Imaging Reagents Research Articles

B-Glycine as a marker for β cell imaging and β cell mass evaluation.

β cell mass (BCM) and function are essential to the diagnosis and therapy of diabetes. Diabetic patients serve β cell loss is, and damage of β cells leads to severe insulin deficiency. Our understanding of the role of BCM in diabetes progression is extremely limited by lacking efficient methods to evaluate BCM in vivo. In vitro methods of labeling islets, including loading of contrast reagent or integration of exogenous biomarker, require artificial manipulation on islets, of which the clinical application is limited. Imaging methods targeting endogenous biomarkers may solve the above problems. However, traditional reagents targeting GLP-1R and VAMT2 result in a high background of adjacent tissues, complicating the identification of pancreatic signals. Here, we report a non-invasive and quantitative imaging technique by using radiolabeled glycine mimics ([18F]FBG, a boron-trifluoride derivative of glycine) to assay islet function and monitor BCM changes in living animals. Glycine derivatives, FBG, FBSa, 2Me-FBG, 3Me-FBG, were successfully synthesized and labeled with 18F. Specificity of glycine derivatives were characterized by in vitro experiment. PET imaging and biodistribution studies were performed in animal models carring GLYT over-expressed cells. In vivo evaluation of BCM with [18F]FBG were performed in STZ (streptozocin) induced T1D (type 1 diabetes) models. GLYT responds to excess blood glycine levels and transports glycine into islet cells to maintain the activity of the glycine receptor (GLYR). Best PET imaging condition was 80min after given a total of 240 ~ 250 nmol imaging reagent (a mixture of [18F]FBG and natural glycine) intravenously. [18F]FBG can detect both endogenous and exogenous islets clearly in vivo. When applied to STZ induced T1D mouse models, total uptake of [18F]FBG in the pancreas exhibited a linear correlation with survival BCM. [18F]FBG targeting the endogenous glycine transporter (GLYT), which is highly expressed on islet cells, avoiding extra modification on islet cells. Meanwhile the highly restricted expression pattern of GLYT excluded the background in adjacent tissues. This [18F]FBG-based imaging technique provides a non-invasive method to quantify BCM in vivo, implying a new evaluation index for diabetic assessment.

Synthesis and characterization of chemosensor: Investigation of cyanide sensing and study as live cell imaging

Chemosensor A , (E)-(4-(2-((2-hydroxynaphthalene-1-yl)methylene)hydrazinyl)benzonitrile has been synthesized by condensation of 4-cyanophenylhydrazine hydrochloride with 2-hydroxy-1-naphthaldehyde in ethanol solvent at ∼80°C. The chemosensor A , has been characterized by FT-IR, ESI-MS and 1H-NMR spectroscopic techniques. The chemosensor A has been investigated for selective cyanide ion sensing ability through UV–visible and fluorescence spectra. Further the chemosensor A was also employed as live cell imaging reagent for intra-cellular detection of cyanide ion in SW480 cells.

Facile synthesis of Gd/Ru-doped fluorescent carbon dots for fluorescent/MR bimodal imaging and tumor therapy

Functional metal doping endows fluorescent carbon dots with richer physical and chemical properties, greatly expanding their potential in the biomedical field. Nonetheless, fabricating carbon dots with integrated functionality for diagnostic and therapeutic modalities remains challenging. Herein, we develop a simple strategy to prepare Gd/Ru bimetallic doped fluorescent carbon dots (Gd/Ru-CDs) via a one-step microwave-assisted method with Ru(dcbpy)3Cl2, citric acid, polyethyleneimine, and GdCl3 as precursors. Multiple techniques were employed to characterize the morphology and properties of the obtained carbon dots. The Gd/Ru-CDs are high mono-dispersity, uniform spherical nanoparticles with an average diameter of 4.2 nm. Moreover, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) confirmed the composition and surface properties of the carbon dots. In particular, the successful doping of Gd/Ru enables the carbon dots not only show considerable magnetic resonance imaging (MRI) performance but also obtain better fluorescence (FL) properties, especially in the red emission area. More impressively, it has low cytotoxicity, excellent biocompatibility, and efficient reactive oxygen species (ROS) generation ability, making it an effective imaging-guided tumor treatment reagent. In vivo experiments have revealed that Gd/Ru-CDs can achieve light-induced tumor suppression and non-invasive fluorescence/magnetic resonance bimodal imaging reagents to monitor the treatment process of mouse tumor models. Thus, this simple and efficient carbon dot manufacturing strategy by doping functional metals has expanded avenues for the development and application of multifunctional all-in-one theranostics.

