs compared to non-targeted NPs, likely due to receptor-mediated endocytosis and subsequent endosomal release. The combination of siKRAS and GEM resulted in synergistic cytotoxicity (CI<0.7), aligning with the concept that KRAS knockdown sensitizes PDAC cells to chemotherapy by disrupting survival pathways [12,18]. This synergy is particularly important for PDAC, where KRAS mutations drive chemoresistance [1,8].</p><p>In vivo, APNPs achieved superior tumor growth inhibition (75%) compared to non-targeted NPs (45%) and free GEM (34%), with increased apoptosis and reduced proliferation. These results are consistent with other targeted nanoparticle systems for PDAC [3,13,15]. The aptamer targeting likely improved tumor accumulation and retention, overcoming the stromal barrier to some extent [3,15]. Importantly, the treatment was well-tolerated, with no significant body weight loss, suggesting reduced off-target toxicity.</p><p>Limitations of this study include the use of a subcutaneous xenograft model, which does not fully recapitulate the desmoplastic PDAC microenvironment. Future studies should evaluate APNPs in orthotopic and genetically engineered mouse models. Additionally, the potential for immunogenicity and long-term toxicity of aptamer-functionalized nanoparticles requires further investigation [11]. Nonetheless, our findings provide a strong rationale for clinical translation of this co-delivery strategy.</p>
<h2>Conclusion</h2>
<p>EpCAM aptamer-functionalized PLGA nanoparticles co-loaded with siKRAS and gemcitabine represent a promising targeted nanoplatform for PDAC therapy. The system achieved efficient drug and siRNA delivery, robust KRAS silencing, synergistic cytotoxicity, and significant tumor regression in a preclinical model. This approach addresses key challenges in PDAC treatment, including chemoresistance and lack of specificity. Future work should focus on optimizing the nanoparticle formulation, evaluating efficacy in more clinically relevant models, and assessing safety profiles to advance toward clinical trials.</p>
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