Supramolecular prodrug of SN38 based on endogenous albumin and SN38 prodrug modified with semaglutide side chain to improve the tumor distribution.

To improve the biodistribution of the drug in the tumor, a supramolecular prodrug of SN38 was fabricated in situ between endogenous albumin and SN38 prodrug modified with semaglutide side chain. Firstly, SN38 was conjugated with semaglutide side chain and octadecanedioic acid via glycine linkers to obtain SI-Gly-SN38 and OA-Gly-SN38 prodrugs, respectively. Both SI-Gly-SN38 and OA-Gly-SN38 exhibited excellent stability in PBS for over 24 h. Due to the strong binding affinity of the semaglutide side chain with albumin, the plasma half-life of SI-Gly-SN38 was 2.7 times higher than that of OA-Gly-SN38. Furthermore, with addition of HSA, the fluorescence intensity of SI-Gly-SN38 was 4 times higher than that of OA-Gly-SN38, confirming its strong binding capability with HSA. MTT assay showed that the cytotoxicity of SI-Gly-SN38 and OA-Gly-SN38 was higher than that of Irinotecan. Even incubated with HSA, the SI-Gly-SN38 and OA-Gly-SN38 still maintained high cytotoxicity, indicating minimal influence of HSA on their cytotoxicity. In vivo pharmacokinetic studies demonstrated that the circulation half-life of SI-Gly-SN38 was twice that of OA-Gly-SN38. SI-Gly-SN38 exhibited significantly reduced accumulation in the lungs, being only 0.23 times that of OA-Gly-SN38. The release of free SN38 in the lungs from SI-Gly-SN38 was only 0.4 times that from OA-Gly-SN38 and Irinotecan. The SI-Gly-SN38 showed the highest accumulation in tumors. The tumor inhibition rate of SI-Gly-SN38 was 6.42% higher than that of OA-Gly-SN38, and 8.67% higher than that of Irinotecan, respectively. These results indicate that the supramolecular prodrug delivery system can be constructed between SI-Gly-SN38 and endogenous albumin, which improves drug biodistribution in vivo, enhances tumor accumulation, and plays a crucial role in tumor growth inhibition.

Investigation of antiproliferative efficacy and apoptosis induction in leukemia cancer cells using irinotecan-loaded liposome-embedded nanofibers constructed from chitosan.

Liposomes and nanofibers have been implemented as efficacious vehicles for delivering anticancer drugs. With this view, this study explores the antiproliferative efficacy and apoptosis induction in leukemia cancer cells utilizing irinotecan-loaded liposome-embedded nanofibers fabricated from chitosan, a biological source. Specifically, we investigate the effectiveness of poly(ε-caprolactone) (PCL)/chitosan (CS) (core)/irinotecan (CPT)nanofibers (termed PCL-CS10 CPT), PCL/chitosan/irinotecan (core)/PCL/chitosan (shell) nanofibers (termed CS/CPT/PCL/CS), and irinotecan-coloaded liposome-incorporated PCL/chitosan-chitosan nanofibers (termed CPT@Lipo/CS/PCL/CS) in releasing irinotecan in a controlled manner and treating leukemia cancer. The fabricated formulations were characterized utilizing Fourier transform infrared analysis, transmission electron microscopy, scanning electron microscopy, dynamic light scattering, zeta potential, and polydispersity index. Irinotecan was released in a controlled manner from nanofibers filled with liposomes over 30 days. The cell viability of the fabricated nanofibrous materials toward Human umbilical vein endothelial cells (HUVECs) non-cancerous cells after 168 h was >98 % ± 1 %. The CPT@Lipo/CS/PCL/CS nanofibers achieved maximal cytotoxicity of 85 % ± 2.5 % against K562 leukemia cancer cells. The CPT@Lipo/CS/PCL/CS NFs exhibit a three-stage drug release pattern and demonstrate significant in vitro cytotoxicity. These findings indicate the potential of these liposome-incorporated core-shell nanofibers for future cancer therapy.

Empowering hydrophobic anticancer drugs by ultrashort peptides: General Co-assembly strategy for improved solubility, targeted efficacy, and clinical application.

