Cell Uptake Assays Research Articles

TAT&RGD Peptide-Modified Naringin-Loaded Lipid Nanoparticles Promote the Osteogenic Differentiation of Human Dental Pulp Stem Cells.

BackgroundNaringin is a naturally occurring flavanone that promotes osteogenesis. Owing to the high lipophilicity, poor in vivo bioavailability, and extensive metabolic alteration upon administration, the clinical efficacy of naringin is understudied. Additionally, information on the molecular mechanism by which it promotes osteogenesis is limited.MethodsIn this study, we prepared TAT & RGD peptide-modified naringin-loaded nanoparticles (TAT-RGD-NAR-NPs), evaluated their potency on the osteogenic differentiation of human dental pulp stem cells (hDPSCs), and studied its mechanism of action through metabolomic analysis.ResultsThe particle size and zeta potential of TAT-RGD-NAR-NPs were 160.70±2.05 mm and –20.77±0.47mV, respectively. The result of cell uptake assay showed that TAT-RGD-NAR-NPs could effectively enter hDPSCs. TAT-RGD-NAR-NPs had a more significant effect on cell proliferation and osteogenic differentiation promotion. Furthermore, in metabolomic analysis, naringin particles showed a strong influence on the glycerophospholipid metabolism pathway of hDPSCs. Specifically, it upregulated the expression of PLA2G3 and PLA2G1B (two isozymes of phospholipase A2, PLA2), increased the biosynthesis of lysophosphatidic acid (LPA).ConclusionThese results suggested that TAT-RGD-NPs might be used for transporting naringin to hDPSCs for modulating stem cell osteogenic differentiation. The metabolomic analysis was used for the first time to elucidate the mechanism by which naringin promotes hDPSCs osteogenesis by upregulating PLA2G3 and PLA2G1B.

Abstract 2477: PD-L1 PET quantifies the pharmacodynamic effects of immunotherapy

Abstract Introduction: Immune checkpoint protein targeted therapeutics have transformed cancer treatment but only a subset of patients derive benefit. To improve efficacy, a variety of combination therapies are being studied. Programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) is used as a biomarker to guide many of those therapies. Non-invasive evaluation of therapy induced changes in PD-L1 levels in the whole body could provide insights to guide those therapies. Here, we investigated the potential of [18F]DK222, a novel PD-L1 binding positron emission tomography (PET) imaging agent, to quantify PD-L1 levels and pharmacodynamics in humanized mouse modes. Methods: Non-small cell lung cancer (NSCLC), urothelial carcinoma (UC) and melanoma cell lines were screened by flow cytometry (FC) for PD-L1 expression and density. In vitro cell uptake assays, in vivo PET imaging and ex vivo biodistribution studies were used to evaluate the specificity of [18F]DK222 to bind PD-L1 (n=5-6/tumor). To quantify immunotherapy induced changes in PD-L1, A375 melanoma xenografts were inoculated in NSG mice humanized with CD34+ cells. Mice were treated with either 5 mg/kg anti- PD-1; (pembrolizumab) or 5 mg/kg of anti-PD-1 + 4 mg/kg of anti- CTLA-4 (ipilimumab) (n=5) and [18F]DK222 PET was acquired before and 7 days after start of treatment. Tumor growth was monitored. FC of tumors was performed to measure changes in TME and immune cell infiltrates. Results: In vitro uptake of [18F]DK222 in NSCLC (H244 > H226 > A549) and UC (BFTC909 > T24 > SCaBER) correlated with PD-L1 density (R2=0.96). [18F]DK222 PET and biodistribution studies in mice with NSCLC and UC xenografts revealed high and variable tumor uptake at 60 min, which was in agreement with PD-L1 levels detected by IHC. These data established sensitivity of [18F]DK222 PET to quantify variable PD-L1 levels in vivo. We next sought to quantify therapy induced changes in PD-L1 levels. We observed higher [18F]DK222 uptake in tumors of mice treated with combination therapy compared to monotherapy (1.83 vs 0.47 %ID/cc). A significant delay in tumor growth was observed with combination therapy compared to monotherapy. Two out of five mice with highest increase in [18F]DK222 uptake in combination therapy group exhibited least tumor growth. Also, final tumor volumes negatively correlated with [18F]DK222 uptake (R2=0.33) irrespective of treatment regimen. FC analysis showed higher immune infiltrates (1320 vs 230 per milligram of tumor), PD-L1+ cells (6005 vs 2739/mg) and ratio of CD8+ to CD4+ cells (1.94 vs 0.47) in tumors treated with combination therapy compared with monotherapy. These observations confirmed that immunotherapy induced changes in PD-L1 levels in tumors can be quantified using [18F]DK222 PET. Conclusion: [18F]DK222 PET can effectively quantify variable PD-L1 levels in tumors. Moreover, [18F]DK222 PET exhibits the sensitivity to quantify PD-L1 pharmacodynamics to inform on tumor response to therapy. Citation Format: Akhilesh Mishra, Dhiraj Kumar, Kuldeep Gupta, Patrick M. Forde, Edward Gabrielson, Sridhar Nimmagadda. PD-L1 PET quantifies the pharmacodynamic effects of immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2477.

