Excellent Serum Stability Research Articles

Folic acid-poly(N-isopropylacrylamide-maltodextrin) nanohydrogels as novel thermo-/pH-responsive polymer for resveratrol breast cancer targeted therapy

BackgroundThe long-term sequelae and serious side effects of conventional breast cancer chemotherapy due to ubiquitous biodistribution and systemic toxicity remain the main obstacle to success in clinic. Recent anticancer research rely on tailoring smart carriers to overcome these drawbacks. In this regard, numerous stimuli-responsive polymers have been assembled and successfully employed as smart carriers various chemotherpeutic agents due to their intrinsic tumor targeting properties. Among which, poly(N-isopropylacrylamide (PNIPAM) has gained increasing attention being amenable to various chemical modifications for tuning its intrinsic thermoresponsivity or endowing additional stimulus responsiveness. This work aims to develop PNIPAM-maltodextrin nanohydrogels (PNIPAM-MD NGs) decorated with folic acid (FA) as smart breast cancer-targeted stimuli-responsive delivery system to the natural polyphenol resveratrol (RSV) suffering from low bioavailaibity. ResultsPNIPAM-MD was synthesized by graft copolymerization, characterized and evaluated for its modulated stimuli-responsivity. The PNIPAM-based copolymer was coupled with FA, the breast cancer active targeting ligand. The FA-PNIPAM-MD NGs core was loaded RSV, then subjected to in vitro and in vivo evaluation. Results showed that the developed RSV-loaded FA-PNIPAM-MD (F3) NGs displayed dual thermo- and pH-responsivities, high drug-loading capacity (8.31 wt%,) and excellent physical and serum stability. The targeted formula F3 exhibited enhanced in vitro anti-breast cancer potency as evident by 7.4 folds reduction in the IC50 of free RSV and significant MCF-7 cellular uptake. In breast cancer-bearing mice, F3 suppressed the tumor growth, lowered VEGF-1 and Ki-67 expression levels, and induced apoptosis via upregulating caspase-3. ConclusionOverall, the PNIPAM-MD NGs as well as its FA-decorated breast cancer-targted NGs could serve as promising smart nanocarrier for efficient RSV brest cancer delivery. It could be safely deduced that the assembled FA-PNIPAM-MD F3 NGs enabled sequestering the therapeutic cargo (RSV) in normal tissue and released it in tumor site, thus guarding against its rapid metabolism and maximizing its accumulation in the cancer tissue as evidenced by the remarkable tumor growth suppression noticed in the targeted RSV/FA-PNIPAM-MD F3 NGs-treated mice compared to that in the free RSV-treated group. Hence, The fabricated nanohydrogels may be further employed for smart delivery of various anti-breast cancer agents.

Human Behavior-Inspired Linchpin-Directed Catalysis for Traceless Precision Labeling of Lysine in Native Proteins.

The complex social ecosystem regulates the spectrum of human behavior. However, it becomes relatively easier to understand if we disintegrate the contributing factors, such as locality and interacting partners. Interestingly, it draws remarkable similarity with the behavior of a residue placed in a social setup of functional groups in a protein. Can it inspire principles for creating a unique environment for the precision engineering of proteins? We demonstrate that localization-regulated interacting partner(s) could render precise and traceless single-site modification of structurally diverse native proteins. The method targets a combination of high-frequency Lys residues through an array of reversible and irreversible reactions. However, excellent simultaneous control over chemoselectivity, site selectivity, and modularity ensures that the user-friendly protocol renders acyl group installation, including post-translational modifications (PTMs), on a single Lys. Besides, it offers a chemically orthogonal handle for the installation of probes. Also, a purification protocol integration delivers analytically pure single-site tagged protein bioconjugates. The precise labeling of a surface Lys residue ensures that the structure and enzymatic activities remain conserved post-bioconjugation. For example, the precise modification of insulin does not affect its uptake and downstream signaling pathway. Further, the method enables the synthesis of homogeneous antibody-fluorophore and antibody-drug conjugates (AFC and ADC; K183 and K249 labeling). The trastuzumab-rhodamine B conjugate displays excellent serum stability along with antigen-specific cellular imaging. Further, the trastuzumab-emtansine conjugate offers highly specific antiproliferative activity toward HER-2 positive SKBR-3 breast cancer cells. This work validates that disintegrate theory can create a comprehensive platform to enrich the chemical toolbox to meet the technological demands at the chemistry, biology, and medicine interface.

