Guest-host doped strategy for constructing ultralong-lifetime near-infrared organic phosphorescence materials for bioimaging.

Organic near-infrared room temperature phosphorescence materials have unparalleled advantages in bioimaging due to their excellent penetrability. However, limited by the energy gap law, the near-infrared phosphorescence materials (>650 nm) are very rare, moreover, the phosphorescence lifetimes of these materials are very short. In this work, we have obtained organic room temperature phosphorescence materials with long wavelengths (600/657-681/732 nm) and long lifetimes (102-324 ms) for the first time through the guest-host doped strategy. The guest molecule has sufficient conjugation to reduce the lowest triplet energy level and the host assists the guest in exciton transfer and inhibits the non-radiative transition of guest excitons. These materials exhibit good tissue penetration in bioimaging. Thanks to the characteristic of long lifetime and long wavelength emissive phosphorescence materials, the tumor imaging in living mice with a signal to background ratio value as high as 43 is successfully realized. This work provides a practical solution for the construction of organic phosphorescence materials with both long wavelengths and long lifetimes.

Mn3+-rich oxide/persistent luminescence nanoparticles achieve light-free generation of singlet oxygen and hydroxyl radicals for responsive imaging and tumor treatment.

X-ray excited persistent luminescence (XEPL) imaging has attracted increasing attention in biomedical imaging due to elimination of autofluorescence, high signal-to-noise ratio and repeatable activation with high penetration. However, optical imaging still suffers from limited for high spatial resolution. Methods: Herein, we report Mn3+-rich manganese oxide (MnOx)-coated chromium-doped zinc gallogermanate (ZGGO) nanoparticles (Mn-ZGGOs). Enhanced XEPL and magnetic resonance (MR) imaging were investigated by the decomposition of MnOx shell in the environment of tumors. We also evaluated the tumor cell-killing mechanism by detection of reactive oxygen (ROS), lipid peroxidation and mitochondrial membrane potential changes in vitro. Furthermore, the in vivo biodistribution, imaging and therapy were studied by U87MG tumor-bearing mice. Results: In the tumor region, the MnOx shell is quickly decomposed to produce Mn3+ and oxygen (O2) to directly generate singlet oxygen (1O2). The resulting Mn2+ transforms endogenous H2O2 into highly toxic hydroxyl radical (·OH) via a Fenton-like reaction. The Mn2+ ions and ZGGOs also exhibit excellent T1-weighted magnetic resonance (MR) imaging and ultrasensitive XEPL imaging in tumors. Conclusion: Both the responsive dual-mode imaging and simultaneous self-supplied O2 for the production of 1O2 and oxygen-independent ·OH in tumors allow for more accurate diagnosis of deep tumors and more efficient inhibition of tumor growth without external activation energy.

NanoBiT System and Hydrofurimazine for Optimized Detection of Viral Infection in Mice-A Novel in Vivo Imaging Platform.

Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing delivery of higher doses for in vivo imaging. The small high-affinity peptide tag (HiBiT), which is only 11 amino-acids in length, was engineered into a clinically used oncolytic adenovirus, and the complementary large protein (LgBiT) was constitutively expressed in tumor cells. Infection of the LgBiT expressing cells with the HiBiT oncolytic virus will reconstitute NanoLuc in the cytosol of the cell, providing strong bioluminescence upon treatment with substrate. This new bioluminescent system served as an early stage quantitative viral transduction reporter in vitro and also in vivo in mice, for longitudinal monitoring of oncolytic viral persistence in infected tumor cells. This platform provides novel opportunities for studying the biology of viruses in animal models.

Exploring the In Vivo and In Vitro Anticancer Activity of Rhenium Isonitrile Complexes.

