Anticancer Drug Gemcitabine Research Articles

Experimental and computational investigation on the binding of anticancer drug gemcitabine with bovine serum albumin

This study reports the experimental and computational investigation on the binding of a common anticancer drug, gemcitabine, with the model plasma protein, bovine serum albumin (BSA). Several experimental and computational methods, such as intrinsic and synchronous fluorescence, UV-visible, and circular dichroism spectroscopies, consensus molecular docking and molecular dynamics simulation have been employed to elucidate the binding mechanism. Gemcitabine altered the UV-visible spectrum of BSA, which is a clear indication of the complex formation between them. The visual inspection of observed fluorescence quenching results at λex = 280 nm and 295 nm has shown the substantial involvement of tyrosine residue, even larger than tryptophan. However, after the correction of inner filter effect of the observed data, it became clear that tyrosine has a negligible role in quenching. A 20-fold decrease in quenching constant was found in the corrected data, as compared to the observed data at λex = 280 nm. There was a 1:1 weak binding between BSA and gemcitabine accompanied by dynamic quenching. The secondary structure of BSA remained almost intact in the presence of gemcitabine. The primary binding site of gemcitabine inside BSA was the drug binding site 2 or DS II, which is located in the subdomain 3 A. MD Simulation results suggested that gemcitabine doesn’t affect or deviate the structure of BSA upon interaction throughout 100 ns time period. The dominating intermolecular forces were hydrophobic forces and hydrogen bonding. A small change in the frontier molecular orbitals of gemcitabine was also observed after its binding with BSA. Communicated by Ramaswamy H. Sarma

The Antimalarial Drug Pyronaridine Inhibits Topoisomerase II in Breast Cancer Cells and Hinders Tumor Progression In Vivo

Breast cancer is the most frequently diagnosed cancer in women worldwide. Pyronaridine (PND), an antimalarial drug, was shown to exert anticancer activity on seventeen different human cancer cells, seven from female breast tissue. Additionally, PND induced apoptosis via mitochondrial depolarization, alteration of cell cycle progression, and DNA intercalation. However, the molecular target of PND in cells was not elucidated. Here, we have further investigated PND's mode of action by using transcriptome analysis. Preclinical studies were also performed to determine whether PND could affect tumor progression in a human breast cancer xenograft in mice. Moreover, we assessed the combined efficacy of PND with well-known anticancer drugs. Transcriptome analyses of PND-treated cancer cells were performed. Topoisomerase II activity was evaluated by an in vitro assay. In addition, daily oral administration of PND was given to mice with human breast cancer xenografts. The differential nuclear staining assay measured in-vitro cell toxicity. The transcriptome signatures suggested that PND might act as a topoisomerase II inhibitor. Thus, topoisomerase inhibition assays were performed, providing evidence that PND is a bona fide topoisomerase II inhibitor. Also, in-vivo studies suggest that PND hinders tumor progression. Besides, combination studies of PND with anticancer drugs cisplatin and gemcitabine revealed higher cytotoxicity against cancer cells than individual drug administration. The findings provide evidence that PND is a topoisomerase II inhibitor and can hinder cancer progression in an animal model, further demonstrating PND's favorable characteristics as a repurposed anticancer drug.

Advanced Nanosystems for Clinical Translation

Advanced Nanosystems for Clinical Translation

Quercetin potentializes the respective cytotoxic activity of gemcitabine or doxorubicin on 3D culture of AsPC-1 or HepG2 cells, through the inhibition of HIF-1α and MDR1.

