Engineering peptide drug therapeutics through chemical conjugation and implication in clinics.

The development of peptide drugs has made tremendous progress in the past few decades because of the advancements in modification chemistry and analytical technologies. The novel-designed peptide drugs have been modified through various biochemical methods with improved diagnostic, therapeutic, and drug-delivery strategies. Researchers found it a helping hand to overcome the inherent limitations of peptides and bring continued advancements in their applications. Furthermore, the emergence of peptide-drug conjugates (PDCs)-utilizes target-oriented peptide moieties as a vehicle for cytotoxic payloads via conjugation with cleavable chemical agents, resulting in the key foundation of the new era of targeted peptide drugs. This review summarizes the various classifications of peptide drugs, suitable chemical modification strategies to improve the ADME (adsorption, distribution, metabolism, and excretion) features of peptide drugs, and recent (2015-early 2024) progress/achievements in peptide-based drug delivery systems as well as their fruitful implication in preclinical and clinical studies. Furthermore, we also summarized the brief description of other types of PDCs, including peptide-MOF conjugates and peptide-UCNP conjugates. The principal aim is to provide scattered and diversified knowledge in one place and to help researchers understand the pinching knots in the science of PDC development and progress toward a bright future of novel peptide drugs.

Peptide-Drug Conjugates: Design, Chemistry, and Drug Delivery System as a Novel Cancer Theranostic.

The emergence of peptide-drug conjugates (PDCs) that utilize target-oriented peptide moieties as carriers of cytotoxic payloads, interconnected with various cleavable/noncleavable linkers, resulted in the key-foundation of the new era of targeted therapeutics. They are capable of retaining the integrity of conjugates in the blood circulatory system as well as releasing the drugs at the tumor microenvironment. Other valuable advantages are specificity and selectivity toward targeted-receptors, higher penetration ability, and drug-loading capacity, making them a suitable candidate to play their vital role as promising carrier agents. In this review, we summarized the types of cell-targeting (CTPs) and cell-penetrating peptides (CPPs) that have broad applications in the advancement of targeted drug-delivery systems (DDS). Moreover, the techniques to overcome the limitations of peptide-chemistry for their extensive implementation to construct the PDCs. Besides this, the diversified breakthrough of linker chemistry, and ample knowledge of various cytotoxic payloads used in PDCs in recent years, as well as the mechanism of action of PDCs was critically discussed. The principal aim is to provide scattered and diversified knowledge in one place and to help researchers understand the pinching knots in the science of PDC development, also their progression toward a bright future for PDCs as novel theranostics in clinical practice.

Identification of a pH-Responsive Peptide-Paclitaxel Conjugate as a Novel Drug with Improved Therapeutic Potential.

A highly sensitive, nontoxic, hydrophilic cell-penetrating peptide (CPP = c[RGDKLAK]) was selected for the construction of an effective peptide-drug conjugate (PDC). A hydrophobic drug paclitaxel (PTX) was successfully conjugated with CPP via ester linkage with succinic acid (SA) as a pH-cleavable linker moiety. The characterization techniques employed in this study indicate the >95% purity of the resulting PDC (CPP-SA-PTX). The in vitro studies show that our proposed PDC exhibits enhanced stability (∼90%) and cytotoxicity (EC50 = 8.32 ± 0.09 nM). Besides the excellent solubility of PDC in water, the PTX effect on positive ß-tubulin-III indicates that the drug releases retained pharmacological properties. Additionally, in vivo, therapeutic-dose treatment reveals the prominent tumor-growth inhibitory effects (2.82-3.24-fold) of PDC in tumor mice models. Subsequently, these observations confirmed that our novel-designed PDC (CPP-SA-PTX) adduct may serve as a promising therapeutic agent to treat glioblastoma.

Multifunctional Iron Oxide Nanocarriers Synthesis for Drug Delivery, Diagnostic Imaging, and Biodistribution Study.