The established platinum-based drugs form covalent DNA adducts to elicit their cytotoxic response. Although they are widely employed, these agents cause toxic side-effects and are susceptible to cancer-resistance mechanisms. To overcome these limitations, alternative metal complexes containing the rhenium(I) tricarbonyl core have been explored as anticancer agents. Based on a previous study ( Chem. Eur. J. 2019, 25, 9206), a series of highly active tricarbonyl rhenium isonitrile polypyridyl (TRIP) complexes of the general formula fac-[Re(CO)3(NN)(ICN)]+, where NN is a chelating diimine and ICN is an isonitrile ligand, that induce endoplasmic reticulum (ER) stress via activation of the unfolded protein response (UPR) pathway are investigated. A total of 11 of these TRIP complexes were synthesized, modifying both the equatorial polypyridyl and axial isonitrile ligands. Complexes with more electron-donating equatorial ligands were found to have greater anticancer activity, whereas the axial ICN ligands had a smaller effect on their overall potency. All 11 TRIP derivatives trigger a similar phenotype that is characterized by their abilities to induce ER stress and activate the UPR. Lastly, we explored the in vivo efficacy of one of the most potent complexes, fac-[Re(CO)3(dmphen)(ptolICN)]+ (TRIP-1a), where dmphen = 2,9-dimethyl-1,10-phenanthroline and ptolICN = para-tolyl isonitrile, in mice. The 99mTc congener of TRIP-1a was synthesized, and its biodistribution in BALB/c mice was investigated in comparison to the parent Re complex. The results illustrate that both complexes have similar biodistribution patterns, suggesting that 99mTc analogues of these TRIP complexes can be used as diagnostic partner agents. The in vivo antitumor activity of TRIP-1a was then investigated in NSG mice bearing A2780 ovarian cancer xenografts. When administered at a dose of 20 mg/kg twice weekly, this complex was able to inhibit tumor growth and prolong mouse survival by 150% compared to the vehicle control cohort.

Outer-Frame-Degradable Nanovehicles Featuring Near-Infrared Dual Luminescence for in Vivo Tracking of Protein Delivery in Cancer Therapy.

In vivo monitoring of cargo protein delivery is critical for understanding the pharmacological efficacies and mechanisms during cancer therapy, but it still remains a formidable challenge because of the difficulty in observing nonfluorescent proteins at high resolution and sensitivity. Here we report an outer-frame-degradable nanovehicle featuring near-infrared (NIR) dual luminescence for real-time tracking of protein delivery in vivo. Upconversion nanoparticles (UCNPs) and fluorophore-doped degradable macroporous silica (DS) with spectral overlap were coupled to form a core-shell nanostructure as a therapeutic protein nanocarrier, which was eventually enveloped with a hyaluronic acid (HA) shell to prevent protein leakage and for recognizing tumor sites. The DS layer served as both a container to accommodate the therapeutic proteins and a filter to attenuate upconversion luminescence (UCL) of the inner UCNPs. After the nanovehicles selectively accumulated at tumor sites and entered cancer cells, intracellular hyaluronidase (HAase) digested the outermost HA protective shell and initiated the outer frame degradation-induced protein release and UCL restoration of UCNPs in the intracellular environment. Significantly, the biodistribution of the nanovehicles can be traced at the 710 nm NIR fluorescence channel of DS, whereas the protein release can be monitored at the 660 nm NIR fluorescence channel of UCNPs. Real-time tracking of protein delivery and release was achieved in vitro and in vivo by NIR fluorescence imaging. Moreover, in vitro and in vivo studies manifest that the protein cytochrome c-loaded nanovehicles exhibited excellent cancer therapeutic efficacy. This nanoplatform assembled by the outer-frame-degradable nanovehicles featuring NIR dual luminescence not only advances our understanding of where, when, and how therapeutic proteins take effect in vivo but also provides a universal route for visualizing the translocation of other bioactive macromolecules in cancer treatment and intervention.

Renal Clearable Luminescent Gold Nanoparticles: From the Bench to the Clinic.

