Structure of the Respiratory Syncytial Virus Polymerase Complex.

Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors.

Deciphering the role of Enterococcus faecium cytidine deaminase in gemcitabine resistance of gallbladder cancer.

Gemcitabine-based chemotherapy is a cornerstone of standard care for gallbladder cancer (GBC) treatment. Still, drug resistance remains a significant challenge, influenced by factors such as tumor-associated microbiota impacting drug concentrations within tumors. Enterococcus faecium, a member of tumor-associated microbiota, was notably enriched in the GBC patient cluster. In this study, we investigated the biochemical characteristics, catalytic activity, and kinetics of the cytidine deaminase of E. faecium (EfCDA). EfCDA showed the ability to convert gemcitabine to its metabolite 2',2'-difluorodeoxyuridine. Both EfCDA and E. faecium can induce gemcitabine resistance in GBC cells. Moreover, we determined the crystal structure of EfCDA, in its apo form and in complex with 2', 2'-difluorodeoxyuridine at high resolution. Mutation of key residues abolished the catalytic activity of EfCDA and reduced the gemcitabine resistance in GBC cells. Our findings provide structural insights into the molecular basis for recognizing gemcitabine metabolite by a bacteria CDA protein and may provide potential strategies to combat cancer drug resistance and improve the efficacy of gemcitabine-based chemotherapy in GBC treatment.

Gemcitabine-Phospholipid Complex Loaded Lipid Nanoparticles for Improving Drug Loading, Stability, and Efficacy against Pancreatic Cancer.

This study aims to encapsulate gemcitabine (GEM) using a phospholipid complex (PLC) in lipid nanoparticles (NPs) to achieve several desirable outcomes, including high drug loading, uniform particle size, improved therapeutic efficacy, and reduced toxicities. The successful preparation of GEM-loaded lipid NPs (GEM-NPs) was accomplished using the emulsification-solidification method, following optimization through Box-Behnken design. The size of the GEM-NP was 138.5 ± 6.7 nm, with a low polydispersity index of 0.282 ± 0.078, as measured by a zetasizer and confirmed by transmission electron and atomic force microscopy. GEM-NPs demonstrated sustained release behavior, surpassing the performance of the free GEM and phospholipid complex. Moreover, GEM-NPs exhibited enhanced cytotoxicity, apoptosis, and cell uptake in Panc-2 and Mia PaCa cells compared to the free GEM. The in vivo pharmacokinetics revealed approximately 4-fold higher bioavailability of GEM-NPs in comparison with free GEM. Additionally, the pharmacodynamic evaluation conducted in a DMBA-induced pancreatic cancer model, involving histological examination, serum IL-6 level estimation, and expression of cleaved caspase-3, showed the potential of GEM-NPs in the management of pancreatic cancer. Consequently, the lipid NP-based approach developed in our investigation demonstrates high stability and uniformity and holds promise for enhancing the therapeutic outcomes of GEM.

Recognition of 8-Oxo-2'-deoxyguanosine in DNA Using the Triphosphate of 2'-Deoxycytidine Connecting the 1,3-Diazaphenoxazine Unit, dCdapTP.

DNA is constantly damaged by various external and internal factors. In particular, oxidative damage occurs in a steady state, and 8-oxo-2'-deoxyguanosine (oxodG) is known as the main oxidative damage. OxodG is a strong genotoxic nucleoside and is thought to be involved in the pathogenesis of cancer and neurological diseases. However, a breakthrough method to detect the position of oxodG in DNA has not yet been developed. Therefore, we attempted to develop a novel method to detect oxodG in DNA using artificial nucleosides. Recently, we have succeeded in the recognition of oxodG in DNA by a single nucleotide elongation reaction using nucleoside derivatives based on a purine skeleton with a 1,3-diazaphenoxazine unit. In this study, we developed a new nucleoside derivative with a pyrimidine skeleton in order to further improve the recognition ability and enzymatic reaction efficiency. We, therefore, designed and synthesized 2'-deoxycytidine-1,3-diazaphenoxazine (Cdap) and its triphosphate derivatives. The results showed that it was incorporated into the primer strand relative to the dG template because of its cytidine skeleton, but it was more effective at the complementary position of the oxodG template. These results indicate that the new nucleoside derivative can be considered as one of the new candidates for the detection of oxodG in DNA.

