Osteoblast-specific down-regulation of NLRP3 inflammasome by aptamer-functionalized liposome nanoparticles improves bone quality in postmenopausal osteoporosis rats.

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood. Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3). Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1ß and IL-18 and increasing the levels of OCN and Runx2. Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.

Development, Optimization, and in vitro Evaluation of Silybin-loaded PLGA Nanoparticles and Decoration with 5TR1 Aptamer for Targeted Delivery to Colorectal Cancer Cells.

Chemotherapeutic agents often lack specificity, intratumoral accumulation, and face drug resistance. Targeted drug delivery systems based on nanoparticles (NPs) mitigate these issues. Poly (lactic-co-glycolic acid) (PLGA) is a well-studied polymer, commonly modified with aptamers (Apts) for cancer diagnosis and therapy. In this study, silybin (SBN), a natural agent with established anticancer properties, was encapsulated into PLGA NPs to control delivery and improve its poor solubility. The field-emission scanning electron microscopy (FE-SEM) showed spherical and uniform morphology of optimum SBN-PLGA NPs with 138.57±1.30nm diameter, 0.202±0.004 polydispersity index (PDI), -16.93±0.45mV zeta potential (ZP), and 70.19±1.63% entrapment efficiency (EE). The results of attenuated total reflectance-Fourier transform infrared (ATR-FTIR) showed no chemical interaction between formulation components, and differential scanning calorimetry (DSC) thermograms confirmed efficient SBN entrapment in the carrier. Then, the optimum formulation was functionalized with 5TR1 Apt for active targeted delivery of SBN to colorectal cancer (CRC) cells in vitro. The SBN-PLGA-5TR1 nanocomplex released SBN at a sustained and constant rate (zero-order kinetic), favoring passive delivery to acidic CRC environments. The MTT assay demonstrated the highest cytotoxicity of the SBN-PLGA-5TR1 nanocomplex in C26 and HT29 cells and no significant cytotoxicity in normal cells. Apoptosis analysis supported these results, showing early apoptosis induction with SBN-PLGA-5TR1 nanocomplex which indicated this agent could cause programmed death more than necrosis. This study presents the first targeted delivery of SBN to cancer cells using Apts. The SBN-PLGA-5TR1 nanocomplex effectively targeted and suppressed CRC cell proliferation, providing valuable insights into CRC treatment without harmful effects on healthy tissues.

Aptamer-Drug conjugates for a targeted and synergistic anticancer Response: Exploiting T30923-5-fluoro-2'-deoxyuridine (INT-FdU) derivatives.

One of the most appealing approaches for cancer treatment is targeted therapy, which is based on the use of drugs able to target cancer cells without affecting normal ones. This strategy lets to overcome the major limitation of conventional chemotherapy, namely the lack of specificity of anticancer drugs, which often leads to severe side effects, decreasing the therapy effectiveness. Delivery of cell-killing substances to tumor cells is one-way targeted drug therapy can work. Generally, monoclonal antibodies are combined with chemotherapeutic drugs, allowing cellular uptake through the binding to their targets on the surface of cancer cells. Aptamer-drug conjugates represent a promising alternative solution to antibodies to minimize off-target effects, considering the remarkable selective binding capabilities of aptamers. In this study, to enhance the therapeutic efficacy of the antineoplastic agent 5-fluoro-2'-deoxyuridine (FdU) in various cancer cells, we focused on the development of a novel conjugate using the antiproliferative aptamer T30923 (INT) as a drug vehicle. Three derivatives composed of T30923 conjugated with a different number of FdU units were synthesized, and their structural and biological properties were thoroughly characterized, highlighting their potential for targeted and synergistic anticancer responses.

Targeted delivery of SN38 to breast cancer using amphiphilic diblock copolymers PHPMA-b-PBAEM as micellar carriers with AS1411 aptamer.

