Integrated Analysis of Noncoding RNAs (PVT-1 and miR-200c) and Their Correlation with STAT4/IL-6 Axis as Reliable Biomarkers for COVID-19 Severity.

Inefficient control of elevated inflammatory mediators in coronavirus disease 2019 (COVID-19) has led to health complications, prompting the exploration of efficient biomarkers for monitoring this condition. We herein sought to investigate the implications of plasmacytoma variant translocation 1 (PVT-1), microRNA-200c (miR-200c), signal transducer and activator of transcription 4 (STAT-4), and interleukin-6 (IL-6), as well as how they correlated with creatinine, C-reactive protein (CRP), and lactate dehydrogenase (LDH) activity to identify biomarkers able to the early prognosis and diagnosis of COVID-19. Our study included a total of 105 infected COVID-19 patients and 35 healthy subjects as controls. Individuals with COVID-19 showed a significant increase in CRP, creatinine, and LDH activity. In addition, COVID-19 patients exhibited significantly higher levels of IL-6. These patients also demonstrated notably elevated expressions of miR-200c and PVT-1. The expression level of STAT4 decreased in the COVID-19 patients, and this decrease was negatively correlated with creatinine and LDH activity. The levels of miR-200c and PVT-1 expressions, and their connections with IL-6 and STAT4 levels, increased significantly with the severity of COVID-19 cases. In addition, receiver operating characteristic analysis showed that PVT-1 and miR-200c could be reliable biomarkers for determining the severity of COVID-19.

Emerging strategies for combating Fusobacterium nucleatum in colorectal cancer treatment: Systematic review, improvements and future challenges.

Colorectal cancer (CRC) is generally characterized by a high prevalence of Fusobacterium nucleatum (F. nucleatum), a spindle-shaped, Gram-negative anaerobe pathogen derived from the oral cavity. This tumor-resident microorganism has been closely correlated with the occurrence, progression, chemoresistance and immunosuppressive microenvironment of CRC. Furthermore, F. nucleatum can specifically colonize CRC tissues through adhesion on its surface, forming biofilms that are highly resistant to commonly used antibiotics. Accordingly, it is crucial to develop efficacious non-antibiotic approaches to eradicate F. nucleatum and its biofilms for CRC treatment. In recent years, various antimicrobial strategies, such as natural extracts, inorganic chemicals, organic chemicals, polymers, inorganic-organic hybrid materials, bacteriophages, probiotics, and vaccines, have been proposed to combat F. nucleatum and F. nucleatum biofilms. This review summarizes the latest advancements in anti-F. nucleatum research, elucidates the antimicrobial mechanisms employed by these systems, and discusses the benefits and drawbacks of each antimicrobial technology. Additionally, this review also provides an outlook on the antimicrobial specificity, potential clinical implications, challenges, and future improvements of these antimicrobial strategies in the treatment of CRC.

Engineering lauric acid-based nanodrug delivery systems for restoring chemosensitivity and improving biocompatibility of 5-FU and OxPt against Fn-associated colorectal tumor.

Colorectal cancer (CRC) occurs in the colorectum and ranks second in the global incidence of all cancers, accounting for one of the highest mortalities. Although the combination chemotherapy regimen of 5-fluorouracil (5-FU) and platinum(IV) oxaliplatin prodrug (OxPt) is an effective strategy for CRC treatment in clinical practice, chemotherapy resistance caused by tumor-resided Fusobacterium nucleatum (Fn) could result in treatment failure. To enhance the efficacy and improve the biocompatibility of combination chemotherapy, we developed an antibacterial-based nanodrug delivery system for Fn-associated CRC treatment. A tumor microenvironment-activated nanomedicine 5-FU-LA@PPL was constructed by the self-assembly of chemotherapeutic drug derivatives 5-FU-LA and polymeric drug carrier PPL. PPL is prepared by conjugating lauric acid (LA) and OxPt to hyperbranched polyglycidyl ether. In principle, LA is used to selectively combat Fn, inhibit autophagy in CRC cells, restore chemosensitivity of 5-FU as well as OxPt, and consequently enhance the combination chemotherapy effects for Fn-associated drug-resistant colorectal tumor. Both in vitro and in vivo studies exhibited that the tailored nanomedicine possessed efficient antibacterial and anti-tumor activities with improved biocompatibility and reduced non-specific toxicity. Hence, this novel anti-tumor strategy has great potential in the combination chemotherapy of CRC, which suggests a clinically relevant valuable option for bacteria-associated drug-resistant cancers.