Fabrication of dopamine conjugated with protein @metal organic framework for targeted drug delivery: A biocompatible pH-Responsive nanocarrier for gemcitabine release on MCF­7 human breast cancer cells.

Metal-organic structures (MOF), modern extremely proliferous materials consisting of metal ions and organic coordinating molecules, has become a promising biomedical material because of its unusual features, including great surface area, wide pore volume, flexible functionality and superior performance for drug loading. In the current investigation, Gemcitabine Hydrochloride (Gem), an anticancer drug, and Amygdalin (Amy) were loaded into a nanocomposite structure formed from bovine serum albumin (BSA) as a center and zeolytic imidazolate framework-8 (ZIF-8) as a pH sensitive protective coating. The formed BSA-Gem@ZIF-8 and BSA-Gem-Amy@ZIF-8 were successively coated by polydopamine, chelated by Au3+ and conjugated via gallic acid (GA), acquired ZIF-8 structure as a multifunctional nanocarrier at the end. It was confirmed by different characterization methods that the nanocarrier was successfully produced. Due to the nature of ZIF-8, pH dependent releases of BSA-Gem@ZIF-8/Dopa/GA and BSA-Gem-Amy@ZIF-8/Dopa/GA were observed in in vitro studies. Cytotoxicity and apoptotic effects of these nanocarriers were evaluated using WST-1 and acridine orange staining in MCF-7 human breast cancer and HUVEC control cell lines. In-vitro cytotoxicity studies showed that both BSA-Gem@ZIF-8/Dopa/GA and BSA-Gem-Amy@ZIF-8/Dopa/GA were more effective than gemcitabine alone in MCF-7 cells with less toxicity in HUVEC cells. Additionally, both pH-responsive nanocarriers induced more apoptotic cell death in MCF-7 cells. We therefore believe that the built multifunctional nanocarrier based on ZIF-8 could be an alternative therapeutic strategy the use of gemcitabine for cancer therapy.

Two Possible Strategies for Drug Modification of Gemcitabine and Future Contributions to Personalized Medicine.

Drug repurposing is an emerging strategy, which uses already approved drugs for new medical indications. One such drug is gemcitabine, an anticancer drug that only works at high doses since a portion is deactivated in the serum, which causes toxicity. In this review, two methods were discussed that could improve the anticancer effect of gemcitabine. The first is a chemical modification by conjugation with cell-penetrating peptides, namely penetratin, pVEC, and different kinds of CPP6, which mostly all showed an increased anticancer effect. The other method is combining gemcitabine with repurposed drugs, namely itraconazole, which also showed great cancer cell inhibition growth. Besides these two strategies, physiologically based pharmacokinetic models (PBPK models) are also the key for predicting drug distribution based on physiological data, which is very important for personalized medicine, so that the correct drug and dosage regimen can be administered according to each patient's physiology. Taking all of this into consideration, it is believed that gemcitabine can be repurposed to have better anticancer effects.

Supramolecular Organization of Polymer Prodrug Nanoparticles Revealed by Coarse-Grained Simulations.

Drug-polymer conjugates that can self-assemble into nanoparticles are promising drug delivery systems that improve the drug bioavailability and allow their controlled release. However, despite the possibility of reaching high drug loadings, the efficiency of the drug release, mediated by cleavage of the drug-polymer linker, is a key parameter to obtain significant anticancer activity. To overcome the limitations of experimental characterizations and to gain a better understanding of such systems, we conducted a coarse-grained molecular dynamics simulation study on four representative drug-polymer conjugates obtained by the "drug-initiated" method and studied their supramolecular organization upon self-assembly. The prodrugs were composed of either a gemcitabine or a paclitaxel anticancer drug, either a propanoate or a diglycolate linker, and a polyisoprene chain. Our simulations gave crucial information concerning the spatial organization of the different components (e.g., drug, linker, polymer, etc.) into the nanoparticles and revealed that the linkers are not fully accessible to the solvent. Notably, some cleavage sites were either poorly hydrated or partially solvated. These observations might account for the low efficiency of drug release from the nanoparticles, particularly when the linker is too short and/or not hydrophilic/solvated enough. We believe that our theoretical study could be adapted to other types of polymer prodrugs and could guide the design of new polymer prodrug nanoparticles with improved drug release efficiency.

Reactivity and DNA Damage by Independently Generated 2'-Deoxycytidin-N4-yl Radical.

Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2'-deoxycytidin-N4-yl radical (dC·) plays a role in DNA damage mediated by one electron oxidants, such as HOCl and ionizing radiation. However, the reactivity of dC· is not well understood. To reduce this knowledge gap, we photochemically generated dC· from a nitrophenyl oxime nucleoside and within chemically synthesized oligonucleotides from the same precursor. dC· formation is confirmed by transient UV-absorption spectroscopy in laser flash photolysis (LFP) experiments. LFP and duplex DNA cleavage experiments indicate that dC· oxidizes dG. Transient formation of the dG radical cation (dG+•) is observed in LFP experiments. Oxidation of the opposing dG in DNA results in hole transfer when the opposing dG is part of a dGGG sequence. The sequence dependence is attributed to a competition between rapid proton transfer from dG+• to the opposing dC anion formed and hole transfer. Enhanced hole transfer when less acidic O6-methyl-2'-deoxyguanosine is opposite dC· supports this proposal. dC· produces tandem lesions in sequences containing thymidine at the 5'-position by abstracting a hydrogen atom from the thymine methyl group. The corresponding thymidine peroxyl radical completes tandem lesion formation by reacting with the 5'-adjacent nucleotide. As dC· is reduced to dC, its role in the process is traceless and is only detectable because of the ability to independently generate it from a stable precursor. These experiments reveal that dC· oxidizes neighboring nucleotides, resulting in deleterious tandem lesions and hole transfer in appropriate sequences.

Gemcitabine Lipid Prodrugs: The Key Role of the Lipid Moiety on the Self-Assembly into Nanoparticles.

A small library of amphiphilic prodrugs has been synthesized by conjugation of gemcitabine (Gem) (a hydrophilic nucleoside analogue) to a series of lipid moieties and investigated for their capacity to spontaneously self-assemble into nanosized objects by simple nanoprecipitation. Four of these conjugates formed stable nanoparticles (NPs), while with the others, immediate aggregation occurred, whatever the tested experimental conditions. Whether such capacity could have been predicted based on the prodrug physicochemical features was a matter of question. Among various parameters, the hydrophilic-lipophilic balance (HLB) value seemed to hold a predictive character. Indeed, we identified a threshold value which well correlated with the tendency (or not) of the synthesized prodrugs to form stable nanoparticles. Such a hypothesis was further confirmed by broadening the analysis to Gem and other nucleoside prodrugs already described in the literature. We also observed that, in the case of Gem prodrugs, the lipid moiety affected not only the colloidal properties but also the in vitro anticancer efficacy of the resulting nanoparticles. Overall, this study provides a useful demonstration of the predictive potential of the HLB value for lipid prodrug NP formulation and highlights the need of their opportune in vitro screening, as optimal drug loading does not always translate in an efficient biological activity.

pH-Sensitive Nanodrug Carriers for Codelivery of ERK Inhibitor and Gemcitabine Enhance the Inhibition of Tumor Growth in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC), a metabolic disorder, remains one of the leading cancer mortality sources worldwide. An initial response to treatments, such as gemcitabine (GEM), is often followed by emergent resistance reflecting an urgent need for alternate therapies. The PDAC resistance to GEM could be due to ERK1/2 activity. However, successful ERKi therapy is hindered due to low ligand efficiency, poor drug delivery, and toxicity. In this study, to overcome these limitations, we have designed pH-responsive nanoparticles (pHNPs) with a size range of 100-150 nm for the simultaneous delivery of ERKi (SCH 772984) and GEM with tolerable doses. These pHNPs are polyethylene glycol (PEG)-containing amphiphilic polycarbonate block copolymers with tertiary amine side chains. They are systemically stable and capable of improving in vitro and in vivo drug delivery at the cellular environment's acidic pH. The functional analysis indicates that the nanomolar doses of ERKi or GEM significantly decreased the 50% growth inhibition (IC50) of PDAC cells when encapsulated in pHNPs compared to free drugs. The combination of ERKi with GEM displayed a synergistic inhibitory effect. Unexpectedly, we uncover that the minimum effective dose of ERKi significantly promotes GEM activities on PDAC cells. Furthermore, we found that pHNP-encapsulated combination therapy of ERKi with GEM was superior to unencapsulated combination drug therapy. Our findings, thus, reveal a simple, yet efficient, drug delivery approach to overcome the limitations of ERKi for clinical applications and present a new model of sensitization of GEM by ERKi with no or minimal toxicity.

