Size- and Shape-controlled Biodegradable Polymer Brushes Based on l-Amino Acid for Intracellular Drug Delivery and Deep-Tissue Penetration.

We report size- and shape-controlled polymer brushes based on l-amino acid bioresource and study the role of polymer topology on the enzymatic biodegradation and deep-tissue penetration under in vitro and in vivo. For this purpose, l-tyrosine-based propargyl-functionalized monomer is tailor-made and polymerized via solvent-free melt polycondensation strategy to yield hydrophobic and clickable biodegradable poly(ester-urethane)s. Postpolymerization click chemistry strategy is applied to make well-defined amphiphilic one-dimensional rodlike and three-dimensional spherical polymer brushes by merely varying the lengths of PEG-azides in the reaction. These core-shell polymer brushes are found to be nontoxic and nonhemolytic and capable of loading clinical anticancer drug doxorubicin and deep-tissue penetrable near-infrared biomarker IR-780. In vitro enzymatic drug-release kinetics and lysotracker-assisted real-time live-cell confocal bioimaging revealed that the rodlike polymer brush is superior than its spherical counterparts for faster cellular uptake and enzymatic biodegradation at the endolysosomal compartments to release DOX at the nucleus. Further, in vivo live-animal bioimaging by IVIS technique established that the IR-780-loaded rodlike polymer brush exhibited efficient deep-tissue penetration ability and emphasized the importance of polymer brush topology control for biological activity. Polymer brushes exhibit good stability in the blood plasma for more than 72 h, they predominately accumulate in the digestive organs like liver and kidney, and they are less toxic to heart and brain tissues. IVIS imaging of cryotome tissue slices of organs confirmed the deep-penetrating ability of the polymer brushes. The present investigation opens opportunity for bioderived and biodegradable polymer brushes as next-generation smart drug-delivery scaffolds.

Zwitterionic Strategy to Stabilize Self-Immolative Polymer Nanoarchitecture under Physiological pH for Drug Delivery In Vitro and In Vivo.

The major bottleneck in using polymer nanovectors for biomedical application, particularly those based on self-immolative poly(amino ester) (PAE), lies in their uncontrolled autodegradation at physiological pH before they can reach the intended target. Here, an elegant triblock-copolymer strategy is designed to stabilize the unstable PAE chains via zwitterionic interactions under physiological pH (pH 7.4) and precisely program their enzyme-responsive biodegradation specifically within the intracellular compartments, ensuring targeted delivery of the cargoes. To achieve this goal, biodegradable polycaprolactone (PCL) platform is chosen, and structure-engineered several di- and triblock architectures to arrive the precise macromolecular geometry. The hydrophobic-PCL core and hydrophilic anionic-PCL block at the periphery shield PAEs against autodegradation, thereby ensuring stability under physiological pH in PBS, FBS, cell culture medium and bloodstream. The clinical anticancer drug doxorubicin and deep-tissue penetrable near-infrared IR-780 biomarker is encapsulated to study their biological actions by in vitro live cancer cells and in vivo bioimaging in live animals. These zwitterions are biocompatible, nonhemolytic, and real-time in vitro live-cell confocal studies have confirmed their internalization and enzymatic biodegradation in the endo-lysosomal compartments to deliver the payload. In vivo bioimaging establishes their prolonged blood circulation for over 72 h, and the biodistribution analysis reveals the accumulation of nanoparticles predominantly in the excretory organs.

Melt Polycondensation Strategy for Amide-Functionalized l-Aspartic Acid Amphiphilic Polyester Nano-assemblies and Enzyme-Responsive Drug Delivery in Cancer Cells.

Aliphatic polyesters are intrinsically enzymatic-biodegradable, and there is ever-increasing demand for safe and smart next-generation biomaterials including drug delivery nano-vectors in cancer research. Using bioresource-based biodegradable polyesters is one of the elegant strategies to meet this requirement; here, we report an l-amino acid-based amide-functionalized polyester platform and explore their lysosomal enzymatic biodegradation aspects to administrate anticancer drugs in cancer cells. l-Aspartic acid was chosen and different amide-side chain-functionalized di-ester monomers were tailor-made having aromatic, aliphatic, and bio-source pendant units. Under solvent-free melt polycondensation methodology; these monomers underwent polymerization to yield high molecular weight polyesters with tunable thermal properties. PEGylated l-aspartic monomer was designed to make thermo-responsive amphiphilic polyesters. This amphiphilic polyester was self-assembled into a 140 ± 10 nm-sized spherical nanoparticle in aqueous medium, which exhibited lower critical solution temperature at 40-42 °C. The polyester nano-assemblies showed excellent encapsulation capabilities for anticancer drug doxorubicin (DOX), anti-inflammatory drug curcumin, biomarkers such as rose bengal (RB), and 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt. The amphiphilic polyester NP was found to be very stable under extracellular conditions and underwent degradation upon exposure to horse liver esterase enzyme in phosphate-buffered saline at 37 °C to release 90% of the loaded cargoes. Cytotoxicity studies in breast cancer MCF 7 and wild-type mouse embryonic fibroblasts cell lines revealed that the amphiphilic polyester was non-toxic to cell lines up to 100 µg/mL, while their drug-loaded polyester nanoparticles were able to inhibit the cancerous cell growth. Temperature-dependent cellular uptake studies further confirmed the energy-dependent endocytosis of polymer NPs across the cellular membranes. Confocal laser scanning microscopy assisted time-dependent cellular uptake analysis directly evident for the endocytosis of DOX loaded polymer NP and their internalization for biodegradation. In a nutshell, the present investigation opens up an avenue for the l-amino acid-based biodegradable polyesters from l-aspartic acids, and the proof of concept is demonstrated for drug delivery in the cancer cell line.