The Implication of Growth Arrest-Specific 5 rs145204276 Polymorphism and Serum Expression of Sirtuin 1, Transforming Growth Factor-Beta, and microRNA-182 in Breast Cancer.

Background: Breast cancer (BC) patients have a higher chance of survival if it is diagnosed at an early stage, which is essential for efficient treatment of the condition. The results of an elevated risk of cancer, including BC, previously associated with the ins/del polymorphism rs145204276 in the promoter region of growth arrest-specific 5 (GAS5) are still up for debate. Thus, this study aimed to appraise the frequency of the GAS5 rs145204276 variant with BC risk and demonstrate the potential impact of the sirtuin 1 (SIRT-1), transforming growth factor-beta (TGF-ß), and microRNA-182 (miR-182) expression and their diagnostic value in BC. Methods: Blood samples of 155 patients with BC and fibroadenoma and 80 healthy controls were analyzed for GAS5 rs145204276 single nucleotide polymorphism (SNP), SIRT-1, TGF-ß, and miRNA-182 expression levels. Results: Ins/ins genotype and ins allele frequencies for GAS5 rs145204276 were considerably higher in BC patients compared with controls. Patients with BC had significantly greater serum levels of TGF-ß, miR-182, and SIRT-1 expression. Conclusions: The SIRT-1, TGF-ß, and miR-182 genes provide novel, noninvasive diagnostic biomarkers for BC.

Remotely Controllable Supramolecular Nanomedicine for Drug-Resistant Colorectal Cancer Therapy Caused by Fusobacterium nucleatum.

Fusobacterium nucleatum (Fn) existing in the community of colorectal cancer (CRC) promotes CRC progression and causes chemotherapy resistance. Despite great efforts that have been made to overcome Fn-induced chemotherapy resistance by co-delivering antibacterial agents and chemotherapeutic drugs, increasing the drug-loading capacity and enabling controlled release of drugs remain challenging. In this study, a novel supramolecular upconversion nanoparticle (SUNP) is constructed by incorporating a positively charged polymer (PAMAM-LA-CD) with Fn inhibition capacity, a negatively charged platinum (IV) oxaliplatin prodrug (OXA-COOH), upconversion nanoparticle (UCNPs) and polyethylene glycol-azobenzene (PEG-Azo) to enhance drug-loading and enable on-demand drug release for drug-resistant CRC treatment. SUNPs exhibit high drug-loading capacity (30.8%) and good structural stability under normal physiological conditions, while disassembled upon exogenous NIR excitation and endogenous azo reductase in the CRC microenvironment to trigger drug release. In vitro and in vivo studies demonstrate that SUNPs presented good biocompatibility and robust performance to overcome chemoresistance, thereby significantly inhibiting Fn-infected cancer cell proliferation. This study leverages multiple dynamic chemical designs to integrate both advantages of drug loading and release in a single system, which provides a promising candidate for precision therapy of bacterial-related drug-resistant cancers.

In situ-activated photothermal nanoplatform for on-demand NO gas delivery and enhanced colorectal cancer treatment.