Abstract A015: Multiplexed live-cell imaging for drug responses in patient-derived organoid models of endometrial cancer

Abstract Patient-derived organoid (PDO) models of endometrial cancer are rapidly emerging as a multifunctional research model to recapitulate in vivo cancer progression and therapeutic response. PDO models are generated by reconstituting patient tumor cells in extracellular basement membrane extract (BME) and culturing as three-dimensional domes supplemented with a specialized niche of growth factors. Despite the rise in popularity of PDO models to assess drug response in vitro, there are limited commercially available reagents that are compatible with BME. In addition, most reagents are endpoint assays that require cell lysis, which is problematic because PDOs are a limited resource. Reducing drug response assessment to a single timepoint misses out on valuable kinetic response data, and samples cannot be used for subsequent downstream analyses, such as RNA sequencing or protein assays. Herein we describe a method to assess drug effects in PDO models using kinetic live-cell imaging. Live-cell imaging is a sophisticated form of automated microscopy that, when combined with fluorescent dyes, has the capacity to quantify multiple cellular responses simultaneously. Importantly, live-cell imaging reagents do not perturb cell viability or induce DNA damage, thereby allowing for accurate repeated assessment of PDO response to treatment over time. In proof-of-concept studies, we first established the utility of this approach to assess the effect of cytotoxic and cytostatic agents in PDO models of endometrial cancer. Specific parameters of interest include PDO number, area, cell health and induction of apoptosis. Agents that produced a cytotoxic effect promoted a decrease in organoid area and number over time, concomitant with an increase in apoptosis as measured by Annexin V staining. Ongoing studies are examining the dose-dependent phenotypic effects of cytostatic agents such as progestins in endometrial cancer PDO models. To evaluate the safety of the compounds, we have also conducted experiments using patient-derived organoids of normal endometrium, which was collected from uninvolved regions of the uterus in cytoreductive surgery. We envision that this automated kinetic imaging platform can be expanded to other phenotypic readouts to better understand diverse therapeutic effects of different classes of drugs under investigation for endometrial cancer. Citation Format: Kaitriana E. Colling, Emily L. Symons, Hiruni K. Sumanasiri, Kristina W. Thiel. Multiplexed live-cell imaging for drug responses in patient-derived organoid models of endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A015.

Amphiphilic Block Copolymers Containing Benzenesulfonyl Azide Groups as Visible Light-Responsive Drug Carriers for Image-Guided Delivery.

Nanoparticles (NPs) containing light-responsive polymers and imaging agents show great promise for controlled drug delivery. However, most light-responsive NPs rely on short-wavelength excitation, resulting in poor tissue penetration and potential cytotoxicity. Moreover, excessively sensitive NPs may prematurely release drugs during storage and circulation, diminishing their efficacy and causing off-target toxicity. Herein, we report visible-light-responsive NPs composed of an amphiphilic block copolymer containing responsive 4-acrylamide benzenesulfonyl azide (ABSA) and hydrophilic N,N'-dimethylacrylamide (DMA) units. The polymer pDMA-ABSA was loaded with the chemotherapy drug dasatinib and zinc tetraphenylporphyrin (ZnTPP). ZnTPP acted as an imaging reagent and a photosensitizer to reduce ABSA upon visible light irradiation, converting hydrophobic units to hydrophilic units and disrupting NPs to trigger drug release. These NPs enabled real-time fluorescence imaging in cells and exhibited synergistic chemophotodynamic therapy against multiple cancer cell lines. Our light-responsive NP platform holds great promise for controlled drug delivery and cancer theranostics, circumventing the limitations of traditional photosensitive nanosystems.

