Design and evaluation of α-helix-based peptide inhibitors for blocking PD-1/PD-L1 interaction.

The current research in tumor immunotherapy indicates that blocking the protein-protein interaction (PPI) between PD-1 and its ligand, PD-L1, may be one of the most effective treatments for cancer patients. The α-helix is a common elements of protein secondary structure and is often involved in protein interaction. Thus, α-helix-based peptides could mimic proteins involved in such interactions and are also capable of modulating PPI in vivo. In this study, starting from a potential α-helix-rich protein, we designed a series of α-helix-based peptide candidates to block PD-1/PD-L1 interaction. These candidates were first screened using molecular docking and molecular dynamics simulations, and then their capacities to inhibit PD-1/PD-L1 interactions and to restore antitumor immune activities were investigated using the HTRF assay, SPR assay, cellular co-culture experiments and animal model experiments. Two peptides exhibited the best anti-tumor effects and the strong ability to restore the immunity of tumor-infiltrating T-cells. Further D-amino acid substitution was employed to improve the serum stability of peptide candidate, making the intravenous administration easier while maintaining the therapeutic efficacy. The resultant peptides showed promise as checkpoint inhibitors for application in tumor immunotherapy. These findings suggested that our strategy for developing peptides starting from an α-helical structure could be used in the design of bioactive inhibitors to potential block protein-protein interactions.

Identification of Potential RBPJ-Specific Inhibitors for Blocking Notch Signaling in Breast Cancer Using a Drug Repurposing Strategy.

Notch signaling is a key parameter in regulating cell fate during tissue homeostasis, and an aberrant Notch pathway can result in mammary gland carcinoma and has been associated with poor breast cancer diagnosis. Although inhibiting Notch signaling would be advantageous in the treatment of breast cancer, the currently available Notch inhibitors have a variety of side effects and their clinical trials have been discontinued. Thus, in search of a more effective and safer Notch inhibitor, inhibiting recombinant signal binding protein for immunoglobin kappaJ region (RBPJ) specifically makes sense, as RBPJ forms a transcriptional complex that activates Notch signaling. From our established database of more than 10,527 compounds, a drug repurposing strategy-combined docking study and molecular dynamic simulation were used to identify novel RBPJ-specific inhibitors. The compounds with the best performance were examined using an in vitro cellular assay and an in vivo anticancer investigation. Finally, an FDA-approved antibiotic, fidaxomicin, was identified as a potential RBPJ inhibitor, and its ability to block RBPJ-dependent transcription and thereby inhibit breast cancer growth was experimentally verified. Our study demonstrated that fidaxomicin suppressed Notch signaling and may be repurposed for the treatment of breast cancer.

Deep learning-based multi-drug synergy prediction model for individually tailored anti-cancer therapies.

While synergistic drug combinations are more effective at fighting tumors with complex pathophysiology, preference compensating mechanisms, and drug resistance, the identification of novel synergistic drug combinations, especially complex higher-order combinations, remains challenging due to the size of combination space. Even though certain computational methods have been used to identify synergistic drug combinations in lieu of traditional in vitro and in vivo screening tests, the majority of previously published work has focused on predicting synergistic drug pairs for specific types of cancer and paid little attention to the sophisticated high-order combinations. The main objective of this study is to develop a deep learning-based approach that integrated multi-omics data to predict novel synergistic multi-drug combinations (DeepMDS) in a given cell line. To develop this approach, we firstly created a dataset comprising of gene expression profiles of cancer cell lines, target information of anti-cancer drugs, and drug response against a large variety of cancer cell lines. Based on the principle of a fully connected feed forward Deep Neural Network, the proposed model was constructed using this dataset, which achieved a high performance with a Mean Square Error (MSE) of 2.50 and a Root Mean Squared Error (RMSE) of 1.58 in the regression task, and gave the best classification accuracy of 0.94, an area under the Receiver Operating Characteristic curve (AUC) of 0.97, a sensitivity of 0.95, and a specificity of 0.93. Furthermore, we utilized three breast cancer cell subtypes (MCF-7, MDA-MD-468 and MDA-MB-231) and one lung cancer cell line A549 to validate the predicted results of our model, showing that the predicted top-ranked multi-drug combinations had superior anti-cancer effects to other combinations, particularly those that were widely used in clinical treatment. Our model has the potential to increase the practicality of expanding the drug combinational space and to leverage its capacity to prioritize the most effective multi-drug combinational therapy for precision oncology applications.

Use of Gamithromycin as a Chiral Selector in Capillary Electrophoresis.