With more and more engineered nanoparticles (NPs) being translated to the clinic, the United States Food and Drug Administration (FDA) has recently issued the latest draft guidance on nanomaterial-containing drug products with an emphasis on understanding their in vivo transport and nano-bio interactions. Following these guidelines, NPs can be designed to target and treat diseases more efficiently than small molecules, have minimum accumulation in normal tissues, and induce minimum toxicity. In this Minireview, we integrate this guidance with our ten-year studies on developing renal clearable luminescent gold NPs. These gold NPs resist serum protein adsorption, escape liver uptake, target cancerous tissues, and report kidney dysfunction at early stages. At the same time, off-target gold NPs can be eliminated by the kidneys with minimum accumulation in the body. Additionally, we identify challenges to the translation of renal clearable gold NPs from the bench to the clinic.

A novel bioluminescence-based method to investigate uptake of bile acids in living cells.

Bile acid transporters, including the ileal apical sodium-dependent bile acid transporter (ASBT) and the hepatic sodium-taurocholate cotransporting polypeptide (NTCP), are crucial for the enterohepatic circulation of bile acids. Our objective was to develop a method for measuring bile acid transporter activity in real time to precisely evaluate rapid changes in their function. We designed a reporter system relying on a novel probe: cholic acid attached to luciferin via a disulfide-containing, self-immolating linker (CA-SS-Luc). Incubation of human embryonic kidney-293 cells coexpressing luciferase and ASBT with different concentrations of CA-SS-Luc (0.01-1 µM) resulted in bioluminescence with an intensity that was concentration- and time-dependent. The bioluminescence measured during incubation with 1 µM CA-SS-Luc was dependent on the levels of ASBT or NTCP expressed in the cells. Coincubation of CA-SS-Luc with natural bile acids enhanced the bioluminescence in a concentration-dependent manner with kinetic parameters for ASBT similar to those previously reported using conventional methods. These findings suggest that this method faithfully assesses ASBT function. Further, incubation with tyrosine phosphatase inhibitor III (PTPIII) led to significantly increased bioluminescence in cells expressing ASBT, consistent with previous studies showing an increase in ASBT function by PTPIII. We then investigated CA-SS-Luc in isolated mouse intestinal epithelial cells. Ileal enterocytes displayed significantly higher luminescence compared with jejunal enterocytes, indicating a transport process mediated by ileal ASBT. In conclusion, we have developed a novel method to monitor the activity of bile acid transporters in real time that has potential applications both for in vitro and in vivo studies. NEW & NOTEWORTHY This article reports the development of a real-time method for measuring the uptake of bile acids using a bioluminescent bile acid-based probe. This method has been validated for measuring uptake via the apical sodium-dependent bile acid transporter and the sodium-taurocholate cotransporting polypeptide in cell culture and ex vivo intestinal models.

Trackable Metallodrugs Combining Luminescent Re(I) and Bioactive Au(I) Fragments.

Hetero-bimetallic and -trimetallic complexes were synthesized by the combination of different metallic fragments, a luminescent Re(I) species, and a bioactive Au(I) derivative. A ditopic P,N-donor ligand (L) was used as linker between both metals, affording six new bipyridine (bipy) Re(I)/Au(I) hetero-metallic complexes of the type fac-[Re(bipy)(CO)3(LAuCl)]+ (4-6) and [(fac-[Re(bipy)(CO)3(L)])2Au]3+ (7-9) after a thorough synthetic procedure. Their emission is associated with a triplet metal-to-ligand charge transfer (Re(dπ) → bipy(π*)) transition and red-shifted in polar solvents with lifetimes in the range of nanoseconds and quantum yield values up to 12.5%. Cytotoxicity values in A549 cells of hetero-trimetallic species are almost twice that for the hetero-bimetallic (ca. 37 vs 69 µM, respectively), being the L-Au fragment the source of the antiproliferative activity. Species 7 and 8 showed similar behavior by fluorescence microscopy, with a nonuniform cytoplasmatic distribution, a clear accumulation in single spots at the edge of the inner cell membrane as well as in areas within the nucleus. Preliminary studies suggest the DNA as one of the targets and passive diffusion as the entrance pathway.

Bioapplications of renal-clearable luminescent metal nanoparticles.