Ultrahighly Sensitive Sandwich-Type Electrochemical Immunosensor for Selective Detection of Tumor Biomarkers

Herein, a novel sandwich-type immunosensor was designed using Pt nanoparticle-decorated SnS2 nanoplates (Pt@SnS2) as a matrix and N,B-doped Eu MOF (N,B-Eu MOF) nanospheres as a signal amplifier. In Pt@SnS2, Pt nanoparticles (NPs) enhance the surface electron transport capability and electrochemiluminescence (ECL) performance of SnS2 nanoplates. The dual "antenna" effect of 5-boronoisophthalic acid (5-bop) and 5-nitroisophthalic acid (5-nop) enables the N,B-Eu MOFs to show very good ECL performance at the cathode. In the presence of the target carcinoembryonic antigen (CEA), the sandwich-type immunosensor provides specific immune responses, and the ECL signal of the immunosensor is greatly amplified by the signal probe N,B-Eu MOFs. In view of the above, the immunosensor was successfully applied for highly sensitive and selective detection of CEA with a detection limit of 0.06 pg·mL-1. This sensor exhibits high sensitivity and specificity, excellent stability, good reproducibility, and good practicability in real human serum.

Synthesis and Evaluation of Two Long-Acting SSTR2 Antagonists for Radionuclide Therapy of Neuroendocrine Tumors.

Somatostatin receptor subtype 2 (SSTR2) has become an essential target for radionuclide therapy of neuroendocrine tumors (NETs). JR11 was introduced as a promising antagonist peptide to target SSTR2. However, due to its rapid blood clearance, a better pharmacokinetic profile is necessary for more effective treatment. Therefore, two JR11 analogs (8a and 8b), each carrying an albumin binding domain, were designed to prolong the blood residence time of JR11. Both compounds were labeled with lutetium-177 and evaluated via in vitro assays, followed by in vivo SPECT/CT imaging and ex vivo biodistribution studies. [177Lu]Lu-8a and [177Lu]Lu-8b were obtained with high radiochemical purity (>97%) and demonstrated excellent stability in PBS and mouse serum (>95%). [177Lu]Lu-8a showed better affinity towards human albumin compared to [177Lu]Lu-8b. Further, 8a and 8b exhibited binding affinities 30- and 48-fold lower, respectively, than that of the parent peptide JR11, along with high cell uptake and low internalization rate. SPECT/CT imaging verified high tumor accumulation for [177Lu]Lu-8a and [177Lu]Lu-JR11 at 4, 24, 48, and 72 h post-injection, but no tumor uptake was observed for [177Lu]Lu-8b. Ex vivo biodistribution studies revealed high and increasing tumor uptake for [177Lu]Lu-8a. However, its extended blood circulation led to an unfavorable biodistribution profile for radionuclide therapy.

Abstract LB527: A novel dimeric small molecule-radio conjugate targeting fibroblast activation protein with high and prolonged tumor uptake

