Cell-Penetrating and Enzyme-Responsive Peptides for Targeted Cancer Therapy: Role of Arginine Residue Length on Cell Penetration and In Vivo Systemic Toxicity.

For the improved delivery of cancer therapeutics and imaging agents, the conjugation of cell-penetrating peptides (CPPs) increases the cellular uptake and water solubility of agents. Among the various CPPs, arginine-rich peptides have been the most widely used. Combining CPPs with enzyme-responsive peptides presents an innovative strategy to target specific intracellular enzymes in cancer cells and when combined with the appropriate click chemistry can enhance theranostic drug delivery through the formation of intracellular self-assembled nanostructures. However, one drawback of CPPs is their high positive charge which can cause nonspecific binding, leading to off-target accumulation and potential toxicity. Hence, balancing cell-specific penetration, toxicity, and biocompatibility is essential for future clinical efficacy. We synthesized six cancer-specific, legumain-responsive RnAANCK peptides containing one to six arginine residues, with legumain being an asparaginyl endopeptidase that is overexpressed in aggressive prostate tumors. When conjugated to Alexa Fluor 488, R1-R6AANCK peptides exhibited a concentration- and time-dependent cell penetration in prostate cancer cells, which was higher for peptides with higher R values, reaching a plateau after approximately 120 min. Highly aggressive DU145 prostate tumor cells, but not less aggressive LNCaP cells, self-assembled nanoparticles in the cytosol after the cleavage of the legumain-specific peptide. The in vivo biocompatibility was assessed in mice after the intravenous injection of R1-R6AANCK peptides, with concentrations ranging from 0.0125 to 0.4 mmol/kg. The higher arginine content in R4-6 peptides showed blood and urine indicators for the impairment of bone marrow, liver, and kidney function in a dose-dependent manner, with instant hemolysis and morbidity in extreme cases. These findings underscore the importance of designing peptides with the optimal arginine residue length for a proper balance of cell-specific penetration, toxicity, and in vivo biocompatibility.

Tumor-tropic Trojan horses: Using mesenchymal stem cells as cellular nanotheranostics.

Various classes of nanotheranostics have been developed for enhanced tumor imaging and therapy. However, key limitations for a successful use of nanotheranostics include their targeting specificity with limited off-site tissue accumulation as well as their distribution and prolonged retention throughout the entire tumor. Due to their inherent tumor-tropic properties, the use of mesenchymal stem cells (MSCs) as a "Trojan horse" has recently been proposed to deliver nanotheranostics more effectively. This review discusses the current status of "cellular nanotheranostics" for combined (multimodal) imaging and therapy in preclinical cancer models. Emphasis is placed on the limited knowledge of the signaling pathways and molecular mechanisms of MSC tumor-tropism, and how such information may be exploited to engineer MSCs in order to further improve tumor homing and nanotheranostic delivery using image-guided procedures.

A Smart Intracellular Self-Assembling Bioorthogonal Raman Active Nanoprobe for Targeted Tumor Imaging.

Inspired by the principle of in situ self-assembly, the development of enzyme-activated molecular nanoprobes can have a profound impact on targeted tumor detection. However, despite their intrinsic promise, obtaining an optical readout of enzyme activity with high specificity in native milieu has proven to be challenging. Here, a fundamentally new class of Raman-active self-assembling bioorthogonal enzyme recognition (nanoSABER) probes for targeted tumor imaging is reported. This class of Raman probes presents narrow spectral bands reflecting their vibrational fingerprints and offers an attractive solution for optical imaging at different bio-organization levels. The optical beacon harnesses an enzyme-responsive peptide sequence, unique tumor-penetrating properties, and vibrational tags with stretching frequencies in the cell-silent Raman window. The design of nanoSABER is tailored and engineered to transform into a supramolecular structure exhibiting distinct vibrational signatures in presence of target enzyme, creating a direct causality between enzyme activity and Raman signal. Through the integration of substrate-specific for tumor-associated enzyme legumain, unique capabilities of nanoSABER for imaging enzyme activity at molecular, cellular, and tissue levels in combination with machine learning models are shown. These results demonstrate that the nanoSABER probe may serve as a versatile platform for Raman-based recognition of tumor aggressiveness, drug accumulation, and therapeutic response.

Targeted Enzyme Activity Imaging with Quantitative Phase Microscopy.