A gemcitabine-based conjugate with enhanced antitumor efficacy by suppressing HIF-1α expression under hypoxia.

Hypoxia is one of the unique features of tumor physiology. Hypoxia inducible factor (HIF-1α), as a major transcription factor in response to hypoxia, has been considered as a promising tumor-specific target for anticancer therapy. The formation of a hypoxic microenvironment in tumors can decrease the curative effect of cytotoxic chemotherapeutic drugs. To promote the antitumor efficacy of chemotherapy by suppressing hypoxia, we designed and prepared a novel gemcitabine-based drug conjugate (GEM-5) containing a HIF-1α inhibitor (YC-1). As expected, GEM-5 showed excellent antiproliferative activity (IC50 = 0.03 µΜ under hypoxia) and remarkably induced the apoptosis of A2780 cells in vitro. Additionally, western blot analysis demonstrated that GEM-5 significantly down-regulated the expression of HIF-1α and up-regulated the expression of tumor suppressor p53. More importantly, GEM-5 effectively inhibited tumor growth in the A2780 xenograft mouse model and significantly ameliorated tumor hypoxia in vivo. This novel, simple, and effective strategy for overcoming tumor hypoxia and enhancing the antitumor effect of chemotherapeutic drugs has great potential in cancer therapy.

Anti-tumor effects and cell motility inhibition of the DN604-gemcitabine combined treatment in human bladder cancer models.

Bladder cancer is one of the major tumors for men in the world, in which therapy the combination of cisplatin and gemcitabine is still fist-line applied to treat with advanced or metastatic bladder cancer. In our early study, we developed a potential Pt(II) agent, DN604, which has anti-tumor effect as potent as cisplatin toward bladder cancers. Herein, we aim at investigating the combinatory application of DN604 with gemcitabine for bladder cancer treatment. In vitro studies proved that the combined treatment of DN604 and gemcitabine could limit cell proliferation by elevating the incidence of DNA damage induced apoptosis. Notably, further researches showed that the DN604-gemcitabine treatment suppressed cell autophagy to inhibit cell motility upon the ROS dependent p38 MAPK signaling pathway, explicating its better anti-tumor activity than single drug treatment or the cisplatin-gemcitabine treatment. In vivo tests confirmed that the DN604-gemcitabine treatment has superior anti-tumor activity with low toxicity to cisplatin or its combination with gemcitabine treatments. DN604 plus gemcitabine, is of great significance for the treatment with human bladder cancer. Our study has provided a potential combination treatment option.

Uric Acid as a Photosensitizer in the Reaction of Deoxyribonucleosides with UV Light of Wavelength Longer than 300 nm: Identification of Products from 2'-Deoxycytidine.

DNA reacts directly with UV light with a wavelength shorter than 300 nm. Although ground surface sunlight includes little of this short-wavelength UV light due to its almost complete absorption by the atmosphere, sunlight is the primary cause of skin cancer. Photosensitization by endogenous substances must therefore be involved in skin cancer development mechanisms. Uric acid is the final metabolic product of purines in humans, and is present at relatively high concentrations in cells and fluids. When a neutral mixed solution of 2'-deoxycytidine, 2'-deoxyguanosine, thymidine, and 2'-deoxyadenosine was irradiated with UV light with a wavelength longer than 300 nm in the presence of uric acid, all the nucleosides were consumed in a uric acid dose-dependent manner. These reactions were inhibited by the addition of radical scavengers, ethanol and sodium azide. Two products from 2'-deoxycytidine were isolated and identified as N4-hydroxy-2'-deoxycytidine and N4,5-cyclic amide-2'-deoxycytidine, formed by cycloaddition of an amide group from uric acid. A 15N-labeled uric acid, uric acid-1,3-15N2, having two 14N and two 15N atoms per molecule, produced N4,5-cyclic amide-2'-deoxycytidine containing both 14N and 15N atoms from uric acid-1,3-15N2. Singlet oxygen, hydroxyl radical, peroxynitrous acid, hypochlorous acid, and hypobromous acid generated neither N4-hydroxy-2'-deoxycytidine nor N4,5-cyclic amide-2'-deoxycytidine in the presence of uric acid. These results indicate that uric acid is a photosensitizer for the reaction of nucleosides by UV light with a wavelength longer than 300 nm, and that an unidentified radical derived from uric acid with a delocalized unpaired electron may be generated.

Intragenomic Decarboxylation of 5-Carboxy-2'-deoxycytidine.