68Ga-Labeled Cystine Knot Peptide Targeting Integrin αvβ6 for Lung Cancer PET Imaging.

Integrin αvβ6 has been considered as a promising biomarker for lung cancer, and its expression is often related to poor prognosis. An αvβ6-binding cystine knot peptide R01-MG was previously engineered and validated. Here, we developed a positron emission tomography (PET) probe of R01-MG for imaging αvβ6-positive lung cancer. Cystine knot peptide R01-MG was synthesized through solid-phase peptide synthesis chemistry and radiolabeled with 68Ga after being conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA). The stability of 68Ga-DOTA-R01-MG was analyzed in phosphate-buffered saline (PBS) (pH 7.4) and fetal bovine serum (FBS). The cell uptake assay of the probe was evaluated using αvβ6-positive (A549 and H1975) and αvβ6-negative (H1299) lung cancer cell lines. In addition, small animal PET imaging and biodistribution studies of 68Ga-DOTA-R01-MG were performed in αvβ6-positive and αvβ6-negative lung cancer models. Our study showed that 68Ga-DOTA-R01-MG exhibited excellent stability in PBS and FBS. Small animal PET imaging and biodistribution data revealed that 68Ga-DOTA-R01-MG displayed rapid and good tumor uptake in animal models with αvβ6-positive lung cancer, and the probe was rapidly cleared from the normal tissues, resulting in good tumor-to-normal tissue contrasts. Meanwhile, no obvious tumor uptake of 68Ga-DOTA-R01-MG was observed in animal models with αvβ6-negative lung cancer, demonstrating specific binding of the probe to integrin αvβ6. In conclusion, 68Ga-DOTA-R01-MG has great potential to be a promising PET tracer for imaging αvβ6-positive lung cancer.

Nectin-4-targeted immunoSPECT/CT imaging and photothermal therapy of triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is more prone to distant metastasis and visceral recurrence in comparison to other breast cancer subtypes, and is related to dismal prognosis. Nevertheless, TNBC has an undesirable response to targeted therapies. Therefore, to tackle the huge challenges in the diagnosis and treatment of TNBC, Nectin-4 was selected as a theranostic target because it was recently found to be highly expressed in TNBC. We developed anti-Nectin-4 monoclonal antibody (mAbNectin-4)-based theranostic pair, 99mTc-HYNIC-mAbNectin-4 and mAbNectin-4-ICG. 99mTc-HYNIC-mAbNectin-4 was applied to conduct immuno-single photon emission computed tomography (SPECT) for TNBC diagnosis and classification, and mAbNectin-4-ICG to mediate photothermal therapy (PTT) for relieving TNBC tumor growth.MethodsNectin-4 expression levels of breast cancer cells (MDA-MB-468: TNBC cells; and MCF-7, non-TNBC cells) were proved by western blot, flow cytometry, and immunofluorescence imagning. Cell uptake assays, SPECT imaging, and biodistribution were performed to evaluate Nectin-4 targeting of 99mTc-HYNIC-mAbNectin-4. A photothermal agent (PTA) mAbNectin-4-ICG was generated and characterized. In vitro photothermal therapy (PTT) mediated by mAbNectin-4-ICG was conducted under an 808 nm laser. Fluorescence (FL) imaging was performed for mAbNectin-4-ICG mapping in vivo. In vivo PTT treatment effects on TNBC tumors and corresponding systematic toxicity were evaluated.ResultsNectin-4 is overexpressed in MDA-MB-468 TNBC cells, which could specifically uptake 99mTc-HYNIC-mAbNectin-4 with high targeting in vitro. The corresponding immunoSPECT imaging demonstrated exceptional performance in TNBC diagnosis and molecular classification. mAbNectin-4-ICG exhibited favourable biocompatibility, photothermal effects, and Nectin-4 targeting. FL imaging mapped biodistribution of mAbNectin-4-ICG with excellent tumor-targeting and retention in vivo. Moreover, mAbNectin-4-ICG-mediated PTT provided advanced TNBC tumor destruction efficiency with low systematic toxicity.ConclusionmAbNectin-4-based radioimmunoimaging provides visualization tools for the stratification and diagnosis for TNBC, and the corresponding mAbNectin-4-mediated PTT shows a powerful anti-tumor effect. Our findings demonstrate that this Nectin-4 targeting strategy offers a simple theranostic platform for TNBC.