The current state of drug delivery systems allows for the resolution of specific issues like inadequate solubility, limited targeting capabilities, and complex preparation processes, requiring tailored designs for different drugs. Yet, the major challenge in clinical application lies in surmounting these obstacles with a universal carrier that is effective for a variety of anticancer drugs. Herein, with the help of computer simulation, we rationally design ultrashort peptides GY and CCYRGD, which can co-assemble with hydrophobic anticancer drugs into nanoparticles with enhanced solubility, targeting ability and anticancer efficacy. Taking 7-ethyl-10-hydroxy camptothecin (SN38) as a model anticancer drug, the co-assembled SN38-GY-CCYRGD nanoparticles significantly enhance the water solubility of SN38 by more than three orders of magnitude. The as-prepared nanoparticles can effectively kill cancer cells, e.g., human small cell lung cancer (A549) cells with a notable cell mortality rate of 71%. Mice experimental results demonstrate the nanoparticles' efficient targeting capability, marked reducing the toxicity to normal tissues while improving antitumor efficacy. This work presents a novel drug delivery method, integrating effective, targeted, and safe strategies into a comprehensive carrier system, designed for the administration of hydrophobic anticancer drugs.

Degradation and mineralization of anti-cancer drugs Capecitabine, Bicalutamide and Irinotecan by UV-irradiation and ozone.

The degradation of three anti-cancer drugs (ADs), Capecitabine (CAP), Bicalutamide (BIC) and Irinotecan (IRI), in ultrapure water by ozonation and UV-irradiation was tested in a bench-scale reactor and AD concentrations were measured through ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). A low-pressure mercury UV (LP-UV) lamp was used and degradation by UV (λ = 254 nm) followed pseudo-first order kinetics. Incident radiation in the reactor was measured via chemical actinometry using uridine. The quantum yields (φ) for the degradation of CAP, BIC and IRI were 0.012, 0.0020 and 0.0045 mol Einstein-1, respectively. Ozone experiments with CAP and IRI were conducted by adding ozone stock solution to the reactor either with or without addition of tert-butanol (t-BuOH) as radical quencher. Using this experimental arrangement, no degradation of BIC was observed, so a semi-batch setup was employed for the ozone degradation experiments of BIC. Without t-BuOH, apparent second order reaction rate constants for the reaction of the ADs with molecular ozone were determined to be 3.5 ± 0.8 ∙ 103 L mol-1 s-1 (CAP), 7.9 ± 2.1 ∙ 10-1 L mol-1 s-1 (BIC) and 1.0 ± 0.3 ∙ 103 L mol-1 s-1 (IRI). When OH-radicals (∙OH) were quenched, rate constants were virtually the same for CAP and IRI. For BIC, a significantly lower constant of 1.0 ± 0.5 ∙ 10-1 L mol-1 s-1 was determined. Of the tested substances, BIC was the most recalcitrant, with the slowest degradation during both ozonation and UV-irradiation. The extent of mineralization was also determined for both processes. UV irradiation was able to fully degrade up to 80% of DOC, ozonation up to 30%. Toxicity tests with Daphnia magna (D. magna) did not find toxicity for fully degraded solutions of the three ADs at environmentally relevant concentrations.

In Silico Study of Camptothecin-Based Pro-Drugs Binding to Human Carboxylesterase 2.

