A Multiepitope Peptide, rOmp22, Encapsulated in Chitosan-PLGA Nanoparticles as a Candidate Vaccine Against Acinetobacter baumannii Infection.

BACKGROUND: The development of vaccines is a promising and cost-effective strategy to prevent emerging multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) infections. The purpose of this study was to prepare a multiepitope peptide nanovaccine and evaluate its immunogenicity and protective effect in BALB/c mice. METHODS: The B-cell and T-cell epitopes of Omp22 from A. baumannii were predicted using bioinformatics methods and identified by immunological experiments. The optimal epitopes were conjugated in series by 6-aminocaproic acid and chemically synthesized multiepitope polypeptide rOmp22. Then, rOmp22 was encapsulated by chitosan (CS) and poly (lactic-co-glycolic) acid (PLGA) to prepare CS-PLGA-rOmp22 nanoparticles (NPs). The immunogenicity and immunoprotective efficacy of the vaccine were evaluated in BALB/c mice. RESULTS: CS-PLGA-rOmp22 NPs were small (mean size of 272.83 nm) with apparently spherical structures, positively charged (4.39 mV) and nontoxic to A549 cells. A high encapsulation efficiency (54.94%) and a continuous slow release pattern were achieved. Compared with nonencapsulated rOmp22, CS-PLGA-rOmp22 immunized BALB/c mice induced higher levels of rOmp22-specific IgG in serum and IFN-γ in splenocyte supernatant. Additionally, lung injury and bacterial burdens in the lung and blood were suppressed, and potent protection (57.14%-83.3%) against acute lethal intratracheal A. baumannii challenge was observed in BALB/c mice vaccinated with CS-PLGA-rOmp22. CONCLUSION: CS-PLGA-rOmp22 NPs elicited specific IgG antibodies, Th1 cellular immunity and protection against acute lethal intratracheal A. baumannii challenge. Our results indicate that this nanovaccine is a desirable candidate for preventing A. baumannii infection.

Identifying metastatic ability of prostate cancer cell lines using native fluorescence spectroscopy and machine learning methods.

Metastasis is the leading cause of mortalities in cancer patients due to the spreading of cancer cells to various organs. Detecting cancer and identifying its metastatic potential at the early stage is important. This may be achieved based on the quantification of the key biomolecular components within tissues and cells using recent optical spectroscopic techniques. The aim of this study was to develop a noninvasive label-free optical biopsy technique to retrieve the characteristic molecular information for detecting different metastatic potentials of prostate cancer cells. Herein we report using native fluorescence (NFL) spectroscopy along with machine learning (ML) to differentiate prostate cancer cells with different metastatic abilities. The ML algorithms including principal component analysis (PCA) and nonnegative matrix factorization (NMF) were used for dimension reduction and feature detection. The characteristic component spectra were used to identify the key biomolecules that are correlated with metastatic potentials. The relative concentrations of the molecular spectral components were retrieved and used to classify the cancer cells with different metastatic potentials. A multi-class classification was performed using support vector machines (SVMs). The NFL spectral data were collected from three prostate cancer cell lines with different levels of metastatic potentials. The key biomolecules in the prostate cancer cells were identified to be tryptophan, reduced nicotinamide adenine dinucleotide (NADH) and hypothetically lactate as well. The cancer cells with different metastatic potentials were classified with high accuracy using the relative concentrations of the key molecular components. The results suggest that the changes in the relative concentrations of these key fluorophores retrieved from NFL spectra may present potential criteria for detecting prostate cancer cells of different metastatic abilities.

Enhanced Safety and Antitumor Efficacy of Switchable Dual Chimeric Antigen Receptor-Engineered T Cells against Solid Tumors through a Synthetic Bifunctional PD-L1-Blocking Peptide.