Cellular DNA is composed of four canonical nucleosides (dA, dC, dG and T), which form two Watson-Crick base pairs. In addition, 5-methylcytosine (mdC) may be present. The methylation of dC to mdC is known to regulate transcriptional activity. Next to these five nucleosides, the genome, particularly of stem cells, contains three additional dC derivatives, which are formed by stepwise oxidation of the methyl group of mdC with the help of Tet enzymes. These are 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC). It is believed that fdC and cadC are converted back into dC, which establishes an epigenetic control cycle that starts with methylation of dC to mdC, followed by oxidation and removal of fdC and cadC. While fdC was shown to undergo intragenomic deformylation to give dC directly, a similar decarboxylation of cadC was postulated but not yet observed on the genomic level. By using metabolic labelling, we show here that cadC decarboxylates in several cell types, which confirms that both fdC and cadC are nucleosides that are directly converted back to dC within the genome by C-C bond cleavage.

Dual Gate-Controlled Therapeutics for Overcoming Bacterium-Induced Drug Resistance and Potentiating Cancer Immunotherapy.

The presence of bacteria in the tumor can cause cancer resistance to chemotherapeutics. To fight against bacterium-induced drug resistance, herein we design self-traceable nanoreservoirs that are simultaneously loaded with gemcitabine (an anticancer drug) and ciprofloxacin (an antibiotic) and are decorated with hyaluronic acid for active tumor targeting. The nanoreservoirs have a pH-sensitive gate and an enzyme-responsive gate that can be opened in the acidic and hyaluronidase-abundant tumor microenvironment to control drug release rates. Moreover, the nanoreservoirs can specifically target the tumor regions without eliciting evident toxicity to normal tissues, kill the intratumoral bacteria, and inhibit the tumor growth even in the presence of the bacteria. Unexpectedly, the nanoreservoirs can activate T cell-mediated immune responses through promoting antigen-presenting dendritic cell maturation and depleting immunosuppressive myeloid-derived suppressor cells in bacterium-infected tumors.

New Mechanism of Gemcitabine and Its Phosphates: DNA Polymerization Disruption via 3'-5' Exonuclease Inhibition.

Gemcitabine (dFdC), a modified deoxycytidine (dC) widely used in tumor treatment, is a prodrug that is phosphorylated to generate mono-, di-, and triphosphates. The triphosphate (dFdCTP) is incorporated into DNA to terminate DNA synthesis in cancer. Some incorporated dFdC nucleotides can be partially removed by the 3'-5' exonuclease activity, namely its editing function, and the others escape the editing. However, whether there is an active mechanism for dFdC to escape the editing remains unclear. We have first discovered that unlike dFdC, its mono-, di-, and triphosphates can inhibit the 3'-5' exonuclease of DNA polymerase I, suppress editing, and allow the active escaping mechanism, whereas its polymerase activity is not remarkably affected. As such, these phosphates can prevent the removal of the incorporated dFdC residue, thereby actively blocking DNA extension and synthesis. The inhibition efficiency of these phosphates follows the increased order of the mono-, di-, and triphosphates of gemcitabine (dFdC < dFdCMP < dFdCDP < dFdCTP). In addition, after the deletion of the 3'-5' exonuclease of cellular DNA polymerase I, the Escherichia coli mutant is more sensitive to dFdCTP than is wild-type E. coli. Our new discovery of the ability of these dFdC phosphates to inhibit exonuclease activity suggests a novel anticancer mechanism of gemcitabine and its phosphate derivatives.

Metformin-Induced Stromal Depletion to Enhance the Penetration of Gemcitabine-Loaded Magnetic Nanoparticles for Pancreatic Cancer Targeted Therapy.