Theranostic FRET Gate to Visualize and Quantify Bacterial Membrane Breaching.

Designing new antimicrobial-cum-probes to study real-time bacterial membrane breaching and concurrently developing inquisitorial image-based analytical tools is essential for the treatment of infectious diseases. An array of aggregation-induced emission (AIE) polymers (donor) consisting of neutral, anionic, and cationic charges were designed and employed as antimicrobial theranostic gatekeepers for the permeabilization of the peptidoglycan layer-adherable crystal violet (CV, acceptor). An AIE-active tetraphenylethylene (TPE)-tagged polycaprolactone biodegradable platform was chosen, and their self-assembled tiny amphiphilic nanoparticles were employed as a gatekeeper in the construction of bacterial membrane-reinforced fluorescent resonance energy transfer (FRET) probes. Electrostatic adhering of the cationic AIE polymer and subsequent gate opening aided fluorescent FRET probe activation on the membrane of Gram-negative bacteria, Escherichia coli. The selective photoexcitation energy transfer process in confocal microscopy experiments facilitated the building of a visualization-based FRET assay for the quantification of bactericidal activity. Nonantimicrobial AIE polymers (neutral and anionic) did not breach the bacterial membrane, resulting in no FRET signal. Detailed photophysical studies were done to establish the FRET probe mechanism, and a proof of concept was established.

Structural Engineering of Star Block Biodegradable Polymer Unimolecular Micelles for Drug Delivery in Cancer Cells.

The present investigation reports the structural engineering of biodegradable star block polycaprolactone (PCL) to tailor-make aggregated micelles and unimolecular micelles to study their effect on drug delivery aspects in cancer cell lines. Fully PCL-based star block copolymers were designed by varying the arm numbers from two to eight while keeping the arm length constant throughout. Multifunctional initiators were exploited for stepwise solvent-free melt ring-opening polymerization of ε-caprolactone and γ-substituted caprolactone to construct star block copolymers having a PCL hydrophobic core and a carboxylic PCL hydrophilic shell, respectively. A higher arm number and a higher degree of branching in star polymers facilitated the formation of unimolecular micelles as opposed to the formation of conventional multimicellar aggregates in lower arm analogues. The dense core of the unimolecular micelles enabled them to load high amounts of the anticancer drug doxorubicin (DOX, ∼12-15%) compared to the aggregated micelles (∼3-4%). The star unimolecular micelle completely degraded leading to 90% release of the loaded drug upon treatment with the lysosomal esterase enzyme in vitro. The anticancer efficacies of these DOX-loaded unimolecular micelles were tested in a breast cancer cell line (MCF-7), and their IC50 values were found to be much lower compared to those of aggregated micelles. Time-dependent cellular uptake studies by confocal microscopy revealed that unimolecular micelles were readily taken up by the cells, and enhancement of the drug concentration was observed at the intracellular level up to 36 h. The present work opens new synthetic strategies for building a next-generation biodegradable unimolecular micellar nanoplatform for drug delivery in cancer research.

l-Amino Acid Based Phenol- and Catechol-Functionalized Poly(ester-urethane)s for Aromatic π-Interaction Driven Drug Stabilization and Their Enzyme-Responsive Delivery in Cancer Cells.

Exploiting aromatic π-interaction for the stabilization of polyaromatic anticancer drugs at the core of the polymer nanoassemblies is an elegant approach for drug delivery in cancer research. To demonstrate this concept, here we report one of the first attempts on enzyme-responsive polymers from aryl-unit containing amino acid bioresources such as l-tyrosine and 3,4-dihydroxy-l-phenylalanine (l-DOPA). A silyl ether protection strategy was adopted to make melt polymerizable monomers, which were subjected to solvent free melt polycondensation to produce silyl-protected poly(ester-urethane)s. Postpolymerization deprotection yielded phenol- and catechol-functionalized poly(ester-urethane)s with appropriate amphiphilicity and produced 100 ± 10 nm size nanoparticles in an aqueous solution. The aromatic π-core in the nanoparticle turns out to be the main driving force for the successful encapsulation of anticancer drugs such as doxorubicin (DOX) and topotecan (TPT). The electron-rich catechol aromatic unit in l-DOPA was found to be unique in stabilizing the DOX and TPT, whereas its l-tyrosine counterpart was found to exhibit limited success. Aromatic π-interactions between l-DOPA and anticancer drug molecules were established by probing the fluorescence characteristics of the drug-polymer chain interactions. Lysosomal enzymatic biodegradation of the poly(ester-urethane) backbone disassembled the nanoparticles and released the loaded drugs at the cellular level. The nascent polymer was nontoxic in breast cancer (MCF7) and WT-MEF cell lines, whereas its DOX and TPT loaded nanoparticles showed remarkable cell growth inhibition. A LysoTracker-assisted confocal microscopic imaging study directly evidenced the polymer nanoparticles' biodegradation at the intracellular level. The present investigation gives an opportunity to design aromatic π-interaction driven drug stabilization in l-amino acid based polymer nanocarriers for drug delivery applications.