Chimeric antigen receptor (CAR)-engineered T cells (CAR-Ts) therapy has an excellent efficacy in cancer treatment, especially its impressive results in hematological malignancies. Unfortunately, its application on solid tumors is challenged by the off-target effects caused by lacking of tumor specific antigens and the immunosuppression caused by the tumor microenvironment. We constructed a switchable dual receptor CAR-T cell (sdCAR-T) whose activity relied upon double antigens (mesothelin and fluorescein isothiocyanate) and was strictly controlled by a "switch" (FPBM) consisting of a PD-L1 blocking peptide conjugated to fluorescein isothiocyanate. SdCAR-T cells were activated only when FPBM and cognate tumor cells expressing both PD-L1 and mesothelin coexist. Importantly, long-term proliferation experiments in vitro and the pharmacodynamic study in vivo showed a stronger antitumor activity of this system compared to the second generation mesothelin CAR-T cells. In view of this novel treatment paradigm being safer and more effective than traditional CAR-T cells, it may become a new strategy for the treatment of solid tumors.

Combined administration of PTX and S-HM-3 in TPGS/Solutol micelle system for oncotarget therapy.

BACKGROUND: S-HM-3 is a tumor angiogenesis inhibitor with short half-life (25 min). In this present, TPGS/Solutol polymeric micelles was prepared to load together insoluble paclitaxel (PTX) and soluble S-HM-3, expecting to together deliver them to the tumor site with long-circulating, targeting function and combating multi-drug resistance (MDR). MATERIALS AND METHODS: PTX and S-HM-3 loaded TPGS/Solutol micelles (PHTSm) were prepared by the method of thin-film evaporation, and characterized by dynamic light scattering, transmission electron microscope (TEM), atomic force microscopy (AFM) and releasing properties. The anticancer effect of the polymeric micelles system was evaluated and confirmed by experiments of in vitro cell uptake study, in vivo pharmacokinetics, and pharmacodynamics studies. RESULTS: Micelles exhibited smooth spherical morphology with 20~30 nm and low critical micelle concentration (CMC) value of 0.000124 mg/mL. Only about 30% of PTX were slowly released from micelles at 48h, which can beneficial to the long circulation in blood. The results of in vitro cell assay proved that S-HM-3 could be easier to get into MDA-MB-231 cell, and its angiogenesis inhibition ability was also enhanced after integrating into micelles. In particular, the results of in vivo studies showed that the half-life of S-HM-3 and PTX was significantly prolonged 25.27 and 5.54 folds, and their AUC0-∞ was enhanced 129.78 and 15.65 times, respectively. Meanwhile 83.05% tumor inhibition rate of PHTSm was achieved compared with 59.99% of PTX. CONCLUSIONS: TPGS and Solutol micelles hold promising potential to resolve the conundrum of combined therapy of cytotoxic drug and angiogenesis inhibitor with different physicochemical property and anticancer mechanism in clinical use.

Recombination of a dual-CAR-modified T lymphocyte to accurately eliminate pancreatic malignancy.

BACKGROUND: The therapeutic application of T cells endowing with chimeric antigen receptors (CARs) is faced with "on-target, off-tumor" toxicity against solid tumors, particularly in the treatment of the pancreatic cancer. To our best knowledge, the pancreatic cancer cell line AsPC-1 often highly expressed some distinct tumor-associated antigens, such as carcino-embryonic antigen (CEA) and mesothelin (MSLN). Therefore, in this research, we have characterized dual-receptor CAR-modified T cells (dCAR-T) that exert effective and safe cytotoxicity against AsPC-1 cells. METHODS: Based on the dual signaling pathway of wild T cells, we designed a novel dCAR diagram specific for CEA and MSLN, which achieved comparable activity relative to that of conventional CAR-T cells (CEA-CAR T or MSLN-CAR T). In this dCAR, a tandem construct containing two physically separate structures, CEA-CD3ζ and MSLN-4/1BB signaling domains were effectively controlled with tumor antigens CEA and MSLN, respectively. Finally, the activity of dCAR-T cells has been verified via in vitro and in vivo experiments. RESULTS: In the presence of cognate tumor cells (AsPC-1) expressing both CEA and MSLN, dCAR-T cells exerted high anti-tumor activity relative to that of other single-receptor CAR-T cells bearing only one signaling pathway (e.g., Cζ-CAR and MBB-CAR). In a xenograft model, dCAR-T cells significantly inhibited the growth of AsPC-1 cells yet no effect on the growth of non-cognate tumor cells. Furthermore, the released cytokines and T cell persistence in mice were comparable with that of conventional CAR-T cells, obtaining specific and controllable cytotoxicity. CONCLUSIONS: A novel type of CAR-T cells, termed dCAR-T, was designed with specific activities, that is, significant cytotoxicity for two antigen-positive tumor cells yet no cytotoxicity for single antigen-positive tumor cells. Dual-targeted CAR-T cells can be precisely localized at the tumor site and can exert high cytotoxicity against tumor cells, alleviating "on-target, off-tumor" toxicity and enabling accurate application of CAR-T cell therapy.