The impact of cancer on lifespan is significantly increasing worldwide. Enhanced activity of drug efflux pumps and the incidences of the tumor microenvironment such as the apparition of a hypoxic gradient inside of the bulk tumor, are the major causes of chemotherapy failure. For instance, expression of Hypoxia-inducible factor (HIF-1α) has been associated with metastasis, resistance to chemotherapy and reduced survival rate. One of the current challenges to fight against cancer is therefore to find new molecules with therapeutic potential that could overcome this chemoresistance. In the present study, we focused on the bioactive plant flavonoid quercetin, which has strong antioxidant and anti-proliferative properties. We examined the efficacy of combined treatments of quercetin and the anti-cancer drugs gemcitabine and doxorubicin, known to specifically act on human pancreatic and hepatic cancer cells, respectively. Moreover, our study aimed to investigate more in-depth the implication of the multidrug transporter MDR1 and HIF-1α n chemoresistance and if quercetin could act on the activity of the drug efflux pumps and the hypoxia-associated effects. We observed that the anti-cancer drugs, were more effective when administered in combination with quercetin, as shown by an increased percentage of dead cells up to 60% in both 2D and 3D cultures. In addition, our results indicated that the combination of anti-cancer drugs and quercetin down-regulated the expression of HIF-1α and increased the expression levels of the regulator of apoptosis p53. Moreover, we observed that quercetin could inhibit the efflux activity of MDR1. Finally, our in vitro study suggests that the efficiency of the chemotherapeutic activity of known anti-cancer drugs might be significantly increased upon combination with quercetin. This flavonoid may therefore be a promising pharmacological agent for novel combination therapy since it potentializes the cytotoxic activity of gemcitabine and doxorubicin on by targeting the chemoresistance developed by the pancreatic and liver cancer cells respectively.

Role of sphingomyelin on the interaction of the anticancer drug gemcitabine hydrochloride with cell membrane models

The fight against drug resistance in chemotherapy requires a molecular-level understanding of the drug interaction with cell membranes, which today is feasible with membrane models. In this study, we report on the interaction of gemcitabine (GEM), a pyrimidine nucleoside antimetabolite used to treat pancreatic cancer, with Langmuir films that mimic healthy and cancerous cell membranes. The cell membrane models were made with eight compositions of a quaternary mixture containing 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), sphingomyelin (SM), and cholesterol (CHOL). The relative concentration of SM was increased so that four of these compositions represented cancerous cells. GEM was found to increase the mean molecular area, also increasing their surface elasticity, with stronger interactions being observed for membranes corresponding to cancerous cells. More specifically, GEM penetrated deepest in the membrane with the highest SM concentration (40 mol%), as inferred from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). This finding was confirmed with molecular dynamics simulations that also indicated how GEM approaches the membrane, which could be useful for guiding the design of drug delivery systems. The experimental and simulation results are consistent with the preferential attachment of GEM onto cancerous cells and highlight the role of SM on drug-cell interactions.

Synthesis and Characterization of Folic Acid Conjugated Gemcitabine Tethered Silver Nanoparticles (FA-GEM-AgNPs) for Targeted Delivery.

Silver nanoparticles (AgNPs) have attracted considerable interest in the medical industry due to their physicochemical properties, small size, and surface plasmon behavior. Their smaller particle size and instability in blood circulation leads to toxicity due to its aggregation as Ag+ ions and accumulation at the deepseated organ. In the present study, we aimed at reducing the toxicity of AgNPs by conjugation with an anticancer drug GEM and to improve their internalization through folate receptors-mediated endocytosis by capping the nanoparticles with folic acid (FA). One-pot facile synthesis of FA capped silver nanoparticles (FA-AgNPs) has been achieved by using FA as a reducing agent. FA-AgNPs were mixed with Gemcitabine (GEM) to obtain tethered FA-GEM-AgNPs. Nanoparticles were characterized by Dynamic Light Scattering (DLS), UV-Visible spectroscopy, Transmission Electron Microscopy (TEM), Energy Dispersive X-ray Analysis (EDAX), Selected Area Electron Diffraction (SAED), and Atomic Absorption Spectroscopy (AAS). The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to determine the cytotoxic effect of the prepared nanoformulations. The apoptotic cell death induced by FA-GEM-AgNPs in breast cancer cells were monitored with Acridine orange (AO)/Ethidium Bromide (EtBr) staining. Compared to GEM and AgNPs, FA-GEM-AgNPs showed enhanced cytotoxic effect and internalization in MDA-MB-453 breast cancer cell line. FA-GEM-AgNPs could be an ideal candidate for targeting cancer cells via folate receptor-mediated endocytosis.