Tertiary-Butylbenzene Functionalization as a Strategy for ß-Sheet Polypeptides.

ß-Sheet forming polypeptides are one of the least explored synthetic systems due to their uncontrolled precipitation in the ring-opening polymerization (ROP) synthetic methodology. Here, a new t-butylbenzene functionalization approach is introduced to overcome this limitation by sterically controlling the propagating polymer chains, and homogeneous polymerization with good control over chain growth was accomplished. New bulky N-carboxyanhydride monomers were designed having t-butylbenzene pendant by multistep organic synthesis, and N-heterocyclic carbene was explored as a catalyst to make high-molecular-weight and narrow polydisperse soluble polypeptides. This ROP process was successfully demonstrated for two ß-sheet forming polypeptides such as poly(l-serine) and poly(l-cysteine). These new t-butylbenzene-functionalized polypeptides were found to be readily soluble in tetrahydrofuran, chloroform, and so forth, and they were produced in high molecular weights having Mn = 32 kDa with dispersity D̵ ≤ 1.3. ROP kinetics were studied by real-time Fourier transform infrared and 1H NMR to determine the actual content of the secondary structures in the propagating chains. These studies established that the α-helical conformational front in the propagation chain was speeding up the polymerization kinetics with good degree of control in the ROP process. Reversible-conformational transitions in the post-polymerization deprotection were found to restore the ß-sheet secondary structures in poly(l-serine)s. The newly developed t-butylbenzene-substituted steric-hindrance approach is valuable in yielding soluble polymers, and this approach could be useful for exploring new polypeptide architectures for long-term impact.

Fluorescent ABC-Triblock Polymer Nanocarrier for Cisplatin Delivery to Cancer Cells.

Monitoring intracellular administration of non-luminescent anticancer drugs like cisplatin is a very challenging task in cancer research. Perylenebisimide (PBI) chromophore tagged fluorescent ABC-triblock polycaprolactone (PCL) nanoscaffold was engineered having carboxylic acid blocks for the chemical conjugation of cisplatin at the core and hydrophilic PEG blocks at the periphery. The amphiphilic ABC triblock Pt-prodrug was self-assembled into <200 nm nanoparticles and exhibited excellent shielding against drug detoxification by the glutathione (GSH) species in the cytosol. In vitro drug release studies confirmed that the Pt-prodrug was stable at extracellular conditions and the PCL block exclusively underwent lysosomal-enzymatic biodegradation at the intracellular level to release the cisplatin drug in the active-form for accomplishing more than 90% cell growth inhibition. Confocal microscopic imaging of the red-fluorescence signals from the perylene chromophores established the simultaneous monitoring and delivery aspects of the Pt-prodrug, and the proof-of-concept was successfully demonstrated in breast and cervical cancer cell lines.

Self-Reporting Polysaccharide Polymersome for Doxorubicin and Cisplatin Delivery to Live Cancer Cells.

We report self-reporting fluorescent polysaccharide polymersome nanoassemblies for enzyme-responsive intracellular delivery of two clinical anticancer drugs doxorubicin (DOX) and cisplatin to study the real-time drug-releasing aspects by fluorescent resonance energy transfer (FRET) bioimaging in live cancer cells. Fluorescent polymersomes were tailor-made by tagging an aggregation-induced emission (AIE) optical chromophore, tetraphenylethylene (TPE), and a plant-based vesicular directing hydrophobic unit through enzyme-biodegradable aliphatic ester chemical linkages in the polysaccharide dextran. The blue-luminescent polymersome self-assembled in water and exhibited excellent encapsulation capability for the red-luminescent anticancer drug DOX. FRET between the AIE polymersome host and DOX guest molecules resulted in a completely turn-off probe. At the intracellular level, the lysosomal enzymatic disassembly of the polymersome restored the dual fluorescent signals from DOX and TPE at the nucleus and the lysosomes, respectively. Live-cell confocal microscopy coupled with selective photoexcitation was employed to study the real-time polymersome disassembly by monitoring the turn-on fluorescent signals in human breast cancer cell lines. Alternatively, carboxylic acid-functionalized AIE polymersomes were also tailor-made for cisplatin stitching to directly monitor Pt drug delivery. The polymersome nanoassemblies exhibited excellent structural tolerance for the chemical conjugation of the Pt drugs, and the fluorescence signals were unaltered. An in vitro drug release study confirmed that the cisplatin-stitched fluorescent polymersomes were very stable under physiological conditions and underwent lysosomal enzymatic degradation to inhibit the cancer cell growth. A lysosomal colocalization experiment using confocal microscopy substantiates the enzyme-responsive degradation of these polymersomes to release both the encapsulated and conjugated drugs at the intracellular level. The present design provides a unique opportunity to deliver more than one anticancer drug from a single polymersome platform in cancer research.