Abstract Imaging procedures based on small molecule-radio conjugates targeting Fibroblast Activation Protein (FAP) are taking the spotlight in clinical practice for the diagnosis of a wide variety of cancer lesions. However, therapeutic applications of FAP-targeting radioligand therapeutics are limited by their short residence time in tumor lesions. We have recently described the discovery of OncoFAP, a novel ultra-high affinity ligand of FAP which has been already validated for PET imaging applications in more than twenty patients with solid tumors. In this abstract, we describe the development and in vivo characterization of BiOncoFAP, a dimeric FAP targeting small molecule based on OncoFAP. In particular, OncoFAP and BiOncoFAP displayed a superimposable affinity for recombinant human FAP (KD = 970 pM and 993 pM, respectively, measured by Fluorescence Polarization). Moreover, the two ligands exhibited a clear selectivity for the target, without significantly interacting with a wide panel of non-target proteins. BiOncoFAP was coupled to a DOTAGA chelator, suitable for radiolabeling with [177Lu]Lu for therapeutic applications. Cold [natLu]Lu-BiOncoFAP-DOTAGA showed an excellent stability in both mouse and human serum, with half-life longer than 5 days, supporting in vivo investigations. In a head-to-head in vivo biodistribution comparative study against [177Lu]Lu-OncoFAP-DOTAGA, [177Lu]Lu-BiOncoFAP-DOTAGA exhibited a more stable and prolonged residence time in FAP-positive tumors implanted in immunodeficient mice (~20% ID/g and ~200-to-1 tumor-to-blood ratio, 24 h after systemic administration). Notably, [177Lu]Lu-BiOncoFAP-DOTAGA did not significantly accumulate in healthy organs, thus showing an outstanding tumor-to-organ ratio (e.g., 12-to-1 tumor-to-kidney and 34-to-1 tumor-to-liver ratio, at the 24 h time point). These findings heighten BiOncoFAP as promising candidate for the development of anti-cancer radioligand therapeutics towards FAP-expressing tumor lesions. Citation Format: Samuele Cazzamalli, Andrea Galbiati, Aureliano Zana, Matilde Bocci, Jacopo Millul, Jacqueline Mock, Abdullah Elsayed, Dario Neri. A novel dimeric small molecule-radio conjugate targeting fibroblast activation protein with high and prolonged tumor uptake [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB527.

Combinational Chemoimmunotherapy for Breast Cancer by Codelivery of Doxorubicin and PD-L1 siRNA Using a PAMAM-Incorporated Liposomal Nanoplatform.

Chemoimmunotherapy can synergistically enhance the therapeutic effects and decrease the side effects by a combined method. However, the effective targeted codelivery of various chemotherapeutic agents and siRNAs remains challenging. Although nanomedicine-based chemoimmunotherapy has shown great potential in cancer treatment in recent years, further effort is needed to simplify the nanocarrier designs and maintain their effective functions. Here, we report a simple but robust multifunctional liposomal nanocarrier that contains a pH-sensitive liposome (LP) shell and a dendritic core for tumor-targeted codelivery of programmed cell death ligand 1 (PD-L1) siRNA and doxorubicin (DOX) (siPD-L1@PM/DOX/LPs). siPD-L1@PM/DOX/LPs had a suitable particle size and zeta potential, excellent stability in serum, and pH-sensitive drug release in vitro. They exhibited significant cell proliferation inhibition compared to free DOX and DOX-loaded LPs and could escape endosomes, effectively release siRNA into the cytoplasm of MCF-7 cells, and significantly reduce the PD-L1 expression on tumor cells. In vivo imaging confirmed high accumulation of siPD-L1@PM/DOX/LPs at the tumor site. More importantly, compared with siPD-L1@PM/LPs or DOX alone, siPD-L1@PM/DOX/LPs were more effective in inhibiting tumor growth and activating cytotoxic T cells in vivo. In conclusion, this nanocarrier may hold promise as a codelivery nanoplatform to improve the treatment of various solid tumors.

Cortisol Detection in Undiluted Human Serum Using a Sensitive Electrochemical Structure-Switching Aptamer over an Antifouling Nanocomposite Layer.

There is a strong and growing need to monitor stress biomarkers in vivo for real-time emotional and wellness assessment. Toward this, we report a reagent-free electrochemical aptasensor with a nanocomposite antifouling layer for sensitive and continuous detection of cortisol in human serum. A thiolated, methylene blue (MB)-tagged conformation-switching aptamer was immobilized over a gold nanowire (AuNW) nanocomposite to capture cortisol and generate a signal proportional to the cortisol concentration. The signal is recorded through differential pulse voltammetry (DPV) and chronoamperometry. The aptasensor exhibited a sensitive response with 0.51 and 0.68 nM detection limits in spiked buffer and undiluted serum samples, respectively. Interference from other structurally similar analogs, namely, epinephrine and cholic acid, was negligible (<10%). The developed nanocomposite-based aptasensor showed excellent stability in undiluted human serum, outperforming several other nanocomposite materials even after prolonged exposure. This work lays the foundation for new biosensor formats such as implantable and wearable sensors.

