Development of Rofecoxib-Based Fluorescent Probes and Investigations on Their Solvatochromism, AIE Activity, Mechanochromism, and COX-2-Targeted Bioimaging.

Cyclooxygenase-2 (COX-2) fluorescent probes are promising tools for early diagnosis of cancer. Traditionally, COX-2 probes were designed by connecting two parts, a fluorophore and a COX-2 binding unit, via a flexible linker. Herein, a new class of COX-2-specific fluorescent probes have been developed via one-step modification from rofecoxib by an integrative approach to combine the binding unit and the fluorophore into one. Among them, several new rofecoxib analogues not only exhibited still potent COX-2 binding ability but also exhibited attractive fluorescence properties, such as tunable blue-red emission, solvatochromism, aggression-induced emission behavior, and mechanochromism. Notably, the emission of 2a16 can be switched between green-yellow in the crystalline state and red-orange in the amorphous state by grinding and fuming treatments. Furthermore, the highly fluorescent compound 2a16 (Φf = 0.94 in powder) displayed a much stronger fluorescence imaging of COX-2 in HeLa cancer cells overexpressing COX-2 than RAW264.7 normal cells with a minimal expression of COX-2. Most importantly, 2a16 can light up human cancer tissues from adjacent normal tissues with a much brighter fluorescence by targeting the COX-2 enzyme. These results demonstrated the potential of 2a16 as a new red fluorescent probe for human cancer imaging in clinical applications.

Design and Synthesis of a Novel NIR Celecoxib-Based Fluorescent Probe for Cyclooxygenase-2 Targeted Bioimaging in Tumor Cells.

Cyclooxygenase-2 (COX-2) imaging agents are potent tools for early cancer diagnosis. Almost all of the COX2 imaging agents using celecoxib as backbone were chemically modified in the position of N-atom in the sulfonamide group. Herein, a novel COX-2 probe (CCY-5) with high targeting ability and a near-infrared wavelength (achieved by attaching a CY-5 dye on the pyrazole ring of celecoxib using a migration strategy) was evaluated for its ability to probe COX-2 in human cancer cells. CCY-5 is expected to have high binding affinity for COX-2 based on molecular docking and enzyme inhibition assay. Meanwhile, CCY-5 caused stronger fluorescence imaging of COX-2 overexpressing cancer cells (Hela and SCC-9 cells) than that of normal cell lines (RAW 264.7 cells). Lipopolysaccharide (LPS) treated RAW264.7 cells revealed an enhanced fluorescence as LPS was known to induce COX-2 in these cells. In inhibitory studies, a markedly reduced fluorescence intensity was observed in cancer cells, when they were co-treated with a COX-2 inhibitor celecoxib. Therefore, CCY-5 may be a selective bioimaging agent for cancer cells overexpressing COX-2 and could be useful as a good monitoring candidate for effective diagnosis and therapy in cancer treatment.

Long Noncoding RNA (lncRNA)-Mediated Competing Endogenous RNA Networks Provide Novel Potential Biomarkers and Therapeutic Targets for Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer and has a high metastasis and reoccurrence rate. Long noncoding RNAs (lncRNAs) play an important role in CRC growth and metastasis. Recent studies revealed that lncRNAs participate in CRC progression by coordinating with microRNAs (miRNAs) and protein-coding mRNAs. LncRNAs function as competitive endogenous RNAs (ceRNAs) by competitively occupying the shared binding sequences of miRNAs, thus sequestering the miRNAs and changing the expression of their downstream target genes. Such ceRNA networks formed by lncRNA/miRNA/mRNA interactions have been found in a broad spectrum of biological processes in CRC, including liver metastasis, epithelial to mesenchymal transition (EMT), inflammation formation, and chemo-/radioresistance. In this review, we summarize typical paradigms of lncRNA-associated ceRNA networks, which are involved in the underlying molecular mechanisms of CRC initiation and progression. We comprehensively discuss the competitive crosstalk among RNA transcripts and the novel targets for CRC prognosis and therapy.

Effect of Retro-Inverso Isomer of Bradykinin on Size-Dependent Penetration of Blood-Brain Tumor Barrier.