The naturally evolved and intestinal pathogenic Fusobacterium nucleatum (Fn)-induced drug resistance profoundly impaired the efficacy of chemotherapy against colorectal cancer (CRC). Alternative treatment modalities against Fn-associated CRC are desperately needed. Herein, we engineer an in situ-activated anti-tumor and antibacterial nanoplatform (Cu2O/BNN6@MSN-Dex) to allow photoacoustic (PA) imaging-guided photothermal and NO gas combinatorial therapy for enhanced Fn-associated CRC treatment. The nanoplatform is constructed by loading cuprous oxide (Cu2O) and nitric oxide (NO) donor (BNN6) into dextran-decorated mesoporous silica nanoparticles (MSN), which is finally surface-functionalized with dextran via dynamic boronate linkage. Cu2O can be sulfuretted in situ by endogenous hydrogen sulfide overexpressed in CRC to produce copper sulfide with remarkable PA and photothermal properties, enabling the generation of NO from BNN6 under 808 nm laser irradiation, which is eventually triggered to release by multiple biological cues in the tumor microenvironment. Cu2O/BNN6@MSN-Dex exhibits superior biocompatibility, as well as H2S-triggered near-infrared-controlled antibacterial and anti-tumor performance in vitro and in vivo via photothermal and NO gas combination therapy. Furthermore, Cu2O/BNN6@MSN-Dex provokes systemic immune responses, thereby promoting anti-tumor efficacy. This study provides a conbinational strategy to effectively inhibit tumors and intratumor pathogens for enhanced CRC treatment.

Development of potent nanosized carbonic anhydrase inhibitor for targeted therapy of hypoxic solid tumors.

Overexpression of two carbonic anhydrase (CA) isoforms, CA IX and XII, in several hypoxic solid tumors provides an extracellular hypoxic microenvironment, interferes with extra- and intracellular pH regulation, thus favoring hypoxic tumor cell survival, proliferation and metastasis. In the current study, a selective inhibitor for human CA isoforms IX and XII (isatin-bearing sulfonamide, WEG-104), was incorporated into nanosized spherical niosomes at high encapsulation efficiency to allow for an enhanced and sustained antitumor activity. In vivo, administration of WEG-104 that is either free (10 mg/kg) or loaded into niosomes (5 mg/kg) into a mice model of Ehrlich ascites solid tumor resulted in comparable efficacy in terms of reduction of tumor weight and volume. Administration of WEG-104-loaded niosomes (10 mg/kg) exhibited superior antitumor activity compared to the free drug, evidenced by reduced tumor weight and volume, marked reduction in the activity of CA IX and XII, and suppression of HIF-1α and MMP-2. Moreover, prominent increase of caspase 3 and pronounced decrease in VEGF immune expression were observed in the treated animals. Hence, loading of molecularly designed compounds that targets CAs in hypoxic solid tumors into nanosized delivery systems provided an auspicious strategy for limiting solid tumor progression and malignancy.

Ameliorative effect of chitosan nanoparticles against carbon tetrachloride-induced nephrotoxicity in Wistar rats.

CONTEXT: Chitosan is a biocompatible polysaccharide that has been widely exploited in biomedical and drug delivery applications. OBJECTIVE: This study explores the renoprotective effect of chitosan nanoparticles in vivo in rats. MATERIALS AND METHODS: Chitosan nanoparticles were prepared via ionotropic gelation method, and several in vitro characterizations were performed, including measurements of particle size, zeta potential, polydispersity index, Fourier transform-infrared spectroscopy, differential scanning calorimetry, and transmission electron microscopy (TEM) imaging. Wistar rats were divided randomly into four groups; negative control, CCl4-induced nephrotoxicity (untreated), and two groups receiving CCl4 + chitosan NPs (10 and 20 mg/kg) orally for 2 weeks. The renoprotective effect was assessed by measuring oxidative, apoptotic, and inflammatory biomarkers, and via histopathological and immunohistochemical examinations for the visualization of NF-κB and COX-2 in renal tissues. RESULTS: Monodisperse spherical nanosized (56 nm) particles were successfully prepared as evidenced by dynamic light scattering and TEM. Oral administration of chitosan nanoparticles (10 and 20 mg/kg) concurrently with CCl4 for 2 weeks resulted in 13.6% and 21.5% reduction in serum creatinine and increase in the level of depleted reduced glutathione (23.1% and 31.8%), respectively, when compared with the positive control group. Chitosan nanoparticles (20 mg/kg) revealed a significant (p ˂ 0.05) decrease in malondialdehyde levels (30.6%), tumour necrosis factor-α (33.6%), interleukin-1ß (31.1%), and caspase-3 (36.6%). CONCLUSIONS: Chitosan nanoparticles afforded significant protection and amelioration against CCl4-induced nephrotoxicity. Thus, chitosan nanoparticles could afford a potential nanotherapeutic system for the management of nephrotoxicity which allows for broadening their role in biomedical delivery applications.