One-Pot Conversion of Free Sialoglycans to Functionalized Glycan Oxazolines and Efficient Synthesis of Homogeneous Antibody-Drug Conjugates through Site-Specific Chemoenzymatic Glycan Remodeling.

Antibody-drug conjugates (ADCs) are an important class of therapeutic agents that harness the highly specific antigen targeting property of antibodies to deliver toxic drugs for targeted cell killing. Site-specific conjugation methods are highly desirable for constructing homogeneous ADCs that possess a well-defined antibody-to-drug ratio, stability, ideal pharmacological profile, and optimal therapeutic index. We report here a facile synthesis of functionalized glycan oxazolines from free sialoglycans that are key donor substrates for enzymatic Fc glycan remodeling and the application of an efficient endoglycosidase mutant (Endo-S2 D184M) for site-specific glycan transfer to construct homogeneous ADCs. We found that by a sequential use of two coupling reagents under optimized conditions, free sialoglycans could be efficiently converted to selectively functionalized glycan oxazolines carrying azide-, cyclopropene-, and norbornene-tags, respectively, in excellent yield and in a simple one-pot manner. We further demonstrated that the recently reported Endo-S2 D184 M mutant was highly efficient for Fc glycan remodeling with the selectively modified glycan oxazolines to introduce tags into an antibody, which required a significantly smaller amount of glycan oxazolines and a much shorter reaction time than that of the Endo-S D233Q-catalyzed reaction, thus minimizing the side reactions. Finally homogeneous ADCs were constructed with three different click reactions. The resulting ADCs showed excellent serum stability, and in vitro cytotoxicity assays indicated that all the three ADCs generated from the distinct click reactions possessed potent and comparable cytotoxicity for targeted cancer cell killing.

Systemic administration of polymersomal oncolytic peptide LTX-315 combining with CpG adjuvant and anti-PD-1 antibody boosts immunotherapy of melanoma

Oncolytic peptide LTX-315 while showing clinical promise in treating solid tumors is limited to intratumoral administration, which is not applicable for inaccessible or metastatic tumors. The cationic and amphipathic nature of oncolytic peptides engenders formidable challenges to developing systems for their systemic delivery. Here, we describe cRGD-functionalized chimaeric polymersomes (cRGD-CPs) as a robust systemic delivery vehicle for LTX-315, which in combination with CpG adjuvant and anti-PD-1 boost immunotherapy of malignant B16F10 melanoma in mice. cRGD-CPs containing 14.9 wt% LTX-315 (cRGD-CPs-L) exhibited a size of 53 nm, excellent serum stability, and strong and selective killing of B16F10 cells (versus L929 fibroblasts) in vitro, which provoked similar immunogenic effects to free LTX-315 as revealed by release of danger-associated molecular pattern molecules. The systemic administration of cRGD-CPs-L gave a notable tumor accumulation of 4.8% ID/g and significant retardation of tumor growth. More interestingly, the treatment of B16F10 tumor-bearing mice was further boosted by co-administration of polymersomal CpG and anti-PD-1 antibody, in which two out of seven mice were cured as a result of strong immune response and long-term immune memory protection. The immunotherapeutic effect was evidenced by secretion of IL-6, IFN-γ and TNF-α, tumor infiltration of CD8+ CTLs and Th, and induction of TEM and TCM in spleen. This study opens a new avenue to oncolytic peptides, which enables durable immunotherapy of tumors via systemic administration.

Enzyme-Agnostic Lysosomal Screen Identifies New Legumain-Cleavable ADC Linkers.