Accurate control of dual-receptor-engineered T cell activity through a bifunctional anti-angiogenic peptide.

BACKGROUND: Chimeric antigen receptors (CARs) presented on T cell surfaces enable redirection of T cell specificity, which has enormous promise in antitumor therapy. However, excessive activity and poor control over such engineered T cells cause significant safety challenges, such as cytokine release syndrome and organ toxicities. To enhance the specificity and controllable activity of CAR-T cells, we report a novel switchable dual-receptor CAR-engineered T (sdCAR-T) cell and a new switch molecule of FITC-HM-3 bifunctional molecule (FHBM) in this study. METHODS: We designed a fusion molecule comprising FITC and HM-3. HM-3, an antitumor peptide including an Arg-Gly-Asp sequence, can specifically target integrin αvß3 that is presented on some tumor cells. Moreover, to improve the specificity of CAR-T cells, we also generated the sdCAR-T cell line against cognate tumor cells expressing human mesothelin (MSLN) and integrin αvß3. Finally, the activity of sdCAR-T cell and FHBM is verified via in vitro and in vivo experiments. RESULTS: In the presence of FHBM, the designed sdCAR-T cells exerted high activity including activation and proliferation and had specific cytotoxicity in a time- and dose-dependent manner in vitro. Furthermore, using a combination of FHBM in nude mice, sdCAR-T cells significantly inhibited the growth of MSLN+ K562 cells and released lower levels of the cytokines (e.g., interleukin-2, interferon γ, interleukin-6, and tumor necrosis factor α) relative to conventional CAR-T cells, obtaining specific, controllable, and enhanced cytotoxicity. CONCLUSIONS: Our data indicate that FHBM can accurately control timing and dose of injected CAR-T cells, and sdCAR-T cells exert significant antitumor activity while releasing lower levels of cytokines for the cognate tumor cells expressing both MSLN and integrin αvß3. Therefore, combination therapies using sdCAR-T cells and the switch molecule FHBM have significant potential to treat malignancies.

Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

Tunable ultrasmall visible-to-extended near-infrared emitting silver sulfide quantum dots for integrin-targeted cancer imaging.

The large size of many near-infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. In this study, we developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from 500 to 1200 nm and a QD core diameter between 1.5 and 9 nm. Conjugation of a tumor-avid cyclic pentapeptide (Arg-Gly-Asp-DPhe-Lys) resulted in monodisperse, water-soluble QDs (hydrodynamic diameter < 10 nm) without loss of the peptide's high binding affinity to tumor-associated integrins (KI = 1.8 nM/peptide). Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and noncytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.

Quantum dots based molecular beacons for in vitro and in vivo detection of MMP-2 on tumor.