Retro-inverso bradykinin (RI-BK) has better metabolic stability and higher affinity for the BK type 2 (B2) receptor, compared with bradykinin. At low doses, RI-BK can selectively enhance the permeability of the blood-brain tumor barrier (BBTB) without harming normal brain tissue. In this study, gold nanoparticles (GNPs) of size ranging from 5 to 90 nm are synthesized to assess the optimal size of nanocarriers that achieves maximum brain accumulation after the treatment of RI-BK. The ability of the GNPs to cross the BBTB is tested in a rat C6 glioma tumor model. The results of inductively coupled plasma-mass spectrometry and transmission electron microscopy indicate that GNPs with size of 70 nm achieve maximum permeability to the glioma. The present study supports the conclusion that RI-BK can enhance the permeability of BBTB and provides fundamental information for further development of nanomedicines or nanoprobes for glioma therapy.

D-Peptides as Recognition Molecules and Therapeutic Agents.

Over recent years, D-peptides have attracted increasing attention. D-peptides increase enzymatic stability, prolong the plasma half-life, improve oral bioavailability, and enhance binding activity and specificity with receptor or target proteins, in comparison with the corresponding L-peptide. Therefore, D-peptides are considered to have potential as recognition molecules and therapeutic agents. This review focuses on the design and application of D-peptides with biological activity.

D-SP5 Peptide-Modified Highly Branched Polyethylenimine for Gene Therapy of Gastric Adenocarcinoma.

Peptide-mediated targeting of tumors has become an effective strategy for cancer therapy. Retro-inverso peptides resist protease degradation and maintain their bioactivity. We used the retro-inverso peptide D(PRPSPKMGVSVS) (D-SP5) as a targeting ligand to develop gene therapy for gastric adenocarcinoma. D-SP5 has a higher affinity for human gastric adenocarcinoma (SGC7901) cells compared with that of its parental peptide, L(SVSVGMKPSPRP) (L-SP5). Polyethylenimine (PEI)/pDNA, polyethylene glycol (mPEG)-PEI/pDNA and D-SP5-PEG-PEI/pDNA were prepared for further study. Quantitative luciferase assays showed the transfection efficiency of D-SP5-PEG-PEI/pGL(4.2) was larger compared with that of mPEG-PEI/pGL(4.2). Flow cytometry assays revealed that the apoptosis rates of SGC7901 cells treated with D-SP5-PEG-PEI/pTRAIL were larger than mPEG-PEI/pTRAIL. Western blot assays indicated that the expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) protein in SGC7901 cells treated with D-SP5-PEG-PEI/pTRAIL was higher compared with that in cells treated with mPEG-PEI/pTRAIL. In vivo pharmacodynamics study revealed that D-SP5-PEG-PEI/pTRAIL could inhibit the growth of gastric adenocarcinoma SGC7901 xenografts in nude mice. Our results demonstrate that D-SP5-PEG-PEI is a safe and efficient gene delivery vector with potential applications in antitumor gene therapy.

Retro-inverso CendR peptide-mediated polyethyleneimine for intracranial glioblastoma-targeting gene therapy.

The development of nonviral gene delivery vectors offers the potential to provide effective treatment for glioblastoma in the form of gene therapy. Here, we report the use of retro-inverso C-end rule (CendR) peptide D(RPPREGR) as a targeting ligand to prepare a D(RPPREGR)-PEG-PEI gene vector. D(RPPREGR) peptide specifically recognized the neuropilin-1 receptor that was overexpressed on U87 glioma cells, and showed enhanced tumor spheroid penetration ability. Compared with parental RGERPPR, D(RPPREGR) possessed improved biological stability and had a higher affinity for U87 glioma cells; it also showed enhanced penetration of the tumor spheroid. mPEG-PEI/pDNA and D(RPPREGR)-PEG-PEI/pDNA complexes were prepared and MTT assay results revealed that the cytotoxicity of D(RPPREGR)-PEG-PEI complexes was significantly lower than that of PEI complexes, with cell survival rates above 80%. Qualitative and quantitative in vitro transfection results revealed that D(RPPREGR)-PEG-PEI complex transfection efficiencies were 1.9 times higher than those of mPEG-PEI. Fluorescent imaging and frozen sections of brain tissue demonstrated that the D(RPPREGR) modification improved the in vivo transfection efficiency of mPEG-PEI in nude mice bearing U87 gliomas. An antiglioblastoma assay revealed that D(RPPREGR)-PEG-PEI carrying the therapeutic gene pORF-hTRAIL significantly prolonged the survival time of intracranial U87 glioma-bearing mice from 25 to 30 days. Therefore, D(RPPREGR)-PEG-PEI appears to be suitable for use as a safe and efficient gene delivery vehicle with potential applications in glioblastoma gene therapy.