The aim of the current study is to design the radiolabeled and drug-loaded nanocarrier with high loading capacity and pH-dependent drug release characteristics that could effectively transport loaded compounds to various organs for efficient diagnostic imaging and chemotherapeutic drug delivery. The aqueous extract of green tea leaves was used to synthesize the small-sized iron oxide nanoparticles (IONPs). The nanoparticles were characterized with UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray analysis (EDX). Iron oxide nanoparticles with sizes smaller than 50 nm were successfully synthesized, making them suitable for in vivo studies. In drug loading trials, 94% of the drug was loaded onto the active surface of iron oxide nanoparticles from the solution. The in vitro drug release study revealed that an acidic environment (pH 4.5) effectively triggers the release of doxorubicin (DOX) from the nanoparticles as compared to a neutral environment (pH 7.4). The gamma-emitting radionuclide 99mTc was successfully labeled with IONPs for biodistribution and imaging studies. The efficiency of radiolabeling was observed to be ≥ 99%. Furthermore, the in vivo biodistribution study of radiolabeled IONPs in rabbit model showed rapid accumulation in various organs such as heart, liver, and kidneys. This work suggested that green synthesized iron oxide nanoparticles are potential nanocarriers for diagnostic imaging and efficiently distributing DOX to specific organs. The aqueous extract of green tea leaves was used for the facile green synthesis of iron oxide nanoparticles (IONPs). Furthermore, the chemotherapeutic drug doxorubicin (DOX) and gamma-emitting radionuclide 99mTc were loaded on these iron oxide nanoparticles to evaluate the in vivo biodistribution and drug delivery studies in the rabbit models.

OFP011 Cyclic Peptide as a Multifunctional Agonist for Opioid/Neuropeptide FF Receptors with Improved Blood-Brain Barrier Penetration.

Mounting evidence indicates that the neuropeptide FF (NPFF) system is involved in the side effects of opioid usage, including antinociceptive tolerance, hyperalgesia, abuse, constipation, and respiratory depression. Our group recently discovered that the multitarget opioid/NPFF receptor agonist DN-9 exhibits peripheral antinociceptive activity. To improve its metabolic stability, antinociceptive potency, and duration, in this study, we designed and synthesized a novel cyclic disulfide analogue of DN-9, OFP011, and examined its bioactivity through in vitro cyclic adenosine monophosphate (cAMP) functional assays and in vivo behavioral experiments. OFP011 exhibited multifunctional agonistic effects at the µ-opioid and the NPFF1 and NPFF2 receptors and partial agonistic effects at the δ- and κ-opioid in vitro, as determined via the cAMP functional assays. Pharmacokinetic and pharmacological experiments revealed improvement in its blood-brain barrier permeability after systemic administration. In addition, subcutaneous OFP011 exhibited potent and long-lasting antinociceptive activity via the central µ- and κ-opioid receptors, as observed in different physiological and pathological pain models. At the highest antinociceptive doses, subcutaneous OFP011 exhibited limited tolerance, gastrointestinal transit, motor coordination, addiction, reward, and respiration depression. Notably, OFP011 exhibited potent oral antinociceptive activities in mouse models of acute, inflammatory, and neuropathic pain. These results suggest that the multifunctional opioid/NPFF receptor agonists with improved blood-brain barrier penetration are a promising strategy for long-term treatment of moderate to severe nociceptive and pathological pain with fewer side effects.

Emerging trends of edible vaccine therapy for combating human diseases especially COVID-19: Pros, cons, and future challenges.

The researchers are still doing efforts to develop an effective, reliable, and easily accessible vaccine candidate to protect against COVID-19. As of the August 2020, nearly 30 conventional vaccines have been emerged in clinical trials, and more than 200 vaccines are in various development stages. Nowadays, plants are also considered as a potential source for the production of monoclonal antibodies, vaccines, drugs, immunomodulatory proteins, as well as used as bioreactors or factories for their bulk production. The scientific evidences enlighten that plants are the rich source of oral vaccines, which can be given either by eating the edible parts of plants and/or by oral administration of highly refined proteins. The use of plant-based edible vaccines is an emerging trend as it possesses minimum or no side effects compared with synthetic vaccines. This review article gives insights into different types of vaccines, the use of edible vaccines, advantages of edible vaccines over conventional vaccines, and mechanism of action of edible vaccines. This review article also focuses on the applications of edible vaccines in wide-range of human diseases especially against COVID-19 with emphasis on future perspectives of the use of edible vaccines.

Facile One-Pot Strategy for Radiosynthesis of 99mTc-Doxycycline to Diagnose Staphylococcus aureus in Infectious Animal Models.