Renal-clearable inorganic nanoparticles (NPs) that hold great potential in the future clinic translations are considered as the next generation of nanomedicine. In the past decade, enormous efforts have been dedicated to the development of renal-clearable NPs with fascinating optical properties, selective disease-targeting capabilities and low nanotoxicities. A further understanding of the design of renal-clearable luminescent metal NPs and their metabolic behavior in the body is important to achieve their clinical transition and extend their bioapplications in disease theranostics. In this review, we discuss the recent synthetic strategies of renal-clearable metal NPs in terms of the considerations of size and composition, surface chemistry and emission wavelength. We also summarize the current disease-related applications of these renal-clearable luminescent metal NPs in tumor targeting, kidney disease and antimicrobial investigations after a discussion of their biological behavior including the pharmacokinetics and biodistribution. Finally, we provide perspectives on the current challenges and upcoming chances for renal-clearable luminescent metal NPs.

Highly Luminescent Folate-Functionalized Au22 Nanoclusters for Bioimaging.

Gold nanoclusters are emerging as new materials for biomedical applications because of promises offered by their ultrasmall size and excellent biocompatibility. Here, the synthesis and optical and biological characterizations of a highly luminescent folate-functionalized Au22 cluster (Au22 -FA) are reported. The Au22 -FA clusters are synthesized by functionalizing the surface of Au22 (SG)18 clusters, where SG is glutathione, with benzyl chloroformate and folate. The functionalized clusters are highly water-soluble and exhibit remarkably bright luminescence with a quantum yield of 42%, significantly higher than any other water-soluble gold clusters protected with thiolate ligands. The folate groups conjugated to the gold cluster give rise to additional luminescence enhancement by energy transfer sensitization. The brightness of Au22 -FA is found to be 4.77 mM-1 cm-1 , nearly 8-fold brighter than that of Au22 (SG)18 . Further biological characterizations have revealed that the Au22 -FA clusters are well-suited for bioimaging. The Au22 -FA clusters exhibit excellent photostability and low toxicity; nearly 80% cell viability at 1000 ppm of the cluster. Additionally, the Au22 -FA clusters show target specificity to folate-receptor positive cells. Finally, the time-course in vivo luminescence images of intravenous-injected mice show that the Au22 -FA clusters are renal-clearable, leaving only 8% of them remained in the body after 24 h post-injection.

Cadmium Telluride Quantum Dots as a Fluorescence Marker for Adipose Tissue Grafts.

Plastic and reconstructive surgeons increasingly apply adipose tissue grafting in a clinical setting, although the anticipation of graft survival is insecure. There are only few tools for tracking transplanted fat grafts in vivo.Murine adipose tissue clusters were incubated with negatively charged, mercaptoproprionic acid-coated cadmium telluride quantum dots (QDs) emitting in the dark red or near infrared. The intracellular localization of QDs was studied by confocal laser scanning microscopy.As a result, the adipose tissue clusters showed a proportional increase in fluorescence with increasing concentrations (1, 10, 16, 30, 50 nM) of cadmium telluride QDs. Laser scanning microscopy demonstrated a membrane bound localization of QDs. Vacuoles and cell nuclei of adipocytes were spared by QDs. We conclude that QDs were for the first time proven intracellular in adult adipocytes and demonstrate a strong fluorescence signal. Therefore, they may play an essential role for in vivo tracking of fat grafts.

Bioluminescent imaging of ABCG2 efflux activity at the blood-placenta barrier.

Physiologic barriers such as the blood placenta barrier (BPB) and the blood brain barrier protect the underlying parenchyma from pathogens and toxins. ATP-binding cassette (ABC) transporters are transmembrane proteins found at these barriers, and function to efflux xenobiotics and maintain chemical homeostasis. Despite the plethora of ex vivo and in vitro data showing the function and expression of ABC transporters, no imaging modality exists to study ABC transporter activity in vivo at the BPB. In the present study, we show that in vitro models of the placenta possess ABCG2 activity and can specifically transport D-luciferin, the endogenous substrate of firefly luciferase. To test ABCG2 transport activity at the BPB, we devised a breeding strategy to generate a bioluminescent pregnant mouse model to demonstrate transporter function in vivo. We found that coadministering the ABCG2 inhibitors Ko143 and gefitinib with D-luciferin increased bioluminescent signal from fetuses and placentae, whereas the control P-gp inhibitor DCPQ had no effect. We believe that our bioluminescent pregnant mouse model will facilitate greater understanding of the BPB and ABCG2 activity in health and disease.

