Molecular Self-Assembly of Bile Acid-Phospholipids Controls the Delivery of Doxorubicin and Mice Survivability

Lipid composition in general determines the drug encapsulation efficacy and release kinetics from liposomes that impact the clinical outcomes of cancer therapy. We synthesized three bile acid phospholipids by conjugating the phosphocholine headgroup to the 3′-hydroxyl group of benzylated lithocholic acid (LCA), deoxycholic acid (DCA), and cholic acid (CA); and investigated the impact of membrane rigidity on drug encapsulation efficacy, drug release kinetics, anticancer effects, and mice survival. Liposomes with a hydrodynamic diameter of 100–110 nm were subsequently developed using these phospholipids. Fluorescence-probe based quantification revealed a more fluidic nature of DCA-PC- and CA-PC-derived liposomes, whereas the LCA-PC-derived ones are rigid in nature. Doxorubicin encapsulation studies showed ∼75% encapsulation and ∼38% entrapment efficacy of doxorubicin using more fluidic DCA-PC and CA-PC derived liposomes as compared to ∼58% encapsulation and ∼18% entrapment efficacy in the case of LCA-PC derived liposomes. <i>In vivo</i> anticancer studies in the murine model confirmed that doxorubicin entrapped CA-PC liposomes compromise mice survival, whereas rigid drug entrapped LCA-PC-derived-liposomes increased mice survival with ∼2-fold decrease in tumor volume. Pharmacokinetic and biodistribution studies revealed an ∼1.5-fold increase in plasma drug concentration and an ∼4.0-fold rise in tumor accumulation of doxorubicin on treatment with drug entrapped LCA-PC liposomes as compared to doxorubicin alone. In summary, this study presents the impact of bile acid derived liposomes with different rigidities on drug delivery and mice survivability

Clathrin-Independent Killing of Intracellular Mycobacteria and Biofilm Disruptions Using Synthetic Antimicrobial Polymers

Current membrane targeting antimicrobials fail to target mycobacteria due to their hydrophobic membrane structure, ability to form drug-resistant biofilms, and their natural intracellular habitat within the confines of macrophages. In this work, we describe engineering of synthetic antimicrobial polymers (SAMPs) derived from biocompatible polyamides that can target drug-sensitive and drug-resistant mycobacteria with high selectivity. Structure–activity relationship studies revealed that reduced hydrophobicity of cationic pendants induces enhanced and selective permeabilization of mycobacterial membranes. The least hydrophobic SAMP (<b>TAC1</b>) was found to be the most active with maximum specificity toward mycobacteria over E. coli, S. aureus, and mammalian cells. Membrane perturbation studies, scanning electron microscopy, and colony PCR confirmed the ability of <b>TAC1</b> to induce membrane lysis and to bind to the genomic material of mycobacteria, thereby inducing mycobacterial cell death. <b>TAC1</b> was most effective in perfusing and disrupting the mycobacterial biofilms and was also able to kill the intracellular mycobacteria effectively without inducing any toxicity to mammalian cells. Cellular uptake studies revealed clathrin independent uptake of <b>TAC1</b>, thereby allowing it to escape hydrolytic lysosomal degradation and effectively kill the intracellular bacteria. Therefore, this manuscript presents the design and selective antimycobacterial nature of polyamide polymers with charged hydrophobic pendants that have ability to disrupt the biofilms and kill intracellular mycobacteria

Design, Synthesis, and Mechanistic Investigations of Bile Acid–Tamoxifen Conjugates for Breast Cancer Therapy

We have synthesized two series of bile acid tamoxifen conjugates using three bile acids lithocholic acid (<b>LCA</b>), deoxycholic acid (<b>DCA</b>), and cholic acid (<b>CA</b>). These bile acid–tamoxifen conjugates possess 1, 2, and 3 tamoxifen molecules attached to hydroxyl groups of bile acids having free acid and amine functionalities at the tail region of bile acids. The <i>in vitro</i> anticancer activities of these bile acid–tamoxifen conjugates show that the free amine headgroup based cholic acid–tamoxifen conjugate (<b>CA-Tam</b>_<b>3</b><b>-Am</b>) is the most potent anticancer conjugate as compared to the parent drug tamoxifen and other acid and amine headgroup based bile acid–tamoxifen conjugates. The cholic acid–tamoxifen conjugate (<b>CA-Tam</b>_<b>3</b><b>-Am</b>) bearing three tamoxifen molecules shows enhanced anticancer activities in both estrogen receptor +ve and estrogen receptor −ve breast cancer cell lines. The enhanced anticancer activity of <b>CA-Tam</b>_<b>3</b><b>-Am</b> is due to more favorable irreversible electrostatic interactions followed by intercalation of these conjugates in hydrophobic core of membrane lipids causing increase in membrane fluidity. Annexin-FITC based FACS analysis showed that cells undergo apoptosis, and cell cycle analysis showed the arrest of cells in sub G₀ phase. ROS assays showed a high amount of generation of ROS independent of ER status of the cell line indicating changes in mitochondrial membrane fluidity upon the uptake of the conjugate that further leads to the release of cytochrome <i>c</i>, a direct and indirect regulator of ROS. The mechanistic studies for apoptosis using PCR and western analysis showed apoptotsis by intrinsic and extrinsic pathways in ER +ve MCF-7 cells and by only an intrinsic pathway in ER −ve cells. <i>In vivo</i> studies in the 4T1 tumor model showed that <b>CA-Tam</b>_<b>3</b><b>-Am</b> is more potent than tamoxifen. These studies showed that bile acids provide a new scaffold for high drug loading and that their anticancer activities strongly depend on charge and hydrophobicity of lipid–drug conjugates