Breast cancer treatment can be challenging, but a targeted drug delivery system (DDS) has the potential to make it more effective and reduce side effects. This study presents a novel nanotherapeutic targeted DDS developed through the self-assembly of an amphiphilic di-block copolymer to deliver the chemotherapy drug SN38 specifically to breast cancer cells. The vehicle was constructed from the PHPMA-b-PEAMA diblock copolymer synthesized via RAFT polymerization. A single emulsion method was then used to encapsulate SN38 within nanoparticles (NPs) formed from the PHPMA-b-PEAMA copolymer. The AS1411 DNA aptamer was covalently bonded to the surface of the micellar NPs, producing a targeted DDS. Molecular dynamics (MD) simulation studies were also performed on the di block polymeric system, demonstrating that SN38 interacted well with the di block. The in vitro results demonstrated that AS1411- decorated SN38-loaded HPMA NPs were highly toxic to breast cancer cells while having a minimal effect on non-cancerous cells. Remarkably, in vivo studies elucidated the ability of the targeted DDS to enhance the antitumor effect of SN38, suppressing tumor growth and improving survival rates compared to free SN38.

L-RNA aptamer-based CXCL12 inhibition combined with radiotherapy in newly-diagnosed glioblastoma: dose escalation of the phase I/II GLORIA trial.

The chemokine CXCL12 promotes glioblastoma (GBM) recurrence after radiotherapy (RT) by facilitating vasculogenesis. Here we report outcomes of the dose-escalation part of GLORIA (NCT04121455), a phase I/II trial combining RT and the CXCL12-neutralizing aptamer olaptesed pegol (NOX-A12; 200/400/600 mg per week) in patients with incompletely resected, newly-diagnosed GBM lacking MGMT methylation. The primary endpoint was safety, secondary endpoints included maximum tolerable dose (MTD), recommended phase II dose (RP2D), NOX-A12 plasma levels, topography of recurrence, tumor vascularization, neurologic assessment in neuro-oncology (NANO), quality of life (QOL), median progression-free survival (PFS), 6-months PFS and overall survival (OS). Treatment was safe with no dose-limiting toxicities or treatment-related deaths. The MTD has not been reached and, thus, 600 mg per week of NOX-A12 was established as RP2D for the ongoing expansion part of the trial. With increasing NOX-A12 dose levels, a corresponding increase of NOX-A12 plasma levels was observed. Of ten patients enrolled, nine showed radiographic responses, four reached partial remission. All but one patient (90%) showed at best response reduced perfusion values in terms of relative cerebral blood volume (rCBV). The median PFS was 174 (range 58-260) days, 6-month PFS was 40.0% and the median OS 389 (144-562) days. In a post-hoc exploratory analysis of tumor tissue, higher frequency of CXCL12+ endothelial and glioma cells was significantly associated with longer PFS under NOX-A12. Our data imply safety of NOX-A12 and its efficacy signal warrants further investigation.

AS1411 aptamer/RGD dual functionalized theranostic chitosan-PLGA nanoparticles for brain cancer treatment and imaging.

Conventional chemotherapy and poor targeted delivery in brain cancer resulting to poor treatment and develop resistance to anticancer drugs. Meanwhile, it is quite challenging to diagnose/detection of brain tumor at early stage of cancer which resulting in severity of the disease. Despite extensive research, effective treatment with real-time imaging still remains completely unavailable, yet. In this study, two brain cancer cell specific moieties i.e., AS1411 aptamer and RGD are decorated on the surface of chitosan-PLGA nanoparticles to improve targeted co-delivery of docetaxel (DTX) and upconversion nanoparticles (UCNP) for effective brain tumor therapy and real-time imaging. The nanoparticles were developed by a slightly modified emulsion/solvent evaporation method. This investigation also translates the successful synthesis of TPGS-chitosan, TPGS-RGD and TPGS-AS1411 aptamer conjugates for making PLGA nanoparticle as a potential tool of the targeted co-delivery of DTX and UCNP to the brain cancer cells. The developed nanoparticles have shown an average particle size <200 nm, spherical in shape, high encapsulation of DTX and UCNP in the core of nanoparticles, and sustained release of DTX up to 72 h in phosphate buffer saline (pH 7.4). AS1411 aptamer and RGD functionalized theranostic chitosan-PLGA nanoparticles containing DTX and UCNP (DUCPN-RGD-AS1411) have achieved greater cellular uptake, 89-fold improved cytotoxicity, enhanced cancer cell arrest even at lower drug conc., improved bioavailability with higher mean residence time of DTX in systemic circulation and brain tissues. Moreover, DUCPN-RGD-AS1411 have greatly facilitated cellular internalization and higher accumulation of UCNP in brain tissues. Additionally, DUCPN-RGD-AS1411 demonstrated a significant suppression in tumor growth in brain-tumor bearing xenograft BALB/c nude mice with no impressive sign of toxicities. DUCPN-RGD-AS1411 has great potential to be utilized as an effective and safe theranostic tool for brain cancer and other life-threatening cancer therapies.

