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.

We report a new pH and enzyme dual responsive biodegradable polymer nanocarrier to deliver multiple anticancer drugs at the intracellular compartment in cancer cells. Natural L-aspartic acid was converted into multifunctional monomer and polymerized to yield new classes of biodegradable aliphatic polyester in-build with pH responsiveness. The transformation of side chain BOC urethanes into cationic NH 1 3 in the acidic endosomal environment disassembled the polymers nanoparticles (pH trigger-1). The biodegradation of aliphatic polyester backbone by esterase enzyme ruptured the nanoassemblies and released the drugs in the cytoplasm (trigger-2). The polymer scaffolds were capable of delivering multiple drugs such as doxorubicin, topotecan, and curcumin (CUR). The cytotoxicity of the nascent and drug-loaded nanoparticles were tested in cervical (HeLa) and breast (MCF-7) cancer cell lines. The nascent polymer nanoscaffolds were found to be nontoxic to cells whereas their drug-loaded nanoparticles exhibited excellent killing. Confocal microscopic images revealed that the drug-loaded polymer nanoparticles were taken up by the cells and the dual degradation process delivered the drugs to nucleus and established the proof-of-concept. The present investigation opens up new platform for L-amino acid based polyester scaffolds, for the first time, in the intracellular drug delivery in cancer treatment.their monomer constituents from un-natural resources. Hence, it would be more appropriate to design biodegradable polymer nanocarriers based on biological monomer resources for cancer treatment and biomedical applications. Among the natural resources, L-amino acids are particularly interesting since their sequence and structure control the three dimensional assemblies of proteins and their enzymatic action in biological system. 24,25 L-Amino acid based synthetic amphiphilic oligopeptides 26 and diblock polypeptides 27-30 were tailor-made and their supramolecular nanoscaffolds were employed for tissue engineering, 31,32 drug and gene delivery, 33,34 etc. Unfortunately, these synthetic peptides were known to encounter difficulty for biodegradation at the intracellular level. 35 Digestive enzymes such as a-chymotrypsin and trypsin (available in intestine) were found to be highly specific in action to cleave peptide sequences in proteins. For example, a-chymotrypsinenzyme selectively cleaves C-terminal of phenyl alanine, tyrosine and tryptophan peptide linkages 36-38 whereas trypsinenzyme cleaves the C-terminal of arginine and lysine in peptides. 7 Hence, it is very difficult to predict or programme the enzymatic biodegradation of synthetic polypeptides in drug Additional Supporting Information may be found in the online version of this article.

The present investigation reports the development of new classes of l-lysine based polyurethanes by a solvent and isocyanate free melt transurethane polycondensation approach. New enzyme and thermo-dual responsive amphiphilic polyurethane nanocarriers were developed for the delivery of drugs both at the intracellular level and at cancer tissue temperature. Multifunctional l-lysine monomers were tailor-made by suitably converting the amine functionalities into urethanes (or carbamates) while masking the carboxylic acid functional unit as amide pendants. The l-lysine monomers underwent melt transurethane polymerization with diols at 150 °C in the presence of catalyst to produce high molecular weight linear polyurethanes. Further, a new amphiphilic l-lysine monomer was designed with a PEG-350 chain as a pendant, and this monomer upon polymerization yielded well-defined amphiphilic aliphatic polyurethanes (APUs). The APU was found to undergo core–shell type self-assembly in aqueous medium to produce nanoparticles of size <175 nm and exhibited excellent encapsulation capabilities for anticancer drugs such as doxorubicin (DOX). The APU nanocarriers showed thermoresponsiveness from clear to turbid solution above the lower critical solution temperature (LCST) at 41–43 °C corresponding to cancer tissue temperature. At the extracellular level, the thermal-stimuli responsiveness (stimuli-1) in the APU nanocarrier was employed as trigger to deliver the DOX at cancer tissue temperature. At the intracellular level, the aliphatic urethane linkages in the APU backbone underwent lysosomal enzymatic-biodegradation (stimuli-2) to deliver DOX. Cytotoxicity studies revealed that the APU nanoparticles were not toxic to cells up to 80.0 μg/mL whereas their DOX-loaded nanoparticles accomplished more than 90% cell death in breast cancer (MCF 7) cells. Confocal microscopy and flow cytometry analysis confirmed that the l-lysine based polymer nanoparticles were readily taken up and internalized in the cancer cells. Live cell imaging using lysotracker was done to prove the intracellular biodegradation of the APU nanocarriers.

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 H 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.

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. Acidcatalyzed 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.