Targeted Tumor Microenvironment Delivery of Floxuridine Prodrug via Soluble Silica Nanoparticles in Malignant Melanoma as a Model for Aggressive Cancer Treatment

Targeted Tumor Microenvironment Delivery of Floxuridine Prodrug via Soluble Silica Nanoparticles in Malignant Melanoma as a Model for Aggressive Cancer Treatment | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Targeted Tumor Microenvironment Delivery of Floxuridine Prodrug via Soluble Silica Nanoparticles in Malignant Melanoma as a Model for Aggressive Cancer Treatment View ORCID Profile Andrés Ramos-Valle , View ORCID Profile Arnau Domínguez , View ORCID Profile Natalia Navarro , View ORCID Profile Ana Márquez-López , View ORCID Profile Anna Aviñó , View ORCID Profile Ramon Eritja , View ORCID Profile Carme Fàbrega , View ORCID Profile Lorena García-Hevia , View ORCID Profile Mónica. L. Fanarraga doi: https://doi.org/10.1101/2025.03.14.643079 Andrés Ramos-Valle a The Nanomedicine Group, Institute Valdecilla-IDIVAL , 39011 Santander, Spain b Molecular Biology Department, Faculty of Medicine, Universidad de Cantabria , 39011 Santander, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andrés Ramos-Valle For correspondence: ramosvallea{at}unican.es fanarrag{at}unican.es Arnau Domínguez c Dpt. Surfactants & Nanobiotechnology, Institute for Advanced Chemistry of Catalonia (IQAC) , CSIC, 08034-Barcelona, Spain d CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine , 08034-Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Arnau Domínguez Natalia Navarro c Dpt. Surfactants & Nanobiotechnology, Institute for Advanced Chemistry of Catalonia (IQAC) , CSIC, 08034-Barcelona, Spain d CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine , 08034-Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Natalia Navarro Ana Márquez-López a The Nanomedicine Group, Institute Valdecilla-IDIVAL , 39011 Santander, Spain b Molecular Biology Department, Faculty of Medicine, Universidad de Cantabria , 39011 Santander, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ana Márquez-López Anna Aviñó c Dpt. Surfactants & Nanobiotechnology, Institute for Advanced Chemistry of Catalonia (IQAC) , CSIC, 08034-Barcelona, Spain d CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine , 08034-Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anna Aviñó Ramon Eritja c Dpt. Surfactants & Nanobiotechnology, Institute for Advanced Chemistry of Catalonia (IQAC) , CSIC, 08034-Barcelona, Spain d CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine , 08034-Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ramon Eritja Carme Fàbrega c Dpt. Surfactants & Nanobiotechnology, Institute for Advanced Chemistry of Catalonia (IQAC) , CSIC, 08034-Barcelona, Spain d CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine , 08034-Barcelona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Carme Fàbrega Lorena García-Hevia a The Nanomedicine Group, Institute Valdecilla-IDIVAL , 39011 Santander, Spain b Molecular Biology Department, Faculty of Medicine, Universidad de Cantabria , 39011 Santander, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lorena García-Hevia Mónica. L. Fanarraga a The Nanomedicine Group, Institute Valdecilla-IDIVAL , 39011 Santander, Spain b Molecular Biology Department, Faculty of Medicine, Universidad de Cantabria , 39011 Santander, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mónica. L. Fanarraga For correspondence: ramosvallea{at}unican.es fanarrag{at}unican.es Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Malignant melanoma presents a significant challenge in oncology due to its aggressive nature and high metastatic potential. Conventional systemic treatments often fail to effectively reach tumor sites, limiting their therapeutic impact. This study introduces a groundbreaking triple-strategy approach for treating malignant melanoma. We developed a novel prodrug, an oligonucleotide, comprising 10 units of Floxuridine (5-fluoro-2’-deoxyuridine) (FdU) nucleoside antimetabolites, to enhance half-life and reduce rapid metabolism. Encapsulated in soluble colloidal silica nanoparticles, this compound is protected and directed towards tumor neovasculature precursor endothelial cell receptors, ensuring local delivery. The strategy focuses on releasing the prodrug in the tumor microenvironment, aiming to eradicate both melanoma cells and their supportive structures. Efficacy was demonstrated in cell culture studies and preclinical models of malignant melanoma, showing a remarkable 50% reduction in tumor size after just three intravenous treatments. These findings underscore the transformative potential of targeting endothelial cell membrane proteins for drug delivery. Our study paves the way for innovative targeted therapies, promising significant advancements in treatment strategies and improved outcomes for patients with metastatic cancers. Key Points Triple-strategy for treating melanoma: FdU 10 prodrug, silica nanoparticle and targeted delivery. Oligonucleotide prodrug (Floxuridine units) enhances half-life and reduces metabolism. Soluble silica nanoparticles protect therapeutic FdU 10 from nucleases and decorated with protein ligands are directed to tumor neovasculature endothelial cells. Significant 50% tumor reduction in preclinical melanoma models after systemic administration with targeted therapies. 1. Introduction Resistance to chemotherapy poses a significant challenge for the systemic treatment of cancer. Despite advancements, many patients experience reduced drug efficacy as cancer cells develop drug resistance. This often necessitates increasing the drug dosages to levels that can threaten a patient’s life. This underscores the urgent need for strategies to overcome resistance and enhance therapeutic outcomes. 