Induction of potent cell growth inhibition by schizophyllan/K-ras antisense complex in combination with gemcitabine

Antisense oligonucleotides (AS-ODNs) specifically hybridize with target mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. In our previous reports, we demonstrated that β-glucan schizophyllan (SPG) can form a complex with AS-ODNs attached with oligo deoxyadenosine dA40 (AS-ODN-dA40/SPG), and that this complex can be recognized by β-glucan receptor Dectin-1 on antigen presenting cells and lung cancer cells. In many types of cancer cell, activating K-ras mutations related to malignancy are frequently observed. In this study, we first designed 78 AS-ODNs for K-ras to optimize the sequence for highly efficient gene suppression. The selected AS-ODN (K-AS07) having dA40 made a complex with SPG. The resultant complex (K-AS07-dA40/SPG) showed an effect of silencing the ras gene in the cells (PC9: human adenocarcinoma differentiated from lung tissue) expressing Dectin-1, leading to the suppression of cell growth. Furthermore, the cytotoxic effect was enhanced when used in combination with the anticancer drug gemcitabine. Gemcitabine, a derivative of cytidine, was shown to interact with dA40 in a sequence-dependent manner. This interaction did not appear to be so strong, with the gemcitabine being released from the complex after internalization into the cells. SPG and the dA40 part of K-AS07-dA40 play roles in carriers for K-AS07 and gemcitabine, respectively, resulting in a strong cytotoxic effect. This combination effect is a novel feature of the AS-ODN-dA40/SPG complexes. These results could facilitate the clinical application of these complexes for cancer treatment.

Solvent-Free Synthesis of Fluorescent Carbon Dots: An Ecofriendly Approach for the Bioimaging and Screening of Anticancer Activity via Caspase-Induced Apoptosis.

Ecofriendly and highly fluorescent carbon dot nanoassemblies have gained prominence for their diverse biological applications, with a focus on combating environmental and human health problems. In this study, we prepared highly fluorescent nitrogen-doped carbon dots from the persimmon fruit (termed as "PCDs"), which was used as a carbon source, via a one-step hydrothermal reaction without any solvent. It is interesting to note that the as-prepared PCDs were highly water-soluble because of the numerous polar functional groups, such as hydroxyl (-OH), amine (-NH2), and carboxylic acid (-COOH) groups, as confirmed by ultraviolet-visible, Fourier-transform infrared, and X-ray photoelectron spectral analysis. These polar functional groups enabled the production of nanohybrids by immobilization of the anticancer drugs doxorubicin and gemcitabine on the surface of the PCDs (PCDs@Dox and PCDs@Gem, respectively) through the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling reaction. Unlike other reports, as-prepared PCDs@Dox and PCDs@Gem nanohybrids were successfully employed for bioimaging and caspase-induced apoptosis applications. The neat PCDs exhibited significant pH-induced cytotoxicity because of the presence of surface carboxylic acid and phenolic moieties. Thus, PCDs@Dox and PCDs@Gem effectively inhibited the proliferation of HeLa cells in a dose-dependent manner, and the cytotoxicity of the nanohybrids in fibroblasts was significantly lower than that of cancerous cells at the same dose. In addition, it is quite certain that promising boi-imaging results were identified from PCDs like as conventional dyes, which may account for the synthesis of high-fluorescent PCDs without using any solvent. The results demonstrate that the nanohybrids mediate the production of reactive oxygen species (ROS), and this mediation is followed by a decrease in the mitochondrial membrane potential through the activation of caspase-3. These results appear to be promising for anticancer drug delivery and bio-imaging engineering applications.