In this short communication, we report the use of a second-generation macrolide antibiotic, gamithromycin (Gam), as a novel chiral selector for enantioseparation in capillary electrophoresis (CE). A preliminary analysis of the experiment results shows that Gam is especially suitable for the separation of chiral primary amines. Factors influencing enantioseparations were systematically investigated including the composition of the background electrolyte (BGE), concentration of Gam, the type and proportion of organic solvents, applied voltage, etc. In particular, N-Methylformamide (NMF) was successfully used as a non-aqueous solvent for Gam, and shown to be extremely effective for the separation of primaquine (PMQ) and 1-aminoindan (AMI) when used alone or mixed with other commonly used non-aqueous solvents (e.g. methanol). To our knowledge this was also the first application of NMF as a non-aqueous solvent for antibiotic chiral selectors in CE. The best separations were obtained with 100 mM Tris, 125 mM H3BO3 and 80 mM Gam in methanol/NMF (25:75) solvent for PMQ and AMI, or 80-100 mM Gam in methanol for the other model analytes. Among the analytes, the resolution (Rs) of amlodipine (AML) reached up to 15.65, which is to our knowledge the highest value ever reported in CE studies for this compound (except for using molecularly imprinted polymers technique).

Reimaging biological barriers affecting distribution and extravasation of PEG/peptide- modified liposomes in xenograft SMMC7721 tumor.

Liposomes, as one of the most successful nanotherapeutics, have a major impact on many biomedical areas. In this study, we performed laser scanning confocal microscope (LSCM) and immunohistochemistry (IHC) assays to investigate the intra-tumor transport and antitumor mechanism of GE11 peptide-conjugated active targeting liposomes (GE11-TLs) in SMMC7721 xenograft model. According to classification of individual cell types in high resolution images, biodistribution of macrophages, tumor cells, cells with high epidermal growth factor receptor (EGFR) expression and interstitial matrix in tumor microenvironment, in addition, their impacts on intra-tumor penetration of GE11-TLs were estimated. Type I collagen fibers and macrophage flooded in the whole SMMC7721 tumor xenografts. Tumor angiogenesis was of great heterogeneity from the periphery to the center region. However, the receptor-binding site barriers were supposed to be the leading cause of poor penetration of GE11-TLs. We anticipate these images can give a deep reconsideration for rational design of target nanoparticles for overcoming biological barriers to drug delivery.

Ratiometric co-encapsulation and co-delivery of doxorubicin and paclitaxel by tumor-targeted lipodisks for combination therapy of breast cancer.

Combination therapy is a promising treatment for certain advanced drug-resistant cancers. Although effective inhibition of various tumor cells was reported in vitro, combination treatment requires improvement in vivo due to uncontrolled ratiometric delivery. In this study, a tumor-targeting lipodisk nanoparticle formulation was developed for ratiometric loading and the transportation of two hydrophobic model drugs, doxorubicin (DOX) and paclitaxel (PTX), in one single platform. Furthermore, a slightly acidic pH-sensitive peptide (SAPSP) incorporated into lipodisks effectively enhanced the tumor-targeting and cell internalization. The obtained co-loaded lipodisks were approximately 30 nm with a pH-sensitive property. The ratiometric co-delivery of two drugs via lipodisks was confirmed in both the drug-resistant MCF-7/ADR cell line and its parental MCF-7 cell line in vitro, as well as in a tumor-bearing mouse model in vivo compared with a cocktail solution of free drugs. Co-loaded lipodisks exerted improved cytotoxicity to tumor cells in culture, particularly to drug-resistant tumor cells at synergistic drug ratios. In an in vivo xenograft mouse model, the anti-tumor ability of co-loaded lipodisks was evidenced by the remarkable inhibitory effect on tumor growth of either MCF-7 or MCF-7/ADR tumors, which may be attributed to the increased and ratiometric accumulation of both drugs in the tumor tissues. Therefore, tumor-specific lipodisks were crucial for the combination treatment of DOX and PTX to completely exert a synergistic anti-cancer effect. It is concluded that for co-loaded lipodisks, cytotoxicity data in vitro could be used to predict their inhibitory activity in vivo, potentially enhancing the clinical outcome of synergistic therapy.

Preparation and optimization of poly (lactic acid) nanoparticles loaded with fisetin to improve anti-cancer therapy.