Posterior eye delivery of angiogenesis-inhibiting RNA nanoparticles via subconjunctival injection.

Neovascularization contributes to various posterior eye segment diseases such as age-related macular degeneration and diabetic retinopathy. RNA nanoparticles were demonstrated previously to enter the corneal and retinal cells after subconjunctival injection for ocular delivery. In the present study, antiangiogenic aptamers (anti-vascular endothelial growth factor (VEGF) and anti-angiopoietin-2 (Ang2) aptamers) were conjugated to RNA nanoparticles. The objectives were to investigate the clearance and distribution of these angiogenesis-inhibiting RNA nanoparticles after subconjunctival injection in vivo and their antiangiogenic effects for inhibiting ocular neovascularization in vitro. The results in the whole-body fluorescence imaging study showed that the clearance of RNA nanoparticles was size-dependent with no significant differences between RNA nanoparticles with and without the aptamers except for pRNA-3WJ. The distribution study of RNA nanoparticles by confocal microscopy of the dissected eye tissues in vivo indicated cell internalization of the larger RNA nanoparticles in the retina and retinal pigment epithelium after subconjunctival injection, and the larger nanoparticles with aptamers showed higher levels of cell internalization than those without. In the cell proliferation assay in vitro, RNA nanoparticles with multiple aptamers had higher antiangiogenic effects. With both longer retention time and high antiangiogenic effect, SQR-VEGF-Ang2 could be a promising RNA nanoparticle for posterior eye delivery.

Aptamer as a targeted approach towards treatment of breast cancer.

Aptamers, a novel type of targeted ligand used in drug delivery, have quickly gained popularity due to their high target specificity and affinity. Different aptamer-mediated drug delivery systems, such as aptamer-drug conjugate (ApDC), aptamer-siRNA, and aptamer-functionalised nanoparticle systems, are currently being developed for the successful treatment of cancer based on the excellent properties of aptamers. These systems can decrease potential toxicity and enhance therapeutic efficacy by targeting the drug moiety. In this review, we provide an overview of recent developments in aptamer-mediated delivery systems for cancer therapy, specifically for breast cancer, and talk about the potential applications and current issues of novel aptamer-based techniques. This study in aptamer technology for breast cancer therapy highlights key aptamers targeting well-established biomarkers such as HER2, oestrogen receptor, and progesterone receptor. Additionally, we explore the potential of aptamers in overcoming various challenges such as drug resistance and improving the delivery of therapeutic agents. This review aims to provide a deeper understanding of the present aptamer-based targeted delivery applications through in-depth analysis to increase efficacy and create new therapeutic approaches that may ultimately lead to better treatment outcomes for cancer patients.

Improving Chemotherapy Effectiveness: Utilizing CuS Nanoparticles Coated with AS1411 Aptamer and Chitosan for Targeted Delivery of Doxorubicin to Cancerous Cells.