The accurate and early diagnosis of infection is an important feature in the biomedical sciences for better treatment and to decrease the rate of morbidity associated with diseases. Doxycycline (DC) is a semisynthetic antibiotic that belongs to tetracycline family and usually prescribed to treat a variety of infections. The objective of the present research work was to develop a new radiopharmaceutical 99mTc-Doxycycline (99mTc-DC), by using SnCl2·2H2O as a reducing agent for diagnostic applications. It was confirmed through this study that 99mTc-DC possessed high radiolabeling yield (95%). In vitro studies were performed by incubating 99mTc-DC in human serum at 37 °C. The in vitro binding interaction of the labeled antibiotic was analyzed with bacterial strain (live Staphylococcus aureus cells), and its stability was further determined. Moreover, for in vivo infection imaging study, the infection was induced with S. aureus (gram positive) cells intramuscularly injected in mice models followed by biodistribution studies for 99mTc-DC that were performed. Biodistribution studies of 99mTc-DC showed that the radiotracer was significantly accumulated at the site of infection and indicated the renal route of excretion. Scintigraphic images obtained as a result of in vivo study showed good uptake of prepared radiotracer (99mTc-DC) in the infectious lesions at 1-, 4-, and 24-h post-injection. Target-to-non-target ratios for 99mTc-DC were significantly different for the infectious lesions and non-infected tissues and remained 2.13 ± 0.3 up to 24-h post-injection of 99mTc-DC. 99mTc-DC showed preferential binding to living bacterial infected sites as compared to other parts of the body, and thus it can be inferred that 99mTc-DC might be a potential candidate to diagnose the infection.

Fabrication of self-assembled peptide nanoparticles for in vitro assessment of cell apoptosis pathway and in vivo therapeutic efficacy.

Near-infrared fluorescent (NIRF) dye-coupled self-assembled RGD-linked proapoptotic peptide nanoparticles have been synthesized with spherical shape and size ~ 30-40 nm diameters. The peptide sequence was coupled with cyanine 5.5 probe as NIRF-dye to introduce optical imaging properties and pH-dependent method was used to design Cy5.5 coupled self-assembled peptide nanoparticles (f-SAPNs). This nanoprobe has the ability to target αvß3-integrin receptor overexpressed on cancer cell's surface with improved internalization capabilities into the mitochondria. The in situ study showed that this peptide sequence has potential to disrupt the mitochondrial membrane efficiently, activating the Caspase-3 enzyme, and ultimately induces cell apoptosis. It has been observed from in vitro study that the degree of apoptosis for f-SAPNs was increased from 25.6% to 96.3%, while decreased degree of necrosis from 51.7% to 0.2% compared with its parent peptide analog (Cy5.5-c[RGDKLAK]; f-CP) occurs. Further investigations revealed that these f-SAPNs showed high uptake in U87MG glioblastoma cells in comparison with PC-3 prostate cancer cells. Moreover, in vivo therapeutic studies represented the prominent decrease in the size of tumor tissue treated with f-CP and f-SAPNs (201 ± 13 mm3 and 104 ± 6 mm3, respectively) compared with untreated tumor tissues (366 ± 18 mm3). These outcomes highlighted the specificity, and efficacy of f-SAPNs toward αvß3-integrin expressing tumor tissue in vivo and suggested that these novel designed f-SAPNs may serve as a potential theranostic drug for brain tumor glioblastoma multiforme. The pH-sensitive method gives NIRF dye-coupled self-assembled peptide nanoparticle (f-SAPNs), enables the tunable synthesis of spherical nanoparticles with high stability towards proteolysis, improved biocompatibility, and promising therapeutic efficacy.

Hybridization of tumor homing and mitochondria-targeting peptide domains to design novel dual-imaging self-assembled peptide nanoparticles for theranostic applications.

A novel hybridized dual-targeting peptide-based nanoprobe was successfully designed by using the cyclic heptapeptide. This peptide has Arg-Gly-Asp-Lys-Leu-Ala-Lys sequence, in which the RGD homing motif and KALK mitochondria-targeting motif were linked via amide bond. The designed peptide probe was further modified through covalent linkage to induce dual-imaging functionality, and self-assembled to form spherical nanoparticles. The novel Cy5.5-SAPD-99mTc nanoparticles were tested for in vitro cytotoxicity, cellular uptake, and apoptosis-inducing functionalities. The cellular internalization, enhanced cytotoxicity and selective receptor binding capabilities against U87MG cells, excellent dual-imaging potential, improved apoptosis-inducing feature by damaging mitochondria, and in vivo preclinical investigations suggested that our newly designed novel hybridized peptide-based dual-imaging nanoparticles may serve as an admirable theranostic probe to treat brain tumor glioblastoma multiforme. This study describes the development of dual-targeting self-assembled peptide nanoparticles followed by modifications using NIRF dye and radiolabeled with 99mTc for dual-imaging and enhanced therapeutic efficacy against brain tumor.