Pro-drugs, which ideally release their active compound only at the site of action, i.e., in a cancer cell, are a promising approach towards an increased specificity and hence reduced side effects in chemotherapy. A popular form of pro-drugs is esters, which are activated upon their hydrolysis. Since carboxylesterases that catalyse such a hydrolysis reaction are also abundant in normal tissue, it is of great interest whether a putative pro-drug is a probable substrate of such an enzyme and hence bears the danger of being activated not just in the target environment, i.e., in cancer cells. In this work, we study the binding mode of carboxylesters of the drug molecule camptothecin, which is an inhibitor of topoisomerase I, of varying size to human carboxylesterase 2 (HCE2) by molecular docking and molecular dynamics simulations. A comparison to irinotecan, known to be a substrate of HCE2, shows that all three pro-drugs analysed in this work can bind to the HCE2 protein, but not in a pose that is well suited for subsequent hydrolysis. Our data suggest, moreover, that for the irinotecan substrate, a reactant-competent pose is stabilised once the initial proton transfer from the putative nucleophile Ser202 to the His431 of the catalytic triad has already occurred. Our simulation work also shows that it is important to go beyond the static models obtained from molecular docking and include the flexibility of enzyme-ligand complexes in solvents and at a finite temperature. Under such conditions, the pro-drugs studied in this work are unlikely to be hydrolysed by the HCE2 enzyme, indicating a low risk of undesired drug release in normal tissue.

Fluorescence Lifetime Nanoscopy of Liposomal Irinotecan Onivyde: From Manufacturing to Intracellular Processing.

Onivyde was approved by the Food and Drug Administration (FDA) in 2015 for the treatment of solid tumors, including metastatic pancreatic cancer. It is designed to encapsulate irinotecan at high concentration, increase its blood-circulation lifetime, and deliver it to cells where it is enzymatically converted into SN-38, a metabolite with 100- to 1000-fold higher anticancer activity. Despite a rewarding clinical path, little is known about the physical state of encapsulated irinotecan within Onivyde and how this synthetic identity changes throughout the process from manufacturing to intracellular processing. Herein, we exploit irinotecan intrinsic fluorescence and fluorescence lifetime imaging microscopy (FLIM) to selectively probe the supramolecular organization of the drug. FLIM analysis on the manufacturer's formulation reveals the presence of two coexisting physical states within Onivyde liposomes: (i) gelated/precipitated irinotecan and (ii) liposome-membrane-associated irinotecan, the presence of which is not inferable from the manufacturer's indications. FLIM in combination with high-performance liquid chromatography (HPLC) and a membrane-impermeable dynamic quencher of irinotecan reveals rapid (within minutes) and complete chemical dissolution of the gelated/precipitated phase upon Onivyde dilution in standard cell-culturing medium with extensive leakage of the prodrug from liposomes. Indeed, confocal imaging and cell-proliferation assays show that encapsulated and nonencapsulated irinotecan formulations are similar in terms of cell-uptake mechanism and cell-division inhibition. Finally, 2-channel FLIM analysis discriminates the signature of irinotecan from that of its red-shifted SN-38 metabolite, demonstrating the appearance of the latter as a result of Onivyde intracellular processing. The findings presented in this study offer fresh insights into the synthetic identity of Onivyde and its transformation from production to in vitro administration. Moreover, these results serve as another validation of the effectiveness of FLIM analysis in elucidating the supramolecular organization of encapsulated fluorescent drugs. This research underscores the importance of leveraging advanced imaging techniques to deepen our understanding of drug formulations and optimize their performance in delivery applications.

Evaluation of In Vivo Prepared Albumin-Drug Conjugate Using Immunoprecipitation Linked LC-MS Assay and Its Application to Mouse Pharmacokinetic Study.

There have been many attempts in pharmaceutical industries and academia to improve the pharmacokinetic characteristics of anti-tumor small-molecule drugs by conjugating them with large molecules, such as monoclonal antibodies, called ADCs. In this context, albumin, one of the most abundant proteins in the blood, has also been proposed as a large molecule to be conjugated with anti-cancer small-molecule drugs. The half-life of albumin is 3 weeks in humans, and its distribution to tumors is higher than in normal tissues. However, few studies have been conducted for the in vivo prepared albumin-drug conjugates, possibly due to the lack of robust bioanalytical methods, which are critical for evaluating the ADME/PK properties of in vivo prepared albumin-drug conjugates. In this study, we developed a bioanalytical method of the albumin-conjugated MAC glucuronide phenol linked SN-38 ((2S,3S,4S,5R,6S)-6-(4-(((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b] quinolin-9-yl)oxy)methyl)(2 (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid) as a proof-of-concept. This method is based on immunoprecipitation using magnetic beads and the quantification of albumin-conjugated drug concentration using LC-qTOF/MS in mouse plasma. Finally, the developed method was applied to the in vivo intravenous (IV) mouse pharmacokinetic study of MAC glucuronide phenol-linked SN-38.