Quantitative phase imaging (QPI) is a powerful optical imaging modality for label-free, rapid, and three-dimensional (3D) monitoring of cells and tissues. However, molecular imaging of important intracellular biomolecules such as enzymes remains a largely unexplored area for QPI. Herein, we introduce a fundamentally new approach by designing QPI contrast agents that allow sensitive detection of intracellular biomolecules. We report a new class of bio-orthogonal QPI-nanoprobes for in situ high-contrast refractive index (RI) imaging of enzyme activity. The nanoprobes feature silica nanoparticles (SiO2 NPs) having higher RI than endogenous cellular components and surface-anchored cyanobenzothiazole-cysteine (CBT-Cys) conjugated enzyme-responsive peptide sequences. The nanoprobes specifically aggregated in cells with target enzyme activity, increasing intracellular RI and enabling precise visualization of intracellular enzyme activity. We envision that this general design of QPI-nanoprobes could open doors for spatial-temporal mapping of enzyme activity with direct implications for disease diagnosis and evaluating the therapeutic efficacy.

In Vivo Imaging of Naked and Microencapsulated Islet Cell Transplantation.

We describe step-by-step methods to label human pancreatic islet cells and murine insulinoma cells and their subsequent transplantation into type I diabetic mouse models with a focus on in vivo imaging using clinically applicable scanners. We also cover islets that are microencapsulated within alginate hydrogels loaded with imaging agents. By following these methods, it is possible to image cell grafts using T1-weighted and T2/T2*-weighted 1H magnetic resonance imaging (MRI), 19F MRI, computed tomography, ultrasound imaging, and bioluminescence imaging in vivo. Considering a myriad of factors that may affect the outcome of proper in vivo detection, we discuss potential issues that may be encountered during and after the process of labeling. The ultimate goal is to use these in vivo imaging approaches to determine and optimize naked and encapsulated islet cell survival, therapeutic function, and engraftment procedures.

A 2D nanotheranostic platform based on graphene oxide and phase-change materials for bimodal CT/MR imaging, NIR-activated drug release, and synergistic thermo-chemotherapy.

Recent years have seen considerable progress in the development of nanomedicine by the advent of 2D nanomaterials serving as ideal platforms to integrate multiple theranostic functions. We synthesized multifunctional stimuli-responsive 2D-based smart nanocomposites (NCs), comprising gold nanoparticles (AuNPs) and superparamagnetic iron oxides (SPIOs) scaffolded within graphene oxide (GO) nanosheets, coated with doxorubicin (DOX)-loaded 1-tetradecanol (TD), and further modified with an alginate (Alg) polymer. TD is a phase-change material (PCM) that confines DOX molecules to the GO surface and melts when the temperature exceeds its melting point (Tm=39 °C), causing the PCM to release its drug payload. By virtue of their strong near-infrared (NIR) light absorption and high photothermal conversion efficiency, GO nanosheets may enable photothermal therapy (PTT) and activate a phase change to trigger DOX release. Upon NIR irradiation of NCs, a synergistic thermo-chemotherapeutic effect can be obtained by GO-mediated PTT, resulting an accelerated and controllable drug release through the PCM mechanism. The biodistribution of these NCs could also be imaged with computed tomography (CT) and magnetic resonance (MR) imaging in vitro and in vivo. Hence, this multifunctional nanotheranostic platform based on 2D nanomaterials appears a promising candidate for multimodal image-guided cancer therapy.

Enzyme-mediated intratumoral self-assembly of nanotheranostics for enhanced imaging and tumor therapy.

Enzyme-mediated intratumoral self-assembled (EMISA) nanotheranostics represent a new class of smart agents for combined imaging and therapy of cancer. Cancer cells overexpress various enzymes that are essential for high metabolism, fast proliferation, and tissue invasion and metastasis. By conjugating small molecules that contain an enzyme-specific cleavage site to appropriate chemical linkers, it is possible to induce self-assembly of nanostructures in tumor cells having the target enzyme. This approach of injecting small theranostic molecules that eventually become larger nanotheranostics in situ avoids some of the major limitations that are encountered when injecting larger, pre-assembled nanotheranostics. The advantage of EMISA nanotheranostics include the avoidance of nonspecific uptake and rapid clearance by phagocytic cells, increased cellular accumulation, reduced drug efflux and prolonged cellular exposure time, all of which lead to an amplified imaging signal and therapeutic efficacy. We review here the different approaches that can be used for preparing EMISA-based organic, inorganic, or organic/inorganic hybrid nanotheranostics based on noncovalent interactions and/or covalent bonding. Imaging examples are shown for fluorescence imaging, nuclear imaging, photoacoustic imaging, Raman imaging, computed tomography imaging, bioluminescent imaging, and magnetic resonance imaging. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Biology-Inspired Nanomaterials > Peptide-Based Structures.