Here, a novel targeted nanostructure complex was designed as an alternative to the traditional treatment approaches for breast cancer. A delivery system utilizing CuS nanoparticles (CuS NPs) was developed for the purpose of targeted administration of doxorubicin (Dox), an anticancer agent. To regulate Dox release, chitosan (CS), a biodegradable and hydrophilic polymer with biocompatible properties, was applied to coat the Dox-loaded CuS NPs. Furthermore, AS1411 aptamer, served as a targeting agent for breast cancer cells (MCF-7 and 4T1 cells), was conjugated with CS-Dox-CuS NPs effectively. To assess the effectiveness of APT-CS-CuS NPs, various methods such as flow cytometry analysis, MTT assay, fluorescence imaging, and in vivo antitumor efficacy were employed. The hollow core and porous surface of CuS NPs improved the Dox loading capacity and entrapment efficiency (almost 100%). The rate of drug release at the tumor site (citrate buffer with pH 5.6) exhibited a marked increase in comparison to that observed within the physiological environment (phosphate buffer with pH 7.4). The targeted formulation (APT-CS-Dox-CuS NPs) significantly increased cytotoxicity of the Dox payload in target cells, including 4T1 (p ≤ 0.0001 (****)) and MCF7 (p ≤ 0.01 (**)) cells compared to CHO cells. Moreover, the ability of tumor growth inhibition of the targeted system was significantly (p ≤ 0.05 (*)) more than free Dox in tumor-bearing mice. The findings indicate that the targeted formulation augmented effectiveness and specificity while minimizing harm to non-targeted cells, signifying its potential as a sophisticated cancer drug delivery system.

Targeted co-delivery of FOXM1 aptamer and DOX by nucleolin aptamer-functionalized pH-responsive biocompatible nanodelivery system to enhance therapeutic efficacy against breast cancer: in vitro and in vivo.

Targeted nanodelivery systems offer a promising approach to cancer treatment, including the most common cancer in women, breast cancer. In this study, a targeted, pH-responsive, and biocompatible nanodelivery system based on nucleolin aptamer-functionalized biogenic titanium dioxide nanoparticles (TNP) was developed for targeted co-delivery of FOXM1 aptamer and doxorubicin (DOX) to improve breast cancer therapy. The developed targeted nanodelivery system exhibited almost spherical morphology with 124.89 ± 12.97 nm in diameter and zeta potential value of - 23.78 ± 3.66 mV. FOXM1 aptamer and DOX were loaded into the nanodelivery system with an efficiency of 100% and 97%, respectively. Moreover, the targeted nanodelivery system demonstrated excellent stability in serum and a pH-responsive sustained drug release profile over a period of 240 h following Higuchi kinetic and Fickian diffusion mechanism. The in vitro cytotoxicity experiments demonstrated that the targeted nanodelivery system provided selective internalization and strong growth inhibition effects of about 45 and 51% against nucleolin-positive 4T1 and MCF-7 breast cancer cell lines. It is noteworthy that these phenomena were not observed in nucleolin-negative cells (CHO). The preclinical studies revealed that a single-dose intravenous injection of the targeted nanodelivery system into 4T1-bearing mice inhibited tumor growth by 1.7- and 1.4-fold more efficiently than the free drug and the non-targeted nanodelivery system, respectively. Our results suggested that the developed innovative targeted pH-responsive biocompatible nanodelivery system could serve as a prospectively potential platform to improve breast cancer treatment.

Utilizing RNA nanotechnology to construct negatively charged and ultrasound-responsive nanodroplets for targeted delivery of siRNA.