Over the past two decades, antibody drug conjugates (ADCs) and small molecule drug conjugates (SMDCs) have widely employed valine-citruline and related cathepsin-cleavable linkers due to their stability in plasma and their rapid cleavage by lysosomal cathepsins. However, a number of recent studies have illustrated that these linkers are subject to cleavage by exogenous enzymes such as Ces1C and neutrophil elastase, thus resulting in off-target release of drug. As such, there is a need to diversify the portfolio of ADC linkers in order to overcome nonspecific drug release. Rather than targeting cathepsins, we began with an "enzyme agnostic" screen in which a panel of 75 peptide FRET pairs were screened for cleavage in lysosomal extracts and in plasma. Unexpectedly, a series of Asn-containing peptides emerged from this screen as being cleaved far more quickly than traditional ValCit-type linkers while retaining excellent stability in plasma. Catabolism studies demonstrated that these linkers were cleaved by legumain, an asparaginyl endopeptidase that is overexpressed in a variety of cancers and is known to be present in the lysosome. MMAE-containing ADCs that incorporated these new linkers were shown to exhibit highly potent and selective cytotoxicity, comparable to analogous ValCit ADCs. Importantly, the Asn-containing linkers were shown to be completely stable to human neutrophil elastase, an enzyme thought to be responsible for the neutropenia and thrombocytopenia associated with ValCitPABC-MMAE ADCs. The legumain-cleavable ADCs were shown to have excellent stability in both mouse and human serum, retaining >85% of the drug after 1 week of incubation. Moreover, the corresponding small molecule FRET pairs exhibited <10% cleavage after 18 h in mouse and human serum. On the basis of these results, we believe that these new linkers (AsnAsn in particular) have significant potential in both ADC and SMDC drug delivery applications.

A novel ‘smart’ PNIPAM-based copolymer for breast cancer targeted therapy: Synthesis, and characterization of dual pH/temperature-responsive lactoferrin-targeted PNIPAM-co-AA

Despite the active research towards introducing novel anticancer agents, the long-term sequelae and side effects of chemotherapy remain the major obstacle to achieving clinical success. Recent cancer research is now utilizing the medicinal chemistry toolbox to tailor novel ‘smart’ carrier systems that can reduce the major limitations of chemotherapy ranging from non-specificity and ubiquitous biodistribution to systemic toxicity. In this aspect, various stimuli-responsive polymers have gained considerable interest due to their intrinsic tumor targeting properties. Among these polymers, poly(N-isopropylacrylamide (PNIPAM) has been chemically modified to tune its thermoresponsivity or even copolymerized to endow new stimulus responsiveness for enhancing tumor targeting. Herein, we set our design rationale to impart additional active targeting entity to pH/temperature-responsive PNIPAM-based polymer for more efficient controlled payloads accumulation at the tumor through cellular internalization via synthesizing novel “super intelligent” lactoferrin conjugated PNIPAM-acrylic acid (LF-PNIPAM-co-AA) copolymer. The synthesized copolymer was physicochemically characterized and evaluated as a smart nanocarrier for targeting breast cancer. In this regard, Honokiol (HK) was utilized as a model anticancer drug and encapsulated in the nanoparticles to overcome its lipophilic nature and allow its parenteral administration, for achieving sustainable drug release with targeting action. Results showed that the developed HK-loaded LF-PNIPAM-co-AA nanohydrogels displayed high drug loading capacity reaching to 18.65 wt.% with excellent physical and serum stability. Moreover, the prepared HK-loaded nanohydrogels exhibited efficient in vitro and in vivo antitumor activities. In vivo, HK-loaded nanohydrogels demonstrated suppression of VEGF-1 and Ki-67 expression levels, besides inducing apoptosis through upregulating the expression level of active caspase-3 in breast cancer-bearing mice. Overall, the developed nanohydrogels (NGs) with pH and temperature responsivity provide a promising nanocarrier for anticancer treatment.

Cyclam-Based Chelators Bearing Phosphonated Pyridine Pendants for 64Cu-PET Imaging: Synthesis, Physicochemical Studies, Radiolabeling, and Bioimaging.