POLYETHYLENE GLYCOL-CHITOSAN NANOPARTICLE GEL FOR THE CONTROLLED NASAL DELIVERY OF NOSCAPINE AGAINST GLIOMA

Objective: To synthesize the PEG surface modulated Chitosan Nanoparticles aiding in Pluronic intranasal gel (PEG-CHT-PL) encapsulating cytotoxic noscapine for enhanced anticancer effect against glioma. Methods: The PEG-CHT-PL was synthesized by inotropic gelation method and evaluated for in vitro characterization parameters such as zeta sizer, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), zeta potential followed by gelling time, Thixotropy and flow index evaluation. The cell uptake assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) evaluation and apoptosis was evaluated on human Glioblastoma U-87 cell line post evaluation by evaluating drug release pattern at 7.4 pH by PBS buffer for stable intranasal brain delivery. Results: The synthesized PEG-CHT-PL showed nanosize range of 110 nm and a smooth spherical shape with a negative zeta potential of27±1.9 mV. The gel showed stable rheology exhibiting negligible normal deviation in viscosity, Thixotropy and flow index on long-term storage. The drug release pattern followed the Higuchi’s model in 48 h at 7.4pH showed sustained noscapine discharge. The in vitro Glioblastoma U-87 cell line studies showed enhanced cell uptake and distribution, with notable cell toxicity by MTT and apoptosis evaluation confirming significant antitumor efficiency via intranasal delivery. Conclusion: The present research has showed promising operational intranasal therapy against brain tumor crossing BBB efficiently and can be subjected for an effective antitumor approach in clinical platform in future drug delivery system.

Synthesis, characterization, molecular docking, antimalarial, and antiproliferative activities of benzyloxy-4-oxopyridin benzoate derivatives.

Background and purpose:Malaria and cancer are two major health issues affecting millions of lives annually. Maltol complexes and derivatives have been extensively investigated as chemotherapeutic and antimalarial activities. In this study, the design, synthesis, biological activities, and docking study of a novel series of pyridinones derivatives were reported.Experimental approach:The chemical structures of synthesized compounds were approved by FTIR, 1HNMR, 13CNMR, and mass spectroscopies. The antimalarial activity was evaluated through β-hematin inhibition assay and the cytotoxicity activities were evaluated against PC12 and fibroblast cell lines via MTT and cell uptake assays. To theoretically investigate the ability of compounds to inhibit hemozoin formation, the synthesized compounds were docked in a heme sheet to explore their binding mode and possible interactions.Findings/Results:β-Hematin inhibition assay showed acceptable activity for 7f, 7c, and 7d compounds and the molecular docking study showed 7h and 7f had effective interactions with the heme sheet. The cytotoxic study revealed compound 4b (IC50 = 18 μM) was significantly more active against PC12 cells than docetaxel (IC50 = 280 μM). The observations of cell uptake images were also shown both cell penetration and monitoring potential of synthesized compounds.Conclusion and implications:The compounds showed a moderate ability to inhibition of heme polymerization and also good interaction with heme through molecular docking was observed. Additionally, some of them have a good cytotoxic effect on the study2 cell line. So further study on these compounds can lead to compounds that can be considered as anti-malarial and/or anticancer agents.

Circumsporozoite Protein of Plasmodium berghei- and George Baker Virus A-Derived Peptides Trigger Efficient Cell Internalization of Bioconjugates and Functionalized Poly(ethylene glycol)-b-poly(benzyl malate)-Based Nanoparticles in Human Hepatoma Cells.

In order to identify the peptides, selected from the literature, that exhibit the strongest tropism towards human hepatoma cells, cell uptake assays were performed using biotinylated synthetic peptides bound to fluorescent streptavidin or engrafted onto nanoparticles (NPs), prepared from biotin-poly(ethylene glycol)-block-poly(benzyl malate) (Biot-PEG-b-PMLABe) via streptavidin bridging. Two peptides, derived from the circumsporozoite protein of Plasmodium berghei- (CPB) and George Baker (GB) Virus A (GBVA10-9), strongly enhanced the endocytosis of both streptavidin conjugates and NPs in hepatoma cells, compared to primary human hepatocytes and non-hepatic cells. Unexpectedly, the uptake of CPB- and GBVA10-9 functionalized PEG-b-PMLABe-based NPs by hepatoma cells involved, at least in part, the peptide binding to apolipoproteins, which would promote NP’s interactions with cell membrane receptors of HDL particles. In addition, CPB and GBVA10-9 peptide–streptavidin conjugates favored the uptake by hepatoma cells over that of the human macrophages, known to strongly internalize nanoparticles by phagocytosis. These two peptides are promising candidate ligands for targeting hepatocellular carcinomas.