Sequential detection of H2S and HOBr with a novel lysosome-targetable fluorescent probe and its application in biological imaging.

Understanding the complex relationship between active small molecules is of great significance in various physiological processes. Herein, we present the design and synthesis of a sequential responsive Lysosome-Naphthalene imide-Azido (lyso-NP-N3) reporter for probing the H2S and HOBr within organelle (lysosome) in living cells. Probe lyso-NP-N3 exhibited high selectivity and sensitivity towards H2S (LOD = 23.5 nM) and HOBr (LOD = 254 nM). Additionally, lyso-NP-N3 possessed an excellent lysosome targeting ability and was utilized to visualize the exogenous/endogenous H2S and HOBr in RAW 264.7, Hela and HepG2 cells. Facilitated by this sequentially activated mechanism, the probe was successfully applied to confirm that the reported scavenger of HOBr, N-acetyl-L-cysteine (NAC) mainly relied on its metabolite H2S to eliminate excess HOBr, thereby playing the role of cell regulation and protection. These results establish the crosstalk between H2S and HOBr in lysosome and provide a promising tool to study metabolite interactions.

A pH-targeted and NIR-responsive NaCl-nanocarrier for photothermal therapy and ion-interference therapy.

Transport ions into cells through nanocarrier to achieve ion-interference therapy provides new inspiration for cancer treatment. In this work, a pH-targeted and NIR-responsive NaCl-nanocarrier is prepared using surfactant Vitamin E-O(EG2-Glu) and modified with polydopamine (PDA) and pH-sensitive zwitterionic chitosan (ZWC). The NaCl-nanocarrier is decorated with NH4HCO3 and IR-780 to introduce near-infrared (NIR)-responsive performance and imaging. Once the NaCl-nanocarrier is exposed to NIR laser, the temperature rises rapidly because of the excellent photothermal conversion ability of PDA, then NH4HCO3 is decomposed into NH3 and CO2, which burst the nanocarrier, resulting in Cl- and Na+ "bomb-like" release. This pH-targeted nanocarrier accumulates more at tumor site and when irradiating the site with NIR light, the temperature rises and excessive Cl- and Na+ are released to destroy the ion homeostasis and inhibit tumor growth effectively. Through this strategy, the unique combination of ion interference therapy and photothermal therapy is achieved.

N-quaternization of heterocyclic compound extended the emission to NIR with large Stokes shift and its application in constructing fluorescent probe.

This is great significant to establish a method that extends the small molecules fluorescence emission wavelength to the near-infrared region (NIR) for in vivo imaging. Hence, we firstly reported a novel fluorogenic scaffold QOH that could extend its fluorescence wavelength from (λem = 555 nm) to NIR (λem = 720 nm) with a large Stokes shift (120 nm) by forming its N-quaternization product (QMOH). In addition, the effect of the introduction of substituent at different modification sites and the properties of substituent on the optical properties of QOH were fully discussed by theoretical calculation. To investigate the possibility of QOH as probe construction, the compound Q-SH and QM-R were synthesized and applied to detect H2S and H2O2 in vitro and in vivo, respectively. This study provided an efficient strategy to extend fluorescence emission to NIR and design fluorescence probes with large ratio variation for accurately imaging biomarkers in biological system.

A feasible self-assembled near-infrared fluorescence sensor for acid phosphatase detection and cell imaging.