Reduction of systemic exposure and side effects by intra-articular injection of anti-inflammatory agents for osteoarthritis: what is the safer strategy?

Osteoarthritis (OA) is a chronic degenerative joint disease associated with pain, inflammation, and cartilage degradation. However, no current treatment can effectively halt the progression of the disease. Therefore, the use of NSAIDs and intra-articular corticosteroids is usually recommended as the primary treatment for OA-associated pain and inflammation. However, there is accumulating evidence that the long-term use of oral NSAIDs and intra-articular corticosteroids can lead to a myriad of negative side effects. Although numerous efforts have been made to develop intra-articular formulations for NSAIDs, the systemic exposure of intra-articular injection of NSAIDs and its potential side effects have not been explicitly investigated. To ascertain the evident and potential side effects of intra-articular injection of anti-inflammatory agents, we have summarised in this review the systemic exposure, local side effects, and systemic side effects of intra-articular injections of anti-inflammatory agents, including NSAIDs and corticosteroids. For developing a safer treatment to fulfil the unmet long-term use needs of patients, a new therapy, which combines the locally active drug and a sustained-release formulation, has been proposed in this review.

Development of Rofecoxib-Based Fluorophores from ACQ to AIE by Positional Regioisomerization.

The development of aggregation-induced emission luminogens (AIEgens) has attracted increasing attention due to their potential applications in various areas in recent years. In this study, a facile conversion from aggregation-caused quenching (ACQ) to aggregation-induced emission (AIE) was achieved by an efficient regioisomerization strategy based on the rofecoxib scaffold. Two compounds, named PYR2 and PYR4, were identified as regioisomers of rofecoxib derivatives to show dramatically different fluorescent properties. Compound PYR2 with an ortho-substituted piperidine group showed typical AIE activity while compound PYR4 with a para-piperidine group exhibited typical ACQ behavior. Notably, compound PYR2 showed polymorphism with two forms of crystals. It was also endowed with reversible mechanochromic luminescence and acidochromic properties. The different fluorescent properties were elucidated by UV/Vis absorption spectroscopy, powder X-ray diffraction, differential scanning calorimetry, and thermogravimetric analyses. Its application as a security ink and in lipid droplets imaging have been demonstrated.

Knowledge mapping concerning applications of nanocomposite hydrogels for drug delivery: A bibliometric and visualized study (2003-2022).