Study on the solubilization effect of 7-ethyl-10-hydroxycamptothecin based on molecular docking and molecular dynamics simulation.

CONTEXT: With the continuous improvement of anticancer drugs, the condition of patients has been controlled to a certain extent, but the problem that still needs to be urgently solved is that most anticancer drug candidates' solubility is low. On the one hand, the low solubility of anticancer drugs may lead to a decrease in the absorption rate of anticancer drugs, poor treatment effect, and even death in severe cases. On the other hand, it will also lead to a waste of medical resources. At the same time, the rapid and scientific screening of ideal anticancer drugs has become a difficult problem that researchers have to face in the research process. In this study, we found two kinds of SN38-ligand complexes that solubilize 7-ethyl-10-hydroxycamptothecin (SN38) through molecular docking and molecular dynamics simulation methods. This process not only provided valuable information on improving the solubility of SN38, but also helped to discover effective potential complexes that solubilize SN38 quickly and scientifically. METHODS: The interaction of the SN38 with folic acid and isoproterenol hydrochloride was rapidly determined by molecular docking and molecular dynamics simulation methods. We used Discovery Studio software to perform molecular docking. And then, we used Gromacs 2019.3 software to perform molecular dynamics, analyzing and comparing the hydrogen bonds, solvent-accessible surface areas, energies, and so on between SN38 and SN38-ligand complexes. And the force field adopted the Gromos 54a7.

On-Demand Activation of a Bioorthogonal Prodrug of SN-38 with Fast Reaction Kinetics and High Releasing Efficiency In Vivo.

Although a myriad of bioorthogonal prodrugs have been developed, very few of them present both fast reaction kinetics and complete cleavage. Herein, we report a new bioorthogonal prodrug strategy with both fast reaction kinetics (k2: ∼103 M-1 s-1) and complete cleavage (>90% within minutes) using the bioorthogonal reaction pair of N-oxide and boron reagent. Distinctively, an innovative 1,6-elimination-based self-immolative linker is masked by N-oxide, which can be bioorthogonally demasked by a boron reagent for the release of both amino and hydroxy-containing payload in live cells. Such a strategy was applied to prepare a bioorthogonal prodrug for a camptothecin derivative, SN-38, resulting in 10-fold weakened cytotoxicity against A549 cells, 300-fold enhanced water solubility, and "on-demand" activation upon a click reaction both in vitro and in vivo. This novel bioorthogonal prodrug strategy presents significant advances over the existing ones and may find wide applications in drug delivery in the future.

The Cisplatin, 5-fluorouracil, Irinotecan, and Gemcitabine Treatment in Resistant 2D and 3D Model Triple Negative Breast Cancer Cell Line: ABCG2 Expression Data.

BACKGROUND: Chemotherapeutics have been commonly used in cancer treatment. OBJECTIVE: In this study, the effects of Cisplatin, 5-fluorouracil, Irinotecan, and Gemcitabine have been evaluated on two-dimensional (2D) (sensitive and resistance) cell lines and three dimensional (3D) spheroid structure of MDA-MB- 231. The 2D cell culture lacks a natural tissue-like structural so, using 3D cell culture has an important role in the development of effective drug testing models. Furthermore, we analyzed the ATP Binding Cassette Subfamily G Member 2 (ABCG2) gene and protein expression profile in this study. We aimed to establish a 3D breast cancer model that can mimic the in vivo 3D breast cancer microenvironment. METHODS: The 3D spheroid structures were multiplied (globally) using the three-dimensional hanging drop method. The cultures of the parental cell line MDA-MB-231 served as the controls. After adding the drugs in different amounts, we observed a clear and well-differentiated spheroid formation for 24 h. The viability and proliferation capacity of 2D (sensitive and resistant) cell lines and 3D spheroid cell treatment were assessed by the XTT assay. RESULTS: Cisplatin, Irinotecan, 5-Fu, and Gemcitabine-resistant MDA-MB-231 cells were observed to begin to disintegrate in a three-dimensional clustered structure at 24 hours. Additionally, RT-PCR and protein assay showed overexpression of ABCG2 when compared to the parental cell line. Moreover, MDA-MB-231 cells grown in 3D showed decreased sensitivity to chemotherapeutics treatment. CONCLUSION: More resistance to chemotherapeutics and altered gene expression profile were shown in 3D cell cultures when compared with the 2D cells. These results might play an important role to evaluate the efficacy of anticancer drugs to explore the mechanisms of MDR in the 3D spheroid forms.