Development of Mesoporous Silica Nanoparticles of Tunable Pore Diameter for Superior Gemcitabine Drug Delivery in Pancreatic Cancer Cells.

Superior delivery of anticancer drug gemcitabine has been achieved with mesoporous silica nanoparticles (MSN), by addressing three challenges in MSN synthesis: (i) MSN was synthesized with particle diameter between 42 to 64 nm, to utilize enhanced permeability and retention effect of small particles, (ii) MSN of larger internal pore diameter (2.5-5.2 nm) was made as a tunable morphological parameter to optimize both drug loading and its release rate, in a controlled, differential manner and (iii) higher drug release at extracellular cancer-cell pH (5.5) was achieved, compared to physiological pH (7.4) of healthy cells. MSN with above features was made by the sol-gel route, with trimethylmethoxysilane as a size-quencher and hexane or decane as a pore expander. Highest gemcitabine loading of 14.92% and a cumulative release of 58% at pH 5.5 could be obtained with the optimum sample having pore diameter of 5.2 nm, in comparison to the desirably low 22% release at pH 7.4. Consequently, we obtained 60% cell growth-inhibition of pancreatic cancer cell-line (MIA Paca-2), via gemcitabine loaded MSN. This was possible because of increased gemcitabine release from MSN with larger pore diameter of 5.2 nm, simultaneously demonstrating good target-selectivity of MSN as a drug-carrier, due to engineering of its pore-size.

Supramolecular Aggregation-Induced Emission Nanodots with Programmed Tumor Microenvironment Responsiveness for Image-Guided Orthotopic Pancreatic Cancer Therapy

Supramolecular nanomaterials as drug carriers have recently received increasing attention due to their intrinsic merits such as high stability, strong inclusion capability, and facile modification of the parental structure; however, intelligent ones with combined capacities of long blood circulation, highly efficient tumor cell uptake, and site-oriented drug release inside tumor cells are still rather limited. Herein, we report a strategy using supramolecular aggregation-induced emission (AIE) nanodots for image-guided drug delivery, which integrate both the advantages of AIE and supramolecular nanomaterials. The supramolecular AIE dots are prepared by the host-guest coassembly of the matrix metalloproteinase-2 (MMP-2) sensitive PEG-peptide (PEG2000-RRRRRRRR (R8)-PLGLAG-EKEKEKEKEKEK (EK6)) and functional α-cyclodextrins (α-CD) derivatives that are conjugated with the anticancer drug gemcitabine (GEM) and a far-red/near-infrared fluorescent rhodanine-3-acetic acid-based AIE luminogen, respectively. The supramolecular AIE dots realize long blood circulation time by virtue of the zwitterionic stealth peptide EK6. After largely accumulating in tumor tissues by the enhanced permeability and retention effect, the supramolecular AIE dots can successively respond to the tumor-overexpressed MMP-2 and intracellular reductive microenvironment, achieving both enhanced cancer cellular uptake and selective GEM release within cancer cells, which thus exhibit excellent tumor inhibition ability in both subcutaneous and orthotopic pancreatic tumor models.

Mechanisms of the interaction of bovine serum albumin with anticancer drug gemcitabine

We investigated quantum-chemical characteristics of gemcitabine (GEM), fluorescence quenching of bovine serum albumin (BSA) in aqueous solutions with the presence of GEM. The static mechanism of the complex formation with moderate binding constant () and number of binding sites was established to take place in the solutions. Studying the energy transfer efficiency we found that molecules of GEM are localized near polar charged amino acid residues of the protein biomolecules at the average distance The calculated thermodynamic parameters demonstrate the presence of both hydrogen bonds and hydrophobic interaction between the protein and ligand molecules.

Pectin-Tannic Acid Nano-Complexes Promote the Delivery and Bioactivity of Drugs in Pancreatic Cancer Cells.