Biodegradable Polymer Theranostic Fluorescent Nanoprobe for Direct Visualization and Quantitative Determination of Antimicrobial Activity.

We report a biodegradable fluorescent theranostic nanoprobe design strategy for simultaneous visualization and quantitative determination of antibacterial activity for the treatment of bacterial infections. Cationic-charged polycaprolactone (PCL) was tailor-made through ring-opening polymerization methodology, and it was self-assembled into well-defined tiny 5.0 ± 0.1 nm aqueous nanoparticles (NPs) having a zeta potential of +45 mV. Excellent bactericidal activity at 10.0 ng/mL concentration was accomplished in Gram-negative bacterium Escherichia coli (E. coli) while maintaining their nonhemolytic nature in mice red blood cells (RBC) and their nontoxic trend in wild-type mouse embryonic fibroblast cells with a selectivity index of >104. Electron microscopic studies are evident of the E. coli membrane disruption mechanism by the cationic NP with respect to their high selectivity for antibacterial activity. Anionic biomarker 8-hydroxy-pyrene-1,3,6-trisulfonic acid (HPTS) was loaded in the cationic PCL NP via electrostatic interaction to yield a new fluorescent theranostic nanoprobe to accomplish both therapeutics and diagnostics together in a single nanosystem. The theranostic NP was readily degradable by a bacteria-secreted lipase enzyme as well as by lysosomal esterase enzymes at the intracellular compartments in <12 h and support their suitability for biomedical application. In the absence of bactericidal activity, the theranostic nanoprobe functions exclusively as a biomarker to exhibit strong green-fluorescent signals in live E. coli. Once it became active, the theranostic probe induces membrane disruption on E. coli, which enabled the costaining of nuclei by red fluorescent propidium iodide. As a result, live and dead bacteria could be visualized via green and orange signals (merging of red+green), respectively, during the course of the antibacterial activity by the theranostic probe. This has enabled the development of a new image-based fluorescence assay to directly visualize and quantitatively estimate the real-time antibacterial activity. Time-dependent bactericidal activity was coupled with selective photoexcitation in a confocal microscope to demonstrate the proof-of-concept of the working principle of a theranostic probe in E. coli. This new theranostic nanoprobe creates a new platform for the simultaneous probing and treating of bacterial infections in a single nanodesign, which is very useful for a long-term impact in healthcare applications.

Development of l-Amino-Acid-Based Hydroxyl Functionalized Biodegradable Amphiphilic Polyesters and Their Drug Delivery Capabilities to Cancer Cells.

Hydroxyl-functionalized amphiphilic polyesters based on l-amino acid bioresources were designed and developed, and their nanoassemblies were explored as intracellular enzyme-biodegradable scaffolds for delivering anticancer drugs and fluorophores to cancer cells. To accomplish this task, acetal-masked multifunctional dicarboxylic ester monomer from l-aspartic acid was tailor-made, and it was subjected to solvent-free melt transesterification polycondensation with commercial diols to produce acetal-functionalized polyesters. Acid-catalyzed postpolymerization deprotection of these acetal-polyesters produced amphiphilic hydroxyl-functionalized polyesters. The amphiphilic polyesters were self-assembled in aqueous medium to produce nanoparticles of size <200 nm. Wide ranges of both water-soluble and water-insoluble anticancer drugs such as doxorubicin (DOX), camptothecin (CPT), and curcumin (CUR) and fluorophores such as Nile red (NR), Rose Bengal (RB), and Congo red (CR) were encapsulated in hydroxyl polyesters nanoparticles. In vitro drug release studies revealed that the aliphatic polyester backbone underwent lysosomal enzymatic-biodegradation to release the loaded cargoes at the intracellular compartments. Lysotracker-assisted live-cell confocal microscopy studies further confirmed the colocalization of the polymer nanoscaffolds in the lysosomes and supported their enzymatic-biodegradation for drug delivery. In vitro cytotoxicity studies showed that the nascent polymers were not toxic, whereas their anticancer drug-loaded nanoparticles exhibited excellent cell killing in cervical cancer (HeLa) cell lines. The drug-loaded (CPT, CUR, and DOX) and the fluorophore-loaded (NR, RB, and CR) polymer nanoparticles were highly luminescent; thus, the encapsulated polymer nanoparticles enabled the multiple color-tunable bioimaging in cancer cells in the entire visible region from blue to deep red. Time-dependent live-cell confocal microscopy studies established that the cellular uptake of drugs and fluorophores was 5 to 10-fold higher while they were delivered from the hydroxyl polyester platform. The hydroxyl polyester nanocarrier design strategy opens up new opportunities in drug delivery to cancer cells from a biodegradable polymer platform based on l-amino acids.