Background: Nanocomposite Hydrogels (NHs) are 3D molecular networks formed by physically or covalently crosslinking polymer with nanoparticles or nanostructures, which are particularly suitable for serving as carriers for drug delivery systems. Many articles pertaining to the applications of Nanocomposite Hydrogels for drug delivery have been published, however, the use of bibliometric and visualized analysis in this area remains unstudied. The purpose of this bibliometric study intended to comprehensively analyze the knowledge domain, research hotspots and frontiers associated with the applications of Nanocomposite Hydrogels for drug delivery. Methods: We identified and retrieved the publications concerning the applications of NHs for drug delivery between 2003 and 2022 from Web of Science Core Collection Bibliometric and visualized analysis was utilized in this investigative study. Results: 631 articles meeting the inclusion criteria were identified and retrieved from WoSCC. Among those, 2,233 authors worldwide contributed in the studies, accompanied by an average annual article increase of 24.67%. The articles were co-authored by 764 institutions from 52 countries/regions, and China published the most, followed by Iran and the United States. Five institutions published more than 40 papers, namely Univ Tabriz (n = 79), Tabriz Univ Med Sci (n = 70), Islamic Azad Univ (n = 49), Payame Noor Univ (n = 42) and Texas A&M Univ (n = 41). The articles were published in 198 journals, among which the International Journal of Biological Macromolecules (n = 53) published the most articles, followed by Carbohydrate Polymers (n = 24) and ACS Applied Materials and Interfaces (n = 22). The top three journals most locally cited were Carbohydrate Polymers, Biomaterials and Advanced materials. The most productive author was Namazi H (29 articles), followed by Bardajee G (15 articles) and Zhang J (11 articles) and the researchers who worked closely with other ones usually published more papers. "Doxorubicin," "antibacterial" and "responsive hydrogels" represent the current research hotspots in this field and "cancer therapy" was a rising research topic in recent years. "(cancer) therapeutics" and "bioadhesive" represent the current research frontiers. Conclusion: This bibliometric and visualized analysis offered an investigative study and comprehensive understanding of publications regarding the applications of Nanocomposite Hydrogels for drug delivery from 2003 to 2022. The outcome of this study would provide insights for researchers in the field of Nanocomposite Hydrogels applications for drug delivery.

Comparison of Absolute Expression and Turnover Number of COX-1 and COX-2 in Human and Rodent Cells and Tissues.

Objective: We aim to quantify the absolute protein expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in various cells and tissues to determine the relative contribution of COX-1 and COX-2 to PGE2 production. Methods: An LC-MS method was developed and validated, then used for quantifying the absolute amounts of COX-1 and COX-2 in recombinant human COX-1 and COX-2, lysates from different cells, tissue microsomes of rodents and humans, Pirc rat colonic polyps, and biopsy specimens from squamous cell carcinoma (SCC) patients. The COX-1 and COX-2 turnover numbers were subsequently calculated based on apparent formation rates of PGE2. Results: A robust LC-MS method for quantification of COX-1 and COX-2 was developed and validated and then used to calculate their apparent turnover numbers. The results showed that COX-1 expression levels were much higher than that of COX-2 in all the tested tissues including the colonic epithelium of F344 (28-fold) and Pirc rats (20-fold), colonic polyps of Pirc rats (8-fold), and biopsy specimens of SCC patients (11-17-fold). In addition, both COX-1 and COX-2 were higher in polyps when compared to adjacent mucosa of Pirc rats. The turnover number of recombinant human COX-2 was 14-fold higher than that of recombinant human COX-1. LPS stimulation increased COX-2 protein expression in three cell lines (Raw 264.7, SCC9 and EOMA) as expected but unexpectedly increased COX-1 protein expression (13.8-fold) in EOMA cells. Conclusion: In human oral cancer tissues and cells as well as Pirc rat colon, COX-1 plays an unexpectedly but more important role than COX-2 in abnormal PGE2 production since COX-1 expression is much higher than COX-2. In addition, COX-1 expression levels are inducible in cells, and higher in polyps than surrounding mucosa in Pirc rat colon. These results indicate that targeted suppression of local COX-1 should be considered to reduce colon-specific PGE2-mediated inflammation.

One-Step Transformation from Rofecoxib to a COX-2 NIR Probe for Human Cancer Tissue/Organoid Targeted Bioimaging.

COX-2 fluorescent probes are promising tools for cancer diagnosis. Such probes have been conventionally designed by conjugating a fluorophore to COX-2 inhibitors through lengthy synthetic processes. Herein, a type of fluorescent probe for COX-2 imaging has been developed using a single-step process from rofecoxib. In total, six rofecoxib analogues were designed using this unique strategy. Several analogues retained comparative COX-2 targeting activity of rofecoxib and also exhibited attractive fluorescent properties, which were investigated using a combination of experimental and theoretical approaches. The most potent analogue, 2a1, displayed strong fluorescent imaging of COX-2 in HeLa cells overexpressing COX-2 compared to Raw 264.7 cells and celecoxib-treated HeLa cells that expressed low levels of COX-2. Notably, our studies indicate that 2a1 can differentiate human cancer tissue from adjacent tissue with much brighter fluorescence either in histological section or cultured 3D organoids. These results illustrate the potential of 2a1 as a COX-2 near infrared fluorescent probe for human cancer imaging in clinical settings.