Clinical magnetic hyperthermia requires integrated magnetic particle imaging.

Magnetic nanomaterials that respond to clinical magnetic devices have significant potential as cancer nanotheranostics. The complexities of their physics, however, introduce challenges for these applications. Hyperthermia is a heat-based cancer therapy that improves treatment outcomes and patient survival when controlled energy delivery is combined with accurate thermometry. To date, few technologies have achieved the needed evolution for the demands of the clinic. Magnetic fluid hyperthermia (MFH) offers this potential, but to be successful it requires particle-imaging technology that provides real-time thermometry. Presently, the only technology having the potential to meet these requirements is magnetic particle imaging (MPI), for which a proof-of-principle demonstration with MFH has been achieved. Successful clinical translation and adoption of integrated MPI/MFH technology will depend on successful resolution of the technological challenges discussed. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Diagnostic Tools > In Vivo Nanodiagnostics and Imaging.

In vivo tracking of unlabelled mesenchymal stromal cells by mannose-weighted chemical exchange saturation transfer MRI.

The tracking of the in vivo biodistribution of transplanted human mesenchymal stromal cells (hMSCs) relies on reporter genes or on the addition of exogenous imaging agents. However, reporter genes and exogenous labels may require bespoke manufacturing and regulatory processes if used in cell therapies, and the labels may alter the cells' properties and are diluted on cellular division. Here we show that high-mannose N-linked glycans, which are abundantly expressed on the surface of hMSCs, can serve as a biomarker for the label-free tracking of transplanted hMSCs by mannose-weighted chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI). For live mice with luciferase-transfected hMSCs transplanted into their brains, post-mortem fluorescence staining with a mannose-specific lectin showed that increases in the CEST MRI signal, which correlated well with the bioluminescence intensity of viable hMSCs for 14 days, corresponded to the presence of mannose. In vitro, osteogenically differentiated hMSCs led to lower CEST MRI signal intensities owing to the concomitantly reduced expression of mannose. The label-free imaging of hMSCs may facilitate the development and testing of cell therapies.

In Vivo Imaging of Implanted Hyaluronic Acid Hydrogel Biodegradation.

Although the use of stem cell therapy for central nervous system (CNS) repair has shown considerable promise, it is still limited by the immediate death of a large fraction of transplanted cells owing to cell handling procedures, injection stress and host immune attack leading to poor therapeutic outcomes. Scaffolding cells in hydrogels is known to protect cells from such immediate death by shielding them from mechanical damage and by averting an immune attack after transplantation. Implanted hydrogels must eventually degrade and facilitate a safe integration of the graft with the surrounding host tissue. Hence, serial monitoring of hydrogel degradation in vivo is pivotal to optimize hydrogel compositions and overall therapeutic efficacy of the graft. We present here methods and protocols to use chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) as a non-invasive, label-free imaging paradigm to monitor the degradation of composite hydrogels made up of thiolated gelatin (Gel-SH), thiolated hyaluronic acid (HA-SH), and poly (ethylene glycol) diacrylate (PEGDA), of which the stiffness and CEST contrast can be fine-tuned by simply varying the composite concentrations and mixing ratios. By individually labeling Gel-S and HA-S with two distinct near-infrared (NIR) dyes, multispectral monitoring of the relative degradation of the components can be used for long-term validation of the CEST MRI findings.

In Vivo MRI Tracking of Tumor Vaccination and Antigen Presentation by Dendritic Cells.

Cancer vaccination using tumor antigen-primed dendritic cells (DCs) was introduced in the clinic some 25 years ago, but the overall outcome has not lived up to initial expectations. In addition to the complexity of the immune response, there are many factors that determine the efficacy of DC therapy. These include accurate administration of DCs in the target tissue site without unwanted cell dispersion/backflow, sufficient numbers of tumor antigen-primed DCs homing to lymph nodes (LNs), and proper timing of immunoadjuvant administration. To address these uncertainties, proton (1H) and fluorine (19F) magnetic resonance imaging (MRI) tracking of ex vivo pre-labeled DCs can now be used to non-invasively determine the accuracy of therapeutic DC injection, initial DC dispersion, systemic DC distribution, and DC migration to and within LNs. Magnetovaccination is an alternative approach that tracks in vivo labeled DCs that simultaneously capture tumor antigen and MR contrast agent in situ, enabling an accurate quantification of antigen presentation to T cells in LNs. The ultimate clinical premise of MRI DC tracking would be to use changes in LN MRI signal as an early imaging biomarker to predict the efficacy of tumor vaccination and anti-tumor response long before treatment outcome becomes apparent, which may aid clinicians with interim treatment management.