Herein we present the preparation of two novel cyclam-based macrocycles (te1pyp and cb-te1pyp), bearing phosphonate-appended pyridine side arms for the coordination of copper(II) ions in the context of 64Cu PET imaging. The two ligands have been prepared through conventional protection-alkylation sequences on cyclam, and their coordination properties have been thoroughly investigated. The corresponding copper complexes have been fully characterized in the solid state (X-ray diffraction analysis) and in solution (EPR and UV-vis spectroscopies). Potentiometric studies combined with spectrometry have also allowed us to determine their thermodynamic stability constants, confirming their high affinity for copper(II) cations. The kinetic inertness of the complexes has been verified by acid-assisted dissociation experiments, enabling their use in 64Cu-PET imaging in mice for the first time. Indeed, the two ligands could be quantitatively radiolabeled under mild conditions, and the resulting 64Cu complexes have demonstrated excellent stability in serum. PET imaging demonstrated a set of features emerging from the combination of picolinates and phosphonate units: high stability in vivo, fast clearance from the body via renal elimination, and most interestingly, very low fixation in the liver. This is in contrast with what was observed for monopicolinate cyclam (te1pa), which had a non-negligible accumulation in the liver, owing probably to its different charge and lipophilicity. These results thus pave the way for the use of such phosphonated pyridine chelators for in vivo 64Cu-PET imaging.

Design of a Biocompatible Hydroxyapatite-Based Nanovehicle for Efficient Delivery of Small Interference Ribonucleic Acid into Mouse Embryonic Stem Cells.

The small interference RNA (siRNA)-assisted RNA interference approach in stem cells for differentiating into cell-specific lineages is gaining importance for its therapeutic potential. An effective gene delivery platform is crucial to achieve this goal. In this context, self-fluorescent, cell-penetrating peptide (CPP)-functionalized hydroxyapatite nanoparticles (R8HNPs) were synthesized by a modified sol gel technique. R8HNPs were crystalline, displayed characteristic bands, and exhibited broad emission spectra from 350 to 750 nm corresponding to green and red fluorescence. The biocompatible R8HNPs displayed robust binding with siRNA and excellent uptake in R1 ESCs. This was attributed to functionalization with CPP. Moreover, the R8HNP-complexed siRNA exhibited excellent serum and room temperature stability. The NPs protected the siRNA from sonication, pH, and temperature-induced stress and efficiently delivered siRNA to trigger 80% silencing of a pluripotency marker gene, Oct4, in R1 ESCs at 48 h. The transient downregulation was also observed at the protein level. Our findings demonstrate R8HNPs as a promising delivery agent for siRNA therapeutics with the potential for lineage-specific differentiation and future applications in regenerative medicine.

Evolution of a highly functional circular DNA aptamer in serum.

Circular DNA aptamers are powerful candidates for therapeutic applications given their dramatically enhanced biostability. Herein we report the first effort to evolve circular DNA aptamers that bind a human protein directly in serum, a complex biofluid. Targeting human thrombin, this strategy has led to the discovery of a circular aptamer, named CTBA4T-B1, that exhibits very high binding affinity (with a dissociation constant of 19 pM), excellent anticoagulation activity (with the half maximal inhibitory concentration of 90 pM) and high stability (with a half-life of 8 h) in human serum, highlighting the advantage of performing aptamer selection directly in the environment where the application is intended. CTBA4T-B1 is predicted to adopt a unique structural fold with a central two-tiered guanine quadruplex capped by two long stem–loops. This structural arrangement differs from all known thrombin binding linear DNA aptamers, demonstrating the added advantage of evolving aptamers from circular DNA libraries. The method described here permits the derivation of circular DNA aptamers directly in biological fluids and could potentially be adapted to generate other types of aptamers for therapeutic applications.

Aryloxy Diester Phosphonamidate Prodrugs of Phosphoantigens (ProPAgens) as Potent Activators of Vγ9/Vδ2 T-Cell Immune Responses.