Nitric Oxide (NO) and NO Synthases (NOS)-Based Targeted Therapy for Colon Cancer.

Colorectal cancer (CRC) is one of the most lethal malignancies worldwide and CRC therapy remains unsatisfactory. In recent decades, nitric oxide (NO)-a free-radical gas-plus its endogenous producer NO synthases (NOS), have attracted considerable attention. NO exerts dual effects (pro- and anti-tumor) in cancers. Endogenous levels of NO promote colon neoplasms, whereas exogenously sustained doses lead to cytotoxic functions. Importantly, NO has been implicated as an essential mediator in many signaling pathways in CRC, such as the Wnt/ß-catenin and extracellular-signal-regulated kinase (ERK) pathways, which are closely associated with cancer initiation, metastasis, inflammation, and chemo-/radio-resistance. Therefore, NO/NOS have been proposed as promising targets in the regulation of CRC carcinogenesis. Clinically relevant NO-donating agents have been developed for CRC therapy to deliver a high level of NO to tumor sites. Notably, inducible NOS (iNOS) is ubiquitously over-expressed in inflammatory-associated colon cancer. The development of iNOS inhibitors contributes to targeted therapies for CRC with clinical benefits. In this review, we summarize the multifaceted mechanisms of NO-mediated networks in several hallmarks of CRC. We review the clinical manifestation and limitations of NO donors and NOS inhibitors in clinical trials. We also discuss the possible directions of NO/NOS therapies in the immediate future.

Retro-inverso bradykinin opens the door of blood-brain tumor barrier for nanocarriers in glioma treatment.

The blood-brain barrier and the blood-brain tumor barrier (BBTB) prevent most drugs entering brain tumors. Complicated preparation procedures of drug delivery systems and damage to normal brain tissue have limited the application of many strategies for the treatment of brain tumor in clinical trials. We have designed a bradykinin analog, retro-inverso bradykinin (RI-BK), which is characterized by resistance to proteolysis and high binding activity with the bradykinin type 2 (B2) receptor. After systemic administration, RI-BK binds to B2 receptors and induces a change in zonula occluden-1 and depolymerization of F-actin to selectively open the BBTB. RI-BK increased the accumulation of drug-loaded nanocarriers in the glioma but not in normal brain. Co-administration with RI-BK enhanced the therapeutic efficiency of drug-loaded nanocarriers for glioma. These findings suggest that RI-BK could be translated into the clinic as an adjunctive treatment for malignant brain tumors.

MPEG-DSPE polymeric micelle for translymphatic chemotherapy of lymph node metastasis.

Lymph node metastasis is one of the major pathways for tumor formation and it is difficult to deliver chemotherapeutics at therapeutic concentrations to lymph node metastasis. This study prepared methyl poly(ethylene glycol)-distearoylphosphatidylethanolamine/doxorubicin (MPEG-DSPE/DOX) micelle for the treatment of lymph node metastasis. The MPEG-DSPE/DOX micelle prepared were of spherical morphology with a particle size of 20 ± 5 nm. The uptake rates of DOX and MPEG-DSPE/DOX micelle by A375 cells were 51.2% and 88.7%, respectively. The phagocytosis rate of MPEG-DSPE/Rhodamine B micelle by RAW264.7 cells was 17.2-fold lower than for Rhodamine B alone. After subcutaneous injection, MPEG-DSPE micelle underwent lymphatic absorption and accumulated in popliteal lymph nodes. MPEG-DSPE/DOX micelle significantly alleviated damage to the subcutaneous tissue of the injection sites compared with DOX alone. We established a model of nude mice bearing lymph node metastasis of A375 cells. After subcutaneous injection, the weights of both the popliteal and iliac lymph nodes of the MPEG-DSPE/DOX micelle group were significantly lower than in the saline and DOX groups. MPEG-DSPE/DOX micelle effectively killed the tumor cells in popliteal and iliac lymph nodes. In conclusion, MPEG-DSPE micelle is a promising drug delivery system for the treatment of lymph node metastasis.