5-Fluorouracil (5-FU) is an essential component of cancer chemotherapy. This drug is effective against a variety of malignancies by inhibiting DNA and RNA synthesis in rapidly dividing cancer cells. [ 1 ] However, like many other drugs, the short half-life and rapid metabolism of 5-FU require frequent dosing to maintain therapeutic levels and close monitoring of liver and kidney function to minimize side effects. [ 2 , 3 ] Various strategies have been explored to enhance the therapeutic outcomes and counter-resistance mechanisms of 5-FU. The combination of this drug with folic acid, leucovorin, irinotecan, and oxaliplatin, [ 4 ] or the encapsulation of 5-FU in carriers such as alginate, chitosan, and carbon nanotubes has shown promising potential. [ 5 – 10 ] While these approaches are hopeful, they present some limitations including drug-loading constraints and the risk of premature degradation. A recent advance involves the synthesis of oligonucleotide strands containing multiple units of 5-fluoro-2’-deoxyuridine (FdU), demonstrating superior efficacy compared to 5-FU alone. [ 11 , 12 ] Notably, the oligonucleotide, composed of 10 units of Floxuridine (FdU 10 ), has shown improved in vitro efficacy when incorporated into DNA-based nanoscaffolds. [ 12 – 14 ] Here we explored the potential of this remarkable compound to enhance therapeutic efficacy through refined encapsulation and targeting strategies, while also addressing potential side effects. To counteract rapid elimination, and shield the drug from degradation, we encapsulated the compound within a novel type of soluble colloidal silica nanoparticles which have been recently validated for the delivery of DNA. [ 15 , 16 ] This approach not only establishes a robust platform for drug delivery but also enables customized ligand-protein attachment directly to the nanoparticles, thereby improving delivery to tumoral tissues. To achieve this, we have developed two novel protein ligands tailored to bind receptors on tumor vascular endothelial cells. This approach circumvents targeting cancer cells, which can develop resistance due to genetic instability and mutational changes that make them unrecognizable to engineered ligands. In contrast, endothelial cells in the tumor microenvironment (TME) are more accessible and play a pivotal role in tumor growth. Concentrating nanomedicines in these cells aids in releasing the prodrug upon dissolution of the nanoparticles in the surrounding tumor environment. This targeted release mechanism is expected to effectively target and eliminate both melanoma cells and their supportive structures. 2. Results 2.1. Synthesis and characterization of the FdU 10 Cy5 oligonucleotide The synthesis of the FdU 10 Cy5 oligonucleotide process involved the preparation of the oligonucleotides using a solid-phase assembly of several units of FdU phosphoramidites obtaining the FdU 10 oligomer, as described in the experimental section ( Figure 1A ). Oligonucleotides were cleaved from the solid support using ammonia treatment. The purity of the oligonucleotides was assessed using High-Performance Liquid Chromatography (HPLC), which confirmed the presence of a predominant product. As an inactive negative control for the study, we synthesized an oligonucleotide consisting of 10 thymidine residues (T 10 ). Both oligonucleotides were synthesized in-house following standard phosphoramidite solid-phase chemistry as previously described and characterized. [ 17 ] The identity of the oligonucleotides was confirmed by MALDI-TOF analysis ( Table 1 , Figure S1A and Figure S1B). To enable tracking of the oligonucleotides in vitro and in vivo , FdU 10 and T 10 oligonucleotides were labeled with Cyanine 5 dye (Cy5) at the 3’ end. Download figure Open in new tab Figure 1. Nanoparticle synthesis and characterization. A. Schematic representation of the chemical synthesis of the FdU 10 oligonucleotide labeled with Cy5. B. General diagram of the synthesis of FdU 10 @SiO 2 particles using a modified Stöber method. C. Overlaid histograms of Cy5 intensity per each nanoparticle synthesis analyzed by flow cytometry (10,000 events per sample). FdU 10 @SiO 2 #04 nanoparticles are indicated (orange). D. Z-size (nm) and ζ-potential (mV) of FdU 10 @SiO 2 #04 particles. E . TEM images of FdU 10 @SiO 2 #04 particle morphology. Inset #1 showing an augmented caption of the particle morphology and the scale adjustment based on the raw image. F. Profile of the release of the prodrug from FdU 10 @SiO 2 #04 particles in PBS at 37°C for 6 days (in red). The dashed green line represents the Korsmeyer-Peppas model fitting to the release kinetics. View this table: View inline View popup Download powerpoint Table 1. Sequences and Molecular Characterization of Synthesized FdU 10 -Cy5 and T 10 -Cy5 Oligonucleotides. The expected (exp) and found (f) molecular weights are provided for comparison, ensuring accuracy in the synthesis process. 