Irinotecan/scFv co-loaded liposomes coaction on tumor cells and CAFs for enhanced colorectal cancer therapy.

BACKGROUND: Cancer-associated fibroblasts (CAFs), as an important component of stroma, not only supply the "soils" to promote tumor invasion and metastasis, but also form a physical barrier to hinder the penetration of therapeutic agents. Based on this, the combinational strategy that action on both tumor cells and CAFs simultaneously would be a promising approach for improving the antitumor effect. RESULTS: In this study, the novel multifunctional liposomes (IRI-RGD/R9-sLip) were designed, which integrated the advantages including IRI and scFv co-loading, different targets, RGD mediated active targeting, R9 promoting cell efficient permeation and lysosomal escape. As expected, IRI-RGD/R9-sLip showed enhanced cytotoxicity in different cell models, effectively increased the accumulation in tumor sites, as well as exhibited deep permeation ability both in vitro and in vivo. Notably, IRI-RGD/R9-sLip not only exhibited superior in vivo anti-tumor effect in both CAFs-free and CAFs-abundant bearing mice models, but also presented excellent anti-metastasis efficiency in lung metastasis model. CONCLUSION: In a word, the novel combinational strategy by coaction on both "seeds" and "soils" of the tumor provides a new approach for cancer therapy, and the prepared liposomes could efficiently improve the antitumor effect with promising clinical application prospects.

Drastic Modulation of Molecular Packing and Intrinsic Dissolution Rates by Meniscus-Guided Coating of Extremely Confined Pharmaceutical Thin Films.

Nanosizing has emerged as one of the most effective formulation strategies for enhancement of dissolution properties of active pharmaceutical ingredients (APIs). In addition to enhancing the specific area of the dissolving solids, nanosizing can also capture and stabilize the metastable form of the API, which can further enhance the solubility by drastic modulation of surface energies. Herein, we employ meniscus-guided coating to fabricate nanothin films of three APIs that show anticancer properties and are poorly soluble:10-HCPT, SN-38, and amonafide. By modulating the coating speed, we systematically deposited the APIs in films ranging from ∼200 nm thickness to extreme confinement of ∼10 nm (<10 molecular layers). In all three APIs, we observe a general order-to-disorder transition with semicrystalline (10-HCPT and amonafide) or amorphous (SN-38) form of API solids trapped in thin films when the thickness decreases below a critical value of ∼25-30 nm. The existence of a critical thickness highlights the importance of nanoconfinement in tuning molecular packing. In the case of 10-HCPT, we demonstrate that the disordered form of the API occurs largely due to lack of incorporation of water molecules in thinner films below the critical thickness, thereby disrupting the three-dimensional hydrogen-bonded network held by water molecules. We further developed a dissolution model that predicts variation of the intrinsic dissolution rate (IDR) with API film thickness, which also closely matched with experimental results. We achieved drastic improvement in the IDR of ∼240% in 10-HCPT by decreasing film thickness alone. Further leveraging the order-to-disorder transition led to 2570% modulation of the IDR for amonafide. Our work demonstrates, for the first time, opportunities to largely modulate API dissolution by precisely controlling the dimensionality of thin films.

A Phenylfurocoumarin Derivative Reverses ABCG2-Mediated Multidrug Resistance In Vitro and In Vivo.

