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A subfield of medical diagnostics known as molecular diagnostics and imaging focuses on visualizing cellular or molecular activities in the body as well as analyzing biological substances. To diagnose illnesses, forecast disease risk, and monitor treatment response, these methods include detecting and analyzing particular biomolecules like DNA, RNA, proteins, or metabolites. Because of the potent tools they provide for illness diagnosis, monitoring, and treatment, molecular imaging and diagnostics are essential to the progress of medical science. While molecular imaging enables the viewing of biological processes in live organisms, molecular diagnostics focuses on the study of biomolecules to guide therapeutic choices. Taken as a whole, they deepen our knowledge of illnesses and help pave the way for customized medicine, which in turn improves treatment methods and outcomes for patients.
The discovery of cell penetrating peptides (CPPs) around twenty-five years ago has been followed by a meteoric rise in their use and the number of tumor diagnostic and therapeutic applications. Because of their tiny size, selective uptake, high stability, fast clearance from non-specific targets, and retention in specific targets, CPPs show promise as a tool for tumor imaging. To capture images of tumors, they can be coupled to radioisotopes, activated probes tagged with fluorophore, polymers, quantum dots, metal chelates, nanoparticles (NPs), and other contrast agents. These imaging agents can be carried, transported, and delivered by CPPs, who can also provide intracellular access and functioning to the imaging cargo. Problems arise with each CPP independently because their chemical characteristics are unique as a result of variations in their amino acid sequence. In addition to its usage in tumor diagnostics, CPP has other in vivo imaging and cell tracking applications in animals.
CPP + imaging agents. (Stiltner J., et al., 2021)
CPPs for delivery of fluorophores and quantum dots. (Sayers E J., et al., 2014)
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The distinctive optical and chemical properties of tiny, fluorescent nanocrystals called quantum dots (Qdots) have garnered a lot of interest. With their attractive luminosity and photostability properties, Qdots offer numerous advantages over standard fluorescent proteins and small molecules. These properties enable long-term visualisation of cellular structures and events, and Qdots have the potential to discriminate individual particles for single particle tracking experiments. Consequently, Qdots are anticipated to have applications in medical diagnostics, such as contrast agents, and are being used more and more as intracellular live-cell probes. To improve Qdot absorption, a nona-arginine peptide rich in cysteine and histidine (C-5H-R9-5H-C) was recently engineered by adjusting the peptide's net hydrophobicity to facilitate membrane interaction. Due to the accumulation of material within cells at 4 ℃, the major route of entry was a direct translocation of the complex through the plasma membrane, even though the peptide improved Qdot uptake at high nanoparticle concentrations (100 nM Qdots complexed with 6 μM peptide) compared to R9 alone or Pas (FFLIP) modified R9.
Despite the widespread use of fluorescent dyes for cellular trace molecule analysis, these chemical compounds have limitations when it comes to long-term imaging, such as a broad emission spectrum, low brightness, and poor light stability. While currently restricted to cellular transduction efficiency, semiconductor nanoparticle quantum dots are gaining traction as great imaging probes thanks to their ability to exhibit fluorescence of varying colors when activated by a single wavelength laser source. After two weeks of following human mesenchymal stem cells (MSCs) in a liver-resection animal model, a Pdot controlled by R8 was able to continue monitoring them normally. Pdots and CPPs demonstrated remarkable biocompatibility and long-term traceability throughout the whole process, which began with localization in the lungs and progressed gradually to the wounded liver. In addition, a different study detailed the use of near-infrared (NIR) fluorescent Pdot to monitor cells throughout the body, even those in deep organs and tissues. The endocytosis of Pdots in MCF-7 tumor cells was greatly improved by R8, as seen by a 100-fold increase in labeling brightness compared to the control group that did not receive CPP. Even after 13 days of incubation, the cytoplasm continued to display a discernible level of fluorescence. With CPP, deep organs are able to take in more Pdot, which should lead to better whole-body fluorescence imaging in the future.