2.2. FdU 10 Cy5 encapsulation and release from FdU 10 @SiO 2 nanoparticles To protect the oligonucleotide from elimination and degradation we opted to encapsulate the FdU 10 Cy5 prodrug within soluble colloidal silica nanoparticles ( Figure 1B ). As demonstrated in our previous investigations, this encapsulation system effectively safeguards DNA integrity and liberates nucleic acids upon dissolution of the silica coating under physiological conditions, both in vitro and in vivo . [ 15 , 16 ] The FdU 10 Cy5 silica nanoparticles (hereafter FdU 10 @SiO 2 ) were prepared following a previously established protocol based on a modified Stöber procedure. [ 15 , 16 ] The single-stranded nature and small size (10 residues) of the FdU 10 oligonucleotides necessitated fine-tuning of the formulation. Therefore, we tested different stoichiometric ratios (Table S1). Among the various formulations evaluated, we observed that increasing the ethanol content promoted the synthesis of smaller and more uniformly sized nanoparticles (Figure S2). This adjustment also resulted in higher encapsulation efficiency of FdU 10 Cy5, as demonstrated by flow cytometry analysis of the loaded nanoparticles ( Figure 1C and Figure S3). Based on these results, the formulation FdU 10 @SiO 2 #04 (henceforth referred to as FdU 10 @SiO 2 ) was selected for the trials. These optimized nanoparticles had a diameter of 190 nm by TEM, a ζ potential of around −27 mV, and a PDI of 0.08 ± 0.06 ( Figure 1D and Figure 1E ). Similar results were observed for the control nanoparticles loaded with the T 10 -Cy5 oligonucleotide (referred to as T 10 @SiO 2 ) (Figure S4). Direct measurement of the encapsulated oligonucleotide within the FdU 10 @SiO 2 silica nanobeads was conducted using thermogravimetric analysis (TGA). The results indicated that 97.7% of the FdU 10 Cy5 total mass added to the synthesis was entrapped in the nanoparticles (Figure S5). This signifies that the embedding efficiency of the FdU 10 @SiO 2 nanoparticles was 97.1% of the initial oligonucleotide included in the mixture, with a 0.44% loading capacity explained by the compact morphology of the particles and the higher density of silica. To complement this study, we assessed the efficiency of oligonucleotide release upon the dissolution of the FdU 10 @SiO 2 nanoparticles in vitro upon incubation in phosphate-buffered saline (PBS) at 37°C. [ 15 , 16 ] Using fluorimetry, we quantified the release kinetics of the FdU 10 Cy5 oligonucleotide into the media (Figure S6). Figure 1F illustrates that approximately 40% of the embedded oligonucleotide was released within the initial 6 hours. Following this, a sustained and prolonged pattern of drug release was observed, persisting for over 6 days. This data was analyzed using the Korsmeyer-Peppas model, revealing a consistent fit with this model, characterized by a release exponent value of 0.14 and an R 2 value of 0.94. The drug release mechanism was determined to be diffusion (Fickian model), with a slope of <0.5 (Table S2). 2.3. Comparative evaluation of free and encapsulated FdU 10 Cy5 in malignant melanoma cell cultures Malignant melanoma is a major challenge in oncology due to its resistance and aggressiveness. With drug resistance mechanisms involving complex pathways, combating melanoma becomes increasingly difficult as it progresses to the metastatic stage. The grim reality is reflected in the statistic that 30% of patients succumb to the disease within five years. [ 18 , 19 ] This underscores the urgent need for continued research and innovative treatment approaches to improve patient outcomes. [ 20 – 23 ] To assess the impact of the FdU 10 oligonucleotide prodrug on B16-F10 malignant melanoma cells, we undertook a comparative analysis concentrating on the efficiency of the decapsulated FdU 10 Cy5 oligonucleotides. We specifically evaluated the efficacy and effects of FdU 10 @SiO 2 nanoparticles compared to the free drug, alongside a control group treated with the inactive control T 10 Cy5, both in free form and encapsulated. Flow cytometry analysis and fluorescence microscopy imaging confirmed the internalization of the nanoparticles by the cells within 2 h of treatment ( Figure 2A and Figure 2B , arrow). Flow cytometry analysis quantitatively demonstrated that the intracellular Mean Fluorescent Intensity (MFI) of the Cy5 fluorophore, representing the total uptake of encapsulated FdU 10 -Cy5 in FdU 10 @SiO2 nanoparticles, was significantly higher compared to the free FdU 10 -Cy5. Specifically, the MFI was 16.6 times higher 2 hours after nanoparticle exposure and 38.5 times higher after 24 hours, highlighting the enhanced cellular uptake and retention of the nanoparticle-encapsulated drug over time. This trend was observed at all the time points analyzed (48 h and 72 h, Figure S7). Previous studies with similar nanoparticles have consistently demonstrated rapid cellular interactions, with internalization occurring within a few hours. [ 24 , 25 ] The intracellular localization of the compounds was confirmed using single-plane confocal microscopy imaging. Melanoma cells treated with free FdU 10 Cy5 oligonucleotide showed Cy5 fluorescence at the perinuclear cytoplasmic region, confirming oligonucleotide internalization, whereas FdU 10 @SiO 2 nanoparticles were scattered throughout the cytoplasm of the cell ( Figure 2B and Figure 2D , arrows). At this time point, no detectable fluorescent drug release was observed using confocal microscopy. Download figure Open in new tab Figure 2. Cellular capture of the free/encapsulated prodrug. A. Mean fluorescence intensity (MFI) of Cy5 in melanoma cells treated with free FdU 10 -Cy5, and FdU 10 @SiO 2 particles at 2 h and 24 h (Data are presented as mean values ± S.D, n =3 (10,000 cells per replica) * p< 0.05, ** p< 0.01, t -test comparisons, see Figure S7). Figures B and C show single Z-plane confocal microscopy images of melanoma cells 2 hours after the indicated treatments. B . The free FdU 10 -Cy5 oligonucleotide (red) is distributed primarily in the perinuclear cytoplasmic region (white arrow). C . FdU 10 @SiO 2 particles display delayed intracellular drug release. Inset #1 shows lateral projections of an intracellular nanoparticle (red arrow), with several other nanoparticles (orange) visible in the same Z-plane. Cy5 fluorescence is depicted in red, while the cell cytoplasm and nucleus are stained green and blue, respectively. At 72 h, the cells treated with both, FdU 10 @SiO 2 or free FdU 10 Cy5 oligonucleotide exhibited noticeable effects, including increased nuclear and cytoplasmic sizes compared to the untreated cells and T 10 @SiO 2 nanoparticle-treated controls ( Figure 3A and Figure 3B ). At this time point, the accumulation of Cy5 fluorescence in the cytoplasm confirmed the presence of a substantial amount of free and encapsulated intracellular oligonucleotides ( Figure 3A , arrows). Download figure Open in new tab Figure 3. In cellulo effects of FdU 10 @SiO 2 particles and controls. A-B. Confocal microscopy images of melanoma cells at 24 and 72 hours post-treatment. Cells treated with PBS and T 10 @SiO 2 nanoparticles exhibited significant proliferation. In contrast, a notable increase in cell size was observed in cultures treated with either the free FdU 10 or the silica-encapsulated FdU 10 prodrug. C . Flow cytometry analysis of the cell cycle in melanoma cultures at 24 and 72 hours post-treatment revealed significant abnormalities following treatment with the drug alone or FdU 10 @SiO 2 nanoparticles. D . Flow cytometry quantification of cell death in untreated (control) melanoma cells versus those exposed to T 10 @SiO 2 , FdU 10 Cy5, and FdU 10 @SiO 2 . Data are presented as mean values ± S.D. ( n =3), with * p <0.05, ** p <0.01, and *** p <0.001 indicating statistical significance. Statistical analysis was performed using one-way ANOVA with multiple t -test comparisons, with a total of 10,000 cells analyzed per flow cytometry replicate. Flow cytometry analysis revealed significant cell cycle disruptions in cells treated with both free and encapsulated FdU 10 -Cy5, showing a clear G0/G1 phase arrest at 24 hours ( Figure 3C and Figure S8). This blockage aligns with the inhibition of DNA synthesis, preventing the cells from entering the S phase. By 72 hours, the cell cycle showed further dysregulation, with a decrease in G0/G1 phase cells and an uncoordinated distribution across other phases. This pattern is consistent with the mechanism of action of 5-FU, [ 1 ] suggesting that the surviving cells transition into a non-proliferative state, as corroborated by Ki67 and p21 immunostaining results (Figure S9). Finally, to demonstrate the cytotoxic effect of these formulations, we quantified tumor cell death ( Figure 3D ). At 24 hours, cells treated with free FdU 10 Cy5 oligonucleotide or FdU 10 @SiO 2 nanoparticles showed death rates of 23% and 33%, respectively compared to untreated control cultures. After 72 hours, cell mortality increased to 65% and 79%, respectively. In contrast, cells exposed to the T 10 @SiO 2 control nanoparticles showed no significant morphological or cell cycle changes. This demonstrates that the observed effect is due to the FdU 10 Cy5 oligonucleotide and not to the nanoparticles themselves. Together, these findings demonstrate that, at least in cell culture, the effect of the FdU 10 Cy5 prodrug can be significantly enhanced when encapsulated in silica nanoparticles. 2.4. Enhanced antitumor efficacy of locally delivered FdU 10 @SiO 2 nanoparticles After the validation of the FdU 10 @SiO 2 nanoparticles in cellulo , we proceeded to investigate the in vivo effects of the treatments through local intratumoral injection. This experiment aimed to evaluate the antitumor efficacy of both free and encapsulated FdU 10 within the tumor microenvironment, independent of their targeting efficiency. The objective was to demonstrate the effective release of the prodrug from the silica particles, the subsequent activation of the drug within the tumor, and its antitumoral effects, regardless of any targeting considerations. To this end, we used immunocompetent tumor-cell-transplanted mice as a preclinical model of malignant melanoma, ( Figure 4A ), a model that has been widely validated in the literature. [ 10 , 26 – 28 ] Transplanted cancer cells produce solid-pigmented melanoma tumors that are easy to monitor, isolate, and assess through anatomopathological examination, ensuring good reproducibility. These in vivo studies enable exploration of the complex interplay between the TME, tumor cells, and tumor-targeted nanomedicines, providing insights into cancer progression and treatment efficacy. Download figure Open in new tab Figure 4. In vivo intratumoral efficacy of the encapsulated compound. A. General scheme and timeline of the melanoma model developed to evaluate the intratumoral efficacy of the treatments. B . In toto Cy5 fluorescence imaging in whole tumors. C . Quantification of Cy5 fluorescence detected in the tumors at sacrifice captured with an IVIS® imaging system (relative radiant efficiency for each n=7 per group, * p< 0.05, t -test analysis). D . Confocal microscopy images of the tumor tissues exposed to the indicated treatments. The fluorescence of the Cy5-labeled prodrug is shown in the red channel (red arrows). Cell cytoplasms and nuclei are shown in green and blue, respectively. A white arrow indicates a macrophage within the tumor that does not contain detectable nanoparticles. E . Quantification of relative tumor weights after intratumoral treatments