Matrix metalloproteinase-2 (MMP-2) is a protease related to tumor invasion and metastasis. It is heavily secreted by malignant tumor cells, allowing the protease to serve as an imaging biomarker of cancer. In this study, a novel sensing system based on fluorescence resonance energy transfer (FRET) from quantum dot (QD, the donor) to organic dye (the acceptor) was constructed for the in vitro and in vivo detection of matrix metalloproteinases-2 via a MMP-2-specific peptide substrate (GPLGVRGKGG). Specifically, 535 nm-emitting CdTe QD were bound to Rhodamine B (RB) through the peptide for in vitro detection of MMP-2, while 720 nm-emitting CdTeS QDs was linked to near infrared dye ICG-Der-02 (MPA) by the peptide for measurement in vivo. When these probes were exposed to MMP-2, the selective cleavage of the peptide resulted in the recovery of fluorescence from QDs. By using the produced 540QD-peptide-RB and 720QD-peptide-MPA probes, we successfully examined MMP-2 in live cells and tumor on nude mouse, respectively. Due to the tunable fluorescence of Qds, this nanosensor can be fine-tuned for a wide range of applications such as the detection of different biomarkers and early diagnosis of disease.

Synthesis of dye conjugates to visualize the cancer cells using fluorescence microscopy.

The clinical diagnosis of most cancers is based on evaluation of histology microscopic slides to view the size and shape of cellular nuclei and morphological structure of tissue. To achieve this goal for in vivo and in-deep tissues, near infrared dyes-bovine serum albumin and immunoglobulin G conjugates were synthesized. The spectral study shows that the absorption and fluorescence of the dye conjugates are in the "tissue optical window" spectral ranges between 650 and 900 nm. The internalization and pinocytosis of the synthesized compounds were investigated at cell level using fluorescence microscopy to obtain the optimal concentration and staining time.

Tryptophan as the fingerprint for distinguishing aggressiveness among breast cancer cell lines using native fluorescence spectroscopy.

Tryptophan is investigated as the key native marker in cells to determine the level of metastasis competence in breast cell lines using native fluorescence spectroscopy. The ratio of fluorescence intensity at 340 nm to intensity at 460 nm is associated with aggressiveness of the cancer cells. We found that the fluorescence of aggressive breast cancer cell has a much higher contribution from tryptophan compared with that from the normal cells and nonaggressive breast cancer cell.

Native fluorescence spectroscopy reveals spectral differences among prostate cancer cell lines with different risk levels.

The spectral changes of native fluorophores among normal fibroblasts and cancer cell lines of different metastatic ability are investigated by fluorescence spectroscopy. The normal (fibroblast), moderately metastatic (DU-145), and advanced metastatic (PC-3) cell lines were each selectively excited at 300 nm, and their fluorescence emission spectra are analyzed using principal component analysis to explore the differences of the relative contents of tryptophan and reduced nicotinamide adenine dinucleotide in these cell lines. The results show that the tryptophan emission featured predominantly in the fluorescence spectra of the advanced metastatic cancer cells in comparison with the moderately metastatic cancer and normal cells.

Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon.

Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery.

Improved targeting of ligand-modified adenovirus as a new near infrared fluorescence tumor imaging probe.

E1/E3-deleted Adenovirus 5 (Ad.5) possesses a great potential in gene therapy because of its high efficacy in gene transfer and low toxicity. Studies have shown that Coxsackie-Adenovirus receptor (CAR) is the determinant factor for the targeting of Adenovirus vectors. To extend the natural targeting of Ad to low CAR expressing tumors, we covalently attached folic acid (FA) to E1/E3-deleted Ad.5 capsids. Near-infrared (NIR) fluorescent dye ICG-Der-02 was subsequently conjugated with FA-Ad particles for in vivo imaging. The cell experiments and acute toxicity studies demonstrated the low toxicity of FA-Ad-ICG02 to normal cell/tissues. The dynamic behavior and targeting ability of FA-Ad-ICG02 to different tumors were investigated by NIR fluorescence imaging. In vitro and in vivo studies demonstrated its high targeting capability to CAR or FR positive tumors. The results support the potential of using ligand-modified Ad probe for tumor diagnosis and targeted therapy.