Applications of CPPs as molecular delivery vehicles. (Xu J., et al., 2019)
Physiological limitations cause limited cellular absorption of luminous probes and nanoparticles, which is problematic for their use as a medium for microscopic viewing of cellular structures or molecular targets. Selecting highly permeable molecules to transmit luminophores is a frequent practice for modifying the luminous probe engraft model, improving cell uptake efficiency, decreasing incubation time, and enabling real-time monitoring. It was reported that a new amphiphilic CPP, F6G6(rR)3R2, which has a hydrophobic N-terminus and a highly hydrophilic C-terminus, could self-assemble into various water-based nanoparticles, with the ability to load three thermally activated delayed fluorescence (TADF) probes, 4CzIPN, NAI-DPAC, and BTZ-DMAC. The nanoparticle, which was made of CPP, showed excellent dispersibility with a narrow polydispersity of less than 0.2 and stability for two weeks at room temperature without precipitation. The co-incubation of Hela cells with the CPP-functionalized NPs revealed that F6G6 (rR) 3R2 had a remarkable ability to transport cells. Fluorescence of several TADF molecules could be seen after only 5 minutes, and flow cytometry confirmed that NP-treated cells had a fluorescence positive rate of >35%, with 4CzIPN showing an even higher rate at > 90%.
Activatable CPPs (ACPPs) are one way to improve tumor imaging. The CPP in this approach has a polycation section that can enter cells and transport cargo, a protease-cleavable linker that can target metalloproteases-2 (MMP-2) and MMP-9, and a polyanion area that inhibits the activity of the cell-penetrating region. The CPP is able to penetrate cells after being triggered by matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9). The theory was that ACPPs would target cancers rather than non-tumor tissues, due to the overexpression of MMP-2 and MMP-9 in many cancer lines. Recent research has shown that colorectal cancer may be targeted and imaged using an ACPP tagged with Cy5. One study used an intravital fluorescent imaging device to conduct in vivo and ex vivo fluorescence imaging in nude HCT-116 xenograft mice. Based on the findings, ACPP-Cy5 shows promise as an imaging agent for the detection of cancers and metastases to the liver, as it accumulates in tumors. Additionally, ACPPs can have targeted cargo attached to them to improve imaging and tumor targeting. In addition to facilitating multimodal imaging, macromolecules provide the following benefits: increasing circulation time and tumor uptake; decreasing background noise via reduced glomerular and synovial filtration; amplifying the quantity of imaging agent on a single peptide; decreasing toxicity; and so on. The polycationic domain of an ACPP was linked to a polyamidoamine dendrimer. Dual labeling of the ACPP dendrimer combination with Cy5 and Gd chelates enabled fluorescence and MRI imaging in tumor-bearing animals. Once again, the data demonstrated that CPPs may be adjusted and mixed in countless ways to improve tumor imaging. They also exhibited greater uptake and tumor selectivity than previously published results.
Schematic diagram of synthetic amphiphilic CPP for imaging. (Xu J., et al., 2019)
Silica mesoporous nanoparticles, a rigid inorganic nanoparticle with high structural stability and echogenic properties, can be applied to highly sensitive ultrasound-enhanced imaging techniques in vivo. Recently, a FITC-TAT conjugated porous silicon nanoparticle (TPSi NP) was confirmed to aggregate in cells. One study pointed out that the CPP-mediated experimental group highly amplified the acoustic scattering of the labeled MSCs, which increased the ultrasound signal by 2.3-fold. The intracellular silicon content of MSC-labeled cells was determined by atomic emission spectroscopy. The results further demonstrated a high labeling efficiency of TAT conjugation: 78.8% of TPSi NPs were localized in cells, and comparatively, 52.7% of TAT-conjugated Psi NPs were internalized by MSC under the same conditions. This type of CPP-mediated TPSi NP ensures sufficient amount of intracellularization and produces a clear ultrasound signal amplification that may contribute to the development of the heart, cancerous sites, and intracranial imaging techniques. Gold nanoparticle (AuNP) is another inorganic nanoparticle with distinct features, such as good biocompatibility and safety, drawing intense interest in biological imaging and clinical diagnostic techniques in recent years. A powerful approach to promote cellular uptake is urgently needed to localize AuNPs into the cytoplasm or specific organelles to achieve the stable biomonitoring potential. The membrane binding and cellular uptake effects of three ultrasmall luminescent gold nanoparticles (CR8-AuNP) with different CPP coverage were explored. Compared to gold nanoparticles coated with glutathione alone, CR-AuNP was reported to be more readily taken up by living Hela cells: at the same ligand density, the minimum time for luminescence observation in cells was 3 h and 1 h, respectively. This result may serve as evidence that CR8 enhances the rate and efficiency of cellular action in membrane binding process and endocytosis.
Cell penetrating peptide conjugated gold nanoparticles for biomedical imaging. (Kumar M., et al., 2018)
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References
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