compared to the PBS-treated control group (Data are presented as mean values ± S.D, n= 10 mice/group, * p< 0.05, ** p< 0.01, *** p< 0.001, One-way ANOVA with multiple t -test comparisons). The treatments comprised a single injection of either the free oligonucleotide FdU 10 Cy5, the encapsulated form in FdU 10 @SiO 2 nanoparticles, or the negative controls (PBS or T 10 @SiO 2 nanoparticles). All the treatments were administered at identical total oligonucleotide doses. Mice were sacrificed three days post-injection (day 10 post-transplant). The local availability of the prodrug in the tumors was monitored for 72 h after treatment using an IVIS® biofluorescence imaging system (Figure S10). Quantification of total Cy5 fluorescence at the tumoral tissues revealed a significant 3-fold increase in FdU 10 Cy5 when encapsulated in nanoparticles compared to the free oligonucleotide ( Figure 4A and Figure 4C ). Confocal microscopy of the tumor tissue showed that the nanoparticles were visible throughout the tumor microenvironment compared to the free prodrug, which was diffusely distributed in the tissue ( Figure 4D , arrows). This suggests improved local drug retention. Consistent with this, tumors treated with FdU 10 Cy5 decreased in size by almost 20% compared to PBS-treated controls, whereas those treated with FdU 10 @SiO 2 were reduced by more than half ( p =0.009, n= 15) ( Figure 4E ). As in the in cellulo study, these results demonstrate that the efficacy of the FdU 10 Cy5 oligonucleotide is significantly improved when administered in the encapsulated form. The high vascularity of the tumor likely contributes to the systemic dispersion and degradation of the free oligonucleotides. In contrast, the FdU 10 @SiO 2 nanoparticles enabled precise deposition and gradual local prodrug release, culminating in a remarkable ca . 50% reduction in the tumor mass following a single injection. 2.5. Systemic FdU 10 @SiO 2 nanoparticle targeting to the neovasculature results in significant antitumoral effects Targeting nanoencapsulated chemotherapeutic drugs to the TME is pivotal in oncology. Unlike traditional methods focusing on cancer cell receptors prone to resistance, TME cells offer stable targets accessible for nanoparticle delivery. Here, our approach focuses on targeting neovascular cells using FdU 10 @SiO 2 nanoparticles. The hypothesis driving this strategy posits that delivering nanoparticles to surface receptors of endothelial cells enables their dissolution and gradual drug release in the vicinity. This process aims to inhibit blood vessel formation and induce cell death in the surrounding tumor microenvironment, indirectly targeting cancer cells. Consequently, this approach exerts a cytotoxic effect that affects both melanoma cells and supportive TME. [ 29 , 30 ] To validate the hypothesis, we explored two distinct tumor neovasculature-targeted ligands. One approach involved targeting the vascular endothelial growth factor receptor (VEGFR), based on its documented success in previous studies for achieving significant antitumoral effects with nanomaterials. [ 31 – 36 ] As a targeting agent we have selected the peptide ligand ATWLPPR, which is an effective antagonist of VEGFR. [ 37 ] For functionalization purposes, we genetically fused the peptide to the carboxyl terminus of GFP, resulting in a protein termed GFP:VEGFRbp ( Figure 5A and Figure S11A). Download figure Open in new tab Figure 5. Neovasculature-targeted treatments via intravenous administration. A. Schematic representation of the protein design for GFP:VEGFRbp and PA17. B . Diagram of the functionalization process to produce FdU 10 @SiO 2 @GFP:VEGFRbp and FdU 10 @SiO 2 @PA17 nanoparticles C . General schematic and timeline for tumor generation and intravenous administration of the experimental treatments to melanoma-bearing mice. D . Quantification of Cy5 fluorescence detected in the tumors at sacrifice (day 12), captured with an IVIS® biofluorescence imaging system, with relative radiant efficiency for each treatment group. Data are presented as mean values ± S.D. ( n =5), with significance indicated as * p <0.05 and ** p <0.01, determined by One-way ANOVA with multiple t -test comparisons. E . Confocal microscopy proyection images of melanoma tumors treated with FdU 10 @SiO 2 , FdU 10 @SiO 2 @GFP:VEGFRbp, and FdU 10 @SiO 2 @PA17. VEGFR and TEM8 receptors are immunostained and visualized in the green channel, cell nuclei in the blue channel, and the prodrug in the red channel. F . Relative tumor masses of the groups treated with T 10 @SiO 2 , free FdU 10 -Cy5, and FdU 10 @SiO 2 compared to the PBS-treated group (in %). Data are presented as mean values ± S.D. ( n =10 mice per group), with significance indicated as * p <0.05, ** p <0.01, and *** p <0.001, analyzed using One-way ANOVA with multiple t -test comparisons. In parallel, we engineered another neovasculature-targeted ligand inspired by the protective antigen protein (PA), which has a high affinity for the tumor endothelial marker 8 (TEM8) predominant in the tumor neovasculature. This receptor is abundantly expressed on the membranes of angiogenic endothelial cells, associated stromal cells, pericytes, cancer stem and invasive cancer cells, and immune cells such as macrophages and cancer-associated fibroblasts. It was also extraordinarily expressed in the murine malignant melanoma (Figure S12). [ 38 – 40 ] As for VEGFR inhibitors, [ 41 , 42 ] the blockade [ 43 ] or knockout [ 44 – 47 ] of TEM8 has been shown to effectively suppress tumor growth while sparing physiological