Famotidine-loaded solid self-nanoemulsifying drug delivery system demonstrates exceptional efficiency in amelioration of peptic ulcer.

Famotidine (FMD) is a highly potent H2-receptor antagonist used in peptic ulcer treatment. However, the drug possesses poor aqueous solubility and permeability. FMD-loaded solid self-nanoemulsifying drug delivery system (FMD-S-SNEDDS) comprised of Labrafil® M 1944 CS, Tween® 20 and PEG 400, adsorbed on Aerosil® 200, has been developed. FMD-S-SNEDDS has demonstrated acceptable micromeritic properties, and upon reconstitution in water, spherical nanosized particles were released, as demonstrated by dynamic light scattering studies and transmission electron microscopy imaging. High encapsulation efficiency of FMD in the developed SNEDDS has been attained, and the saturated solubility of the drug has increased by 20-fold when it was incorporated in the SNEDDS. Several in vitro characterizations have been carried out, including, Fourier transform-infrared spectroscopy, differential scanning calorimetry, scanning electron microscopy, and drug dissolution studies. In vivo, upon administration of the free drug suspension, marketed product (FAMOTIN®) and FMD-S-SNEDDS (40 mg/kg) in peptic ulcer rat models, FMD-S-SNEDDS and the marketed FMD demonstrated 12.5- and 4.7-fold reduction in ulcers number, and 28.7- and 7.2-fold reduction in ulcer severity, respectively, compared to the control untreated animals. FMD-S-SNEDDS showed a significant (p < 0.05) increase in the levels of depleted glutathione and endothelial nitric oxide synthase, and significantly (p < 0.05) reduced the elevated level of malondialdehyde, as compared to the free and marketed FMD. Only FMD-S-SNEDDS could restore the elevated proton pump activity and cyclic adenosine monophosphate RNA expression to their normal levels. Hence, FMD-S-SNEDDS provides a great potential as a nanotherapeutic system for treatment of peptic ulcer.

A dendritic polyamidoamine supramolecular system composed of pillar[5]arene and azobenzene for targeting drug-resistant colon cancer.

Fusobacterium nucleatum caused drug-resistant around tumor sites often leads to the failure of chemotherapy during colorectal cancer (CRC) treatment. Multifunctional cationic quaternary ammonium materials have been widely used as broad-spectrum antibacterial agents in antibacterial and anticancer fields. Herein, we design a smart supramolecular quaternary ammonium nanoparticle, namely quaternary ammonium PAMAM-AZO@CP[5]A (Q-P-A@CP[5]A), consisting of azobenzene (AZO)-conjugated dendritic cationic quaternary ammonium polyamidoamine (PAMAM) as the core and carboxylatopillar[5]arene (CP[5]A)-based switch, for antibacterial and anti-CRC therapies. The quaternary ammonium-PAMAM-AZO (Q-P-A) core endows the supramolecular system with enhanced antibacterial and anticancer properties. -N+CH3 groups on the surface of Q-P-A are accommodated in the CP[5]A cavity under normal conditions, which significantly improves the biocompatibility of Q-P-A@CP[5]A. Meanwhile, the CP[5]A host can be detached from -N+CH3 groups under pathological conditions, achieving efficient antibacterial and antitumor therapies. Furthermore, azoreductase in the tumor site can break the -NN- bonds of AZO in Q-P-A@CP[5]A, leading to the morphology recovery of supramolecular nanoparticles and CRC therapy through inducing cell membrane rupture. Both in vitro and in vivo experiments demonstrate that Q-P-A@CP[5]A possesses good biocompatibility, excellent antibacterial effect, and CRC treatment capability with negligible side effects. This supramolecular quaternary ammonium system provides an effective treatment method to overcome chemotherapy-resistant cancer caused by bacteria.

Morphologic design of sugar-based polymer nanoparticles for delivery of antidiabetic peptides.

