Abnormal tumor vessels impede the transport and distribution of chemotherapeutics, resulting in low drug concentration at tumor sites and compromised drug efficacy. Normalizing tumor vessels can modulate tumor vascular permeability, alleviate tumor hypoxia, increase blood perfusion, attenuate interstitial fluid pressure, and improve drug delivery. Herein, a novel strategy combining cediranib, a tumor vessel normalizing agent, with an enzyme responsive size-changeable gold nanoparticle (AuNPs-A&C) was developed. In vivo photoacoustic and fluorescence imaging showed that oral pretreatment with 6 mg/kg/day of cediranib for two consecutive days significantly enhanced the retention of AuNPs-A&C in 4T1 tumor. In vivo photoacoustic imaging for hemoglobin (Hb) and oxyhemoglobin (HbO2), Evans blue assay, and immunofluorescence assay showed that cediranib pretreatment markedly increased tumor vascular permeability and tumor oxygenation, while distinctly decreased the tumor microvessel density, demonstrating normalized tumor vessels and favorably altered microenvironment. Additionally, the combination strategy considerably elevated the tumor targeting capacity of different nanoparticle formulations (AuNPs-PEG, AuNPs-A&C), while coadministration of cediranib and AuNPs-A&C achieved prevailing tumor targeting and antitumor efficacy in 4T1 tumor bearing mouse model. In conclusion, we report a novel combined administration strategy to further improve tumor diagnosis and treatment.Keywords: 4T1 tumor; cediranib; drug delivery; gold nanoparticles; vessel normalization;

Glioblastoma (GBM), one of the most lethal cancers, remains as a hard task to handle. The major hurdle of nanostructured therapeutic agents comes from the limited retention at the GBM site and poor selectivity. In this study, we reported dual-functional gold nanoparticles (AuNPs) to figure out the biological barrier and improve their accumulation in GBM. The nanoparticles, AuNP-A&C-R, were composed of two functional particles: one was Ala-Ala-Asn-Cys-Asp (AK) and R8-RGD-comodified AuNPs (AuNP-AK-R) and the other was 2-cyano-6-amino-benzothiazole and R8-RGD-comodified AuNPs (AuNP-CABT-R). AuNP-A&C-R could aggregate in the presence of legumain, resulting in a size increase from 41.4 ± 0.6 to 172.9 ± 10.2 nm after 8 h incubation. After entering the circulatory system, AuNP-A&C-R actively targeted the integrin αvβ3 receptor on blood–brain barrier (BBB), mediated transcytosis of particles across BBB, and then targeted the receptor on the GBM cells. Once AuNP-A&C-R entered into GBM, they formed further aggregates with increased size extracellularly or intracellularly because of the overexpressed legumain, which in turn blocked their backflow to the bloodstream or limited their exocytosis by cells. In vivo optical imaging demonstrated that AuNP-A&C-R were efficiently delivered to the GBM site and retained with high selectivity. We further confirmed that AuNP-A&C-R acquired a higher accumulation at the GBM site than AuNP-A&C and AuNP-R because of the synergistic effect. More importantly, the doxorubicin (DOX)-loaded AuNP-A&C-R showed an improved chemotherapeutic effect to C6 GBM-bearing mice, which significantly prolonged the median survival time by 1.22-fold and 1.27-fold compared with the DOX-loaded AuNP-A&C and the DOX-loaded AuNP-R, respectively. These results suggested that the dual-functional nanoplatform is promising for the GBM treatment.Keywords: click cycloaddition; enhanced accumulation; gold nanoparticles; legumain; R8-RGD;

Brain tumor remains one of the most serious threats to human beings. Different from peripheral tumors, drug delivery to brain tumor is largely restricted by the blood brain barrier (BBB). To fully conquer this barrier and specifically deliver drugs to brain tumor, dual targeting delivery systems were explored, which are functionalized with two active targeting ligands: one to the BBB and the other to the brain tumor. The development of dual targeting delivery system is still in its early stage, and attentions need to be paid to issues and concerns that remain unresolved in future studies.

Tumor microenvironment (TME) plays a critical role in tumorigenesis, tumor invasion and metastasis. TME is composed of stroma, endothelial cells, pericytes, fibroblasts, smooth muscle cells, and immune cells, which is characterized by hypoxia, acidosis, and high interstitial fluid pressure. Due to the important role of TME, we firstly reviewed the composition of TME and discussed the impact of TME on tumor progression, drug and nanoparticle delivery. Next, we reviewed current strategies developed to modulate TME, including modulating tumor vasculature permeability, tumor associated macrophage phenotypes, tumor associated fibroblasts, tumor stroma components, tumor hypoxia, and multiple interventions simultaneously. Also, potential problems and future directions of TME modulation strategy have been discussed.