Ultrasound nanodroplets (NDs) have been reported as a promising nanocarrier for siRNA delivery depending on its unique strengths of sonoporation. Presently, common means for NDs-mediated siRNA delivery is through electrostatic interaction, but challenges like cationic toxicity still exist. In this study, we demonstrated a novel strategy to construct negatively charged and ultrasound (US)-responsive O-carboxymethyl chitosan (O-CMS) NDs as a siRNA targeted delivery system through three-way junction of bacteriophage phi29 DNA packaging motor (3WJ-pRNA) nanotechnology. 39nt A10-3.2 aptamer targeting prostate specific membrane antigen (PSMA) and 21nt siRNA against cationic amino acid transporter 1 (siCAT-1) were annealed to 3WJ-pRNA scaffold via complementation with an extended sequence. The cholesterol molecule attached to one branch facilitates the 3WJ-pRNA nanoparticles anchoring onto NDs. The desired O-CMS NDs with siRNA-loading and RNA-aptamer modification (A10-3.2/siCAT-1/3WJ-NDs) were successfully prepared, which were with spherical shapes, core-shell structures and uniform in sizes (198 nm with PDI 0.3). As a main proportion of shell, O-CMC showed a certain anti-tumor effects. In vitro studies demonstrated that A10-3.2/siCAT-1/3WJ-NDs exhibited good contrast-enhanced US imaging, buffering capacity and high bio-safety, were able to deliver siCAT-1 to PSMA-overexpressed prostate cancer cells under US irradiation, thus silence the CAT-1 expression, and consequently suppressing 22RV1 cell proliferation and migration. Taken overall, our findings provide a promising strategy to develop negatively charged and US-responsive NDs for tumor-targeted siRNA delivery.

Aptamer-Aptamer Chimera for Targeted Delivery and ATP-Responsive Release of Doxorubicin into Cancer Cells.

Aptamers offer a great opportunity to develop innovative drug delivery systems that can deliver cargos specifically into targeted cells. In this study, a chimera consisting of two aptamers was developed to deliver doxorubicin into cancer cells and release the drug in cytoplasm in response to adenosine-5'-triphosphate (ATP) binding. The chimera was composed of the AS1411 anti-nucleolin aptamer for cancer cell targeting and the ATP aptamer for loading and triggering the release of doxorubicin in cells. The chimera was first produced by hybridizing the ATP aptamer with its complementary DNA sequence, which is linked with the AS1411 aptamer via a poly-thymine linker. Doxorubicin was then loaded inside the hybridized DNA region of the chimera. Our results show that the AS1411-ATP aptamer chimera was able to release loaded doxorubicin in cells in response to ATP. In addition, selective uptake of the chimera into cancer cells was demonstrated using flow cytometry. Furthermore, confocal laser scanning microscopy showed the successful delivery of the doxorubicin loaded in chimeras to the nuclei of targeted cells. Moreover, the doxorubicin-loaded chimeras effectively inhibited the growth of cancer cell lines and reduced the cytotoxic effect on the normal cells. Overall, the results of this study show that the AS1411-ATP aptamer chimera could be used as an innovative approach for the selective delivery of doxorubicin to cancer cells, which may improve the therapeutic potency and decrease the off-target cytotoxicity of doxorubicin.

AS1411 Nucleolin-Specific Binding Aptamers Reduce Pathological Angiogenesis through Inhibition of Nucleolin Phosphorylation.

Proliferative retinopathies produces an irreversible type of blindness affecting working age and pediatric population of industrialized countries. Despite the good results of anti-VEGF therapy, intraocular and systemic complications are often associated after its intravitreal use, hence novel therapeutic approaches are needed. The aim of the present study is to test the effect of the AS1411, an antiangiogenic nucleolin-binding aptamer, using in vivo, ex vivo and in vitro models of angiogenesis and propose a mechanistic insight. Our results showed that AS1411 significantly inhibited retinal neovascularization in the oxygen induced retinopathy (OIR) in vivo model, as well as inhibited branch formation in the rat aortic ex vivo assay, and, significantly reduced proliferation, cell migration and tube formation in the HUVEC in vitro model. Importantly, phosphorylated NCL protein was significantly abolished in HUVEC in the presence of AS1411 without affecting NFκB phosphorylation and -21 and 221-angiomiRs, suggesting that the antiangiogenic properties of this molecule are partially mediated by a down regulation in NCL phosphorylation. In sum, this new research further supports the NCL role in the molecular etiology of pathological angiogenesis and identifies AS1411 as a novel anti-angiogenic treatment.