Synthesis and biological evaluation of a <sup>99m</sup>Tc-labeled glucose derivative for tumor imaging

<p indent="0mm">The development of novel tumor-imaging agents for single-photon-emission-computed tomography (SPECT) with good performance, simple preparation and low price is a hot topic in the field of radiopharmaceutical research. ^99mTc is the most widely used SPECT nuclide in clinic. In order to develop a novel ^99mTc-labeled tumor radiopharmaceuticals, in this study, a glucose derivative (CN6DG) containing isonitriles was synthesized and it was radiolabeled with ^99mTc to obtain the ^99mTc-CN6DG complex with high radiochemical purity (&gt;95%). The complex is hydrophilic (log<italic>D</italic>=−2.52±0.33) and has good stability <italic>in vitro </italic>and<italic> in vivo</italic>. <italic>In vitro</italic> cell-uptake assay showed that tumor cells uptake of the complex was related to glucose transporters. In S180 tumor-bearing mice <sc>2 h</sc> after injection, the tumor-uptake value was 0.75±0.12 %ID/g and the tumor/muscle, tumor/blood ratios were 4.41 and 18.75, respectively. In A549 tumor-bearing mice after <sc>2 h</sc> of post-injection, the tumor-uptake value was 2.62±0.23 %ID/g, which was higher than that of ^99mTc-CN5DG (1.48±0.23 %ID/g). Its tumor/blood (52.4 <italic>vs</italic>. 49.33) ratios and tumor/muscle ratios (6.89 <italic>vs</italic>. 24.67) were as good as that of ^99mTc-CN5DG. The SPECT/CT imaging results in S180 and A549 tumor-bearing mice showed that it had significant tumor uptakes, indicating that it is a promising tumor-imaging agent.

99mTc-labeled peptide targeting interleukin 13 receptor α 2 for tumor imaging in a cervical cancer mouse model.

Pep-1 (CGEMGWVRC) can potently bind to interleukin 13 receptor α 2 (IL-13Rα2), a tumor-restricted receptor found to be expressed in various malignancies. In this study, we intended to prepare a 99mTc-labeled probe and evaluate its in vivo tumor accumulation properties in a cervical cancer xenograft model. The Pep-1 was designed and radiolabeled with 99mTc by conjugation with mercaptoacetyl-triglycine (MAG3). The labeling yield, radiochemical purity and stability were characterized in vitro. Cell uptake assays and fluorescence imaging were conducted for qualitative and quantitative evaluation of the specificity and affinity of Pep-1. Flow cytometry and tissue immunofluorescence were used to confirm the IL-13Rα2 expression in cervical cancer. Biodistribution and in vivo imaging were performed periodically to evaluate the imaging value of 99mTc-MAG3-Pep-1 in cervical cancer xenograft model. 99mTc-MAG3-Pep-1 was successfully prepared with a high labeling yield and radiochemical purity (> 95%). Specific cell uptake was demonstrated by scramble control and unlabeled MAG3-Pep-1 blockade. Flow cytometry and tissue immunofluorescence also confirmed the mild IL-13Rα2 expression of HeLa. In the gamma imaging study and biodistribution, the tumors were imaged clearly at 2-6h after injection of 99mTc-MAG3-Pep-1 and the accumulation of 99mTc-MAG3-Pep-1 in tumor was significantly higher than that in the blocking and scramble controls, demonstrating ligand-receptor binding specificity. This work demonstrated that 99mTc-MAG3-Pep-1 can bind to cervical cancer with high affinity and specificity. MAG3-Pep-1 may be a prospective precursor for IL-13Rα2-expressing cancer therapy.

Preliminary evaluation of [11C]MAGL-0519 as a promising PET ligand for the diagnosis of Hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prevalent liver malignancy, which ranks third in the cancer-related cause of deaths in worldwide and ninth in the United States. Currently, HCC is typically diagnosed by ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) scan at its late stage and the survival of HCC patients after diagnosis is usually very poor. Therefore, the development of novel and effective tool for early diagnosis, characterization and staging of HCC patients is of critical importance. Recent studies have demonstrated correlation of HCC with MAGL. In HCC cells, upregulation of MAGL activity enhanced cell invasiveness ability, while pharmacological blockade of MAGL led to significant inhibition of this trend. In this study, we aim to visualize the expression and activity of hepatic MAGL in different HCC cells and HCC patients’ samples by taking advantage of positron emission tomography (PET) imaging with our previously developed MAGL radioligand [11C]MAGL-0519. As a result, [11C]MAGL-0519 exhibited higher radioactivity accumulation in HepaG2 and Hepa 1–6 cell lines compared with that of normal liver cells (AML-12 and LX-2), indicating higher MAGL expression levels in these HCC cells. This rationale was then validated by Western blot and immunofluorescent staining analysis. Furthermore, HCC patients’ liver sections exhibited significantly increased uptake of [11C]MAGL-0519, which was consistent with the results in cell uptake assays. Taking together, these results provided a biological rationale and built a foundation to use [11C]MAGL-0519 as a potential and effective PET ligand for the diagnosis of HCC.