Fisetin is a natural flavonoid with promising antitumor activity, whereas its clinical application is limited by its hydrophobic property. In this study, we aimed to load fisetin into poly(lactic acid) (PLA) nanoparticles to increase fisetin's solubility and therapeutic efficacy. Based on spontaneous emulsification solvent diffusion (SESD) method, the formulation of PLA nanoparticles was optimized by two successive experimental designs. One-factor-at-a-time variation experiments were first applied to investigate the effects of four process variables on three responses, including drug encapsulation efficiency, average particles size and cumulative drug release ratio, followed by determining the possible ranges of these variables. Subsequently, the combinations of four variables at best levels were evaluated using a Taguchi orthogonal array design with regard to the same three responses. Eventually, the nanoparticle prepared by optimized procedure showed a narrow size distribution around 226.85 ±â€¯4.78 nm with a high encapsulation efficiency of 90.35%. The incorporation of fisetin in nanoparticles was subsequently confirmed by FT-IR and DSC spectroscopy. Furthermore, cytotoxicity assay against HCT116 colon cancer cells in vitro and antitumor test in a xenograft 4T1 breast cancer model in vivo demonstrated that the antitumor effect of drug-loaded nanoparticles was superior to that of free drug solution.

Tumor-specific delivery of doxorubicin through conjugation of pH-responsive peptide for overcoming drug resistance in cancer.

The major obstacles opposed to doxorubicin (Dox) based chemotherapy are the induction of drug resistance, together with non-specific toxicities for healthy cells. In this study, we prepared a peptide-Dox conjugate aimed at offering Dox molecules a tumor-specific functionality and improving the therapeutic effects of Dox against resistant tumor cells. A slightly acidic pH-sensitive peptide (SAPSP) with high selectivity for cancer cells was attached to Dox to obtain SAPSP-Dox prodrug. The structures and properties of this prodrug were characterized, confirming several merits, including desirable pH-sensitive property, good serum stability and favorable release behavior. Cellular uptake studies demonstrated that SAPSP-Dox was preferably accumulated in cancer cells (Dox-sensitive MCF-7 and Dox-resistant MCF-7/ADR), followed by displaying 26-fold less toxic toward noncancerous MCF-10A cells than free Dox do. The conjugated peptides enabled Dox to escape the efflux effect of P-glycoprotein mediated pump via endocytotic pathway, giving rise to remarkable cytotoxicity and apoptotic effect on MCF-7/ADR cells. Moreover, the superior inhibition efficacy of SAPSP-Dox in vivo was more evident in the both drug-sensitive and drug-resistant xenograft tumor animal models, which enabled Dox to primarily accumulated in tumor. The conjugates also demonstrated a longer half-life in plasma and a lower side effect, for example, reduced cardiac toxicity. Evidence of this study suggests that SAPSP-Dox has the potential to be a potent prodrug for treating drug resistant cancers.

Simultaneous delivery of anti-miR21 with doxorubicin prodrug by mimetic lipoprotein nanoparticles for synergistic effect against drug resistance in cancer cells.

The development of drug resistance in cancer cells is one of the major obstacles to achieving effective chemotherapy. We hypothesized that the combination of a doxorubicin (Dox) prodrug and microRNA (miR)21 inhibitor might show synergistic antitumor effects on drug-resistant breast cancer cells. In this study, we aimed to develop new high-density lipoprotein-mimicking nanoparticles (HMNs) for coencapsulation and codelivery of this potential combination. Dox was coupled with a nuclear localization signal (NLS) peptide to construct a prodrug (NLS-Dox), thereby electrostatically condensing miR21 inhibitor (anti-miR21) to form cationic complexes. The HMNs were formulated by shielding these complexes with anionic lipids and Apo AI proteins. We have characterized that the coloaded HMNs had uniformly dispersed distribution, favorable negatively charged surface, and high coencapsulation efficiency. The HMN formulation effectively codelivered NLS-Dox and anti-miR21 into Dox-resistant breast cancer MCF7/ADR cells and wild-type MCF7 cells via a high-density-lipoprotein receptor-mediated pathway, which facilitated the escape of Pgp drug efflux. The coloaded HMNs consisting of NLS-Dox/anti-miR21 demonstrated greater cytotoxicity with enhanced intracellular accumulation in resistant MCF7/ADR cells compared with free Dox solution. The reversal of drug resistance by coloaded HMNs might be attributed to the suppression of miR21 expression and the related antiapoptosis network. Furthermore, the codelivery of anti-miR21 and NLS-Dox by HMNs showed synergistic antiproliferative effects in MCF7/ADR-bearing nude mice, and was more effective in tumor inhibition than other drug formulations. These data suggested that codelivery of anti-miR21 and chemotherapeutic agents by HMNs might be a promising strategy for antitumor therapy, and could restore the drug sensitivity of cancer cells, alter intracellular drug distribution, and ultimately enhance chemotherapeutic effects.

Targeted Biomimetic Nanoparticles for Synergistic Combination Chemotherapy of Paclitaxel and Doxorubicin.