The single signal amplification strategy is significant for detecting various disease biomarkers but is restricted by its limited accuracy. The multi-signal and multi-mode methods have overcome this deficiency. Acid phosphatase (ACP) is an important intracellular enzyme but one-step cell imaging material-based probes are scarce for ACP. Herein, we designed a one-step self-assembled polymer probe using neutral red (NR), modified-(pyridoxal-5'-phosphate (PLP)) and Eu3+. The polymer exhibited non-emission and excellent stability. Upon the catalytic hydrolysis reaction of ACP, the polymer exhibited two strong fluorescence signals at 373 nm and 613 nm and an appreciable decline of absorbance at 395 nm. The probe has excellent selectivity and higher sensitivity with a limit of detection as low as 0.02 mU mL-1. It possesses favorable biocompatibility and has been successfully used to detect and image intracellular ACP in several living cells.

Radiosynthesis and in silico bioevaluation of 131 I-Sulfasalazine as a highly selective radiotracer for imaging of ulcerative colitis.

This study demonstrated the tracking of ulcerative colitis, which is considered a stressful immune disease. Although there are many ways to test for this disease including dependence on gases, dyes, and painful anal endoscopy, these treatment modalities have many disadvantages. Hence, it is the utmost need of time to discover new methods to detect this chronic immune disease and to avoid the defects of traditional methodologies. Sulfasalazine (SSD) was labeled with iodine-131 (half-life: 8 days, Energy: 971 keV) under optimum reaction conditions including the amount of reducing agent, pH factor, chloramine-T (Ch-T) amount, and incubation period. Characterization was performed using 1 H/ 13 C-NMR, ESI-MS, and HPLC (UV/ Radio) techniques. The biodistribution study was performed in normal and ulcerative mice models, and in silico molecular docking study was performed to evaluate the possible mechanism of action to target peroxisome proliferator-activated receptor gamma (PPARγ). The high radiolabeling yield of [131 I]-sulfasalazine ([131 I]-SSD) was achieved ≥90% through the direct labeling method with radioactive iodine-131 in the presence of chloramine-T (100 µg). The radiotracer [131 I]-SSD was observed to be stable in normal saline and freshly eluted serum up to 12 hr at ambient temperature (37℃ ± 2℃). The radiotracer [131 I]-SSD showed the highest uptake in the targeted organ (i.e., ulcerative colon) which was observed to be ≥75% injected dose per gram (% ID/g) organ for 24 hr postinjection (p.i). Furthermore, in silico data collected from molecular modeling analysis of SSD and [131 I]-SSD with antimicrobial protein (PDB code: 3KEG) and peroxisome proliferator-activated receptor gamma (PPARγ) (PDB code: 4XTA) showed azoreductase activity and high binding potential for PPAR-γ site, respectively. The results of biological studies obtained in this study enlighten the usefulness of radiotracer [131 I]-SSD as a potential imaging agent for ulcerative colitis.

Evaluation and validation of tungsten fiducial marker-based image-guided radiotherapy.

In this research work, a simple homemade cubic phantom was designed to validate the Image-Guided Radiotherapy (IGRT) set up and verified with the help of tungsten fiducial markers (size 2-3 mm) inserted into the cubic phantom. Phantom made up of Styrofoam, was scanned with the help of 16 slice Toshiba CT scanner where each slice was of 1 mm thickness and HU level set to -1000. A radio-opaque contrast medium was rubbed on the phantom to visualize the scanner images. Once the iso-center had been marked on a phantom with the help of in-room positioning laser and the fields (RT-LAT and AP) were applied on the contoured body of the phantom in Varian's ARIA-11 Eclipse dosimeter software, the same position of the phantom was reproduced on Varian's Linear Accelerator DHX. Known shifts of 3.0 to 30.0 mm from the marked iso-center were applied on the phantom by moving the couch in all six directions one by one. On each applied couch shift, an x-ray image of the phantom was acquired with the help of an MV portal imager of Linac in AP and RT-LAT direction. This image was superimposed with a reference image of phantom and shift accuracy calculated by ARIA-11 software was noted down. It turned out that irrespective of the position of the phantom on the couch, the calculated corrected shift and deviation from reference position was always between ± 1-2 mm which is the required accuracy for IGRT according to International Atomic Energy Agency (IAEA). This process was repeated 40 times and each time, the corrected shift came out to be ± 1-2 mm. We can conclude that our system is safe and accurate enough to perfectly position the actual patient for IGRT.

Multifunctional self-assembled peptide nanoparticles for multimodal imaging-guided enhanced theranostic applications against glioblastoma multiforme.