In Vivo Imaging of Allografted Glial-Restricted Progenitor Cell Survival and Hydrogel Scaffold Biodegradation.

Transplanted glial-restricted progenitor (GRP) cells have potential to focally replace defunct astrocytes and produce remyelinating oligodendrocytes to avert neuronal death and dysfunction. However, most central nervous system cell therapeutic paradigms are hampered by high initial cell death and a host anti-graft immune response. We show here that composite hyaluronic acid-based hydrogels of tunable mechanical strengths can significantly improve transplanted GRP survival and differentiation. Allogeneic GRPs expressing green fluorescent protein and firefly luciferase were scaffolded in optimized hydrogel formulations and transplanted intracerebrally into immunocompetent BALB/c mice followed by serial in vivo bioluminescent imaging and chemical exchange saturation transfer magnetic resonance imaging (CEST MRI). We demonstrate that gelatin-sensitive CEST MRI can be exploited to monitor hydrogel scaffold degradation in vivo for ∼5 weeks post transplantation without necessitating exogenous labeling. Hydrogel scaffolding of GRPs resulted in a 4.5-fold increase in transplanted cell survival at day 32 post transplantation compared to naked cells. Histological analysis showed significant enhancement of cell proliferation as well as Olig2+ and GFAP+ cell differentiation for scaffolded cells compared to naked cells, with reduced host immunoreactivity. Hence, hydrogel scaffolding of transplanted GRPs in conjunction with serial in vivo imaging of cell survival and hydrogel degradation has potential for further advances in glial cell therapy.

Furin-Mediated Self-Assembly of Olsalazine Nanoparticles for Targeted Raman Imaging of Tumors.

Olsalazine (Olsa) is a broad-spectrum anti-cancer agent acting as a DNA-methylation inhibitor. When conjugated to 2-cyano-6-aminobenzothiazole and a peptide substrate specific for the tumor-overexpressed enzyme furin, it can self-assemble into nanoparticles that can be detected by chemical-exchange saturation-transfer magnetic-resonance imaging (CEST MRI). We report here that these nano-assemblies can also be detected with high specificity in furin-overexpressing tumor cells by Raman spectroscopy with a distinct scattering signature and demonstrate the utility of this sensing mechanism in vitro and in vivo. Our findings suggest that Raman spectroscopy could be used for high-resolution image-guided surgery to precisely delineate tumor margins during and after resection in real-time as well as to determine microscopic tumor invasion and multifocal locoregional tumor spread, which are currently impossible to visualize with available imaging technologies, including CEST MRI.

Multifunctional Theranostic Graphene Oxide Nanoflakes as MR Imaging Agents with Enhanced Photothermal and Radiosensitizing Properties.

The integration of multiple therapeutic and diagnostic functions into a single nanoplatform for image-guided cancer therapy has been an emerging trend in nanomedicine. We show here that multifunctional theranostic nanostructures consisting of superparamagnetic iron oxide (SPIO) and gold nanoparticles (AuNPs) scaffolded within graphene oxide nanoflakes (GO-SPIO-Au NFs) can be used for dual photo/radiotherapy by virtue of the near-infrared (NIR) absorbance of GO for photothermal therapy (PTT) and the Z element radiosensitization of AuNPs for enhanced radiation therapy (RT). At the same time, this nanoplatform can also be detected by magnetic resonance (MR) imaging because of the presence of SPIO NPs. Using a mouse carcinoma model, GO-SPIO-Au NF-mediated combined PTT/RT exhibited a 1.85-fold and 1.44-fold higher therapeutic efficacy compared to either NF-mediated PTT or RT alone, respectively, resulting in a complete eradication of tumors. As a sensitive multifunctional theranostic platform, GO-SPIO-Au NFs appear to be a promising nanomaterial for enhanced cancer imaging and therapy.

CT and CEST MRI bimodal imaging of the intratumoral distribution of iodinated liposomes.