Codelivery of multiple chemotherapeutics has become a versatile strategy in recent cancer treatment, but the antagonistic behavior of combined drugs limited their application. We developed a recombinant high-density lipoprotein (rHDL) nanoparticle for the precise coencapsulation and codelivery of two established drugs and hypothesized that they could act synergistically to improve anticancer efficacy. The coloaded rHDL was formulated by passively incorporating hydrophobic paclitaxel (PTX), and subsequently remotely loading hydrophilic doxorubicin (Dox) into the same nanoparticles. The resultant rHDL system restored targeted delivery function toward cancer cells via scavenger receptor class B (SR-BI), as confirmed by in vitro confocal imaging and flow cytometry. These coloaded rHDL nanoparticles were remarkably effective in increasing the ratiometric accumulation of drugs in cancer cells and enhancing antitumor response at synergistic drug ratios. In particular, they exhibited more efficacious anticancer effects in an in vitro cytotoxicity evaluation and in a xenograft tumor model of hepatoma compared with free drug cocktail solutions. These results confirm that the coloaded rHDL nanoparticles are promising candidates for the synergistic delivery of drugs with diverse physicochemical properties in cancer treatment integrating efficiency and safety considerations.

Targeting and liposomal drug delivery to CD40L expressing T cells for treatment of autoimmune diseases.

CD40L is considered as an important target for the treatment of autoimmune diseases. There have been many efforts devoted to the development of antibodies and other molecules to disrupt CD40/CD40L interaction for therapeutic benefits. In this study, we designed a CD40L specific peptide ligand - A25 based on CD40L crystal structure and molecular docking studies. Its binding affinity and specificity to CD40L were confirmed by Surface Plasmon Resonance (SPR) measurements. The peptide A25 was then conjugated on the surface of liposomes and shown to be able to mediate specific liposomal drug delivery to CD40L+ cells. Loaded with the cytostatic drug methotrexate (MTX), the A25 modified liposome could significantly reduce the CD40L+ cell ratios in the experimental autoimmune encephalomyelitis (EAE) mice, resulting in great improvement in clinical scores. Since CD40L+ cells are involved in the pathological development of many auto-immune diseases, A25 conjugated drug targeting systems may be useful for developing therapies that are more efficacies and with less side effects.

Effects of surface displayed targeting ligand GE11 on liposome distribution and extravasation in tumor.

Targeting ligands displayed on liposome surface had been used to mediate specific interactions and drug delivery to target cells. However, they also affect liposome distribution in vivo, as well as the tissue extravasation processes after IV injection. In this study, we incorporated an EGFR targeting peptide GE11 on liposome surfaces in addition to PEG at different densities and evaluated their targeting properties and antitumor effects. We found that the densities of surface ligand and PEG were critical to target cell binding in vitro as well as pharmacokinetic profiles in vivo. The inclusion of GE11-PEG-DSPE and PEG-DSPE at 2% and 4% mol ratios in the liposome formulation mediated a rapid accumulation of liposomes within 1 h after IV injection in the tumor tissues surrounding neovascular structures. This is in addition to the EPR effect that was most prominently described for surface PEG modified liposomes. Therefore, despite the fact that the distribution of liposomes into interior tumor tissues was still limited by diffusion, GE11 targeted doxorubicin loaded liposomes showed significantly better antitumor activity in tumor bearing mice as a result of the fast active-targeting efficiency. We anticipate these understandings can benefit further optimization of targeted drug delivery systems for improving efficacy in vivo.

Recombinant high density lipoprotein nanoparticles for target-specific delivery of siRNA.

PURPOSE: Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for treatments of numerous diseases. However, the progress towards broad application of siRNA requires the development of safe and effective vectors that target to specific cells. In this study, we developed a novel recombinant high density lipoprotein (rHDL) vector with high siRNA encapsulation efficiency. METHODS: They were prepared by condensing siRNA with various commercial cationic polymers and coating the polyplex with a layer of lipids and apolipoprotein AI (apo AI). The rHDL nanoparticles were used to transfect SMMC-7721 hepatoma cells with stable luciferase expression. The uptake and intracellular trafficing of siRNA were also investigated. RESULTS: Characterization studies revealed these rHDL nanoparticles had similar physical properties as natural HDLs. The various rHDL formulations had high silencing efficiency (more than 70% knockdown) in hepatocytes with minimum cytotoxicity. Moreover, the uptake of rHDL by SMMC-7721 was confirmed to be mediated through the natural HDL uptake pathway. CONCLUSIONS: The work described here demonstrated the optimized rHDL nanoparticles may offer a promising tool for siRNA delivery to the liver.