The synthesis of self-assembled peptide nanoparticles using a facile one-pot synthesis approach is gaining increasing attention, allowing therapy in combination with diagnosis. Their drawback is limited diagnostic potential, which can be improved after necessary modifications and efficacious functionalization. Herein, a cyclic heptapeptide having the Arg-Gly-Asp-Lys-Leu-Ala-Lys sequence was modified by conjugation of the ε-amino group of the terminal lysine residue with diethylenetriamine pentaacetic acid (DTPA) as a bifunctional chelating agent (BFC) for radiolabeling with a γ-emitting radionuclide (99mTc, half-life 6.01 h; energy 140 keV). Further, the free amino group of the middle lysine residue was successfully conjugated with near-infrared fluorescence (NIRF) dye Cyanine5.5 N-succinimidyl ester (Ex/Em = 670/701 nm) by a co-assembly method to form newly designed novel NIRF dye conjugated self-assembled peptide-DTPA (Cy5.5@SAPD) nanoparticles. The fluorescent nanoparticle formation was confirmed by using a fluorescence spectrophotometer (Ex/Em = 650/701 nm), and the transmission electron microscope (TEM) images showed a size of ∼ 40 nm with a lattice fringe distance of 0.294 nm. Cytotoxicity and confocal laser scanning microscopy (CLSM) studies showed that these nanoparticles possess a high affinity for the αvß3-integrin receptor overexpressed on brain tumor glioblastoma with an EC50 = 20 µM. Moreover, these nanoparticles were observed to have potential to internalize into U87MG cells more prominently than HEK-293 cancer cells and induce apoptosis. The apoptosis assay showed 79.5% apoptotic cells after 24 h treatment of Cy5.5@SAPD nanoparticles. Additionally, these nanoparticles were also radiolabeled with 99mTc for the single photon emission computed tomography (SPECT) imaging study in tumor-bearing female Balb/c mice. The excellent imaging feature of Cy5.5@SAPD-99mTc nanoparticles as a multimodal (SPECT/NIRF) diagnostic probe, as well as noteworthy therapeutic potential was observed. The results suggested that our newly designed novel dual-targeting dual-imaging nanoparticles may serve as an admirable theranostic probe to treat brain tumor glioblastoma multiforme.

Synthesis of 99mTc-labeled 2-Mercaptobenzimidazole as a novel radiotracer to diagnose tumor hypoxia.

Discovery of 99mTc-labeled imidazole derivatives as a potential radiotracer for hypoxic tumor imaging is considered to be of great interest because of non-invasive detection capabilities. 2-Mercaptobenzimidazole (2-MBI) was successfully synthesized, characterized and radiolabeled with [99mTc (CO)3(H2O)3]+ intermediate to form 99mTc-2-MBI complex with radiochemical purity of ≥95% yield as observed by instant-thin layer chromatography (ITLC) and radio-high performance liquid chromatography (radio-HPLC). The 99mTc-2-MBI complex was observed to be stable in saline and serum with no noticeable decomposition at room temperature and 37 °C, respectively, over a time period of 24 h. Biodistribution results in Balb/c mice bearing S180 tumor show that 99mTc-2-MBI highly internalized in tumor tissue, also possess preferably high tumor/muscle and tumor/blood ratios 4.14 ±â€¯0.77 and 3.91 ±â€¯0.63, respectively at 24 h incubation. Scintigraphic imaging study shows 99mTc-2-MBI is visibly accumulated in hypoxic tumor tissue, suggesting it would be a promising radiotracer for early stage diagnosis of tumor hypoxia.

Fluorescent RGD-based pro-apoptotic peptide conjugates as mitochondria-targeting probes for enhanced anticancer activities.

We have designed 2-domain anticancer peptides with RGD-based KLAK bi-functional short motifs (linear and cyclic analogues). RGD tripeptide acts as tumor blood vessel 'homing' motif while KLAK tetrapeptide internalized in mitochondria and causes cell apoptosis. All three peptides (RGDKLAK; HM, cyclic-RGDKLAK; HMC-1, and RGD-cyclic-KLAK; HMC-2) were conjugated with fluorescein isothiocyanate isomer-I (5-FITC; F) for in-vivo and in-vitro optical imaging studies. These fluorescent-peptide (FL-peptide) analogues were analyzed to possess αvß3-integrin targeting affinity, high uptake in in-vitro cell binding assays followed by in-vivo tumor xenograft mice studies. Pharmacological profile reveals that F-HMC-1 analogue exhibited selectively and specifically higher affinity for αvß3-integrin than other analogues in U87MG cells in comparison with HeLa cells. The subcutaneous U87MG tumor xenograft mice models clearly visualized the uptake of F-HMC-1 in tumor tissue in contrast with normal tissues with tumor-to-normal tissue ratio (T/NT = 15.9 ±â€¯1.1) at 2 h post-injection. These results suggested that F-HMC-1 peptide has potential diagnostic applications for targeting αvß3-integrin assessed by optical imaging study in U87MG tumor xenograft mice models.