angiogenesis. This approach presents a promising new direction in cancer therapy with minimal adverse effects. [ 48 – 50 ] The engineered PA-inspired ligand, which we named PA17 ( Figure 5A and Figure S11B), had a molecular weight of 17 kDa and had a high affinity for the TEM8 receptor. [ 51 ] The two engineered ligand-proteins were produced recombinantly and purified according to standard procedures (Methods). They were subsequently used to electrostatically functionalize the FdU 10 @SiO 2 and control T 10 @SiO 2 nanoparticles following an established protocol ( Figure 5B ). [ 52 ] The correct surface functionalization with the ligand proteins was confirmed using SDS-PAGE (Figure S13). The functionalized nanoparticles were named FdU 10 @SiO 2 @GFP:VEGFRbp and FdU 10 @SiO 2 @PA17. The validation of these targeted nanomedicines was performed in preclinical immunocompetent animal models of malignant melanoma. Systemic treatment involved three intravenous injections on days 4, 7, and 10 post-transplantation, as detailed in Figure 5C (Methods). A total amount of 120 µg of the encapsulated FdU 10 Cy5 oligonucleotide was applied both, in bare nanoparticles (non-functionalized) as control, and particles functionalized with the targeting ligands. Regular monitoring and weighing of mice throughout the treatment period showed no evidence of any toxic effects (Figure S14). Treated mice were euthanized on day 12 post-transplantation. A semi-quantitative assessment of oligonucleotide accumulation in toto using an IVIS® biofluorescence detection system confirmed a significant increase in FdU 10 Cy5 accumulation in tumors. This was the case for nanoparticles coated with both GFP:VEGFRbp and PA17 ligands, with 1.7 and 1.6 times more than naked FdU 10 @SiO 2 particles, respectively ( Figure 5D and S15). The effective delivery of the FdU 10 Cy5 fluorescent oligonucleotide was corroborated by confocal microscopy imaging of the fresh tumor stroma. Notably, distinct Cy5 fluorescence patterns indicating prodrug distribution were observed in tumors of mice treated with both targeted nanoparticle variants. In mice treated with FdU 10 @SiO 2 @VEGFRbp nanoparticles, prodrug accumulation appeared as red spots predominantly located near VEGFR-positive tissue. In contrast, tumors treated with FdU 10 @SiO 2 @PA17 nanoparticles showed increased fluorescence primarily within and around the periphery of the tumor vasculature ( Figure 5E , arrows). Quantification of the antitumoral effect revealed a significant 50% reduction in tumor size using targeted nanoparticles compared to PBS-treated controls ( Figure 5F ). The T 10 @SiO 2 nanoparticles functionalized with the ligand proteins showed limited effectiveness in reducing tumor mass, despite some intrinsic ligand activity (Figure S16). Bare FdU 10 @SiO 2 nanoparticles used as controls exhibited only a modest 5% reduction. These results highlight the significantly greater efficacy of the targeted encapsulated drug treatment. Finally, to confirm the effectiveness of the targeting mechanism in reducing tumor neovasculature, we conducted histochemical evaluations of the tumoral tissues. As depicted in Figure S17, treatment with the two endothelial cell-targeting nanoparticles significantly reduced the intratumoral neovasculature in approximately a 75% reduction compared to control tumors treated with PBS or the non-functionalized FdU 10 @SiO 2 nanoparticles. 3. Discussion Systemic pharmacological treatments are effective but often have short durations of action and require high doses, leading to severe side effects. Nanoencapsulation of drugs offers a promising solution by enabling controlled release and prolonged drug performance. However, only 1-2% of administered nanomedicines reach the tumor, [ 53 , 54 ] posing significant challenges in aggressive cancers like malignant melanoma. A pivotal aspect of this study revolves around synthesizing and delivering antiproliferative oligonucleotides as a prodrug. Encapsulation within soluble silica nanoparticles protects the prodrug from degradation and swift elimination, while novel protein ligands of VEGF and TEM8 receptors precisely target these nanoparticles to the tumor neovasculature and TME. Upon reaching the tumor tissues, the FdU 10 oligonucleotide undergoes decapsulation and exonuclease activation to release active 5’-phosphate nucleotide derivatives, thereby reducing potential systemic toxicity. This targeted approach aims to trigger an effect on both cancer cells and supportive microenvironmental cells. The utilization of an immunocompetent ( wild type ) animal model has also been instrumental in enabling drug delivery strategies tailored to the cellular components of the TME. Most trials and proof-of-concept tests assessing the efficacy of nanomedicine have utilized nude animals with severely compromised immune systems. Hence, the effects observed for many drug nanodelivery studies in these animals are notably reduced when wild-type immunocompetent animals are employed. In summary, the combination of FdU 10 with silica particles and a targeted strategy directed at the tumor neovasculature has shown great potential. The achievement of 50% antitumor activity after only three intravenous treatments highlights the critical importance of targeting the microenvironment in aggressive cancer therapy. This study paves the way for more effective and less toxic treatment options for aggressive cancers such as malignant melanoma. 