[Study on the spectrophotometric determination of microamounts of iron with 1-(2-pyridylazo)-2-naphthol-6-sulphonic acid].

A new spectrophotometric method has been developed for the highly selective determination of Fe2+ with 1-(2-pyridylazo)-2-naphthol-6-sulphonic Acid. At pH 4.0, Fe2+ an be selectively determined at the wavelength of 760 nm. The calibration curve is liner in a concentration range of 0-10 micrograms/10 ml, relative coefficient is 0.9997, the apparent molar absorptivity is 1.57 x 104 L.mol-1.cm-1, RSD of within-run and between-day were 1.66% and 2.51%, the average rate of recovery 101.1%. The method has been applied to the direct determination of iron in milk powder, common coexistent substances don't interfere with the determination.

Isomeric folate-conjugated polymeric micelles bind to folate receptors and display anticancer effects.

The present study aimed to prepare and evaluate polymeric micelles conjugated with folic acid through α- or γ-carboxyl groups for antitumor efficacy. The isomeric block copolymers, α- and γ-folate-polyethyleneglycol- distearoyl phosphatidylethanolamine (α- and γ-Fol-PEG-DSPE), were produced by solid phase peptide synthesis. Three types of doxorubicin (DOX)-loaded polymeric micelles (MPEG-DSPE-DOX and α- / γ-Fol-PEG-DSPE- DOX micelles) were prepared via the film formation method. Compared with MPEG-DSPE-DOX micelles, the α- / γ-Fol-PEG-DSPE-DOX micelles presented a higher cellular uptake behavior in the live cell study. Cell viability percentages were 81.8%, 57.3%, 56.6% at 2 hours for MPEG-DSPE-DOX, α- and γ-Fol-PEG-DSPE- DOX micelles, respectively (p<0.05). Using the KB xenograft tumor model, both α- and γ-folate-conjugated micelles were found to have better antitumor effects with lower toxicity in comparison with MPEG-DSPE-DOX micelles. No difference in in vivo antitumor efficacy was found between α-and γ-Fol-PEG-DSPE-DOX micelles. The folate-conjugated micelles might be a potentially useful strategy for tumor targeting of therapeutic agents, whether grafting with folic acid through α- or γ-carboxyl groups.

Targeted gene delivery to glioblastoma using a C-end rule RGERPPR peptide-functionalised polyethylenimine complex.

Safe and efficient systems capable of specifically targeting brain tumour cells represent a promising approach for the treatment glioblastoma multiforme. Neuropilin-1 (NRP-1) is over-expressed in U87 glioma cells. In the current study, the tumour specific peptide RGERPPR, which binds specifically to NRP-1, was used as a targeting ligand in a gene delivery strategy for glioblastoma. The RGERPPR peptide was coupled to branched polyethylenimine (PEI, 25kDa) using heterobifunctional Mal-PEG-NHS, resulting in a novel gene delivery polymer. Polymer/plasmid DNA (pDNA) complexes were formed and their sizes and zeta potentials were measured. Compared with the unmodified mPEG-PEI/pDNA complexes, the RGERPPR-PEG-PEI/pDNA complex led to a significant enhancement in intracellular gene uptake and tumour spheroid penetration. Furthermore, the RGERPPR-PEG-PEI/pDNA complex facilitated enhanced transfection efficiency levels, as well as a reduction in cytotoxicity when tested in U87 glioma cells in vitro. Most significantly of all, when complexes formed with pDsRED-N1 were injected into the tail vein of intracranial U87 tumour-bearing nude mice, the RGERPPR-PEG-PEI complexes led to improved levels of red fluorescence protein expression in the brain tissue. Taken together, the results show that RGERPPR-PEG-PEI could be used as a safe and efficient gene delivery vehicle with potential applications in glioblastoma gene delivery.