Enzyme-Responsive Theranostic FRET Probe Based on l-Aspartic Amphiphilic Polyester Nanoassemblies for Intracellular Bioimaging in Cancer Cells.

The present study reports one of the first attempts on the design and development of an enzymatic-biodegradable theranostic fluorescence resonance energy transfer (FRET) probe constructed on l-amino acid polymer nanoassemblies and demonstrates the proof-of-concept in live cell bioimaging. l-Aspartic acid was converted into amide or carbamate pendants containing bis-carboxylic acid ester monomers, and they were subjected to melt polymerization along with commercial diols to produce amphiphilic aliphatic polyesters. Nanoparticles of size <200 nm were obtained because of self-assembly of these amphiphilic polyesters in an aqueous medium. These nanoparticles exhibited excellent encapsulation capability for green-fluorescent anti-inflammatory drug curcumin (CUR) and highly luminescent red-fluorophore Nile red (NR) to yield a CUR-NR theranostic FRET probe. Detailed photophysical studies were carried out to demonstrate photoexcitation energy transfer from CUR to NR for the occurrence of the FRET phenomena. The theranostic FRET probe was found to be very stable at extracellular environment and underwent biodegradation at the intracellular regions for delivery of the loaded cargoes. As a result, the theranostic FRET probe functioned as turn-on at the extracellular level and became turn-off at the intracellular level under lysosomal enzyme-responsiveness. The polymer nanoparticle was nontoxic to cells, whereas its CUR encapsulated nanoparticle showed relatively good cytotoxicity in breast cancer cell lines. Live cell confocal microscopy studies using lysotracker staining confirmed the colocalization of CUR as well as NR within the polymer nanoparticles in the lysosomes for enzymatic-biodegradation. Selective photoexcitation experiments in the confocal microscope were carried out to study the FRET probe action in cancer cells. Time-dependent FRET imaging directly supported the occurrence of FRET at the intracellular level and enabled the real-time drug release studies. The present approach opens natural resource-based biodegradable theranostic FRET probes for bioimaging application.

Biotin-Tagged Polysaccharide Vesicular Nanocarriers for Receptor-Mediated Anticancer Drug Delivery in Cancer Cells.

Biotin-conjugated multistimuli-responsive polysaccharide vesicular nanocarriers are designed and developed, for the first time, to accomplish receptor-mediated endocytosis in cancer cells and to deliver anticancer drugs to intracellular compartments. For this purpose, a new renewable hydrophobic unit was custom designed with redox-degradable disulfide and enzyme-biodegradable aliphatic ester chemical linkages, and it was conjugated along with biotin on the dextran backbone. The dextran derivative self-assembled into nanovesicles of <200 nm in size, which were characterized by dynamic and static light scattering, electron, and atomic force microscopes. Avidin-HABA assay established the high affinity of biotin-tagged dextran vesicles toward membrane-receptors up to 25 nM concentration. Doxorubicin-hydrochloride (DOX.HCl)-loaded dextran vesicles exhibited stable formulation in phosphate-buffered saline (PBS) and fetal bovine serum (FBS). Redox-degradation by glutathione (GSH) showed 60% drug release, whereas lysosomal esterase enzyme enabled >98% drug release in 12 h. Confocal microscope and flow cytometry-assisted time-dependent cellular uptake studies revealed that the biotin-receptors overexpressed in cervical cancer cells (HeLa) exhibited larger drug accumulation through the receptor-assisted endocytosis process. This process enabled the delivery of higher amount of DOX and significantly enhanced the killing in cancer cells (HeLa) compared to wild-type mouse embryonic fibroblast cells (WT-MEF, normal cells). Control experiments such as biotin pretreatment in cancer cells and energy-suppressed cellular uptake at 4 °C further supported the occurrence of receptor-mediated endocytosis by the biotin-tagged polymer vesicles. This report provides first insights into the targeted polysaccharide vesicle platform, and the proof-of-concept is successfully demonstrated in biotin receptor-overexpressed cervical cancer cells.

Polymer Nanovesicle-Mediated Delivery of MLN8237 Preferentially Inhibits Aurora Kinase A To Target RalA and Anchorage-Independent Growth in Breast Cancer Cells.