BACKGROUND: To develop liposomes loaded with iodinated agents as nanosized CT/MRI bimodal contrast agents for monitoring liposome-mediated drug delivery. METHODS: Rhodamine-labeled iodixanol (VisipaqueTM)-loaded liposomes (IX-lipo) were prepared and tested for their properties as a diamagnetic CEST contrast agent in vitro. Mice bearing subcutaneous CT26 colon tumors were injected i.v. with 1 g/kg (535 mg iodine/kg) IX-lipo, and in vivo CT and CEST MR images were acquired on day 3. CT and CEST MR images were also acquired for tumor-bearing mice co-injected with IX-lipo and tumor necrosis factor (TNF-α). RESULTS: In addition to CT contrast, IX-lipo exhibited a strong CEST contrast similar to its non-liposomal form, with a detectability of ~2 nM per liposome. Both CT imaging and CEST MRI showed that i.v. injection of IX-lipo resulted in a rim enhancement of CT26 tumors with a heterogeneous central distribution. In contrast, co-injection of TNF-α caused a significantly augmented CT/MRI contrast in the tumor center. The intratumoral biodistribution of IX-lipo correlated well to the rhodamine patterns observed with fluorescence microscopy. CONCLUSIONS: We have developed a CT/MRI bimodal imaging approach for monitoring the delivery and biodistribution of liposomes by loading them with the clinically approved X-ray/CT contrast agent iodixanol. Our approach may be easily adapted for other-FDA approved iodinated agents and thus has great translational potential.

Furin-mediated intracellular self-assembly of olsalazine nanoparticles for enhanced magnetic resonance imaging and tumour therapy.

Among the strategies used for enhancement of tumour retention of imaging agents or anticancer drugs is the rational design of probes that undergo a tumour-specific enzymatic reaction preventing them from being pumped out of the cell. Here, the anticancer agent olsalazine (Olsa) was conjugated to the cell-penetrating peptide RVRR. Taking advantage of a biologically compatible condensation reaction, single Olsa-RVRR molecules were self-assembled into large intracellular nanoparticles by the tumour-associated enzyme furin. Both Olsa-RVRR and Olsa nanoparticles were readily detected with chemical exchange saturation transfer magnetic resonance imaging by virtue of exchangeable Olsa hydroxyl protons. In vivo studies using HCT116 and LoVo murine xenografts showed that the OlsaCEST signal and anti-tumour therapeutic effect were 6.5- and 5.2-fold increased, respectively, compared to Olsa without RVRR, with an excellent 'theranostic correlation' (R2 = 0.97) between the imaging signal and therapeutic response (normalized tumour size). This furin-targeted, magnetic resonance imaging-detectable platform has potential for imaging tumour aggressiveness, drug accumulation and therapeutic response.

Development of Zinc-Specific iCEST MRI as an Imaging Biomarker for Prostate Cancer.

The healthy prostate contains the highest concentration of mobile zinc in the body. As this level decreases dramatically during the initial development of prostate cancer, in vivo detection of prostate zinc content may be applied for diagnosis of prostate cancer. Using 19 F ion chemical exchange saturation transfer magnetic resonance imaging (iCEST MRI) and TF-BAPTA as a fluorinated Zn-binding probe with micromolar sensitivity, we show that iCEST MRI is able to differentiate between normal and malignant prostate cells with a 10-fold difference in contrast following glucose-stimulated zinc secretion in vitro. The iCEST signal decreased in normal prostate cells upon downregulation of the ZIP1 zinc transporter. In vivo, using an orthotopic prostate cancer mouse model and a transgenic adenocarcinoma of the mouse prostate (TRAMP) model, a gradual decrease of >300 % in iCEST contrast following the transition of normal prostate epithelial cells to cancer cells was detected.

Carbon Dots as a New Class of Diamagnetic Chemical Exchange Saturation Transfer (diaCEST) MRI Contrast Agents.

While carbon dots (C-dots) have been extensively investigated pertaining to their fluorescent, phosphorescent, electrochemiluminescent, optoelectronic, and catalytic features, their inherent chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) properties are unknown. By virtue of their hydrophilicity and abundant exchangeable protons of hydroxyl, amine, and amide anchored on the surface, we report here that C-dots can be adapted as effective diamagnetic CEST (diaCEST) MRI contrast agents. As a proof-of-concept demonstration, human glioma cells were labeled with liposomes with or without encapsulated C-dots and implanted in mouse brain. In vivo CEST MRI was able to clearly differentiate labeled cells from non-labeled cells. The present findings may encourage new applications of C-dots for in vivo imaging in deep tissues, which is currently not possible using conventional fluorescent (near-infrared) C-dots.