The treatment of brain tumors remains a challenge due to the limited accumulation of drugs and nanoparticles. Here, we triggered the aggregation of gold nanoparticles (AuNPs) using legumain to enhance the retention of chemotherapeutics in brain tumors. This nanoplatform, AuNPs-A&C, is comprised of Ala-Ala-Asn-Cys-Lys modified AuNPs (AuNPs-AK) and 2-cyano-6-aminobenzothiazole modified AuNPs (AuNPs-CABT). AuNPs-AK could be hydrolyzed to expose the 1,2-thiolamino groups on AuNPs-AK in the presence of legumain, which occurs by a click cycloaddition with the contiguous cyano group on AuNPs-CABT, resulting in formation of AuNPs aggregates. This strategy led to an enhanced retention of the AuNPs in glioma cells both in vitro and in vivo due to the blocking of nanoparticle exocytosis and minimizing nanoparticle backflow to the bloodstream. After conjugation of doxorubicin (DOX) via a pH-sensitive linker to AuNPs-A&C, the efficiency for treating glioma was improved. The median survival time for the DOX-linked AuNPs-A&C increased to 288% in comparison to the saline group. We further show the use of the AuNPs-A&C for optical imaging applications. In conclusion, we provide a strategy to increase nanoparticle tumor accumulation with the potential to improve therapeutic outcome.Keywords: brain tumors; click cycloaddition; gold nanoparticles; legumain; tumor microenvironment

To improve glioma targeting delivery efficiency and to monitor drug delivery and treatment outcome, a novel tumor microenvironment sensitive size-shrinkable theranostic system was constructed and evaluated. The G-AuNPs-DC-RRGD system was constructed by fabricating small sized gold nanoparticles (AuNPs) onto matrix metalloproteinase-2 (MMP-2) degradable gelatin nanoparticles (GNPs), doxorubicin (DOX) and Cy5.5 were decorated onto AuNPs through a hydrazone bond to enable the system with pH triggered cargoes release, and RRGD, a tandem peptide of RGD and octarginine was surface-modified onto the system to enable it with glioma active targeting ability. In vitro, the size of G-AuNPs-DC-RRGD could effectively shrink from 188.2 nm to 55.9 nm after incubation with MMP-2, while DOX and Cy5.5 were released in a pH dependent manner. Cellular uptake demonstrated that G-AuNPs-DC-RRGD could be effectively taken up by cells with higher intensity than G-AuNPs-DC-PEG. A study of tumor spheroids further demonstrated that the particles with smaller size showed better penetration ability, while RRGD modification could further improve permeability. In vivo, G-AuNPs-DC-RRGD displayed the best glioma targeting and accumulation efficiency, with good colocalization with neovessels. Cy5.5 also was colocalized well with DOX, indicating that Cy5.5 could be used for imaging of DOX delivery.

Despite the great achievements that nanomedicines have obtained so far, deep penetration of nanomedicines into tumors is still a major challenge in tumor treatment. The enhanced permeability and retention (EPR) effect was the main theoretical foundation for using nanomedicines to treat solid tumor. However, the antitumor efficiency is modest because the tumor is heterogeneous, with dense collagen matrix, abnormal tumor vasculature, and lymphatic system. Nanomedicines could only passively accumulate near leaky site of tumor vessels, and they cannot reach the deep region of tumor. To enhance further the tumor penetration efficiency, we developed a novel strategy of coadministering cell-homing penetration peptide iRGD with size-shrinkable and tumor-microenvironment-responsive multistage system (DOX-AuNPs-GNPs) to overcome these barriers. First, iRGD could specifically increase the permeability of tumor vascular and tumor tissue, leading to more DOX-AuNPs-GNPs leaking out from tumor vasculature. Second, the multistage system passively accumulated in tumor tissue and shrank from 131.1 to 46.6 nm to reach the deep region of tumor. In vitro, coadministering iRGD with DOX-AuNPs-GNPs showed higher cellular uptake and apoptosis ratio. In vivo, coadministering iRGD with DOX-AuNPs-GNPs presented higher penetration and accumulation in tumor than giving DOX-AuNPs-GNPs alone, leading to the best antitumor efficiency in 4T1 tumor-bearing mouse model.Keywords: deep penetration; iRGD; multistage; size-shrinkable; tumor microenvironment sensitive