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

Investigation of the strategies for targeting of the afterglow nanoparticles to tumor cells.

Afterglow nanoparticles have been widely investigated as new agents for cancer imaging and as a light source for photodynamic activation for cancer treatment. For both applications, the targeting of the afterglow nanoparticles to tumor cells is an important and challenging issue. Here we report the strategies for targeting Sr3MgSi2O8:Eu(2+),Dy(3+) afterglow nanoparticles to tumor cells by conjugating with variety of targeting molecules such as folic acid, RGD peptide, and R-11 peptide. For folic acid targeting, experimental observations were conducted on PC-3 cells (folate receptor negative), MCF-7 (folate receptor positive), and KB cells (folate receptor positive) to compare the cellular uptake and confirm targeted delivery. For the cyclic RGDfK peptide, experiments were carried out on the integrin αvß3 positive MDA-MB-231 breast cancer cell line and the integrin αvß3 negative MCF-7 breast cancer cell lines in order to compare the cellular uptakes. As for R11-SH peptide, cellular uptake of the afterglow nanoparticles was observed on LNCaP and PC3 prostate cancer cell lines. All the observations showed that the cellular uptakes of the nanoparticles were enhanced by conjugation to variety of targeting molecules which are specific for breast and prostate cancer cells.

In vivo bioluminescence imaging of transplanted mesenchymal stem cells as a potential source for pancreatic regeneration.

Stem cell therapy has been studied intensively as a promising therapeutic strategy toward a cure for diabetes. To study the effect of mesenchymal stem cell (MSC) transplantation for pancreatic regeneration, we monitored the localization and distribution of transplanted MSCs by bioluminescence imaging in a mouse model. Bone marrow MSCs were isolated and transfected with a highly sensitive firefly luciferase reporter gene. To assess the efficiency of MSC transplantation, a partially pancreatectomized (PPx) mouse model was used. Transplanted MSCs were monitored by confocal microscopy and in vivo bioluminescence imaging. Daily blood glucose levels and glucose tolerance were measured. Insulin-secreting beta cells were immunostained, and insulin levels were measured via enzyme-linked immunosorbent assay. Bioluminescence signals were clearly detected from the transplanted MSCs in the pancreatic region regardless of injection route. However, locally injected MSCs exhibited more rapid proliferation than ductally injected MSCs. PPx mice harboring transplanted MSCs gradually recovered from impaired glucose tolerance. Although insulin secretion was not observed in MSCs, transplanted MSCs facilitate the injured pancreas to recover its function. In vivo optical imaging of transplanted MSCs using a highly sensitive luciferase reporter enables the assessment of MSC transplantation efficiency in a PPx mouse model.

Bioluminescent imaging of drug efflux at the blood-brain barrier mediated by the transporter ABCG2.

ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood-brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that D-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter.

Functional near infrared-emitting Cr3+/Pr3+ co-doped zinc gallogermanate persistent luminescent nanoparticles with superlong afterglow for in vivo targeted bioimaging.