Vγ9/Vδ2 T-cells are activated by pyrophosphate-containing small molecules known as phosphoantigens (PAgs). The presence of the pyrophosphate group in these PAgs has limited their drug-like properties because of its instability and polar nature. In this work, we report a novel and short Grubbs olefin metathesis-mediated synthesis of methylene and difluoromethylene monophosphonate derivatives of the PAg (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBP) as well as their aryloxy diester phosphonamidate prodrugs, termed ProPAgens. These prodrugs showed excellent stability in human serum (t1/2 > 12 h) and potent activation of Vγ9/Vδ2 T-cells (EC50 ranging from 5 fM to 73 nM), which translated into sub-nanomolar γδ T-cell-mediated eradication of bladder cancer cells in vitro. Additionally, a combination of in silico and in vitro enzymatic assays demonstrated the metabolism of these phosphonamidates to release the unmasked PAg monophosphonate species. Collectively, this work establishes HMBP monophosphonate ProPAgens as ideal candidates for further investigation as novel cancer immunotherapeutic agents.

In Vitro Evaluation of Radiolabeled Amphotericin B for Molecular Imaging of Mold Infections.

Invasive pulmonary aspergillosis and mucormycosis are life-threatening complications in immunocompromised patients. A rapid diagnosis followed by early antifungal treatment is essential for patient survival. Given the limited spectrum of biomarkers for invasive mold infections, recent studies have proposed the use of radiolabeled siderophores or antibodies as molecular probes to increase the specificity of radiological findings by nuclear imaging modalities. While holding enormous diagnostic potential, most of the currently available molecular probes are tailored to the detection of Aspergillus species, and their cost-intensive and sophisticated implementation restricts their accessibility at less specialized centers. In order to develop cost-efficient and broadly applicable tracers for pulmonary mold infections, this study established streamlined and high-yielding protocols to radiolabel amphotericin B (AMB) with the gamma emitter technetium-99m (99mTc-AMB) and the positron emitter gallium-68 (68Ga-AMB). The radiochemical purity of the resulting tracers consistently exceeded 99%, and both probes displayed excellent stability in human serum (>98% after 60 to 240 min at 37°C). The uptake kinetics by representative mold pathogens were assessed in an in vitro Transwell assay using infected endothelial cell layers. Both tracers accumulated intensively and specifically in Transwell inserts infected with Aspergillus fumigatus, Rhizopus arrhizus, and other clinically relevant mold pathogens compared with their accumulation in uninfected inserts and inserts infected with bacterial controls. Inoculum-dependent enrichment was confirmed by gamma counting and autoradiographic imaging. Taken together, this pilot in vitro study proposes 99mTc-AMB and 68Ga-AMB to be facile, stable, and specific probes, meriting further preclinical in vivo evaluation of radiolabeled amphotericin B for molecular imaging in invasive mycoses.

Functionally Versatile and Highly Stable Chelator for 111In and 177Lu: Proof-of-Principle Prostate-Specific Membrane Antigen Targeting.

Here, we present the synthesis and characterization of a new potentially nonadentate chelator H4pypa and its bifunctional analogue tBu4pypa-C7-NHS conjugated to prostate-specific membrane antigen (PSMA)-targeting peptidomimetic (Glu-urea-Lys). H4pypa is very functionally versatile and biologically stable. Compared to the conventional chelators (e.g., DOTA, DTPA), H4pypa has outstanding affinities for both 111In (EC, t1/2 ≈ 2.8 days) and 177Lu (β-,γ, t1/2 ≈ 6.64 days). Its radiolabeled complexes were achieved at >98% radiochemical yield, RT within 10 min, at a ligand concentration as low as 10-6 M, with excellent stability in human serum over at least 5-7 days (<1% transchelation). The thermodynamic stabilities of the [M(pypa)]- complexes (M3+ = In3+, Lu3+, La3+) were dependent on the ionic radii, where the smaller In3+ has the highest pM value (30.5), followed by Lu3+ (22.6) and La3+ (19.9). All pM values are remarkably higher than those with DOTA, DTPA, H4octapa, H4octox, and H4neunpa. Moreover, the facile and versatile bifunctionalization enabled by the p-OH group in the central pyridyl bridge of the pypa scaffold (compound 14) allows incorporation of a variety of linkers for bioconjugation through easy nucleophilic substitution. In this work, an alkyl linker was selected to couple H4pypa to a PSMA-targeting pharmacophore, proving that the bioconjugation sacrifices neither the tumor-targeting nor the chelation properties. The biodistribution profiles of 111In- and 177Lu-labeled tracers are different, but promising, with the 177Lu analogue particularly outstanding.