The ATP-binding cassette subfamily G member 2 (ABCG2) transporter is involved in the development of multidrug resistance in cancer patients. Many inhibitors of ABCG2 have been reported to enhance the chemosensitivity of cancer cells. However, none of these inhibitors are being used clinically. The aim of this study was to identify novel ABCG2 inhibitors by high-throughput screening of a chemical library. Among the 5812 compounds in the library, 23 compounds were selected in the first screening, using a fluorescent plate reader-based pheophorbide a (PhA) efflux assay. Thereafter, to validate these compounds, a flow cytometry-based PhA efflux assay was performed and 16 compounds were identified as potential inhibitors. A cytotoxic assay was then performed to assess the effect these 16 compounds had on ABCG2-mediated chemosensitivity. We found that the phenylfurocoumarin derivative (R)-9-(3,4-dimethoxyphenyl)-4-((3,3-dimethyloxiran-2-yl)methoxy)-7H-furo [3,2-g]chromen-7-one (PFC) significantly decreased the IC50 of SN-38 in HCT-116/BCRP colon cancer cells. In addition, PFC stimulated ABCG2-mediated ATP hydrolysis, suggesting that this compound interacts with the substrate-binding site of ABCG2. Furthermore, PFC reversed the resistance to irinotecan without causing toxicity in the ABCG2-overexpressing HCT-116/BCRP cell xenograft mouse model. In conclusion, PFC is a novel inhibitor of ABCG2 and has promise as a therapeutic to overcome ABCG2-mediated MDR, to improve the efficiency of cancer chemotherapy.

pH-responsive and folate-coated liposomes encapsulating irinotecan as an alternative to improve efficacy of colorectal cancer treatment.

Irinotecan (IRN) is a semisynthetic derivative of camptothecin that acts as a topoisomerase I inhibitor. IRN is used worldwide for the treatment of several types of cancer, including colorectal cancer, however its use can lead to serious adverse effects, as diarrhea and myelosuppression. Liposomes are widely used as drug delivery systems that can improve chemotherapeutic activity and decrease side effects. Liposomes can also be pH-sensitive to release its content preferentially in acidic environments, like tumors, and be surface-functionalized for targeting purposes. Herein, we developed a folate-coated pH-sensitive liposome as a drug delivery system for IRN to reach improved tumor therapy without potential adverse events. Liposomes were prepared containing IRN and characterized for particle size, polydispersity index, zeta potential, concentration, encapsulation, cellular uptake, and release profile. Antitumor activity was investigated in a murine model of colorectal cancer, and its toxicity was evaluated by hematological/biochemical tests and histological analysis of main organs. The results showed vesicles smaller than 200 nm with little dispersion, a surface charge close to neutral, and high encapsulation rate of over 90%. The system demonstrated prolonged and sustained release in pH-dependent manner with high intracellular drug delivery capacity. Importantly, the folate-coated pH-sensitive formulation had significantly better antitumor activity than the pH-dependent system only or the free drug. Tumor tissue of IRN-containing groups presented large areas of necrosis. Furthermore, no evidence of systemic toxicity was found for the groups investigated. Thus, our developed nanodrug IRN delivery system can potentially be an alternative to conventional colorectal cancer treatment.

Molecular level precision and high molecular weight peptide dendrimers for drug-specific delivery.

Peptide dendrimers have a broad application in biomedical science due to their biocompatibility, diversity, and multifunctionality, but the precision synthesis of high-molecule weight peptide dendrimers remains challenging. We here report the facile and liquid-phase synthesis of molecular level precision and amino-acid built-in polylysine (PLL) dendrimers with molecular weights as high as ∼60 kDa. Three types of polyhedral oligosilsesquioxane (POSS)-cored PLL dendrimers with phenylalanine, tyrosine, or histidine as building blocks were synthesized. The precise structures of the dendrimers were confirmed by MALDI-TOF MS, GPC, and 1H NMR spectroscopy. The interior functionalized peptide dendrimers improved the encapsulation capability of SN38 and sustained the release profiles. Enhanced molecular interactions between the peptide dendrimers and drugs were explored by both NMR experiments and computer simulations. The peptide dendrimer/SN38 formulations showed potent antitumor activity against multiple cancer cell lines. We believe that this strategy can be applied to the synthesis of tailor-made functional peptide dendrimers for drug-specific delivery and other diverse biomedical applications.