Zwitterionic polymer nanoparticles of diverse morphologies (spherical, cylindrical, and platelet-like) constructed from biocompatible sugar-based polymers are designed to extend the pharmacological activities of short- and long-acting insulin peptides, thereby providing potential for therapeutic systems capable of reducing the frequency of administration and improving patient compliance. Amphiphilic block copolymers composed of zwitterionic poly(d-glucose carbonate) and semicrystalline polylactide segments were synthesized, and the respective block length ratios were tuned to allow formation of nanoscopic assemblies having different morphologies. Insulin-loaded nanoparticles had similar sizes and morphologies to the unloaded nanoparticle counterparts. Laser scanning confocal microscopy imaging of three-dimensional spheroids of vascular smooth muscle cells and fibroblasts after treatment with LIVE/DEAD® stain and FITC-insulin-loaded nanoparticles demonstrated high biocompatibility for the nanoconstructs of the various morphologies and significant intracellular uptake of insulin in both cell lines, respectively. Binding of short-acting insulin and long-acting insulin glargine to nanoparticles resulted in extended hypoglycemic activities in rat models of diabetes. Following subcutaneous injection in diabetic rats, insulin- and insulin glargine-loaded nanoparticles of diverse morphologies had demonstrated up to 2.6-fold and 1.7-fold increase in pharmacological availability, in comparison to free insulin and insulin glargine, respectively. All together, the negligible cytotoxicity, immunotoxicity, and minimal cytokine adsorption onto nanoparticles (as have been demonstrated in our previous studies) provide exciting and promising evidence of biocompatible nanoconstructs that are poised for further development toward the management of diabetes.

Engineering of smart nanoconstructs for delivery of glucagon-like peptide-1 analogs.

Glucagon-like peptide-1 (GLP-1) receptor agonists are being increasingly exploited in clinical practice for management of type 2 diabetes mellitus due to their ability to lower blood glucose levels and reduce off-target effects of current therapeutics. Nanomaterials had viewed myriad breakthroughs in protecting peptides against degradation and carrying therapeutics to targeted sites for maximizing their pharmacological activity and overcoming limitations associated with their application. This review highlights the latest advances in designing smart multifunctional nanoconstructs and engineering targeted and stimuli-responsive nanoassemblies for delivery of GLP-1 receptor agonists. Furthermore, advanced nanoconstructs of sophisticated supramolecular assembly yet efficient delivery of GLP-1/GLP-1 analogs, nanodevices that mediate intrinsic GLP-1 secretion per se, and nanomaterials with capabilities to load additional moieties for synergistic antidiabetic effects, are demonstrated.

Multiple analyte profiling (MAP) index as a powerful diagnostic and therapeutic monitoring tool.

A robust data mining algorithm is presented as a critical solution to the challenge of managing intensive data generated from the recently developed multiplexing techniques, which allow simultaneous detection of up to 500 biomarkers in a few microliters of a single sample. Furthermore, detailed methodology is provided for exploiting the new algorithm along with examples for description of the first application as a powerful diagnostic and therapeutic monitoring tool in the management of breast cancer, as a disease model.

Effects of Glutathione and Histidine on NO Release from a Dimeric Dinitrosyl Iron Complex (DNIC).

Rates of NO release from synthetic dinitrosyl iron complexes (DNICs) are shown to be responsive to coordination environments about iron. The effect of biologically relevant cellular components, glutathione and histidine, on the rate of NO release from a dimeric, "Roussin's Red Ester", DNIC with bridging µ-S thioglucose ligands, SGlucRRE or [(µ-SGluc)Fe(NO)2]2 (SGluc = 1-thio-ß-d-glucose tetraacetate), was investigated. From the Griess assay and X-band EPR data, decomposition of the product from the histidine-cleaved dimer, [(SGluc)(NHis)Fe(NO)2], generated Fe(III) and increased the NO release rate in aqueous media when compared to the intact SGlucRRE precursor. In contrast, increasing concentrations of exogenous glutathione generated the stable [(SGluc)(GS)Fe(NO)2]- anion and depressed the rate of NO release. Both of the cleaved, monomeric intermediates were characterized with ESI-MS, EPR, and FT-IR spectroscopies. On the basis of the Griess assay coupled with data from an intracellular fluorometric probe, both the monomeric DNICs and dimeric SGlucRRE diffuse into smooth muscle cells, chosen as appropriate archetypes of vascular relaxation, and release their NO payload. Ultimately, this work provides insight into tuning NO release beyond the design of DNICs, through the incubation with safe, accessible biological molecules.