Fluorescent carbonaceous nanodots (CDs) are suitable for biomedical applications owing to their excellent photoluminescence properties. However, their toxicity has not been sufficiently evaluated. In this study, we demonstrated that both CDs and PEGylated CDs (PEG-CDs) displayed low cytotoxicity to several cell lines with a concentration as high as 1 mg mL−1, and that autophagy might be involved in the cytotoxicity. In vivo, continuous administration of CDs or PEG-CDs showed no significant alteration on the hematology, blood biochemistry indexes and cell morphology of the main organs. In a zebrafish embryo model, CDs led to a considerably higher mortality and abnormality frequency than PEG-CDs, which demonstrated that PEGylation could decrease the toxicity of CDs. In conclusion, CDs displayed low systemic toxicity but considerable developmental toxicity, and PEGylation could reduce the toxicity, while autophagy may be involved in the toxic mechanisms of the CDs and PEG-CDs. Thus, our studies were exceedingly encouraging and provided some possibility for the clinical application of CDs, while developmental toxicity should be paid much more attention when evaluating the toxicity of nanomaterials.

Fluorescent carbonaceous dots (CDs) have attracted much attention due to their unique properties. However, their application in non-invasive imaging of diseased tissues was restricted by the short excitation/emission wavelength and the poor targeting efficiency of CDs. In this study, CDs were prepared from sucrose and glutamic acid with a particle size of 57.5 nm. An obvious emission could be observed at 600 nm to 700 nm when excited at around 500 nm. This property enabled CDs with a capacity for deep tissue imaging with low background adsorption. RGD, a ligand which could target most tumor and neovasculature cells, was anchored onto CDs after PEGylation. The product, RGD–PEG–CDs could accumulate in MCF-7/ADR xenografts at high intensity, which was 1.65-fold higher than that of PEG–CDs. Furthermore, RGD–PEG–CDs showed good colocalization with neovasculature. Thus, RGD–PEG–CDs could be used for non-invasive MCF-7/ADR tumor imaging. CDs functionalized with other ligands may also be used as a non-invasive probe for many kinds of tumor imaging.

Targeting delivery and deep penetration have been attracting tremendous attention in triple-negative breast cancer (TNBC) theranostics. Herein, we reported a novel multistage system (G-AuNPs-DOX-RRGD) with an active targeting effect and size-changeable property to inhibit tumor growth and metastasis in 4T1 xenograft bearing mice. The system was constructed through fabricating small-size gold nanoparticles (AuNPs) onto matrix metalloproteinase-2 (MMP-2) degradable gelatin nanoparticles (GNPs). Doxorubicin (DOX) was tethered onto AuNPs via a pH sensitive hydrazone bond, and RRGD, a tandem peptide of RGD and octarginine, was surface-decorated onto the system to improve its tumor targeting efficiency. In vitro, the G-AuNPs-DOX-RRGD could shrink from 185.9 nm to 71.2 nm after 24 h incubation with MMP-2 and the DOX was released in a pH-dependent manner. Tumor spheroid penetration and collagen diffusion demonstrated G-AuNPs-DOX-RRGD possessed best penetrating efficiency. In vivo, the G-AuNPs-DOX-RRGD actively targeted to the 4T1 tumor and then penetrated through the interstitial matrix, resulted in enhanced accumulation in the deep tumor region. Therefore, the G-AuNPs-DOX-RRGD could approach excellent anti-tumor capacity owing to the synergistic effect of RRGD and the size-changeable property.

Angiopep-2 modified and doxorubicin loaded carbonaceous nanodots (AN-PEG-DOX-CDs) were synthesized for glioma cell targeting, delivery and redox-responsive release of doxorubicin. Our results provided the possibility of using CDs to construct smart drug delivery systems.

A multistage drug delivery system was designed, which showed MMP-2 sensitive shrinkage and enhanced penetration properties.