Synergistic Interactions of Cannabidiol with Chemotherapeutic Drugs in MCF7 Cells: Mode of Interaction and Proteomics Analysis of Mechanisms.

Cannabidiol (CBD), a nonpsychoactive phytocannabinoid, has recently emerged as a potential cytotoxic agent in addition to its ameliorative activity in chemotherapy-associated side effects. In this work, the potential interactions of CBD with docetaxel (DOC), doxorubicin (DOX), paclitaxel (PTX), vinorelbine (VIN), and 7-ethyl-10-hydroxycamptothecin (SN-38) were explored in MCF7 breast adenocarcinoma cells using different synergy quantification models. The apoptotic profiles of MCF7 cells after the treatments were assessed via flow cytometry. The molecular mechanisms of CBD and the most promising combinations were investigated via label-free quantification proteomics. A strong synergy was observed across all synergy models at different molar ratios of CBD in combination with SN-38 and VIN. Intriguingly, synergy was observed for CBD with all chemotherapeutic drugs at a molar ratio of 636:1 in almost all synergy models. However, discording synergy trends warranted the validation of the selected combinations against different models. Enhanced apoptosis was observed for all synergistic CBD combinations compared to monotherapies or negative controls. A shotgun proteomics study highlighted 121 dysregulated proteins in CBD-treated MCF7 cells compared to the negative controls. We reported the inhibition of topoisomerase II ß and α, cullin 1, V-type proton ATPase, and CDK-6 in CBD-treated MCF7 cells for the first time as additional cytotoxic mechanisms of CBD, alongside sabotaged energy production and reduced mitochondrial translation. We observed 91 significantly dysregulated proteins in MCF7 cells treated with the synergistic combination of CBD with SN-38 (CSN-38), compared to the monotherapies. Regulation of telomerase, cell cycle, topoisomerase I, EGFR1, protein metabolism, TP53 regulation of DNA repair, death receptor signalling, and RHO GTPase signalling pathways contributed to the proteome-wide synergistic molecular mechanisms of CSN-38. In conclusion, we identified significant synergistic interactions between CBD and the five important chemotherapeutic drugs and the key molecular pathways of CBD and its synergistic combination with SN-38 in MCF7 cells. Further in vivo and clinical studies are warranted to evaluate the implementation of CBD-based synergistic adjuvant therapies for breast cancer.

Tumor vasculature-targeting PEGylated peptide-drug conjugate prodrug nanoparticles improve chemotherapy and prevent tumor metastasis.

Metastasis is the main cause of death in cancer patients; therefore, new strategies or technologies that can inhibit the growth of primary tumors and their metastatic spread are extremely valuable. In this study, we selected an E-selectin-binding peptide as a targeting ligand and an inhibitor of metastasis, and conjugated this peptide with SN38 and PEG to produce an amphiphilic PEGylated peptide-drug conjugate (PDC). Novel self-assembled nanoparticles were then formed by the amphiphilic conjugate. The particles were actively targeted to the tumor vasculature by the peptide and passively to the tumor site by the enhanced permeability and retention (EPR) effect. As a nano-prodrug, this multifunctional conjugate (PEG-Pep-SN38) could reduce tumor growth, with an effect similar to that of irinotecan. Moreover, it could prolong the survival of mice bearing primary HCT116 tumors, which was not observed for its parent drug, SN38, nor the clinical prodrug of SN38 (irinotecan). Furthermore, this PDC prodrug prevented B16-F10 colonization in the lungs of mice. This study describes a new tumor vasculature-targeting PDC nano-prodrug with convenient preparation and high potential for cancer therapy, with the potential to be applied to other chemotherapeutic drugs.

"Sweet tooth"-oriented SN38 prodrug delivery nanoplatform for targeted gastric cancer therapy.