Near infrared (NIR)-emitting persistent luminescent nanoparticles (PLNPs) have great potential for in vivo bioimaging with the advantages of no need for in situ excitation, high signal-to-noise ratio, and deep tissue penetration. However, functional NIR-emitting PLNPs with long afterglow for long-term in vivo imaging are lacking. Here, we show the synthesis of NIR-emitting long-persistent luminescent nanoparticles (LPLNPs) Zn2.94Ga1.96Ge2O10:Cr(3+),Pr(3+) by a citrate sol-gel method in combination with a subsequent reducing atmosphere-free calcination. The persistent luminescence of the LPLNPs is significantly improved via codoping Pr(3+)/Cr(3+) and creating suitable Zn deficiency in zinc gallogermanate. The LPLNP powder exhibits bright NIR luminescence in the biological transparency window with a superlong afterglow time of over 15 days. A persistent energy transfer between host and Cr(3+) ion in the LPLNPs is observed and its mechanism is discussed. PEGylation greatly improves the biocompatibility and water solubility of the LPLNPs. Further bioconjugation with c(RGDyK) peptide makes the LPLNPs promising for long-term in vivo targeted tumor imaging with low toxicity.

A luminescent and biocompatible photoCORM.

The water-soluble rhenium(I) complex fac-[Re(bpy)(CO)(3)(thp)](+) (1) [CF(3)SO(3)(-) salt; bpy = 2,2'-bipyridine, thp = tris(hydroxymethyl)phosphine] is both strongly luminescent and photoactive toward carbon monoxide release. It is stable in aerated aqueous media, is incorporated into cells from the human prostatic carcinoma cell line PPC-1, and shows no apparent cytotoxicity. Furthermore, the solvated Re(I) photoproduct of CO release (2) is also luminescent, a feature that allows one to track the transformation of 1 to 2 inside such cells using confocal fluorescence microscopy. In this context, 1 is a very promising candidate as a photoactivated CO releasing moiety (photoCORM) with potential therapeutic applications.

Effect of core diameter, surface coating, and PEG chain length on the biodistribution of persistent luminescence nanoparticles in mice.

A growing insight toward optical sensors has led to several major improvements in the development of convenient probes for in vivo imaging. Efficient optical detection using quantum dots (QDs) as well as near-infrared organic dyes relies on several key driving principles: the ability to lower background absorption or autofluorescence from tissue, a good photostability of the probe, and a high quantum yield. In this article, we report the real-time biodistribution monitoring of lanthanide-doped persistent luminescence nanoparticles (PLNP), emitting in the near-infrared window, in healthy and tumor-bearing mice. We focused on the influence of hydrodynamic diameter, ranging from 80 to 180 nm, and polyethylene glycol (PEG) surface coating on the behavior of our probes. Tissue distribution was found to be highly dependent on surface coverage as well as core diameter. The amount of PLNP in the blood was highly increased for small (d < 80 nm) and stealth particles. On the opposite, PEG shield molecular weight, ranging from 5 to 20 kDa, had only negligible influence on the in vivo biodistribution of our silicate-based material.

Tetrameric far-red fluorescent protein as a scaffold to assemble an octavalent peptide nanoprobe for enhanced tumor targeting and intracellular uptake in vivo.

Relatively weak tumor affinities and short retention time in vivo hinder the application of targeting peptides in tumor molecular imaging. Multivalent strategies based on various scaffolds have been utilized to improve the ability of peptide-receptor binding or extend the clearance time of peptide-based probes. Here, we use a tetrameric far-red fluorescent protein (tfRFP) as a scaffold to create a self-assembled octavalent peptide fluorescent nanoprobe (Octa-FNP) using a genetic engineering approach. The multiligand connecting, fluorophore labeling and nanostructure formation of Octa-FNP were performed in one step. In vitro studies showed Octa-FNP is a 10-nm fluorescent probe with excellent serum stability. Cellular uptake of Octa-FNP by human nasopharyngeal cancer 5-8F cells is 15-fold of tetravalent probe, ∼80-fold of monovalent probe and ∼600-fold of nulvalent tfRFP. In vivo enhanced tumor targeting and intracellular uptake of Octa-FNP were confirmed using optical imaging and Western blot analysis. It achieved extremely high contrast of Octa-FNP signal between tumor tissue and normal organs, especially seldom Octa-FNP detected in liver and spleen. Owing to easy preparation, precise structural and functional control, and multivalent effect, Octa-FNP provides a powerful tool for tumor optical molecular imaging and evaluating the targeting ability of numerous peptides in vivo.