Toward the Optimization of Dinitrosyl Iron Complexes as Therapeutics for Smooth Muscle Cells.

In this study, dinitrosyl iron complexes (DNICs) are shown to deliver nitric oxide (NO) into the cytosol of vascular smooth muscle cells (SMCs), which play a major role in vascular relaxation and contraction. Malfunction of SMCs can lead to hypertension, asthma, and erectile dysfunction, among other disorders. For comparison of the five DNIC derivatives, the following protocols were examined: (a) the Griess assay to detect nitrite (derived from NO conversion) in the absence and presence of SMCs; (b) the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay for cell viability; (c) an immunotoxicity assay to establish if DNICs stimulate immune response; and (d) a fluorometric assay to detect intracellular NO from treatment with DNICs. Dimeric Roussin's red ester (RRE)-type {Fe(NO)2}9 complexes containing phenylthiolate bridges, [(µ-SPh)Fe(NO)2]2 or SPhRRE, were found to deliver NO with the lowest effect on cell toxicity (i.e., highest IC50). In contrast, the RRE-DNIC with the biocompatible thioglucose moiety, [(µ-SGlu)Fe(NO)2]2 (SGlu = 1-thio-ß-d-glucose tetraacetate) or SGluRRE, delivered a higher concentration of NO to the cytosol of SMCs with a 10-fold decrease in IC50. Additionally, monomeric DNICs stabilized by a bulky N-heterocyclic carbene (NHC), namely, 1,3-bis(2,4,6-trimethylphenyl)imidazolidene (IMes), were synthesized and yielded the DNIC complexes SGluNHC, [IMes(SGlu)Fe(NO)2], and SPhNHC, [IMes(SPh)Fe(NO)2]. These oxidized {Fe(NO)2}9 NHC DNICs have an IC50 of ∼7 µM; however, the NHC-based complexes did not transfer NO into the SMC. Per contra, the reduced, mononuclear {Fe(NO)2}10 neocuproine-based DNIC, neoDNIC, depressed the viability of the SMCs, as well as generated an increase of intracellular NO. Regardless of the coordination environment or oxidation state, all DNICs showed a dinitrosyl iron unit (DNIU)-dependent increase in viability. This study demonstrates a structure-function relationship between the DNIU coordination environment and the efficacy of the DNIC treatments.

Functional, Degradable Zwitterionic Polyphosphoesters as Biocompatible Coating Materials for Metal Nanostructures.

A zwitterionic polyphosphoester (zPPE), specifically l-cysteine-functionalized poly(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane (zPBYP), has been developed as a poly(ethylene glycol) (PEG) alternative coating material for gold nanoparticles (AuNPs), the most extensively investigated metal nanoparticulate platform toward molecular imaging, photothermal therapy, and drug delivery applications. Thiol-yne conjugation of cysteine transformed an initial azido-terminated and alkynyl-functionalized PBYP homopolymer into zPBYP, offering hydrolytic degradability, biocompatibility, and versatile reactive moieties for installation of a range of functional groups. Despite minor degradation during purification, zPPEs were able to stabilize AuNPs presumably through multivalent interactions between combinations of the side chain zwitterions (thioether and phosphoester groups of the zPPEs with the AuNPs). 31P NMR studies in D2O revealed ca. 20% hydrolysis of the phosphoester moieties of the repeat units had occurred during the workup and purification by aqueous dialysis at pH 3 over ca. 1 d, as observed by the 31P signal of the phosphotriesters resonating at ca. -0.5 to -1.7 shifting downfield to ca. 1.1 to -0.4 ppm, attributed to transformation to phosphates. Further hydrolysis of side chain and backbone units proceeded to an extent of ca. 75% over the next 2 d in nanopure water (pH 5-6). The NMR degradation results were consistent with the broadening and red-shift of the surface plasmon resonance (SPR) observed by UV-vis spectroscopy of the zPPE-coated AuNPs in water over time. All AuNP formulations in this study, including those with citrate, PEG, and zPPE coatings, exhibited negligible immunotoxicity, as determined by cytokine overexpression in the presence of the nanostructures relative to those in cell culture medium. Notably, the zPPE-coated AuNPs displayed superior antifouling properties, as assessed by the extent of cytokine adsorption relative to both the PEGylated and citrate-coated AuNPs. Taken together, the physicochemical and biological evaluations of zPPE-coated AuNPs in conjunction with PEGylated and citrate-coated analogues indicate the promise of zPPEs as favorable alternatives to PEG coatings, with negligible immunotoxicity, good antifouling performance, and versatile reactive groups that enable the preparation of highly tailored nanomaterials for diverse applications.