Design, Synthesis, and Mechanistic Investigations of Bile Acid–Tamoxifen Conjugates for Breast Cancer Therapy

We have synthesized two series of bile acid tamoxifen conjugates using three bile acids lithocholic acid (<b>LCA</b>), deoxycholic acid (<b>DCA</b>), and cholic acid (<b>CA</b>). These bile acid–tamoxifen conjugates possess 1, 2, and 3 tamoxifen molecules attached to hydroxyl groups of bile acids having free acid and amine functionalities at the tail region of bile acids. The <i>in vitro</i> anticancer activities of these bile acid–tamoxifen conjugates show that the free amine headgroup based cholic acid–tamoxifen conjugate (<b>CA-Tam</b>_<b>3</b><b>-Am</b>) is the most potent anticancer conjugate as compared to the parent drug tamoxifen and other acid and amine headgroup based bile acid–tamoxifen conjugates. The cholic acid–tamoxifen conjugate (<b>CA-Tam</b>_<b>3</b><b>-Am</b>) bearing three tamoxifen molecules shows enhanced anticancer activities in both estrogen receptor +ve and estrogen receptor −ve breast cancer cell lines. The enhanced anticancer activity of <b>CA-Tam</b>_<b>3</b><b>-Am</b> is due to more favorable irreversible electrostatic interactions followed by intercalation of these conjugates in hydrophobic core of membrane lipids causing increase in membrane fluidity. Annexin-FITC based FACS analysis showed that cells undergo apoptosis, and cell cycle analysis showed the arrest of cells in sub G₀ phase. ROS assays showed a high amount of generation of ROS independent of ER status of the cell line indicating changes in mitochondrial membrane fluidity upon the uptake of the conjugate that further leads to the release of cytochrome <i>c</i>, a direct and indirect regulator of ROS. The mechanistic studies for apoptosis using PCR and western analysis showed apoptotsis by intrinsic and extrinsic pathways in ER +ve MCF-7 cells and by only an intrinsic pathway in ER −ve cells. <i>In vivo</i> studies in the 4T1 tumor model showed that <b>CA-Tam</b>_<b>3</b><b>-Am</b> is more potent than tamoxifen. These studies showed that bile acids provide a new scaffold for high drug loading and that their anticancer activities strongly depend on charge and hydrophobicity of lipid–drug conjugates

Array-Based Sensing of Metastatic Cells and Tissues Using Nanoparticle–Fluorescent Protein Conjugates

Rapid and sensitive methods of discriminating between healthy tissue and metastases are critical for predicting disease course and designing therapeutic strategies. We report here the use of an array of gold nanoparticle–green fluorescent protein elements to rapidly detect metastatic cancer cells (in minutes), as well as to discriminate between organ-specific metastases and their corresponding normal tissues through their overall intracellular proteome signatures. Metastases established in a <i>new</i> preclinical non-small-cell lung cancer metastasis model in athymic mice were used to provide a challenging and realistic testbed for clinical cancer diagnosis. Full differentiation between the analyte cell/tissue was achieved with as little as 200 ng of intracellular protein (∼1000 cells) for each nanoparticle, indicating high sensitivity of this sensor array. Notably, the sensor created a distinct fingerprint pattern for the normal and metastatic tumor tissues. Moreover, this array-based approach is unbiased, precluding the requirement of <i>a priori</i> knowledge of the disease biomarkers. Taken together, these studies demonstrate the utility of this sensor for creating fingerprints of cells and tissues in different states and present a generalizable platform for rapid screening amenable to microbiopsy samples

Tethering of Chemotherapeutic Drug/Imaging Agent to Bile Acid-Phospholipid Increases the Efficacy and Bioavailability with Reduced Hepatotoxicity

Weakly basic drugs display poor solubility and tend to precipitate in the stomach’s acidic environment causing reduced oral bioavailability. Tracing of these orally delivered therapeutic agents using molecular probes is challenged due to their poor absorption in the gastrointestinal tract (GIT). Therefore, we designed a gastric pH stable bile acid derived amphiphile where Tamoxifen (as a model anticancer drug) is conjugated to lithocholic acid derived phospholipid (LCA-Tam-PC). <i>In vitro</i> studies suggested the selective nature of LCA-Tam-PC for cancer cells over normal cells as compared to the parent drug. Fluorescent labeled version of the conjugate (LCA-Tam-NBD-PC) displayed an increased intracellular uptake compared to Tamoxifen. We then investigated the antitumor potential, toxicity, and median survival in 4T1 tumor bearing BALB/c mice upon LCA-Tam-PC treatment. Our studies confirmed a significant reduction in the tumor volume, tumor weight, and reduced hepatotoxicity with a significant increase in median survival on LCA-Tam-PC treatment as compared to the parent drug. Pharmacokinetic and biodistribution studies using LCA-Tam-NBD-PC witnessed the enhanced gut absorption, blood circulation, and tumor site accumulation of phospholipid–drug conjugate leading to improved antitumor activity. Therefore, our studies revealed that conjugation of chemotherapeutic/imaging agents to bile acid phospholipid can provide a new platform for oral delivery and tracing of chemotherapeutic drugs