Selection of DNA Aptamers Recognizing EpCAM-Positive Prostate Cancer by Cell-SELEX for in vitro and in vivo MR Imaging.

BACKGROUND: The sensitive and specific detection of pathogenic cells is important in tumor diagnosis at an early stage. Aptamers are short single-stranded oligonucleotides evolved from systematic evolution of ligands by exponential enrichment (SELEX). It has been proved that aptamers can interact with cognate target molecules with high affinity and specificity and have great potential in the development of medical imaging at molecular level. PURPOSE: To select epithelial cell adhesion molecule (EpCAM) specific aptamers targeting prostate cancer and further to conjugate aptamers with GoldMag nanoparticles (a typical iron oxide core/gold shell structure) to construct magnetic molecular probes for medical imaging. METHODS: EpCAM-specific aptamers were selected by Cell-SELEX. The enrichment of specific aptamer candidates was monitored by flow cytometric analysis. Aptamers were further conjugated with GoldMag nanoparticles to construct magnetic molecular probes. The affinity and specificity of aptamer candidates and aptamer-conjugated GoldMag nanoparticles were evaluated. The MR imaging of aptamer-conjugated GoldMag nanoparticles to prostate cancer was further explored in vitro and in vivo. RESULTS: After 12 rounds of selection, aptamer candidates Eppc6 and Eppc14 could specifically target three types of prostate cancer cells, revealing a high affinity of Eppc6 and Eppc14. Moreover, aptamer-conjugated GoldMag nanoparticles not only exhibited good affinity to different prostate cancer cells but also produced strong T2WI signal intensity reduction distinguished from peritumoral tissue in MRI, indicating that the molecular probes possess both the affinity properties of EpCAM-specific aptamer and the superparamagnetic features of iron oxide. CONCLUSION: Our study indicates that aptamer Eppc6 and Eppc14 can recognize prostate cancer cells and tissues. The aptamer-conjugated GoldMag nanoparticles constructed in the study can be used as a molecular imaging agent for detection of PCa in MRI.

CRYßB2 enhances tumorigenesis through upregulation of nucleolin in triple negative breast cancer.

Expression of ß-crystallin B2 (CRYßB2) is elevated in African American (AA) breast tumors. The underlying mechanisms of CRYßB2-induced malignancy and the association of CRYßB2 protein expression with survival have not yet been described. Here, we report that the expression of CRYßB2 in breast cancer cells increases stemness, growth, and metastasis. Transcriptomics data revealed that CRYßB2 upregulates genes that are functionally associated with unfolded protein response, oxidative phosphorylation, and DNA repair, while down-regulating genes related to apoptosis. CRYßB2 in tumors promotes de-differentiation, an increase in mesenchymal markers and cancer-associated fibroblasts, and enlargement of nucleoli. Proteome microarrays identified a direct interaction between CRYßB2 and the nucleolar protein, nucleolin. CRYßB2 induces nucleolin, leading to the activation of AKT and EGFR signaling. CRISPR studies revealed a dependency on nucleolin for the pro-tumorigenic effects of CRYßB2. Triple-negative breast cancer (TNBC) xenografts with upregulated CRYßB2 are distinctively sensitive to the nucleolin aptamer, AS-1411. Lastly, in AA patients, higher levels of nucleolar CRYßB2 in primary TNBC correlates with decreased survival. In summary, CRYßB2 is upregulated in breast tumors of AA patients and induces oncogenic alterations consistent with an aggressive cancer phenotype. CRYßB2 increases sensitivity to nucleolin inhibitors and may promote breast cancer disparity.

Tumor-Specific Delivery of 5-Fluorouracil-Incorporated Epidermal Growth Factor Receptor-Targeted Aptamers as an Efficient Treatment in Pancreatic Ductal Adenocarcinoma Models.