The small GTPase RalA is a known mediator of anchorage-independent growth in cancers and is differentially regulated by adhesion and aurora kinase A (AURKA). Hence, inhibiting AURKA offers a means of specifically targeting RalA (over RalB) in cancer cells. MLN8237 (alisertib) is a known inhibitor of aurora kinases; its specificity for AURKA, however, is compromised by its poor solubility and transport across the cell membrane. A polymer nanovesicle platform is used for the first time to deliver and differentially inhibit AURKA in cancer cells. For this purpose, polysaccharide nanovesicles made from amphiphilic dextran were used as nanocarriers to successfully administer MLN8237 (VMLN) in cancer cells in 2D and 3D microenvironments. These nanovesicles (<200 nm) carry the drug in their intermembrane space with up to 85% of it released by the action of esterase enzyme(s). Lysotracker experiments reveal the polymer nanovesicles localize in the lysosomal compartment of the cell, where they are enzymatically targeted and MLN released in a controlled manner. Rhodamine B fluorophore trapped in the nanovesicles hydrophilic core (VMLN+RhB) allows us to visualize its uptake and localization in cells in a 2D and 3D microenvironment. In breast cancer, MCF-7 cells VMLN inhibits AURKA significantly better than the free drug at low concentrations (0.02-0.04 µM). This ensures that the drug in VMLN at these concentrations can specifically inhibit up to 94% of endogenous AURKA without affecting AURKB. This targeting of AURKA causes the downstream differential inhibition of active RalA (but not RalB). Free MLN8237 at similar concentrations and conditions failed to affect RalA activation. VMLN-mediated inhibition of RalA, in turn, disrupts the anchorage-independent growth of MCF-7 cells supporting a role for the AURKA-RalA crosstalk in mediating the same. These studies not only identify the polysaccharide nanovesicle to be an improved way to efficiently deliver low concentrations of MLN8237 to inhibit AURKA but, in doing so, also help reveal a role for AURKA and its crosstalk with RalA in anchorage-independent growth of MCF-7 cells.

Multistimuli-Responsive Amphiphilic Poly(ester-urethane) Nanoassemblies Based on l-Tyrosine for Intracellular Drug Delivery to Cancer Cells.

Multistimuli-responsive l-tyrosine-based amphiphilic poly(ester-urethane) nanocarriers were designed and developed for the first time to administer anticancer drugs in cancer tissue environments via thermoresponsiveness and lysosomal enzymatic biodegradation from a single polymer platform. For this purpose, multifunctional l-tyrosine monomer was tailor-made with a PEGylated side chain at the phenolic position along with urethane and carboxylic ester functionalities. Under melt dual ester-urethane polycondensation, the tyrosine monomer reacted with diols to produce high molecular weight amphiphilic poly(ester-urethane)s. The polymers produced 100 ± 10 nm spherical nanoparticles in aqueous medium, and they exhibited thermoresponsiveness followed by phase transition from clear solution into a turbid solution in heating/cooling cycles. Variable temperature transmittance, dynamic light scattering, and 1H NMR studies revealed that the polymer chains underwent reversible phase transition from coil-to-expanded chain conformation for exhibiting the thermoresponsive behavior. The lower critical solution temperature of the nanocarriers was found to correspond to cancer tissue temperature (at 42-44 °C), which was explored as an extracellular trigger (stimuli-1) for drug delivery through the disassembly process. The ester bond in the poly(ester-urethane) backbones readily underwent enzymatic biodegradation in the lysosomal compartments that served as intracellular stimuli (stimuli-2) to deliver drugs. Doxorubicin (DOX) and camptothecin (CPT) drug-loaded polymer nanocarriers were tested for cellular uptake and cytotoxicity studies in the normal WT-MEF cell line and cervical (HeLa) and breast (MCF7) cancer cell lines. In vitro drug release studies revealed that the polymer nanoparticles were stable under physiological conditions (37 °C, pH 7.4) and they exclusively underwent disassembly at cancer tissue temperature (at 42 °C) and biodegradation by lysosomal-esterase enzyme to deliver 90% of DOX and CPT. Drug-loaded polymer nanoparticles exhibited better cytotoxic effects than their corresponding free drugs. Live cell confocal microscopy imaging experiments with lysosomal tracker confirmed the endocytosis of the polymer nanoparticles and their biodegradation in the lysosomal compartments in cancer cells. The increment in the drug content in the cells incubated at 42 °C compared to 37 °C supported the enhanced drug uptake by the cancer cells under thermoresponsive stimuli. The present work creates a new platform for the l-amino acid multiple-responsive polymer nanocarrier platform for drug delivery, and the proof-of-concept was successfully demonstrated for l-tyrosine polymers in cervical and breast cancer cells.

π-Conjugate Fluorophore-Tagged and Enzyme-Responsive l-Amino Acid Polymer Nanocarrier and Their Color-Tunable Intracellular FRET Probe in Cancer Cells.