A new type of carbon dots (CD-Asp) with targeting function toward brain cancer glioma was synthesized via a straightforward pyrolysis route by using d-glucose and l-aspartic acid as starting materials. The as-prepared CD-Asp exhibits not only excellent biocompatibility and tunable full-color emission, but also significant capability of targeting C6 glioma cells without the aid of any extra targeting molecules. In vivo fluorescence images showed high-contrast biodistribution of CD-Asp 15 min after tail vein injection. A much stronger fluorescent signal was detected in the glioma site than that in normal brain, indicating their ability to freely penetrate the blood–brain barrier and precisely targeting glioma tissue. However, its counterparts, the CDs synthesized from d-glucose (CD-G), l-asparic acid (CD-A), or d-glucose and l-glutamic acid (CD-Glu) have no or low selectivity for glioma. Therefore, CD-Asp could act as a fluorescence imaging and targeting agent for noninvasive glioma diagnosis. This work highlights the potential application of CDs for constructing an intelligent nanomedicine with integration of diagnostic, targeting, and therapeutic functions.Keywords: brain cancer glioma; carbon dots; diagnosis; full-color emission; targeted imaging;

Fluorescent carbon dots (CD) possess impressive potential in bioimaging because of their low photobleaching, absence of optical blinking and good biocompatibility. However, their relatively short excitation/emission wavelengths restrict their application in in vivo imaging. In the present study, a kind of CD was prepared by a simple heat treatment method using glycine as the only precursor. The diameter of CD was lower than 5 nm, and the highest emission wavelength was 500 nm. However, at 600 nm, there was still a relatively strong fluorescent emission, suggesting CD could be used for in vivo imaging. Additionally, several experiments demonstrated that CD possessed good serum stability and low cytotoxicity. In vitro, CD could be taken up into C6 glioma cells in a time- and concentration-dependent manner, with both endosomes and mitochondria involved. In vivo, CD could be used for non-invasive glioma imaging because of its high accumulation in the glioma site of the brain, which was demonstrated by both in vivo imaging and ex vivo tissue imaging. Furthermore, the fluorescent distribution in tissue slices also showed CD distributed in glioma with high intensity, while with a low intensity in normal brain tissue. In conclusion, CD were prepared using a simple method with relatively long excitation and emission wavelengths and could be used for non-invasive glioma imaging.

Fluorescent carbon nanoparticles (CNP) have gained much attention due to their unique fluorescent properties and safety. In this study, we evaluated the potential application of CNP and PEGylated CNP (PEG-CNP) in noninvasive heart imaging. CNP was prepared by hydrothermal treatment of silk. The particle size and zeta potential of CNP were 121.8 nm and −3.7 mV, respectively, which did not change significantly after PEGylation with a PEG density of 4.43 ± 0.02 μg/mg CNP. FTIR and XPS showed that CNP possessed several functional groups, such as −COOH, −OH, and NH2, which could be utilized for PEGylation and other modifications. CNP displayed strong blue fluorescence after excitation at the wavelength of 375 nm. PEG-CNP displayed better serum stability compared to CNP. The hemolysis rate of PEG-CNP was lower than that of CNP, suggesting PEGylation could enhance the hemocompatibility of CNP. Both CNP and PEG-CNP showed higher uptake capacity by H9c2 cells (a heart cell line) than that by human umbilical vein endothelial cells (HUVEC), suggesting the particles tend to be selectively taken up by heart cells. Both CNP and PEG-CNP were proven to be taken up through endosome-mediated pathway, and the colocalization of nanoparticles with mitochondria was also observed. In vivo results demonstrated that CNP could target heart with much higher fluorescent intensity than liver and spleen. Although PEGylation could decrease the distribution in heart, it remained high for PEG-CNP. In conclusion, CNP could be used for heart imaging, and moreover, PEGylation could improve the stability and biocompatibility of CNP.

Fluorescent carbonaceous nanodots (CDs) have attracted much attention due to their unique properties. However, their application in noninvasive imaging of diseased tissues was restricted by the short excitation/emission wavelengths and the low diseased tissue accumulation efficiency. In this study, CDs were prepared from glucose and glutamic acid with a particle size of 4 nm. Obvious emission could be observed at 600 to 700 nm when CDs were excited at around 500 nm. This property enabled CDs with capacity for deep tissue imaging with low background adsorption. Angiopep-2, a ligand which could target glioma cells, was anchored onto CDs after PEGylation. The product, An-PEG-CDs, could target C6 glioma cells with higher intensity than PEGylated CDs (PEG-CDs), and endosomes were involved in the uptake process. In vivo, An-PEG-CDs could accumulate in the glioma site at higher intensity, as the glioma/normal brain ratio for An-PEG-CDs was 1.73. The targeting effect of An-PEG-CDs was further demonstrated by receptor staining, which showed An-PEG-CDs colocalized well with the receptors expressed in glioma. In conclusion, An-PEG-CDs could be successfully used for noninvasive glioma imaging.