Discovery and Mechanistic Study of Tailor-Made Quinoline Derivatives as Topoisomerase 1 Poison with Potent Anticancer Activity.

To overcome chemical limitations of camptothecin (CPT), we report design, synthesis, and validation of a quinoline-based novel class of topoisomerase 1 (Top1) inhibitors and establish that compound 28 ( N-(3-(1 H-imidazol-1-yl)propyl)-6-(4-methoxyphenyl)-3-(1,3,4-oxadiazol-2-yl)quinolin-4-amine) exhibits the highest potency in inhibiting human Top1 activity with an IC50 value of 29 ± 0.04 nM. Compound 28 traps Top1-DNA cleavage complexes (Top1ccs) both in the in vitro cleavage assays and in live cells. Point mutation of Top1-N722S fails to trap compound 28-induced Top1cc because of its inability to form a hydrogen bond with compound 28. Unlike CPT, compound 28 shows excellent plasma serum stability and is not a substrate of P-glycoprotein 1 (permeability glycoprotein) advancing its potential anticancer activity. Finally, we provide evidence that compound 28 overcomes the chemical instability of CPT in human breast adenocarcinoma cells through generation of persistent and less reversible Top1cc-induced DNA double-strand breaks as detected by γH2AX foci immunostaining after 5 h of drug removal.

A TRAIL-Delivered Lipoprotein-Bioinspired Nanovector Engineering Stem Cell-Based Platform for Inhibition of Lung Metastasis of Melanoma.

Genetically engineered mesenchymal stem cells (MSCs), as non-viral gene delivery platforms, are rapidly evolving in tumor therapy due to their low immunogenicity and natural tumor-homing capacity.Methods: In this paper, we selected reconstituted high-density lipoprotein (rHDL), a lipoprotein-bioinspired nanovector with specific binding ability to scavenger receptor B type I (SR-BI) expressed on MSCs, as a transfection agent to genetically modify MSCs. pDNA encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was used as a functional gene to be transfected into the nucleus of MSCs for TRAIL expression. Lauric acid-coupled polyethyleneimine (PEI-LA) as an amphiphilic cationic polymer was synthesized to electrostatically bind to pDNA, and then incorporated into rHDL to form rHDL/PEI-LA/pDNA nanoparticles.Results: The nanoparticles exhibited homogenous particle size and excellent serum stability in vitro. Meanwhile, this SR-BI-targeted rHDL performed efficient intracellular gene delivery, specific lysosome-independent mechanism of cellular uptake and high transfection of pDNA towards MSCs. Moreover, high TRAIL expression in MSCs was detected after rHDL-mediated transfection. In vitro and in vivo results indicated that genetically engineered MSCs could accurately target to B16F10 cells, thereby producing significant apoptosis-inducing effect on aggressive melanoma.Conclusion: TRAIL-expressing MSCs engineered by rHDL nanovector was an efficient and hypotoxic method for stem cells-based pulmonary melanoma metastasis-targeting therapy.

Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of Fc-fused lectins and antibodies.

The utilization of immuno-magnetic nanoparticles (MNPs) for the selective capture, enrichment, and separation of specific glycoproteins from complicated biological samples is appealing for the discovery of disease biomarkers. Herein, MNPs were designed and anchored with abundant boronic acid (BA) and photoreactive alkyl diazirine (Diaz) functional groups to obtain permanently tethered Fc-fused Siglec-2 and antiserum amyloid A (SAA) mAb with the assistance of reversible boronate affinity and UV light activation in an orientation-controlled manner. The Siglec-2-Fc-functionalized MNPs showed excellent stability in fetal bovine serum (FBS) and excellent efficiency in the extraction of cell membrane glycoproteins. The anti-SAA mAb-functionalized MNPs maintained active Ab orientation and preserved antigen recognition capability in biological samples. Thus, the BA-Diaz-based strategy holds promise for the immobilization of glycoproteins, such as antibodies, with the original protein binding activity maintained, which can provide better enrichment for the sensitive detection of target proteins.