Characterization of Gold Nanoparticles Synthesized with Zingiber zerumbet Extracts

Gold nanoparticles have been used as drug carriers and imaging reagents. The plant extract-capped gold nanoparticles provide better properties than chemically synthesized gold nanoparticles. In this study, Zingiber zerumbet extract-mediated green production of gold nanoparticles (Z. zerumbet@Au NPs) was established. Based on the UV-visible spectroscopy and transmission electron microscope (TEM) results, most Z. zerumbet@Au NPs were spherical. When more Z. zerumbet extracts were added, more spherical nanoparticles were formed. The hydrodynamic size changed slightly along with time, and the average size was approximately 170 nm. Capping of Z. zerumbet extract on the surface of gold nanoparticles was confirmed by Fourier transform infrared spectroscopy (FTIR). In general, phenolic compounds or flavonoid compounds were considered to be the reducing agent. However, zerumbone was identified as the reducing agent in this study. The resulting oxidized products were characterized by high-performance liquid chromatography-mass spectrometry (HPLC-MS). As a result, the solvent was proven to be involved in nanoparticle synthesis. Overall, Z zerumbet@Au NPs showed great potential to be used in cosmetic- or biomedicine-related fields.

Control of the fluorescence lifetime in dye based nanoparticles.

Fluorescent dye based nanoparticles (NPs) have received increased interest due to their high brightness and stability. In fluorescence microscopy and assays, high signal to background ratios and multiple channels of detection are highly coveted. To this end, time-resolved imaging offers suppression of background and temporal separation of spectrally overlapping signals. Although dye based NPs and time-resolved imaging are widely used individually, the combination of the two is uncommon. This is likely due to that dye based NPs in general display shortened and non-mono-exponential lifetimes. The lower quality of the lifetime signal from dyes in NPs is caused by aggregation caused quenching (ACQ) and energy migration to dark states in NPs. Here, we report a solution to this problem by the use of the small-molecule ionic isolation lattices (SMILES) concept to prevent ACQ. Additionally, incorporation of FRET pairs of dyes locks the exciton on the FRET acceptor providing control of the fluorescence lifetime. We demonstrate how SMILES NPs with a few percent rhodamine and diazaoxatriangulenium FRET acceptors imbedded with a cyanine donor dye give identical emission spectra and high quantum yields but very different fluorescence lifetimes of 3 ns and 26 ns, respectively. The two spectrally identical NPs are easily distinguished at the single particle level in fluorescence lifetime imaging. The doping approach for dye based NPs provides predictable fluorescence lifetimes and allows for these bright imaging reagents to be used in time-resolved imaging detection modalities.

Green Synthesis of Carboxymethyl Chitosan-Based CuInS2 QDs with Luminescent Response toward Pb2+ Ion and Its Application in Bioimaging.

Polysaccharide-based QDs have attracted great attention in the field of biological imaging and diagnostics. How to get rid of the high heavy metal toxicity resulting from conventional Cd- and Pb-based QDs is now the main challenge. Herein, we offer a simple and environmentally friendly approach for the "direct" interaction of thiol-ending carboxymethyl chitosan (CMC-SH) with metal salt precursors, resulting in CuInS2 QDs based on polysaccharides. A nucleation-growth mechanism based on the LaMer model can explain how CMC-CuInS2 QDs are formed. As-prepared water-soluble CMC-CuInS2 QDs exhibit monodisperse particles with sizes of 5.5-6.5 nm. CMC-CuInS2 QDs emit the bright-green fluorescence at 530 nm when excited at 466 nm with the highest quantum yield of ∼18.0%. Meanwhile, the fluorescence intensity of CMC-CuInS2 QD aqueous solution is quenched with the addition of Pb2+ and the minimal limit of detection is as little as 0.4 nM. Furthermore, due to its noncytotoxicity, great biocompatibility, and strong biorecognition ability, CMC-CuInS2 QDs can be exploited as a possible cell membrane imaging reagent. The imaging studies also demonstrate that CMC-CuInS2 QDs are suitable for Pb2+ detection in live cells and living organisms (zebrafish). Thus, this work offers such an efficient, green, and practical method for creating low-toxicity and water-soluble QD nanosensors for a sensitive and selective detection of toxic metal ion in live cells and organisms.