Pancreatic ductal adenocarcinoma, as one of the most aggressive cancers, is characterized by rich desmoplastic stroma that forms a physical barrier for anticancer drugs. To address this issue, we herein report a two-step sequential delivery strategy for targeted therapy of pancreatic cancer with gemcitabine (GEM). In this sequential strategy, metformin (MET) was first administrated to disrupt the dense stroma, based on the fact that MET downregulated the expression of fibrogenic cytokine TGF-ß to suppress the activity of pancreatic stellate cells (PSCs), through the 5'-adenosine monophosphate-activated protein kinase pathway of PANC-1 pancreatic cancer cells. In consequence, the PSC-mediated desmoplastic reactions generating α-smooth muscle actin and collagen were inhibited, which promoted the delivery of GEM and pH (low) insertion peptide (pHLIP) comodified magnetic nanoparticles (denoted as GEM-MNP-pHLIP). In addition, pHLIP largely increased the binding affinity of the nanodrug to PANC-1 cells. The targeted delivery and effective accumulation of MET/GEM-MNP-pHLIP in vivo were confirmed by magnetic resonance imaging enhanced by the underlying magnetic nanoparticles. The tumor growth inhibition of the sequential MET and GEM-MNP-pHLIP treatment were investigated on both subcutaneous and orthotopic tumor mice models. A remarkably improved therapeutic efficacy, for example, up to 91.2% growth inhibition ratio over 30 d of treatment, well-exemplified the novel cascade treatment for pancreatic cancer and the innovative use of MET.

5-Formylcytosine to cytosine conversion by C-C bond cleavage in vivo.

Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. However, whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unanswered question. Here we report the incorporation of synthetic isotope- and (R)-2'-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. Therefore, we conclude that fdC undergoes C-C bond cleavage in stem cells, leading to the direct re-installation of unmodified dC.

Chitosan Hydrogels for Synergistic Delivery of Chemotherapeutics to Triple Negative Breast Cancer Cells and Spheroids.

PURPOSE: This study aimed to develop a hydrogel system for treating aggressive triple negative breast cancer (TNBC) via kinetically-controlled delivery of the synergistic drug pair doxorubicin (DOX) and gemcitabine (GEM). A 2D assay was adopted to evaluate therapeutic efficacy by determining combination index (CI), and a 3D assay using cancer spheroids was implemented to assess the potential for translation in vivo. METHODS: The release of DOX and GEM from an acetylated-chitosan (ACS, degree of acetylation χAc = 40 ± 5%) was characterized to identify a combined drug loading that affords release kinetics and dose that are therapeutically synergistic. The selected DOX/GEM-ACS formulation was evaluated in vitro with 2-D and 3-D models of TNBC to determine the combination index (CI) and the tumor volume reduction, respectively. RESULTS: Therapeutically desired release dosages and kinetics of GEM and DOX were achieved. When evaluated with a 2-D model of TNBC, the hydrogel afforded a CI of 0.14, indicating a stronger synergism than concurrent administration of DOX and GEM (CI = 0.23). Finally, the therapeutic hydrogel accomplished a notable volume reduction of the cancer spheroids (up to 30%), whereas the corresponding dosages of free drugs only reduced growth rate. CONCLUSIONS: The ACS hydrogel delivery system accomplishes drug release kinetics and molar ratio that affords strong therapeutically synergism. These results, in combination with the choice of ACS as affordable and highly abundant source material, provide a strong pre-clinical demonstration of the potential of the proposed system for complementing surgical resection of aggressive solid tumors.

Nanoformulation of Apolipoprotein E3-Tagged Liposomal Nanoparticles for the co-Delivery of KRAS-siRNA and Gemcitabine for Pancreatic Cancer Treatment.

PURPOSE: KRAS is the most frequently mutated gene in human cancers, and ~ 90% of pancreatic cancers exhibit KRAS mutations. Despite the well-known role of KRAS in malignancies, directly inhibiting KRAS is challenging. METHODS: In this study, we successfully synthesized apolipoprotein E3-based liposomes for the co-delivery of gemcitabine (GEM) and a small interfering RNA targeting KRAS (KRAS-siRNA) to improve the efficacy of pancreatic cancer treatment. RESULTS: Apolipoprotein E3 self-assembly on the liposome surface led to a substantial increase in its internalization in PANC1 human pancreatic cancer cells. KRAS-siRNA led to downregulated KRAS protein expression and KRAS-dependent carcinogenic pathways, resulting in the inhibition of cell proliferation, cell cycle arrest, increased apoptosis, and suppression of tumor progression. The combination of KRAS-siRNA and GEM induced a synergistic improvement in cell apoptosis and significantly lower cell viability compared with single-agent therapy. The low IC50 value of A3-SGLP might be attributed to potentiation of the anticancer effect of GEM by siRNA-mediated silencing of KRAS mutations, thereby inducing synergistic effects on cancer cells. CONCLUSION: A3-SGLP led to a marked decrease in the overall tumor burden and did not show any signs of toxicity. Therefore, the combination of KRAS-siRNA and GEM holds great potential for the treatment of pancreatic cancer.