Resistance of primary breast cancer cells with enhanced pluripotency and stem cell activity to sex hormonal stimulation and suppression.

Female sex steroid hormones have a fundamental role in breast cancer. Meanwhile, current evidence supports the contribution of breast cancer stem cells in carcinogenesis, metastasis, and resistance to cytotoxic chemotherapy. Nevertheless, the interaction between breast cancer stem cells with sex hormones or key hormonal antagonists remains elusive. OBJECTIVE: To investigate the effect of diverse sex hormonal stimulation and suppression regimens on the proliferation of a primary human breast cancer cells with stem cell activity. METHODS: Cells were exposed to estradiol, progesterone, letrozole, ulipristal acetate, or a combination of ulipristal acetate-letrozole, continually for 6 months. Additionally, nanoparticle-linked letrozole and ulipristal acetate formulations were included in a subsequent short-term exposure study. Phenotypic, pathologic, and functional characteristics of unexposed cells were investigated. RESULTS: The proliferation of breast cancer cells was comparable among all hormonal stimulation and suppression groups (P= 0.8). In addition, the nanoparticle encapsulated hormonal antagonists were not able to overcome the observed resistance of cells. Cell characterization showed a mesenchymal-like phenotype overexpressing three master pluripotency markers (Oct 4, SOX2, and Nanog), and 92% of cells were expressing ALDH1A1. Notably, the CD44 high/CD24 low cell population presented only 0.97%-5.4% over repeat analyses. Most cells lacked the expression of mesenchymal markers; however, they showed differentiation into osteogenic and adipogenic lineages. Upon transfer to serum-free culture, the long-term maintained mesenchymal-like cancer cells showed remarkable morphologic plasticity as they switched promptly into an epithelial-like phenotype with significant mammosphere formation capacity (P= 0.008). CONCLUSION: Breast cancer cells can develop a pluripotent program with enhanced stemness activity that may together contribute to universal resistance to sex hormonal stimulation or deprivation. Isolation and characterization of patient-derived breast cancer stem cells in large clinical studies is therefore crucial to identify new targets for endocrine therapies, potentially directed towards stemness and pluripotency markers. Such direction may help overcoming endocrine resistance and draw attention to breast cancer stem cells' behaviour under endogenous and exogenous sex hormones throughout a woman's reproductive life.

A Vinyl Ether-Functional Polycarbonate as a Template for Multiple Postpolymerization Modifications.

A highly-reactive vinyl ether-functionalized aliphatic polycarbonate and its block copolymer were developed as templates for multiple post-polymerization conjugation chemistries. The vinyl ether-functional six-membered cyclic carbonate monomer was synthesized by a well-established two-step procedure starting from 2,2-bis(hydroxymethyl)propionic acid. An organobase-catalyzed ring-opening polymerization of the synthesized monomer afforded polycarbonates with pendant vinyl ether functionalities (PMVEC). The vinyl ether moieties on the resulting polymers were readily conjugated with hydroxyl- or thiol-containing compounds via three different post-polymerization modification chemistries - acetalization, thio-acetalization, and thiol-ene reaction. Acetal-functionalized polycarbonates were studied in depth to exploit their acid-labile acetal functionalities. Acetalization of the amphiphilic diblock copolymer of poly(ethylene glycol) methyl ether (mPEG) and PMVEC, mPEG113-b-PMVEC13, with the model hydroxyl compound 4- methylbenzyl alcohol resulted in a maximum of 42% acetal and 58% hydroxyl side chain groups. Nonetheless, the amphiphilicity of the block polymer allowed for its self-assembly in water to afford nanostructures, as characterized via dynamic light scattering and transmission electron microscopy. The kinetics of acetal cleavage within the block polymer micelles were examined in acidic buffered solutions (pH 4 and 5). In addition, mPEG-b-PMVEC and its hydrolyzed polymer mPEG-b-PMHEC (i.e., after full cleavage of acetals) exhibited minimal cytotoxicity to RAW 264.7 mouse macrophages, indicating that this polymer system represents a biologically non-hazardous material with pH-responsive activity.