A Flexible Bivalent Approach to Comprehensively Improve the Performances of Stilbazolium Dyes as Amyloid-β Fluorescent Probes.

Exploring new ways to reconstruct the structure and function of inappropriate organic fluorophores for improving amyloid-β (Aβ) fluorescent imaging performance is desired for precise detection and early diagnosis of Alzheimer's disease (AD). With stilbazolium dyes as examples, here, we present a multipronged approach to comprehensively improved the Aβ fluorescent imaging performance through a flexible bivalent method, where a flexible carbon chain was introduced to link two monomers to form a homodimer. Our results reveal a mechanism wherein the flexible linker creates a well-defined probe with specific orientations and distinct photophysical properties. Applying this approach in combination with theoretical simulation, the homodimers exhibited a comprehensive improvement of the Aβ fluorescent imaging performance of the dye monomers, including better photostability and higher signal-to-noise (S/N) ratio, higher "off-on" near-infrared fluorescence (NIRF) response sensitivity, higher specificity and affinity to Aβ deposits, and more reasonable lipophilicity for blood-brain barrier (BBB) penetrability. The results demonstrate that flexible homodimers offer a multipronged approach to obtaining high-performance NIRF imaging reagents for the detection of Aβ deposits both in vitro and in vivo.

Boron Clusters Alter the Membrane Permeability of Dicationic Fluorescent DNA-Staining Dyes.

Membrane-permeable fluorescent dyes that stain DNA are useful reagents for microscopic imaging, as they can be introduced into living cells to label DNA. However, the number of these dyes, such as Hoechst 33342, is limited. Here, we show that the icosahedral dodecaborate B12Br122-, a superchaotropic carrier that delivers different types of molecules into cells, functions as an excellent carrier for membrane-impermeable fluorescent dyes. Propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI), dicationic membrane-impermeable fluorescent dyes that stain DNA, can permeate cell membranes in the presence of boron clusters. Methyl green (MG), a dicationic dye used in the histological and fluorescent staining of DNA, permeated cell membranes in the presence of boron clusters. In contrast, monocationic membrane-permeable fluorescent dyes, such as acridine orange and pyronin Y, exhibited reduced fluorescence in cells in the presence of boron clusters. Boron clusters do not quench dicationic fluorescent dyes in water in vitro but have quenching effects on monocationic fluorescent dyes. We have demonstrated that the addition of B12Br122- to impermeable dicationic fluorescent DNA-staining dyes, such as DAPI, PI, and MG, which have been widely used for numerous years, imparts membrane permeability to introduce these dyes into living cells.

Thermally activated delayed fluorescence and high-contrast mechanochromism of anthrone-based donor-acceptor systems.

The development of materials that emit in the deep-red to near-infrared region of the spectrum has attracted significant attention due to their potential as optical sensing and imaging reagents in biology. Herein, we report the synthesis and optoelectronic characterization of four anthraquinone-based emitters, T-tBuCz-AQ, T-MeOCz-AQ, C-tBuCz-AQ, and C-MeOCz-AQ, and two pyrazoloanthrone-based emitters, tBuCz-PA and DMAC-PA. Depending on the donor, these compounds emit in the spectral range between 640 and 750nm in the neat film, while the emission of the 10 wt% doped films in poly(methyl methacrylate) (PMMA) is blue-shifted between 600 and 700nm and has low photoluminescence quantum yields between 2.6% and 6.6%. Of these compounds, T-tBuCz-AQ, T-MeOCz-AQ, and C-tBuCz-AQ exhibited thermally activated delayed fluorescence (TADF) in 10 wt% doped films in PMMA, while the crystals of T-tBuCz-AQ also showed TADF. Compound tBuCz-PA showed a high-contrast and reversible photoluminescence (PL) response upon mechanical grinding and hexane fuming.