Pancreatic cancer (PanCa) is a lethal disease. Conventional chemotherapies for PanCa offer severe systemic toxicities. Thus, the development of a successful nanomedicine-based therapeutic regimen with augmented therapeutic efficacy is highly sought. Naturally occurring pectin and modified pectin-based drug delivery systems exhibit remarkable self-targeting ability via galactose residues to various cancer cells. Herein, we developed and used an innovative approach of highly stable nanocomplexes based on modified pectin and tannic acid (MPT-NCs). The nanocomplex formation was enabled by strong intermolecular interactions between pectin and tannic acid under very mild conditions. These nanocomplexes were characterized by particle size and morphology (DLS, TEM, and SEM), FT-IR spectroscopy, and zeta potential measurements. Additionally, MPT-NCs were capable of encapsulating anticancer drugs (5-fluorouracil, gemcitabine, and irinotecan) through tannic acid binding. The in vitro bioactivity of these drug MPT-NCs were evaluated in pancreatic cancer adenocarcinoma (PDAC) cell lines (HPAF-II and PANC-1). A dose-dependent internalization of nanocomplexes was evident from microscopy and flow cytometry analysis. Both proliferation and colony formation assays indicated the anticancer potential of pectin drug nanocomplexes against PDAC cells compared to that of free drug treatments. Together, the pectin-based nanocomplexes could be a reliable and efficient drug delivery strategy for cancer therapy.

Gemcitabine induces Parkin-independent mitophagy through mitochondrial-resident E3 ligase MUL1-mediated stabilization of PINK1

Mitophagy plays an important role in the maintenance of mitochondrial homeostasis. PTEN-induced kinase (PINK1), a key regulator of mitophagy, is degraded constitutively under steady-state conditions. During mitophagy, it becomes stabilized in the outer mitochondrial membrane, particularly under mitochondrial stress conditions, such as in treatment with uncouplers, generation of excessive mitochondrial reactive oxygen species, and formation of protein aggregates in mitochondria. Stabilized PINK1 recruits and activates E3 ligases, such as Parkin and mitochondrial ubiquitin ligase (MUL1), to ubiquitinate mitochondrial proteins and induce ubiquitin-mediated mitophagy. Here, we found that the anticancer drug gemcitabine induces the stabilization of PINK1 and subsequent mitophagy, even in the absence of Parkin. We also found that gemcitabine-induced stabilization of PINK1 was not accompanied by mitochondrial depolarization. Interestingly, the stabilization of PINK1 was mediated by MUL1. These results suggest that gemcitabine induces mitophagy through MUL1-mediated stabilization of PINK1 on the mitochondrial membrane independently of mitochondrial depolarization.

Long-term cultivation using ineffective MDM2 inhibitor concentrations alters the drug sensitivity profiles of PL21 leukaemia cells

AbstractAcquired MDM2 inhibitor resistance is commonly caused by loss-of-function TP53 mutations. In addition to the selection of TP53-mutant cells by MDM2 inhibitors, MDM2 inhibitor-induced DNA damage may promote the formation of TP53 mutations. Here, we cultivated 12 sublines of the intrinsically MDM2 inhibitor-resistant TP53 wild-type acute myeloid leukaemia cell line PL21 for 52 passages in the presence of ineffective concentrations of the MDM2 inhibitor nutlin-3 but did not observe loss-of-function TP53 mutations. This suggests that MDM2 inhibitors select TP53-mutant cells after mutations have occurred, but do not directly promote TP53 mutations. Unexpectedly, many sublines displayed increased sensitivity to the anti-cancer drugs cytarabine, doxorubicin, or gemcitabine. Consequently, therapies can affect the outcome of next-line treatments, even in the absence of a therapy response. This finding is conceptually novel. A better understanding of such processes will inform the design of improved therapy protocols in the future.