4. Conclusion The developed treatment strategy for malignant melanoma has yielded remarkable outcomes through the integration of the potent FdU 10 prodrug compound with a unique soluble silica nanoparticle encapsulation system and two distinct tumor endothelial cell-targeted ligands. This approach has led to a notable reduction in tumor size, approximately 50%, emphasizing the effectiveness of targeting cells within the TME rather than solely focusing on cancer cell receptors. These results establish a foundation for tailored therapies and bring hope to patients battling malignant melanoma and other aggressive metastatic cancers. By specifically targeting the neovasculature with FdU 10 -loaded nanoparticles, our study offers a promising avenue for future therapeutic interventions. 5. Methods Oligonucleotide synthesis and characterization Briefly, oligonucleotide synthesis was performed at a 1 µmol scale using phosphoramidite solid-phase protocols as described previously. [ 55 ] After ammonia deprotection, oligonucleotides were desalted using Sephadex G-25 columns and used without additional purification. The purity of the oligonucleotides was analyzed by HPLC and MALDI-TOF mass spectroscopy (more details in Supplementary Methods). Synthesis, characterization, and prodrug loading of FdU 10 @SiO 2 nanoparticles In a 1.5 ml microtube, the corresponding 4.5 µg of FdU 10 -Cy5 (see Table S2, for particles #4 = 4.5 µg) were dispersed in 18 µl of nuclease-free ddH 2 O (2.5 M). This FdU 10 -Cy5 solution was poured into a 1.5 ml microtube containing 236 µl of absolute EtOH (for particles FdU 10 @SiO 2 #4, rest see Table S1). The mixture was stirred at 1,200 r.p.m for 5 min. Subsequently, 17.3 µl of ammonia 25% (0.31 M) and 5.6 µl TEOS (0.06 M) were added to this mixture. The reaction mixture was then vortexed for 2 h at 1,200 r.p.m. FdU 10 @SiO 2 spheres were centrifuged at 6,500 r.p.m, washed three times with absolute EtOH, and stored in 100 µl EtOH. TEM images were obtained with a JEM1011 equipped with a high-resolution Gatan digital camera (JEOL) and analyzed using ImageJ software. DLS size distribution and Z-potential measurements were performed in triplicate using a Malvern Panalytical Ultra Zetasizer, with data presented as mean ± standard deviation (SD). Oligonucleotide loading was quantified using flow cytometry and thermogravimetric analysis (TGA) in a dynamic oxygen atmosphere with a heating ramp of 250°C (5 min) to 1000°C at a rate of 10°C/min. A Pfeiffer Vacuum ThermoStar mass spectrometer was used to measure MS signals: m/z = 18, m/z = 46, m/z = 44, and m/z = 15. Prodrug release was performed in vitro by stirring FdU 10 @SiO2 particles in PBS for 72 hours at 37°C. Samples were extracted at various time points and analyzed using fluorescence spectroscopy with an Edinburgh Inst. FLSP920 spectrofluorometer. The FdU 10 Cy5 release in PBS was fitted to a Korsmeyer–Peppas kinetic model. In cellulo studies Murine malignant melanoma B16-F10 cells (Innoprot) were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Panbiotech) supplemented with 10% fetal bovine serum (Fisher Scientific; Waltham, MA, USA) and antibiotics. FdU 10 Cy5@SiO 2 particles were resuspended in the medium at 100 µg NPs/ml. At 2-, 24-, and 72-hours post-addition, cells were washed twice with PBS, fixed in 4% paraformaldehyde, stained, mounted, and analyzed using a Nikon AIR confocal microscope or flow cytometry with a CytoFLEX8® (Beckman Coulter) system. Cell cycle analysis was performed on cultures treated with T 10 @SiO 2 and FdU 10 Cy5@SiO 2 particles. At 24, 48, and 72 hours, cells were harvested, washed, fixed in 70% cold EtOH, and stained with Propidium Iodide. Immunofluorescence for Ki67 (Abcam) and p21 (Abcam) was conducted on cells after 72 h treatment (100 µg NPs / mL) and fixed with 4% paraformaldehyde 72 hours post-treatment, using anti-Ki67 and anti-p21 polyclonal antibodies, and visualized with Alexa Fluor 488 and Cy3 conjugated secondary antibodies (Invitrogen). All images were pseudocolored. Preclinical animal models In vivo experiments were designed to minimize animal use, with ethical approval obtained from the Gobierno de Cantabria, Consejería de Medio Rural, Pesca y Alimentación (accreditation reference: PI-05-23). C57BL/6 mice (8 weeks old) were purchased from Janvier Labs. Animals were maintained, handled, and sacrificed following directive 2010/63/UE. Local in vivo treatments involved intra-scapular subcutaneous transplanting CD1 neonate mice with 1 × 10 5 B16-F10 cells resuspended in 10 μl of IMDM containing antibiotics as previously described. [ 26 ] After 7 days, mice were randomly divided into four groups receiving PBS, T 10 @SiO 2 , free FdU 10 Cy5, or FdU 10 @SiO 2 at 300 µg FdU 10 Cy5/kg. Mice were euthanized 3 days post-treatment, and the tumors were weighed and evaluated in the IVIS® Biofluorescence equipment to quantify the Cy5 labeled prodrug using excitation/emission wavelengths of 650/670 nm during a 1-min exposure. The fluorescence intensity was quantified as the mean radiant efficiency (photons s⋅cm -2 ⋅sr)/(μW⋅cm 2 ). Confocal microscopy imaging when used for quantification, was conducted with identical settings for both control and experimental samples, utilizing a Nikon AIR microscope. Systemic in vivo treatments involved intra-scapular subcutaneous transplanting with 2 × 10 5 B16-F10 cells resuspended in 10 μl of IMDM-containing antibiotics as previously described. [ 26 ] For the treatment phase, mice were randomly divided into 4 groups, each consisting of 10 mice. These groups received PBS, FdU 10 @SiO 2 , FdU 10 @SiO 2 @GFP:VEGFRbp, FdU 10 @SiO 2 @PA17. Mice were treated 3 times on days 