Physalis Mottle Virus-Like Nanocarriers with Expanded Internal Loading Capacity.

An ongoing challenge in precision medicine is the efficient delivery of therapeutics to tissues/organs of interest. Nanoparticle delivery systems have the potential to overcome traditional limitations of drug and gene delivery through improved pharmacokinetics, tissue targeting, and stability of encapsulated cargo. Physalis mottle virus (PhMV)-like nanoparticles are a promising nanocarrier platform which can be chemically targeted on the exterior and interior surfaces through reactive amino acids. Cargo-loading to the internal cavity is achieved with thiol-reactive small molecules. However, the internal loading capacity of these nanoparticles is limited by the presence of a single reactive cysteine (C75) per coat protein with low inherent reactivity. Here, we use structure-based design to engineer cysteine-added mutants of PhMV VLPs that display increased reactivity toward thiol-reactive small molecules. Specifically, the A31C and S137C mutants show a greater than 10-fold increased rate of reactivity towards thiol-reactive small molecules, and PhMV Cys1 (A31C), PhMV Cys2 (S137C), and PhMV Cys1+2 (double mutant) VLPs display up to three-fold increased internal loading of the small molecule chemotherapeutics aldoxorubicin and vcMMAE and up to four-fold increased internal loading of the MRI imaging reagent DOTA(Gd). These results further improve upon a promising plant virus-based nanocarrier system for use in targeted delivery of small-molecule drugs and imaging reagents in vivo.

Spying on parahydrogen-induced polarization transfer using a half-tesla benchtop MRI and hyperpolarized imaging enabled by automation

Nuclear spin hyperpolarization is a quantum effect that enhances the nuclear magnetic resonance signal by several orders of magnitude and has enabled real-time metabolic imaging in humans. However, the translation of hyperpolarization technology into routine use in laboratories and medical centers is hampered by the lack of portable, cost-effective polarizers that are not commercially available. Here, we present a portable, automated polarizer based on parahydrogen-induced hyperpolarization (PHIP) at an intermediate magnetic field of 0.5 T (achieved by permanent magnets). With a footprint of 1 m2, we demonstrate semi-continuous, fully automated 1H hyperpolarization of ethyl acetate-d6 and ethyl pyruvate-d6 to P = 14.4% and 16.2%, respectively, and a 13C polarization of 1-13C-ethyl pyruvate-d6 of P = 7%. The duty cycle for preparing a dose is no more than 1 min. To reveal the full potential of 1H hyperpolarization in an inhomogeneous magnetic field, we convert the anti-phase PHIP signals into in-phase peaks, thereby increasing the SNR by a factor of 5. Using a spin-echo approach allowed us to observe the evolution of spin order distribution in real time while conserving the expensive reagents for reaction monitoring, imaging and potential in vivo usage. This compact polarizer will allow us to pursue the translation of hyperpolarized MRI towards in vivo applications further.

Wide dynamic range and real-time reagent identification and imaging using multi-wavelength terahertz parametric generation and machine learning

In this study, we propose a technique for identifying and imaging reagents through shielding over a wide dynamic range using a real-time terahertz (THz) spectroscopy system with multi-wavelength THz parametric generation/detection and machine learning. To quickly identify reagents through shielding, the spectral information of the “detection Stokes beam” is used for reagent recognition via machine learning. In general THz wave-based reagent identification, continuous spectra are acquired and analyzed quantitatively by post-processing. In actual applications, however, such as testing for illicit drugs in mail, the technology must be able to quickly identify reagents as opposed to quantifying the amount present. In multi-wavelength THz parametric generation/detection, THz spectral information can be measured instantly using a “multi-wavelength detection Stokes beam” and near-infrared (NIR) camera. Moreover, machine learning enables reagent identification in real-time and over a wide dynamic range. Furthermore, by plotting the identification results as pixel values, the spatial distribution of reagents can be imaged at high speed without the need for post-processing.