Detection of DNA 3'-phosphatase activity based on exonuclease III-assisted cascade recycling amplification reaction.

DNA 3'-phosphatase is an essential enzyme, which plays a pivotal role in repairing DNA damage. The peculiar activity of DNA 3'-phosphatase has been proved to associate with a variety of human pathologies. Therefore, sensitive determination of DNA 3'-phosphatase is necessary for clinical diagnosis and therapy. Here, we develop a simple, sensitive, and specific fluorescent biosensor including three DNA chains of hairpin DNA1, hairpin DNA2 and fluorescence probe DNA (FP) for detecting the activity of DNA 3'-phosphatase. First, biotin-modified hairpin DNA1 binds with streptavidin-modified magnetic beads (MB) to get MB-DNA1. DNA 3'-phosphatase can hydrolyze phosphate groups on MB-DNA1 to form hydroxyl groups, which leads to the polymerization extension and nicking endonuclease cleavage reaction to obtain the trigger DNA1 fragment (tDNA1). Next, two cyclic amplification reactions are designed. In cycle I, the tDNA1 hybridizes with the hairpin DNA2, which leads the hairpin structure of DNA2 opened and the fluorescence signal of 6-carboxy-fluorescein (FAM) labeled on hairpin DNA2 turned on. This cyclic reaction is amplified by exonuclease III (Exo III). At the same time, the trigger DNA2 fragment (tDNA2) is obtained. In cycle II, similarly, the tDNA2 hybridizes with FP. Thus, the fluorescence signal of FAM labeled on FP released, which multiplies with the fluorescence signal from cycle I. Finally, this strategy is applied to determine two typical DNA 3'-phosphatases including T4 polynucleotide kinase (T4 PNK) and alkaline phosphatase (ALP) with the detection limit (LOD) of 0.0033 and 0.00037 U/mL, respectively. The method provides a promising platform to evaluate the DNA 3'-phosphatase activity in the complicated biological samples and can be potentially applied in the relevant fields such as biomedical research, drug discovery and clinical diagnosis.

Synthesis of 99m Tc-roxithromycin: A novel diagnostic agent to discriminate between septic and aseptic inflammation.

Roxithromycin is a second-generation macrolide antibiotic derived from erythromycin. In the current study, roxithromycin (ROX) was successfully labeled with technetium-99m for early diagnosis of bacterial infection and discrimination between septic and aseptic inflammation. The highest radiochemical purity of ≥95% was achieved by investigating different labeling parameters such as pH, ligand/reducing agent concentration, temperature, and amount of stabilizing agent. For this purpose, 0.3-0.5 mg ligand, 2-6 µg SnCl2 ·2H2 O as a reducing agent at basic pH (8-10 pH) and 2 mg mannitol used as a stabilizing agent, in the end, 370 MBq 99m Tc added into the reaction vials and incubated for a wide range of temperature (-4 to 65°C). The percent radiochemical purity of 99m Tc-roxithromycin was assessed with the help of the radio-thin-layer chromatography technique. The characterization studies were carried out using electrophoresis and Radio-HPLC techniques as well as saline stability and serum stability studies were also performed. Furthermore, biodistribution study was also performed in an inflamed animal model to discriminate between septic (heat-killed Staphylococcus aureus) and aseptic (turpentine oil) inflammatory lesions. The results were elaborated that 99m Tc-roxithromycin (99m Tc-ROX) was clearly bounded at the septic inflammation site (T/NT ratio of 7.08 ± 1.14) at 30 min postadministration, and maximum accumulation was seen in heart, lungs, liver, stomach, kidneys, and intestine. The results were suggested that 99m Tc-ROX might be used to discriminate between septic and aseptic inflammatory lesions at an early stage.