BACKGROUNDS & AIMS: Fluoropyrimidine c (5-fluorouracil [5FU]) increasingly represents the chemotherapeutic backbone for neoadjuvant, adjuvant, and palliative treatment of pancreatic ductal adenocarcinoma (PDAC). Even in combination with other agents, 5FU efficacy remains transient and limited. One explanation for the inadequate response is insufficient and nonspecific delivery of 5FU to the tumor. METHODS: We designed, generated, and characterized 5FU-incorporated systematic evolution of ligands by exponential enrichment (SELEX)-selected epidermal growth factor receptor (EGFR)-targeted aptamers for tumor-specific delivery of 5FU to PDAC cells and tested their therapeutic efficacy in vitro and in vivo. RESULTS: 5FU-EGFR aptamers reduced proliferation in a concentration-dependent manner in mouse and human pancreatic cancer cell lines. Time-lapsed live imaging showed EGFR-specific uptake of aptamers via clathrin-dependent endocytosis. The 5FU-aptamer treatment was equally effective in 5FU-sensitive and 5FU-refractory PDAC cell lines. Biweekly treatment with 5FU-EGFR aptamers reduced tumor burden in a syngeneic orthotopic transplantation model of PDAC, in an autochthonously growing genetically engineered PDAC model (LSL-KrasG12D/+;LSL-Trp53flox/+;Ptf1a-Cre [KPC]), in an orthotopic cell line-derived xenograft model using human PDAC cells in athymic mice (CDX; Crl:NU-Foxn1nu), and in patient-derived organoids. Tumor growth was significantly attenuated during 5FU-EGFR aptamer treatment in the course of follow-up. CONCLUSIONS: Tumor-specific targeted delivery of 5FU using EGFR aptamers as the carrier achieved high target specificity; overcame 5FU resistance; and proved to be effective in a syngeneic orthotopic transplantation model, in KPC mice, in a CDX model, and in patient-derived organoids and, therefore, represents a promising backbone for pancreatic cancer chemotherapy in patients. Furthermore, our approach has the potential to target virtually any cancer entity sensitive to 5FU treatment by incorporating 5FU into cancer cell-targeting aptamers as the delivery platform.

Novel CD123 polyaptamer hydrogel edited by Cas9/sgRNA for AML-targeted therapy.

CD123 targeting molecules have been widely applied in acute myelocytic leukemia (AML) therapeutics. Although antibodies have been more widely used as targeting molecules, aptamer have unique advantages for CD123 targeting therapy. In this study, we constructed an aptamer hydrogel termed as SSFH which could be precisely cut by Cas9/sgRNA for programmed SS30 release. To construct hydrogel, rolling-circle amplification (RCA) was used to generate hydrogel containing CD123 aptamer SS30 and sgRNA-targeting sequence. After incubation with Cas9/sgRNA, SSFH could lose its gel property and liberated the SS30 aptamer sequence, and released SS30 has been confirmed by gel electrophoresis. In addition, SS30 released from SSFH could inhibit cell proliferation and induce cell apoptosis in vitro. Moreover, SSFH could prolong survival rate and inhibit tumor growth via JAK2/STAT5 signaling pathway in vivo. Additionally, molecular imaging revealed SSFH co-injected with Cas9/sgRNA remained at the injection site longer than free aptamer. Furthermore, once the levels of cytokines were increasing, the complementary sequences of aptamers injection could neutralize SS30 and relieve side effect immediately. This study suggested that CD123 aptamer hydrogel SSFH and Cas9/sgRNA system has strong potential for CD123-positive AML anticancer therapy.

Preparation, characterization, and in vitro tumor-suppressive effect of anti-miR-21-equipped RNA nanoparticles.