Development of Fully Degradable Phosphonium-Functionalized Amphiphilic Diblock Copolymers for Nucleic Acids Delivery.

To expand the range of functional polymer materials to include fully hydrolytically degradable systems that bear bioinspired phosphorus-containing linkages both along the backbone and as cationic side chain moieties for packaging and delivery of nucleic acids, phosphonium-functionalized polyphosphoester- block-poly(l-lactide) copolymers of various compositions were synthesized, fully characterized, and their self-assembly into nanoparticles were studied. First, an alkyne-functionalized polyphosphoester- block-poly(l-lactide) copolymer was synthesized via a one pot sequential ring opening polymerization of an alkyne-functionalized phospholane monomer, followed by the addition of l-lactide to grow the second block. Second, the alkynyl side groups of the polyphosphoester block were functionalized via photoinitiated thiol-yne radical addition of a phosphonium-functionalized free thiol. The polymers of varying phosphonium substitution degrees were self-assembled in aqueous buffers to afford formation of well-defined core-shell assemblies with an average size ranging between 30 and 50 nm, as determined by dynamic light scattering. Intracellular delivery of the nanoparticles and their effects on cell viability and capability at enhancing transfection efficiency of nucleic acids (e.g., siRNA) were investigated. Cell viability assays demonstrated limited toxicity of the assembly to RAW 264.7 mouse macrophages, except at high polymer concentrations, where the polymer of high degree of phosphonium functionalization induced relatively higher cytotoxicity. Transfection efficiency was strongly affected by the phosphonium-to-phosphate (P+/P-) ratios of the polymers and siRNA, respectively. The AllStars Hs Cell Death siRNA complexed to the various copolymers at a P+/P- ratio of 10:1 induced comparable cell death to Lipofectamine. These fully degradable nanoparticles might provide biocompatible nanocarriers for therapeutic nucleic acid delivery.

Poly(glycerol methacrylate)-based degradable nanoparticles for delivery of small interfering RNA.

Nucleic acids therapeutic efficiency is generally limited by their low stability and intracellular bioavailability, and by the toxicity of the carriers used to deliver them to the target sites. Aminated poly(glycerol methacrylate) polymers are biodegradable and pH-sensitive polymers that have been used previously to deliver antisense oligonucleotide and show high transfection efficiency. The purpose of this study is to compare the efficiency and toxicity of aminated linear poly(glycerol methacrylate) (ALT) biodegradable polymer to the most commonly used cationic degradable (i.e. chitosan) and non-degradable (i.e. polyethylenimine (PEI)) polymers for delivery of short interfering RNA (siRNA). ALT, PEI and chitosan polymers were able to form nanosized particles with siRNA. Size, size-distribution and zeta-potential were measured over a wide range of nitrogen-to-phosphate (N/P) ratios, and the stability of the formed nanoparticles in saline and upon freeze-drying was also assessed. No significant cytotoxicity at the range of the tested concentrations of ALT and chitosan nanoparticles was observed, whereas the non-degradable PEI showed significant toxicity in huh-7 hepatocyte-derived carcinoma cell line. The safety profiles of the degradable polymers (ALT and chitosan) over non-degradable PEI were demonstrated in vitro and in vivo. In addition, ALT nanoparticles were able to deliver siRNA in vivo with significantly higher efficiency than chitosan nanoparticles. The results in the present study give evidence of the great implications of ALT nanoparticles in biomedical applications due to their biocompatibility, low cytotoxicity, high stability and simple preparation method.