Arylamino‐substituted Rhodamine as a Fluorogenic Molecular Rotor for the Wash‐free Imaging of Non‐catalytic Proteins in Live Cells

AbstractFluorescent probes are valuable tools to visualize non‐catalytic proteins in live cells. Currently, the majority of imaging reagents for non‐catalytic proteins are based on “always‐on” fluorophores and the use of these reagents usually necessitate a wash step to remove unbounded fluorophores before microscope imaging. Herein, we report the use of arylamino‐substituted rhodamine as an activatable fluorophore for the imaging of non‐catalytic protein in live cells. We have shown the induction of an arylamino to structurally rigid rhodamine could significantly reduce the fluorescent emission in aqueous medium but the ligand‐directed binding of this molecule to protein receptor could effective restrict its intramolecular motion and thus lead to enhancement in fluorescence intensity at 590 nm over 30‐fold. With fluorescent probes based on this fluorophore, we could visualize integrin αvβ3 and azido‐functionalized glycans in living cells with high contrast in a wash‐free manner.

Lysosome-targeted silicon quantum dots theranostics for simultaneous fluorescent imaging and photodynamic therapy

As an emerging treatment method, photodynamic therapy (PDT) has attracted considerable interest due to the characteristics of non-invasiveness, repeatable treatment, high spatiotemporal resolution and few side effects. However, the life span (<40 ns) and diffusion distance (<20 nm) of reactive oxygen species such as singlet oxygen (1O2) in tumor cells are extremely short, which has seriously limited therapeutic efficacy of PDT. The enrichment site of photosensitizers in cancer cells is usually the first site of PDT action, which will not only affect the biological signaling pathway of cancer cell death, but also is closely related to the final therapeutic effect. Therefore, the design and preparation of photosensitizers targeting specific subcellular organelles can directly break the biological function of the organelle and trigger the corresponding cell death signaling pathway, which can significantly improve the efficacy of PDT. Herein, a lysosome-targeted silicon quantum dots (L-Si QDs) was first made by diethylene glycol-mediated synthetic route as a multicolor fluorescent imaging reagents and a new photosensitizer. The as-prepared L-Si QDs exhibit bright fluorescence with excellent pH stability and time stability, excitation-dependent emission, and good biocompatibility. Furthermore, the results of cell experiments showed that L-Si QDs was accumulated in lysosomes after being taken up by cancer cells, and can efficiently produce 1O2 upon 635 nm laser irradiation, which can damage lysosomes, up-regulate cleavage caspase-3, increase Bax release, down-regulate Bcl-2 and induce cell apoptosis finally. This study significantly broadens the biomedical applications of silicon quantum dots and provides excellent nanomaterials candidates for tumor phototherapy.

Viral Nanoparticles‐Mediated Delivery of Therapeutic Cargo

AbstractThe rapid advancement of nanotechnology in recent years has opened new avenues of investigation for biomedical sciences. Viral nanoparticles (VNPs) are formulated from plant viruses, mammalian viruses, or bacteriophages. Based on their structure, viruses, and synthetic carriers have been utilized to design bio‐inspired nanocarriers, which serve as building blocks for innovative therapeutic applications. Scientists can chemically or genetically engineer VNPs to encompass various properties, such as enhancing their functionalization with therapeutic molecules and imaging reagents, enabling targeted delivery to specific ligands. The implementation of these novel nanocarrier platforms can revolutionize treatments for cancer, infectious diseases, and chronic illnesses. The primary goal of drug delivery systems is to localize cargo to the specific target site, increasing therapeutic benefits and minimizing off‐target effects. This review critically evaluates the major virus species used as nanocarriers, their applications in therapeutics, and their advantages and disadvantages.

Detection of oxytetracycline in milk using a novel carbon dots-based fluorescence probe via facile pyrolysis synthesis.