MicroRNAs play an irreplaceable role in gene expression regulation. Upregulation of several miRNAs increases the risk of invasion and metastasis of breast cancer cells. An oncogenic miRNA, miR-21, is highly expressed in triple-negative breast cancer (TNBC) and is associated with tumor proliferation, invasion, carcinogenesis, prognosis, and therapeutic resistance. However, targeted delivery of therapeutic anti-miRNAs into cancer cells remains challenging, especially for TNBC. In this study, we report the application of an RNA nanotechnology-based platform for the targeted delivery of anti-miR-21 by epidermal growth factor receptor (EGFR) aptamer in vitro to TNBC and chemical-resistant breast cancer cells. RNA nanoparticles reduced cell viability and sensitized breast cancer cells to doxorubicin (DOX) treatment in vitro. Inhibition of miR-21 by RNA nanoparticles suppressed TNBC cell invasion, migration, and colony formation. The results indicate the potential application of nanotechnology-based delivery platforms in clinical anti-cancer therapeutics.

Nucleolin-Targeting AS1411 Aptamer-Modified Micelle for the Co-Delivery of Doxorubicin and miR-519c to Improve the Therapeutic Efficacy in Hepatocellular Carcinoma Treatment.

BACKGROUND: Multidrug resistance (MDR) has emerged to be a major hindrance in cancer therapy, which contributes to the reduced sensitivity of cancer cells toward chemotherapeutic drugs mainly owing to the over-expression of drug efflux transporters. The combination of gene therapy and chemotherapy has been considered as a potential approach to improve the anti-cancer efficacy by reversing the MDR effect. MATERIALS AND METHODS: The AS1411 aptamer-functionalized micelles were constructed through an emulsion/solvent evaporation strategy for the simultaneous co-delivery of doxorubicin and miR-519c. The therapeutic efficacy and related mechanism of micelles were explored based on the in vitro and in vivo active targeting ability and the suppression of MDR, using hepatocellular carcinoma cell line HepG2 as a model. RESULTS: The micelle was demonstrated to possess favorable cellular uptake and tumor penetration ability by specifically recognizing the nucleolin in an AS1411 aptamer-dependent manner. Further, the intracellular accumulation of doxorubicin was significantly improved due to the suppression of ABCG2-mediated drug efflux by miR-519c, resulting in the efficient inhibition of tumor growth. CONCLUSION: The micelle-mediated co-delivery of doxorubicin and miR-519c provided a promising strategy to obtain ideal anti-cancer efficacy through the active targeting function and the reversion of MDR.

Effect of Topical Instillation of Pegaptanib Sodium Upon Inflammatory Corneal Neovascularization in Rabbits.

Purpose: To evaluate the effect of topical instillation of pegaptanib sodium upon inflammatory angiogenesis induced in the rabbit cornea by alkaline cauterization. Methods: Inflammatory angiogenesis was induced by alkaline (sodium hydroxide) cauterization in the corneas of 29 male New Zealand rabbits. The animals were divided into 4 groups: a control group treated with 0.5% carboxymethylcellulose sodium eye drops, a group treated with 1.0% prednisolone acetate eye drops, a group treated with 0.5% pegaptanib sodium diluted in 15 mL 0.5% carboxymethylcellulose sodium, and a group treated with 1.0% pegaptanib sodium diluted in 15 mL 0.5% carboxymethylcellulose sodium. After cauterization, eye drops were administered every 12 hours for 21 days. The animals were evaluated every 3 days after cauterization, and the newly formed vessels were quantified from photographs. The treatment effectiveness was analyzed with 3 parameters of antiangiogenic response: neovascularization area (NA), total vascular length (TVL), and number of blood vessels (BVN). Results: Average NA, TVL, and BVN values were significantly higher in both pegaptanib groups than in the prednisolone group. A nonstatistically significant reduction in parameters on days 18 and 21 was the minimum achieved in both pegaptanib groups. The efficacy of the treatments in relation to the control was significantly greater in the prednisolone group than in the 0.5% pegaptanib group or the 1.0% pegaptanib group (P < 0.001). Conclusion: Topical instillation of 0.5% and 1.0% pegaptanib sodium diluted in 15 mL 0.5% carboxymethylcellulose sodium had no inhibitory effect on corneal neovascularization in this rabbit model.