Recombinant high-density lipoprotein nanoparticles containing gadolinium-labeled cholesterol for morphologic and functional magnetic resonance imaging of the liver.

BACKGROUND: Natural high-density lipoproteins (HDL) possess important physiological functions to the transport of cholesterol from the peripheral tissues to the liver for metabolic degradation and excretion in the bile. METHODS AND RESULTS: In this work, we took advantage of this pathway and prepared two different gadolinium (Gd)-DTPA-labeled cholesterol-containing recombinant HDL nanoparticles (Gd-chol-HDL) and Gd-(chol)(2)-HDL as liver-specific magnetic resonance imaging (MRI) contrast agents. The reconstituted HDL nanoparticles had structural similarity to native HDL, and could be taken up by HepG2 cells via interaction with HDL receptors in vitro. In vivo MRI studies in rats after intravenous injections of 10 µmol gadolinium per kg of recombinant HDL nanoparticles indicated that both nanoparticles could provide signal enhancement in the liver and related organs. However, different T(1)-weighted image details suggested that they participated in different cholesterol metabolism and excretion pathways in the liver. CONCLUSION: Such information could be highly useful to differentiate functional changes as well as anatomic differences in the liver. These cholesterol-derived contrast agents and their recombinant HDL preparations may warrant further development as a new class of contrast agents for MRI of the liver and related organs.

In vitro and in vivo study of a nanoliposomal cisplatin as a radiosensitizer.

OBJECTIVE: To investigate the in vitro and in vivo radiosensitization effect of an institutionally designed nanoliposome encapsulated cisplatin (NLE-CDDP). MATERIALS AND METHODS: NLE-CDDP was developed by our institute. In vitro radiosensitization of NLE-CDDP was evaluated by colony forming assay in A549 cells. In vivo radiosensitization was studied with tumor growth delay (TGD) in Lewis lung carcinoma. The radiosensitization for normal tissue was investigated by jejunal crypt survival. The radiosensitization studies were carried out with a 72 h interval between drug administration and irradiation. The mice were treated with 6 mg/kg of NLE-CDDP or CDDP followed by single doses of 2 Gy, 6 Gy, 16 Gy, and 28 Gy. Sensitization enhancement ratio (SER) was calculated by D(0)s of cell survival curves for A549 cells, doses needed to yield TGD of 20 days in Lewis lung carcinoma, or D(0)s of survival curves in crypt cells in radiation alone and radiation plus drug groups. RESULTS: Our NLE-CDDP could inhibit A549 cells in vitro with half maximal inhibitory concentration of 1.12 µg/mL, and its toxicity was 2.35 times that observed in CDDP. For in vitro studies of A549 cells, SERs of NLE-CDDP and CDDP were 1.40 and 1.14, respectively, when combined with irradiation. For in vivo studies of Lewis lung carcinoma, the strongest radiosensitization was found in the 72 h interval between NLE-CDDP and irradiation. When given 72 h prior to irradiation, NLE-CDDP yielded higher radiosensitization than CDDP (SER of 4.92 vs 3.21) and slightly increased injury in jejunal crypt cells (SER of 1.15 vs 1.19). Therefore, NLE-CDDP resulted in a higher TGF than did CDDP (4.28 vs 2.70) when SERs were compared between experiments in vivo and in jejunal crypt cell studies. CONCLUSIONS: Our NLE-CDDP was demonstrated to have radiosensitization with TGF of 4.28 when administrated 72 h prior to irradiation.

A synthetic peptide mediated active targeting of cisplatin liposomes to Tie2 expressing cells.

Tie2 receptor is a receptor tyrosine kinase that plays important roles in vascular angiogenesis, and also highly expressed by a number of cancer cells. In this study, we reported an active targeting liposome system directed by a novel peptide ligand PH1 that can improve drug efficacies specifically to Tie2 expressing cells. The PH1 peptide (TMGFTAPRFPHY) was selected by phage display library screening combined with surface plasmon resonance binding assays. It was covalently conjugated to the distal end of DSPE-PEG(2000)-Maleimide lipid and loaded onto liposome membranes as the targeting ligand. These PH1-PEG-liposomes containing the anticancer drug cisplatin were showed to bind tightly to Tie2 positive cells, mediate active endocytosis of the drug containing liposomes, and result in much higher cell specific cytoxicities than mPEG coated liposomes. They can be used not only to target vascular endothelial cells for anti-angiogenesis effects, but also to improve drug delivery and release in Tie2 expressing cancer cells. Such liposome formulation may be developed into a very useful agent for metronomic chemotherapy.