Hyaluronic Acid Modified Curcumin-Loaded Chitosan Nanoparticles Inhibit Chondrocyte Apoptosis to Attenuate Osteoarthritis via Upregulation of Activator Protein 1 and RUNX Family Transcription Factor 2.

Hyaluronic acid (HA) and curcumin (CUR) have been previously utilized for osteoarthritis (OA) treatment. CUR-loaded chitosan nanoparticles (CUR@CS NPs) and HA CUR@CS NPs were synthesized in our research to ascertain the synergistic impacts of HA and CUR-loaded NPs on OA treatment. CUR@CS NPs and HA CUR@CS NPs were synthesized with evaluation of their particle size, potential, PDI, encapsulation efficiency, drug loading and surface coating as well as HA binding rate. The in vitro CUR release curve and stability of HA-CUR@CS NPs were measured. Chondrocytes were isolated from the cartilages of OA patients, followed by cell uptake assay. The chondrocyte viability and apoptosis were determined. Subsequently, the knee OA model was established, followed by H&E, Safranin O/Fast green staining and micro-CT. HA CUR@CS NPs improved CUR stability and bioavailability. CUR@CS NPs and HA-CUR@CS NPs were successfully characterized and could further be internalized by chondrocytes. CUR@CS NPs promoted tBHP-induced chondrocyte viability and inhibited chondrocyte apoptosis. HA-CUR@CS NPs upregulated the AP-1 and RUNX2 transcription levels to activate Hedgehog pathway, which subsequently blocked the Notch pathway. Mechanically, HA-CUR@CS NPs sustained release and long-lasting effect and long-term retention in the joint cavity and downregulated the expression of several pro-inflammatory cytokines in vivo. HA-CUR@CS NPs exhibited superior effects in the preceding experiments than CUR@CS NPs. Altogether, HA-CUR@CS NPs may restrict inflammation and chondrocyte apoptosis in OA through upregulation of AP-1 and RUNX2.

Evans blue-modified radiolabeled fibroblast activation protein inhibitor as long-acting cancer therapeutics.

Rationale: Fibroblast activation protein (FAP) targeted molecular imaging radiotracers have shown promising preclinical and clinical results in tumor diagnosis. However, rapid clearance and inadequate tumor retention of these molecules have hindered them for further clinical translation in cancer therapy. In this study, we aimed to develop a series of albumin binder-truncated Evans blue (EB) modified FAP targeted radiotracers, and optimize the pharmacokinetic (PK) characteristics to overcome the existing limitations in order to apply in the radionuclide therapy of cancer.Methods: A series of compounds with the general structure of EB-FAPI-Bn were synthesized based on a FAP inhibitor (FAPI) variant (FAPI-02) and radiolabeled with 177LuCl3. To verify the binding affinity and FAP targeting specificity of these tracers in vitro, U87MG cell uptake and competition assays were performed. Preclinical PK was evaluated in U87MG tumor-bearing mice using SPECT imaging and biodistribution studies. The lead compound EB-FAPI-B1 was selected and cancer therapeutic efficacy of 177Lu-EB-FAPI-B1 was assessed in U87MG tumor-bearing mice.Results: 177Lu-EB-FAPI-B1, B2, B3, B4 were stable in PBS (pH 7.4) and saline for at least 24 h. EB-FAPI-B1 showed high binding affinity (IC50 = 16.5 nM) to FAP in vitro, which was comparable with that of FAPI-02 (IC50 = 10.9 nM). SPECT imaging and biodistribution studies of 177Lu-EB-FAPI-B1, B2, B3, B4 have proved their prominently improved tumor accumulation and retention at 96 h post-injection, especially for 177Lu-EB-FAPI-B1, high tumor uptake and low background signal make it the optimal compound. Compared to the saline group, noteworthy tumor growth inhibitions of 177Lu-EB-FAPI-B1 have been observed after administration of different dosages.Conclusion: In this study, several EB modified FAPI-02 related radiopharmaceuticals have been synthesized successfully and evaluated. High binding affinity and FAP targeting specificity were identified in vitro and in vivo. Remarkably enhanced tumor uptake and retention of EB-FAPI-B1 were found over the unmodified FAPI-02. 177Lu-EB-FAPI-B1 showed remarkable tumor growth suppression in U87MG tumor model with negligible side effects, indicating that 177Lu-EB-FAPI-B1 is promising for clinical application and transformation.

18F-labeled Dimer-Sansalvamide A Cyclodecapeptide: A Novel Diagnostic Probe to Discriminate Pancreatic Cancer from Inflammation in a Nude Mice Model.