Amphiphilic blue-fluorescence carbon dots (B-CDs) were synthesized via pyrolysis method with citric acid and oleamine as precursors. B-CDs are monodispersed in ethanol, toluene, and ultrapure water with the average particle sizes of 3.33nm, 2.05nm, and 4.12nm, respectively. The maximum emission wavelength of the B-CDs excitation at 370nm is located at 459nm. The B-CDs have good optical properties with excellent photostability. The fluorescence quantum yield (QY) of the as-prepared CDs is as high as 30.17%. The fluorescence of B-CDs is quenched because of static quenching by oxytetracycline. A high selective and sensitive fluorescence probe for detecting oxytetracycline was constructed with a linear range of 1.52-27.60µg/mL and the detection limit of 0.33µg/mL. The B-CDs-based fluorescence probe can be applied to analyze oxytetracycline in milk; the recoveries and relative standard are satisfactory. Furthermore, the B-CDs were exploited for imaging of SH-SY5Y cells. The results demonstrate that as-synthesized CDs can serve as a cellular imaging reagent owing to remarkable bioimaging performance. This work provides a new strategy for the detection of oxytetracycline in food.

The Potency of Cowpea Mosaic Virus Particles for Cancer In Situ Vaccination Is Unaffected by the Specific Encapsidated Viral RNA.

Plant virus nanoparticles can be used as drug carriers, imaging reagents, vaccine carriers, and immune adjuvants in the formulation of intratumoral in situ cancer vaccines. One example is the cowpea mosaic virus (CPMV), a nonenveloped virus with a bipartite positive-strand RNA genome with each RNA packaged separately into identical protein capsids. Based on differences in their densities, the components carrying RNA-1 (6 kb) denoted as the bottom (B) component or carrying RNA-2 (3.5 kb) denoted as the middle (M) component can be separated from each other and from a top (T) component, which is devoid of any RNA. Previous preclinical mouse studies and canine cancer trials used mixed populations of CPMV (containing B, M, and T components), so it is unclear whether the particle types differ in their efficacies. It is known that the CPMV RNA genome contributes to immunostimulation by activation of TLR7. To determine whether the two RNA genomes that have different sizes and unrelated sequences cause different immune stimulation, we compared the therapeutic efficacies of B and M components and unfractionated CPMV in vitro and in mouse cancer models. We found that separated B and M particles behaved similarly to the mixed CPMV, activating innate immune cells to induce the secretion of pro-inflammatory cytokines such as IFNα, IFNγ, IL-6, and IL-12, while inhibiting immunosuppressive cytokines such as TGF-β and IL-10. In murine models of melanoma and colon cancer, the mixed and separated CPMV particles all significantly reduced tumor growth and prolonged survival with no significant difference. This shows that the specific RNA genomes similarly stimulate the immune system even though B particles have 40% more RNA than M particles; each CPMV particle type can be used as an effective adjuvant against cancer with the same efficacy as native mixed CPMV. From a translational point of view, the use of either B or M component vs the mixed CPMV formulation offers the advantage that separated B or M alone is noninfectious toward plants and thus provides agronomic safety.

Proteinoid Polymers and Nanocapsules for Cancer Diagnostics, Therapy and Theranostics: In Vitro and In Vivo Studies

Proteinoids-simple polymers composed of amino acids-were suggested decades ago by Fox and coworkers to form spontaneously by heat. These special polymers may self-assemble in micrometer structures called proteinoid microspheres, presented as the protocells of life on earth. Interest in proteinoids increased in recent years, in particular for nano-biomedicine. They were produced by stepwise polymerization of 3-4 amino acids. Proteinoids based on the RGD motif were prepared for targeting tumors. Nanocapsules form by heating proteinoids in an aqueous solution and slowly cooling to room temperature. Proteinoid polymers and nanocapsules suit many biomedical applications owing to their non-toxicity, biocompatibility and immune safety. Drugs and/or imaging reagents for cancer diagnostic, therapeutic and theranostic applications were encapsulated by dissolving them in aqueous proteinoid solutions. Here, recent in vitro and in vivo studies are reviewed.