Transferrin-Conjugated Polymer-Coated Mesoporous Silica Nanoparticles Loaded with Gemcitabine for Killing Pancreatic Cancer Cells

In order to achieve superior delivery of the anticancer drug gemcitabine to Mia PaCa-2 pancreatic cancer cell lines, we have synthesized transferrin (Tf)-conjugated, polymer-coated, mesoporous sili...

Sensitive Nucleic Acid Detection at NH2‐MWCNTs Modified Glassy Carbon Electrode and its Application for Monitoring of Gemcitabine‐DNA Interaction

AbstractAn electrochemical dsDNA nanobiosensor was fabricated using amino‐functionalized multi walled carbon nanotubes modified glassy carbon electrode (NH2fMWCNTs/GCE) for the sensitive detection of DNA bases and electrochemical monitoring of drug‐DNA interaction. The influence of functional groups on MWCNT was studied by MWCNT functionalized with NH2 (NH2fMWCNTs) and COOH (COOHfMWCNT) groups based on the signal of DNA bases. The modified electrodes were characterized by scanning electron microscopy. One layer of calf thymus double stranded deoxyribonucleic acid (ct‐dsDNA) was immobilized onto the NH2fMWCNTs/GCE (dsDNA/NH2fMWCNTs/GCE). The dsDNA/NH2fMWCNTs/GCE were used to investigate the interaction between the dsDNA and the anticancer drug gemcitabine by differential pulse voltammetry in acetate buffer of pH 4.70. For the confirmation of interaction, the lowering in intensity of the current signals of guanine and adenine was considered as an indicator. Electrochemical impedance spectroscopy studies were performed for the comparison of the modified surfaces. In order to define and visualize the interaction mechanism between gemcitabine and dsDNA/NH2fMWCNTs/GCE at the molecular level, in silico methods including docking and molecular dynamics simulations were employed.

Biodistribution and Pharmacokinetic Study of Gemcitabine Hydrochloride Loaded Biocompatible Iron-Based Metal Organic Framework

This study was designed to improve bioavailability and therapeutic efficacy of Gemcitabine (GEM) with reduced side effects using MOF MIL-100 as cargo. MIL-100 was synthesized, and characterized by microscopic and spectroscopic techniques. Impregnation approach was used for encapsulation of GEM inside the MIL-100 (i.e., MIL100-GEM). In-vitro release studies of MIL100-GEM was carried out in different media (PBS, deionized water and Tris buffer, pH = 7.4, 9.5 mM) to find out the drug release mechanism. Cytotoxicity and apoptosis assays were evaluated using MTT and fluorescence-activated cell sorting (FACS) assay in MiaPaCa-2 pancreatic cancer cell lines. Biocompatibility, pharmacokinetic and biodistribution studies of MIL100-GEM were assessed in Wistar rats. MIL100-GEM exhibited high encapsulation efficiency (78.6 ± 0.5%) and maximum payload (23.6 ± 1%). PXRD confirmed crystallinity of MIL-100, and did not show any effect on its structural integrity after encapsulation of GEM. In-vitro release studies revealed a biphasic release pattern in PBS buffer which followed Higuchi diffusion kinetics. In-vitro cytotoxicity studies showed low IC50 value for MIL100-GEM (3.50 ± 1.33 µg/ml) compared to GEM (6.22 ± 1.55 µg/ml), ensuring adequate cell proliferation after 72 h. Hemolysis study showed that MIL100-GEM (14.54 ± 1.3%) had better biocompatibility than the native GEM (30.52 ± 1.67%). Furthermore, pharmacokinetic and biodistribution studies exhibited ∼17-fold increased bioavailability, ∼20-fold increased distribution half-life and ∼15-folds elimination half-life of GEM with less accumulation of drug in the kidneys. MIL-100 MOF was synthesized and characterized to address the metabolic degradation issue of GEM. Biocompatible, MIL100-GEM demonstrated efficient drug (GEM) loading and enhanced cytotoxic activity in pancreatic cancer cell line with augmented bioavailability, providing MIL-100 a promising drug cargo. MIL-100 was synthesized using a microwave-assisted method. The anticancer drug Gemcitabine Hydrochloride (GEM) was loaded into the MIL-100 using the impregnation method. Encapsulation protected the drug from its metabolic inactivation to enhance bioavailability in target organs and reduce side effects.