Early detection of pancreatic cancer has been a long-standing challenge. Inflammatory mass is the main source of false-positive findings in 18F-labeled fluorodeoxyglucose (18F-FDG) positron emission tomography / computed tomography (PET/CT). Heat shock protein 90 (Hsp90) is an established biomarker overexpressed in pancreatic cancer. We modified a Dimer-Sansalvamide A cyclodecapeptide by conjugating it with the bifunctional chelator NOTA (1,4,7-triazacyclononane-1,4,7-trisacetic acid), yielding 18F-NOTA-Dimer-Sansalvamide A cyclodecapeptide (18F-NOTA-Dimer-San A). The binding specificity of the probe was confirmed by in vitro cell uptake assays in Hsp90-positive PL45 pancreatic cancer cells. Hsp90 expression was imaged via MicroPET in pancreatic cancer xenografts and inflammation in mice. All of the mice received an intravenous injection of 18F-NOTA-Dimer-San A, and images were acquired at 1 and 2 hour time points. The novel probe demonstrated prominent tumor uptake in the pancreatic cancer xenografts (4.00 ± 0.88 %ID/g, 5.80 ± 0.94 %ID/g), and the inflammatory thigh showed minimal uptake (0.85 ± 0.01 %ID/g, 1.50 ± 0.20 %ID/g) at 1 and 2 hours after injection, respectively. The activity accumulation between the two groups was significantly different (P < 0.05), and the biodistribution data was consistent with the images. Moreover, immunohistochemistry (IHC) confirmed that the expression of Hsp90 was positive in PL45 pancreatic cancer but negative in the muscles next to the tumor and inflammatory muscles. We concluded that 18F-NOTA-Dimer-San A PET might allow non-invasive imaging for Hsp90 expression in tumors and has the potential to discriminate pancreatic cancer from inflammatory mass.

A pH-responsive complex based on supramolecular organic framework for drug-resistant breast cancer therapy

Chemotherapy is one of the main ways to treat breast cancer clinically. However, the multidrug resistance to anti-tumor drugs limits their clinical use. To overcome these drawbacks, development of drug delivery systems (DDSs) has attracted more and more attention in cancer therapy. At present, the preparation and purification process are complicated for many reported DDSs, while clinic calls for new DDSs that are more convenient for preparation. Here, a new pH-responsive supramolecular organic framework drug delivery complex loading doxorubicin (DOX) is fabricated. Anti-tumor activity of the system in vitro was investigated by cell cytotoxicity, uptake assay, and cell apoptosis analysis. The anti-tumor activity in vivo was investigated by inspecting nude mice body weight, tumor volume, and weight, also a preliminary mechanism probe was conducted by HE and TUNEL staining. The DOX@SOF displayed high stability, good biocompatibility, and pH regulated drug release. At acid condition, the hydrazone bonds would be broken, which result in the dissociation of SOF, and then the drugs would be released from the system. Furthermore, DOX@SOF enhanced cellular internalization. Both in vitro and in vivo experiments reflected that DOX@SOF could enhance the anti-tumor activity of DOX for the MCF-7/ADR tumor cells and tumors. This study provides a highly efficient strategy to prepare stimulus-responsive supramolecular drug delivery complex for treatment of drug-resistant cancer, the results presented inspiring scientific interests in exploring new drug delivery strategy and reversing multi-drug resistance for clinical chemotherapy.

A pH-responsive complex based on supramolecular organic framework for drug-resistant breast cancer therapy

Chemotherapy is one of the main ways to treat breast cancer clinically. However, the multidrug resistance to anti-tumor drugs limits their clinical use. To overcome these drawbacks, the development of drug delivery systems (DDSs) has attracted more and more attention in cancer therapy. At present, the preparation and purification process are complicated for many reported DDSs, while the clinic calls for new DDSs that are more convenient for preparation. Here a new pH-responsive supramolecular organic framework drug delivery complex loading doxorubicin (DOX) is fabricated. Anti-tumor activity of the system in vitro was investigated by cell cytotoxicity, uptake assay, and cell apoptosis analysis. The anti-tumor activity in vivo was investigated by inspecting nude mice body weight, tumor volume and weight, also a preliminary mechanism probe was conducted by HE and TUNEL staining. The DOX@SOF displayed high stability, good biocompatibility and pH-regulated drug release. At acid condition, the hydrazone bonds would be broken, which result in the dissociation of SOF, and then the drugs would be released from the system. Furthermore, DOX@SOF enhanced cellular internalization. Both in vitro and in vivo experiments reflected that DOX@SOF could enhance the anti-tumor activity of DOX. for the MCF-7/ADR tumor cells and tumors. This study provides a highly efficient strategy to prepare a stimulus-responsive supramolecular drug delivery complex for the treatment of drug-resistant cancer, the results presented inspiring scientific interests in exploring new drug delivery strategies and reversing multi-drug resistance for clinical chemotherapy.