4, 7 and 10 after cell transplant at a concentration of 120 μg/kg of FdU 10 -Cy5 /dose. The mice were euthanized 12 days after the transplant and their tissues were collected and fixed in formalin. Tumors were weighed and evaluated in the IVIS® Biofluorescence equipment to quantify the Cy5 labeled prodrug using excitation/emission wavelengths of 650/670 nm during a 1-min exposure. The fluorescence intensity was quantified as the mean radiant efficiency (photons s⋅cm -2 ⋅sr)/(μW⋅cm 2 ). Confocal microscopy imaging was conducted with identical settings for both control and experimental samples, utilizing a Nikon AIR microscope. VEGF and TEM8 receptors were immunostained with anti-VEGF Receptor 1 (Thermofisher) and TEM8/ATR (Abcam), antibodies, and were visualized with an Alexa Fluor 488 conjugated secondary antibody (Invitrogen). Tumors were evaluated using an IVIS® Biofluorescence system and confocal microscopy using a Nikon AIR microscope. All images were pseudocolored. Design, production, purification, and functionalization of the ligand proteins Recombinant gene constructs encoding GFP:VEGFRbp were cloned into pET 15b plasmid systems (Novagen) by General Biosystems, Inc. (Morrisville, USA). Both ligand proteins were fused to a 10-histidine tag at their amino-terminus (Figure S13) for electrostatic particle functionalization as in previous studies. [ 52 , 55 ] Constructs were transformed and expressed in One Shot™ BL21 (DE3) E. coli (Thermo Fisher Scientific). Proteins were purified using Ni-TED columns (Protino® Ni-TED, Macherey-Nagel) following standard biochemical procedures. Protein analysis was conducted using SDS-PAGE with Coomassie-stained gels analyzed using the BioRad GelDoc EZ system software. FdU 10 @SiO 2 functionalization with both ligands was based on a previous protocol of our group. In brief, 100 μg of particles were immersed in 500 μL PBS containing saturating amounts of the tagged protein (ca. 0.5 mg/mL) at room temperature. The mixture was sonicated in a water bath for 5 min at 4 °C. Unbound protein was removed by repeated centrifugation. SDS-PAGE electrophoresis was used to quantify the protein captured on the surfaces of the particles. These were stripped in Laemmli sample buffer (BioRad) at 90 °C. The stripped protein was loaded in precast Mini-Protean® TGX™, BioRad gels for SDS-PAGE analysis. The semi-quantification of the total amount of protein on the surface of the particles was performed on Coomasie-stained gels using the software of the BioRad GelDoc EZ system. Statistics Results are expressed as mean values along with their corresponding standard deviations (SD). Statistical differences in means were assessed using a t-test or ANOVA, conducted through Graphpad Prism, and significance was established at p < 0.05. In cases where ANOVA yielded significant results, pairwise comparisons were carried out. The total number of events and the confidence levels achieved in the experiment (n) are all specified in each figure caption for reference. Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Author Contributions ARV, AD, NN, AML, AA, and LGH, performed the experiments. All authors discussed the results and wrote the manuscript. MLF, RE, and CF obtained the funding. All authors have approved the final version of the manuscript. Conflict of Interest Statement The authors reports no conflicts of interest in this work. Data Availability Statement The data presented in this study are available on request from the corresponding author. This research introduces a novel approach for treating malignant melanoma using a triple-strategy therapy. A Floxuridine-based oligonucleotide prodrug is encapsulated in silica nanoparticles, targeting tumor neovasculature. Results from in vitro and preclinical models show significant tumor reduction, highlighting the potential of this targeted delivery system in improving treatment outcomes for metastatic melanoma. Download figure Open in new tab Acknowledgements We acknowledge the financial support from the Spanish Instituto de Salud Carlos III, under Project ref. DTS24/000237, PI22/00030, and PI23/00261 co-funded by the European Regional Development Fund, “Investing in your future,” Grant TED2021-129248 BeI00 the Spanish Ministerio de Ciencia e Innovación (MICINN) Projects PID2020-118145RB-I00 and TED2021-129248B-100, co-funded by the European Union FEDER funds; the Gobierno Regional de Cantabria and IDIVAL for the project Refs INNVAL21/19, NEXTVAL 22/12, and AR IDI-020-022 fellowship; N.N. held a predoctoral contract grant (PRE2021-097856). We also thank Oligonucleotide synthesis was performed by the ICTS ‘‘NANBIOSIS” (CIBER BBN) and specifically by the https://www.nanbiosis.es/portfolio/u29-oligonucleotide-synthesis-platform-osp/ oligonucleotide synthesis platform (OSP) U29 at IQAC-CSIC. We gratefully acknowledge Ms. Débora Muñoz and Claudia Zamarrón for their technical help, Drs. Iñigo Casafont and Juan Carlos Acosta for their assistance with the senescence experiments, and Dr. Rafel Valiente for his help with fluorimetry experiments. The figures and graphs have been created with BioRender software (BioRender.com, License ID: 9519A1C8-0002). We would like to express our gratitude to the COST Action DARTER 17103 for providing a collaborative environment that facilitated the development of this research. References [1]. ↵ D. B. Longley , D. P. Harkin , P. G. Johnston , Nature Reviews Cancer 2003 3:5 2003 , 3 , 330 . OpenUrl CrossRef PubMed Web of Science [2]. ↵ R. B. Diasio , B. E. 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