•A simple one step method was developed to prepare CNP from biomaterials.•Cell uptake of CNP is a concentration-dependent manner.•CNP showed poor colocalization with endosomes and mitochondria.•CNP could adsorb serum protein.•CNP showed well hemocompatibility and low cytotoxicity.Highly fluorescent carbon nanospheres with a quantum yield of 17.6% have been prepared by a one-step method with hydrothermal treatment of spider silk. Due to the high photostability, low toxicity and well blood compatibility, these carbon nanospheres could be used as an excellent probes for cancer cell imaging.

•CNP possessed low cytotoxicity and well hemocompatibility.•CNP was taken up into A549 cells mainly through clathrin-mediated endocytosis.•CNP distributed highest in heart than in other tissues.Carbon nanospheres (CNP) possess several unique properties that render CNP superior to traditional organic dyes and quantum dots in the biological application. However, the interaction of CNP with biological systems was far from well-known. In this study, a simple method using cocoon silk was used to synthesize photoluminescent CNP. The particle size of CNP was 100.6 nm with well dispersity. The excitation/emission wavelength was 340 nm and 442 nm. Cellular uptake demonstrated the uptake of CNP by A549 cells was a time-, concentration- and energy-dependent procedure. Endosome was involved in the uptake rather than mitochondria. Through several uptake inhibitors, it showed the uptake was energy-dependent and mainly mediated by clathrin-mediated endocytosis. In vivo, CNP were mainly distributed in heart and lung, while only a modest amount of CNP was distributed in spleen, liver and kidney. The distribution in tumor was relatively low, which made CNP a candidate for heart cell imaging. At as high as 2 mg/mL, CNP showed no obvious toxicity to cells. The hemolysis rate of CNP was also lower than 10%. These results suggested CNP was relatively safe in biological application.

•A new kind of fluorescent carbonaceous nanospheres (CDs) was prepared using glutamic acid and glucose as the precursors.•The prepared CDs possessed excellent optical properties and good biocompatibility.•CDs were used as biological probe for cell imaging in vitro.•We report the application of CDs in non-invasive brain imaging.Fluorescent carbonaceous nanospheres (CDs) have generated much excitement in bioimaging because of their impressive fluorescent properties and good biocompatibility. In this study, we evaluated the potential application of CDs in noninvasive brain imaging. A new kind of CDs was prepared by a heat treating method using glutamic acid and glucose as the precursors. The hydrated diameter and zeta potential of CDs were 101.1 nm (PDI = 0.110) and −22.4 mV respectively. Palpable emission spectrum could be observed from 400 nm to 600 nm when excited at corresponding wavelength, suggesting CDs could be used as a noninvasive bio-probe for in vivo imaging. Additionally, several experiments indicated that CDs possess good serum stability and hemocompatibility with low cytotoxicity. In vitro, the CDs could be efficiently taken up by bEnd.3 cells in a concentration- and time-dependent manner. In vivo, CDs could be used for noninvasive brain imaging due to its high accumulation in brain region, which was demonstrated by in vivo imaging and ex vivo tissue imaging. Moreover, the fluorescent distribution in tissue slice showed CDs accumulated in brain with high intensity. In conclusion, CDs were prepared using a simple one-step method with unique optical and good biological properties and could be used for noninvasive brain imaging.

Carbonaceous dots exhibit increasing applications in diagnosis and drug delivery due to excellent photostability and biocompatibility properties. However, relative short excitation and emission of melanin carbonaceous dots (MCDs) limit the applicability in fluorescence bioimaging. Furthermore, the generally poor spatial resolution of fluorescence imaging limits potential in vivo applications. Due to a variety of beneficial properties, in this study, MCDs were prepared exhibiting great potential in fluorescence and photoacoustic dual-mode bioimaging. The MCDs exhibited a long excitation peak at 615 nm and emission peak at 650 nm, further highlighting the applicability in fluorescence imaging, while the absorbance peak at 633 nm renders MCDs suitable for photoacoustic imaging. In vivo, the photoacoustic signal of MCDs was linearly correlated with the concentration of MCDs. Moreover, the MCDs were shown to be taken up into triple negative breast cancer cell line 4T1 in both a time- and concentration-dependent manner. In vivo fluorescence and photoacoustic imaging of subcutaneous 4T1 tumor demonstrated that MCDs could passively target triple negative breast cancer tissue by enhanced permeability and retention effects and may therefore be used for tumor dual-mode imaging. Furthermore, fluorescence distribution in tissue slices suggested that MCDs may distribute in 4T1 tumor with high efficacy. In conclusion, the MCDs studied offer potential application in fluorescence and photoacoustic dual-mode imaging.