The present investigation accounts one of the first example of enzyme-responsive and π-conjugate-tagged l-amino acid amphiphilic polymer and their fluorescence resonance energy transfer (FRET) probes for color-tunable intracellular bioimaging in cancer cells. Melt polymerizable oligo-phenylenevinylene (OPV) π-conjugated diol was tailor-made and subjected to thermo-selective melt transesterification reaction with multifunctional l-aspartic acid monomer to yield OPV-tagged amphiphilic luminescent polyesters. These amphiphilic polyesters self-assembled through strong aromatic π-π stacking and hydrophilic/hydrophobic noncovalent forces into <200 nm size blue-luminescent nanoparticles in aqueous medium. The OPV-tagged polymer nanoparticles served as FRET donor and encapsulated water insoluble Nile Red (NR) fluorophore as a FRET acceptor. Detail photophysical studies revealed that both the OPV and NR were confined within Förster distance in the polymer nanocontainer and the nanodomains provided appropriate geometry for efficient excitation energy transfer from OPV to NR. Cytotoxicity studies in breast cancer (MCF 7), cervical cancer (HeLa) and normal (Wild-type MEF) cell lines revealed that both the nascent luminescent OPV nanoparticles and OPV-NR FRET probes were nontoxic to cells up to 100 µg/mL. Confocal microscope images confirmed the efficient transportation of polymer and FRET probes across the cell membranes and their preferable accumulation in the cytoplasm of the cells. Lysosomal tracker assisted live cell imaging provided direct evidence for the localization of the polymer nanoparticles at the lysosomal compartments in the cytoplasm. In vitro enzyme-responsive studies revealed that the aliphatic polyester backbone in the polymer nanoparticles was readily biodegradable by lysosomal enzymes like esterase, chymotrypsin, trypsin, and also redox GSH species in the cytoplasm. Selective photoexcitation in confocal microscope exhibited bright OPV blue-luminescence and strong red-emission from NR followed by the excitation energy transfer and occurrence of FRET process at the intracellular environment in cancer cell lines. Both the polymer design and the biodegradable polymer FRET concept are completely new; thus, the present approach opens up new platform of research opportunities for natural l-amino acid based luminescent polymer probes for color-tunable bioimaging in cancer cells.

Development of l-Tyrosine-Based Enzyme-Responsive Amphiphilic Poly(ester-urethane) Nanocarriers for Multiple Drug Delivery to Cancer Cells.

New classes of enzymatic-biodegradable amphiphilic poly(ester-urethane)s were designed and developed from l-tyrosine amino acid resources and their self-assembled nanoparticles were employed as multiple drug delivery vehicles in cancer therapy. The amine and carboxylic acid functional groups in l-tyrosine were converted into dual functional ester-urethane monomers and they were subjected to solvent free melt polycondensation with hydrophilic polyethylene glycols to produce comb-type poly(ester-urethane)s. The phenolic unit in the l-tyrosine was anchored with hydrophobic alkyl side chain to bring appropriate amphiphilicity in the polymer geometry to self-assemble them as stable nanoscaffolds in aqueous medium. The topology of the polymer was found to play a major role on the glass transition, crystallinity, and viscoelastic rheological properties of l-tyrosine poly(ester-urethane)s. The amphiphilic polymers were self-assembled as 200 ± 10 nm nanoparticles and they exhibited excellent encapsulation capabilities for anticancer drugs such as doxorubicin (DOX) and camptothecin (CPT). In vitro drug release studies revealed that the drug-loaded l-tyrosine nanoparticles were stable at extracellular conditions and they underwent enzymatic-biodegradation exclusively at the intracellular level to release the drugs. Cytotoxicity studies in the cervical cancer (HeLa) and normal WT-MEFs cell lines revealed that the nascent l-tyrosine nanoparticles were nontoxic, whereas the CPT and DOX drug-loaded polymer nanoparticles exhibited excellent cell killing in cancer cells. Confocal microscopic imaging confirmed the cellular internalization of drug-loaded nanoparticles. The drugs were taken up by the cells much higher quantity while delivering them from l-tyrosine nanoparticle platform compared to their free state. Flow cytometry analysis showed that the DOX-loaded polymer nanoscaffolds internalized the drugs 8-10× higher compared to free DOX. Both the synthesis of new classes of poly(ester-urethane)s via melt polycondensation approach and the enzyme-responsive drug delivery concept were accomplished for the first time. Thus, the present investigation is expected to open up new opportunities for l-tyrosine polymeric materials in biomaterial and thermoplastic applications.

Cisplatin-Stitched Polysaccharide Vesicles for Synergistic Cancer Therapy of Triple Antagonistic Drugs.