Initial biological evaluation of a novel microtubule‐based PET ligand in rodent and NHP models of AD

AbstractBackgroundMicrotubule (MT) integrity is critical for cell function and viability. Abnormal MT stability is commonly attributed to hyperphosphorylation of the MT‐associated protein, tau. However, the time course of MT instability in disease progression remains unknown. In vivo MT imaging offers an opportunity to gain critical information on MT changes in relation to staging of ADRDs. Our lab reported the first brain‐penetrant MT PET ligand, [11C]MPC‐6827 and evaluated in vivo imaging in normal rodents and non‐human primates (NHP). Herein we report its initial biological evaluations in (a) cells, (b) tau knock‐out mice, and (c) aged NHPs (Caribbean‐origin vervets) with changes in AD‐related biomarkers.MethodIn vitro [11C]MPC‐6827 cell uptake assay was performed in patient‐derived SH‐SY5Y cells with different MT destabilizing and stabilizing agents. Radioactive binding was determined as %ID(injected dose)/mg of protein present in each well. Dynamic 0‐60 min brain microPET/CT imaging was performed in tau knock‐out and wild‐type mice (n=4). Four vervets (20‐29y) with different cerebrospinal fluid (CSF) Aβ42 levels (2 high levels‐1461, 1614 and 2 low levels‐579,732) underwent 0‐120 min brain PET/CT imaging with [11C]MPC‐6827. ROIs were drawn on the whole‐brain and SUVR values were calculated from co‐registered PET/MR images.ResultIn vitro cell uptake assays with [11C]MPC‐6827 in SH‐SY5Y cells displayed high uptake with MT destabilizing agents and low uptake with stabilizing agents. MicroPET imaging demonstrated higher radioactive brain uptake in tau knockout over wild‐type mice. Vervets with low CSF Aβ42 levels showed high radioactive brain uptake and the youngest vervet (20y) displayed lowest brain uptake.ConclusionConsistent with our in vitro cell uptake and in vivo rodent imaging experiments, [11C]MPC‐6827 increased with MT destabilizations. Radioactive uptake was inversely related to CSF Aβ levels and directly related to age in a NHP model of AD (Fig 1). Collectively our data suggest that [11C]MPC‐6827 is more selective towards destabilized MTs. On‐going experiments include rigorous PET imaging analyses, determination of metabolic plasma‐blood parameters, and correlations with Aβ42 and tau levels. Nonetheless, our promising results support the high translational utility of [11C]MPC‐6827 to image MTs in ADRDs.

Initial biological evaluations of a novel microtubule‐based PET ligand in Alzheimer’s disease

AbstractBackgroundMicrotubule (MT) integrity is critical for cell function and viability. Abnormal MT‐stability in AD is commonly attributed to hyperphosphorylation of the MT‐associated protein, tau. However, the time course of MT instability in disease progression remains unknown. In vivo MT imaging offers an opportunity to gain critical information on MT changes in relation to staging of ADRDs. Our lab reported the first brain‐penetrant MT PET ligand, [11C]MPC‐6827 and evaluated in vivo imaging in normal rodents and non‐human primates (NHP). Herein we report its initial biological evaluations in (a) neuronal cells, (b) tau knock‐out mice, and (c) aged NHPs (Caribbean‐origin vervets) with changes in AD‐related biomarkers.MethodIn vitro [11C]MPC‐6827 cell uptake assay was performed in patient‐derived SH‐SY5Y cells with different MT destabilizing and stabilizing agents. Radioactive binding efficiency was determined as %ID (injected dose)/mg of protein present in each well. Dynamic 0‐60 min brain microPET/CT imaging was performed in tau knock‐out and wild type mice (n=4). Four vervets (20‐29y) with different cerebrospinal fluid (CSF) Aβ42 levels (2 high levels‐1461, 1614 and 2 low levels‐579,732) underwent 0‐120 min brain PET/CT imaging with [11C]MPC‐6827. ROIs were drawn on the whole‐brain and SUVR values were calculated from co‐registered PET/MR images.ResultIn vitro cell uptake assays with [11C]MPC‐6827 in SH‐SY5Y cells displayed high uptake with MT destabilizing agents and low uptake with stabilizing agents. MicroPET imaging demonstrated higher radioactive brain uptake in tau knockout over wild‐type mice. Vervets with low CSF Aβ42 levels showed high radioactive brain uptake and the youngest vervet (20y) displayed lowest brain uptake.ConclusionConsistent with our in vitro cell uptake and in vivo rodent imaging experiments, [11C]MPC‐6827 increased with MT destabilizations. Radioactive uptake was inversely related to CSF Aβ levels and directly related to age in a NHP model of AD (Fig 1). Collectively our data suggest that [11C]MPC‐6827 is more selective towards destabilized MTs. On‐going experiments include rigorous PET imaging analyses, determination of metabolic plasma‐blood parameters, and correlations with Aβ42 and tau levels. Nonetheless, our promising results support the high translational utility of [11C]MPC‐6827 to image MTs in ADRDs.