Most cancer cells employ overexpression of glucose transports (GLUTs) to satisfy glucose demand ("Sweet Tooth") for increased aerobic glycolysis rates. GLUT1, one of the most widely expressed GLUTs in numerous cancers, was identified as a prognosis-related biomarker of gastric cancer via tissue array analysis. Herein, a "Sweet Tooth"-oriented SN38 prodrug delivery nanoplatform (Glu-SNP) was developed for targeted gastric cancer therapy. For this purpose, a SN38-derived prodrug (PLA-SN38) was synthesized by tethering 7-ethyl-10-hydroxycamptothecin (SN38) to biocompatible polylactic acid (PLA) with the appropriate degree of polymerization (n = 44). The PLA-SN38 conjugate was further assembled with glycosylated amphiphilic lipid to obtain glucosamine-decorated nanoparticles (Glu-SNP). Glu-SNP exhibited potent antitumor efficiency both in vitro and in vivo through enhanced cancer cell-specific targeting associated with the overexpression of GLUT1, which provides a promising approach for gastric cancer therapy.

Reducing PD-L1 expression with a self-assembled nanodrug: an alternative to PD-L1 antibody for enhanced chemo-immunotherapy.

The binding between the immune checkpoints, programmed cell death ligand 1 (PD-L1) and programmed cell death 1 (PD-1), compromises T-cell-mediated immune surveillance. Immune checkpoint therapy using immune checkpoint inhibitors (ICIs) to block PD-L1 on cancer cell membrane or PD-1 on activated T cell membrane can restore antitumor function of T cell. However, the intracellular expression of PD-L1 and its active redistribution to cancer cell membrane may impair the therapeutic benefits of ICIs. To address this issue, herein we develop a nanodrug (MS NPs) capable of reducing PD-L1 expression and enhancing antitumor effects. Methods: The nanodrug was self-assembled from immunoadjuvant metformin (Met, an old drug) and anticancer agent 7-ethyl-10-hydroxycamptothecin (SN38) via hydrogen bonds and electrostatic interactions. A series of experiments, including the characterization of MS NPs, the validation of MS NPs-mediated down-regulation of PD-L1 expression and in vitro therapeutic effect, the MS NPs-mediated in vivo chemo-immunotherapy and tumor metastasis inhibition were carried out. Results: Different from ICIs that conformationally block PD-L1 on cancer cell membrane, MS NPs directly reduced the PD-L1 level via metformin to achieve immunotherapy. Therefore, MS NPs showed enhanced chemo-immunotherapy effect than its counterparts. MS NPs were also effective in inhibiting tumor metastasis by remodeling the extracellular matrix and restoring immune surveillance. Additionally, no obvious toxicity was observed in major organs from MS NPs-treated mice and a high survival rate of mice was obtained after MS NPs treatment. Conclusion: We have designed nanodrug MS NPs by self-assembly of the immunoadjuvant Met and the anticancer agent SN38 for combined immunotherapy and chemotherapy. MS NPs might break the deadlock of antibody-based ICIs in immunotherapy, and repurposing old drug might provide a new perspective on the development of novel ICIs.

An Advanced Computational Evaluation for the Most Biologically Active Enantiomers of Chiral Anti-Cancer Agents.

BACKGROUND: Cancer is a very dangerous disease whose treatment can be improved by removing the factors that cause side effects if the drugs prescribed for cancer are chiral in nature. OBJECTIVES: A computational evaluation for the most biologically active enantiomeric form of chiral drugs attacking the DNA of the cell, was made for the first time, and compared with the experimental work done by others previously. METHODS: All the enantiomeric structures of the drugs taken in the present study were obtained using Marvin sketch, and the structure of DNA to be docked with enantiomers, was obtained from the protein data bank. After that, all the enantiomers of the chiral drugs were docked with DNA one by one for the evaluation of the most biologically active enantiomeric form. RESULTS: The docking study showed that the different enantiomers interacted with DNA differently because of having different arrangements of atoms/groups. The binding affinity of one of the two enantiomeric forms was higher than that of another. CONCLUSION: R-methotrexate for breast cancer; R-mitotane for adrenocortical cancer; R-duvelisib for blood cancer, and S-irinotecan for colon cancer would be a suitable drug with less toxicity as well as other side effects.