Curvature increases permeability of the plasma membrane for ions, water and the anti-cancer drugs cisplatin and gemcitabine

In this work the permeability of a model asymmetric plasma membrane, for ions, water and the anti-cancer drugs cisplatin and gemcitabine is studied by means of all-atom molecular dynamics simulations. It is shown for the first time that permeability of the highly curved membrane increases from one to three orders of magnitude upon membrane bending depending on the compound and the sign of curvature. Our results suggest that the membrane curvature could be an important factor of drug translocation through the membrane.

Establishment of a novel experimental model for muscle‐invasive bladder cancer using a dog bladder cancer organoid culture

In human and dogs, bladder cancer (BC) is the most common neoplasm affecting the urinary tract. Dog BC resembles human muscle‐invasive BC in histopathological characteristics and gene expression profiles, and could be an important research model for this disease. Cancer patient‐derived organoid culture can recapitulate organ structures and maintains the gene expression profiles of original tumor tissues. In a previous study, we generated dog prostate cancer organoids using urine samples, however dog BC organoids had never been produced. Therefore we aimed to generate dog BC organoids using urine samples and check their histopathological characteristics, drug sensitivity, and gene expression profiles. Organoids from individual BC dogs were successfully generated, expressed urothelial cell markers (CK7, CK20, and UPK3A) and exhibited tumorigenesis in vivo. In a cell viability assay, the response to combined treatment with a range of anticancer drugs (cisplatin, vinblastine, gemcitabine or piroxicam) was markedly different in each BC organoid. In RNA‐sequencing analysis, expression levels of basal cell markers (CK5 and DSG3) and several novel genes (MMP28,CTSE,CNN3,TFPI2,COL17A1, and AGPAT4) were upregulated in BC organoids compared with normal bladder tissues or two‐dimensional (2D) BC cell lines. These established dog BC organoids might be a useful tool, not only to determine suitable chemotherapy for BC diseased dogs but also to identify novel biomarkers in human muscle‐invasive BC. In the present study, for the 1st time, dog BC organoids were generated and several specifically upregulated organoid genes were identified. Our data suggest that dog BC organoids might become a new tool to provide fresh insights into both dog BC therapy and diagnostic biomarkers.

Hydroxyapatite-poly(vinyl alcohol) core-shell nanoparticles for dual delivery of methotrexate and gemcitabine for bone cancer treatment

The delivery of multiple drugs with bone substitutes is the current strategy for the treatment of bone diseases. In this study, hydroxyapatite nanoparticles (HA NPs) as a core and poly(vinyl alcohol) (PVA) as the corona were developed for the dual delivery of anti-cancer drugs methotrexate (MTX) and gemcitabine (GEM). MTX was conjugated to PVA through ester linkages with a high payload of around 20%. GEM was then physically loaded onto the MTX conjugated-PVA-HA NPs with a loading efficiency of 83%. In vitro drug release studies of GEM-loaded-MTX conjugated-PVA-HA NPs in phosphate buffered saline (pH 7.4) showed a combined release of both MTX (25%) and GEM (60%) at the end of 10 days. Physically loaded MTX showed a burst release of around 50% compared to 16% by conjugated MTX within 24 h which supports the significance of conjugating drug molecules. Cell viability studies using osteosarcoma MG-63 cells confirmed that polymer grafted NPs were non-cytotoxic, while the drug-loaded NPs showed dose-dependent cytotoxicity. The synthesized NPs will be a promising bone-substitute material for the co-delivery of many therapeutic molecules for the treatment of bone diseases.