Doxorubicin-loaded natural daptomycin micelles with enhanced targeting and anti-tumor effect invivo.

Development of a simple method to enhance targeting and anti-tumor effect of the chemotherapeutic agents invivo is a major problem. Amphipathic and natural daptomycin is biocompatible antibacterial polypeptide used in clinical practice. Herein, doxorubicin (DOX) was stabilized by zwitterionic daptomycin (Dap) micelles in aqueous solution to form a zwitterionic nanodrug (Dap-DOX micelles). The hydrodynamic size and zeta potential of Dap-DOX micelles were 85nm and-10 mV, respectively. The study on the controlled release showed that more DOX molecules were released from Dap-DOX micelles at acidic condition of tumor tissue than that at neutral condition of normal tissue which was due to pH responsiveness of Dap-DOX micelles. Dap-DOX micelles exhibited good stability in fibrinogen solution. Moreover, MTT studies showed that Dap-DOX micelles had higher cytotoxicity than free DOX. Notably, the results of flow cytometry indicated that the average fluorescence intensity of Dap-DOX micelle-treated cells was higher than that of free DOX-treated cells, and acidic conditions were more favorable for Dap-DOX micelles than normal pH in cell uptake assay. More importantly, Dap-DOX micelles were biocompatible invivo based on the changes of weight and blood indexes of mice. Dap-DOX micelles were selectively accumulated at tumor sites invivo through EPR effect, which reduced the toxicity of free DOX and achieved excellent tumor inhibition effect. The tumor inhibition rate of Dap-DOX micelles reached 96%. Dap-DOX micelles also effectively inhibited the growth of bacterial. Taken together, Dap-based drug delivery systems are promising and effective in cancer therapy.

Honokiol Prodrug Nanoparticles Based on In Situ Albumin Binding for Long Circulation and High Tumor Uptake.

Honokiol (HK) has antiproliferation effects against numerous cancer cells, but its low solubility and bioavailability impede its application. In this study, a prodrug of HK (HP) featuring a maleimide group was synthesized and then mixed with tocopherol polyethylene glycol succinate to prepare prodrug nanoparticles (HP-NPs). In vitro albumin binding experiments showed that HP rapidly reacted with the cysteine thiols of albumin to form a covalent conjugate that released HK slowly in the LLC tumor cell line. In vitro cell apoptosis and uptake assays showed that the cellular uptake of the HK increased into the LLC cells as the albumin concentration increased. Strikingly, in vivo pharmacokinetics and pharmacodynamics measurements demonstrated that the HP-NPs significantly prolonged the circulation and increased tumor accumulation. Taken together, our study demonstrated, both in vitro and in vivo, that the albumin-based HP-NPs delivery system holds significant potential toward the treatment of lung cancer in clinical studies.

Arginine-Arginine-Leucine Peptide Targeting Heat Shock Protein 70 for Cancer Imaging.

Arg-Arg-Leu (RRL) is a potent tumor-homing tripeptide. However, the binding target is unclear. In this study, we intended to identify the binding target of RRL and evaluate the tumor targeting of 99mTc-MAG3-RRL in vivo. Biotin-RRL, 5-TAMRA-RRL, and 99mTc-MAG3-RRL were designed to trace the binding target and tumor lesion. Immunoprecipitation-mass spectrometry was conducted to identify the candidate proteins and determination of the subcellular localization was also performed. A pull-down assay was performed to demonstrate the immunoprecipitate. Fluorescence colocalization and cell uptake assays were performed to elucidate the correlation between the selected binding protein and RRL, and the internalization mechanism of RRL. Biodistribution and in vivo imaging were performed to evaluate the tumor accumulation and targeting of 99mTc-MAG3-RRL. The target for RRL was screened to be heat shock protein 70 (HSP70). The prominent uptake distribution of RRL was concentrated in the membrane and cytoplasm. A pull-down assay demonstrated the existence of HSP70 in the biotin-RRL captured complex. Regarding fluorescence colocalization and cell uptake assays, RRL may interact with HSP70 at the nucleotide-binding domain (NBD). Clathrin-dependent endocytosis and macropinocytosis could be a vital internalization mechanism of RRL. In vivo imaging and biodistribution both demonstrated that 99mTc-MAG3-RRL can trace tumors with satisfactory accumulation in hepatoma xenograft mice. The radioactive signals accumulated in tumor lesions can be blocked by VER-155008, which can bind to the NBD of HSP70. Our findings revealed that RRL may interact with HSP70 and that 99mTc-MAG3-RRL could be a prospective probe for visualizing overexpressed HSP70 tumor sections.