New cisplatin-stitched polysaccharide vesicular nanocarrier is developed for combination therapy of three clinical important antagonistic drugs together to accomplish synergistic cancer therapy in breast cancer treatment. Carboxylic functionalized dextran was tailor-made for the chemical conjugation of cisplatin, and a renewable hydrophobic unit was anchored in the backbone to interdigitize the chains to self-assemble as cisplatin-stitched polysaccharide nanovesicles. Water-soluble DNA-intercalating drug doxorubicin·HCl (DOX) and water insoluble topoisomerase type I inhibitor drug camptothecin (CPT) were encapsulated in these vesicles to produce dual or triple drug-loaded vesicular nanocarrier. This unique cisplatin, DOX and CPT triple drug-loaded dextran vesicles were stable in aqueous medium, and the vesicular geometry acted as a shield for Pt-polymer drug conjugate against glutathione (GSH) detoxification under physiological conditions. Lysosomal enzymes ruptured the nanovesicle exclusively at the intracellular compartments to deliver the combination of all three drugs simultaneously to maximize the therapeutic efficacies. In vitro cytotoxicity studies revealed that free cisplatin was highly detoxified by the GSH in breast cancer cells, whereas the enhanced stability of Pt-stitched dextran vesicle against GSH facilitated ∼99% cell killing in breast cancer cells. Combination therapy studies revealed that the free cisplatin, DOX, and CPT were found to be antagonistic to each other. Dual drug-loaded vesicles exhibited synergistic cancer cell killing while delivering these antagonistic drugs from a dextran vesicular platform. Remarkable synergistic cell killing was accomplished in cisplatin, DOX, and CPT triple drug-loaded vesicles at nanogram concentrations in breast cancer cells. The internalization of drugs and cellular uptake were confirmed by confocal microscope and flow cytometry analysis. The drugs were taken by the cancer cells in large amounts while delivering them from dextran vesicles compared to their free form. These spectacular results opened new opportunities for synergistic cancer therapy for GSH-overexpressed breast cancer using triple drug-loaded polysaccharide vesicular nanocarriers.

Fluorescent-Tagged Biodegradable Polycaprolactone Block Copolymer FRET Probe for Intracellular Bioimaging in Cancer Cells.

The present investigation reports a new fluorophore-tagged biodegradable polycaprolactone (PCL) block copolymer FRET-probe for intracellular imaging in cancer therapy. A hydroxyl functionalized π-conjugated oligo-phenylenevinylene (OPV) chromophore was tailor-made, and it was incorporated in a t-butyl ester substituted polycaprolactone block copolymer via ring opening polymerization. This blue-luminescent OPV-PCL triblock self-assembled as <200 nm spherical nanoparticles (FRET donor), and it encapsulated water insoluble Nile red (NR, FRET acceptor) to yield an OPV-NR FRET probe. Selective photo excitation of the OPV chromophore in block nanoassemblies enabled the excitation energy transfer from the OPV to NR and facilitated the efficient FRET process in aqueous medium. Time-correlated fluorescent decay dynamics and detailed photophysical studies were carried out to estimate the Förster distance, donor-acceptor distance, and the excitation energy transfer efficiency. These parameters confirmed the occurrence of the FRET process within the confined nanoparticle environment. The PCL chains in the FRET probe were susceptible to enzymatic biodegradation in intracellular environments, and the degradation process controlled the FRET on/off mechanism. Cytotoxicity studies revealed that the FRET probe was biocompatible and nontoxic to cells, and the FRET-probe was found to be readily taken up by the cancer cells, and it was internalized in the cytoplasm and peri-nuclear environment. Selective photoexcitation of the OPV chromophore in a confocal microscope exhibited dual emission from the FRET probe. The cancer cells exhibited blue luminescence (self-emission) with respect to the OPV chromophore (in the blue channel) and bright red-luminescence from the NR dye followed by the FRET process at the cellular level (in the red channel). The dual luminescence characteristics, biodegradation and biocompatibility, make the newly designed PCL-OPV-NR FRET probe an excellent biomedical nanodevice for bioimaging applications, and the proof-of-concept was established in cervical (HeLa) and breast cancer (MCF 7) cell lines.

Triple Block Nanocarrier Platform for Synergistic Cancer Therapy of Antagonistic Drugs.

A unique biodegradable triple block nanocarrier (TBN) is designed and developed for synergistic combination therapy of antagonistic drugs for cancer treatment. The TBN was built with hydrophilic polyethylene glycol (PEG) outer shell; a middle hydrophobic and biodegradable polycaprolactone (PCL) block for encapsulating anthracycline anticancer drug like doxorubicin (DOX), and an inner carboxylic-functionalized polycaprolactone (CPCL) core for cisplatin (CP) drug conjugation. TBN-cisplatin drug conjugate self-assembled as stable nanoparticles in saline (also in PBS) wherein the hydrophobic PCL block functions as a shield for Pt-drug stability against GSH detoxification. Enzymatic-biodegradation of TBN exclusively occurred at the intracellular environment to deliver both cisplatin (CP) and doxorubicin (DOX) simultaneously to the nucleus. As a result, the TBN-cisplatin conjugate and its DOX-loaded nanoparticles accomplished 100% cell growth inhibition in GSH overexpressed breast cancer cells. Combination therapy revealed that free drugs were antagonistic to each other, whereas the dual drug-loaded TBN exhibited excellent synergistic cell killing at much lower drug concentrations in breast cancer cells. Confocal microscopic analysis confirmed the localization of drugs in the cytoplasm and at peri-nuclear site. Flow cytometry analysis revealed that the drugs were taken up 4-fold better while delivering them from TBN platform compared to free form. The TBNs approach is a perfect platform to overcome the GSH detoxification in Pt-drugs and enable the codelivery of antagonistic drugs like cisplatin and DOX from single polymer dose to accomplish synergistic killing in breast cancer cells.