Sustainable Biodegradable Polymers
Engineering macromolecular architectures from natural resources and to restore the constituent elements back to the nature at end-of life is an essential prerequisite for the development of sustainable polymer products for both commercial and biomedical applications. A new eco-friendly melt polycondensation approach was developed by our group for L-amino acid resources under solvent-free conditions. In this new process, L-amino acids were readily converted into dual ester-urethane monomers and polycondensed with diols under melt conditions to produce high molecular weight polymers as shown in Figure 1a. The methodology is readily applicable to a wide range of L-amino acid monomers. Under the melt polycondensation process, these monomers produce new entities of poly(ester-urethane)s that are thermally stable and high molecular weights associated with tuneable thermal properties. Structural engineering of L-tyrosine monomer with suitable hydrophilic and hydrophobic side chains enabled the development of thermo-responsive polymers along with biodegradation to deliver anticancer drugs at the tumour specific temperature range. L-Aspartic acid was masked with amide-functionalized acetal to carryout exclusively melt transesterification to yield acetal-masked polyester. Recently, this stagey is expanded to two bio-resources namely L-amino acids and sugars in a single synthetic process to build long-chain amphiphilic polymers that render complete biodegradation under lysosomal enzyme-stimuli while administrating the desired cargoes at the intracellular compartments and reinstate the feedstock with 100 % atom economy. D-Mannitol (sugar) was readily converted into melt-polymerizable five-membered and six-membered bicyclic diacetalized diols and polymerized with L-amino acid monomers to yield new classes of aliphatic and hybrid poly(ester-urethane)s. In vitro release kinetics established more than 90% of the loaded cargoes under the lysosomal enzymatic-biodegradation of the polymers into L-amino acid and sugar monomeric units while the polymers exhibited very good biocompatibility up to 250 mg/mL in wild-type MEFs cell lines. The above L-amino acid polymer based synthetic methodology opens a new platform that supports the concept for the need for the development of fully bio-resource based polymers for long-term application.
Figure 1: Development of New Polymer Chemistry: (a) Development of melt polycondensation strategy for L-Amino acid-based enzymatic-biodegradable non-peptide polymers. Real-time intracellular biodegradation of the above polymer nano-assemblies and delivery of DOX to the nucleus in live-cells confocal microscope studies. (b) Development of steric-hindrance ring opening polymerization strategy to access high molecular weight β-sheet polypeptides and their unexplored block copolymer polyelectrolyte architectures.
β-Sheet forming polypeptides are one of the least explored synthetic systems and this main restriction is associated with the uncontrollable-precipitation of the polymer chains during their synthesis. As a result, the b-sheet polymerization was found to be “non-living” with dead propagation chains and they were inefficient for the synthesis of block copolymer architectures. This important problem in polypeptide area is addressed by us by developing new steric-hindrance ring opening polymerization strategy by controlling the helicity of the propagating polymer chains as shown in Figure 1b. New bulky N-carboxyanhydride (NCA) monomers were designed having t-butylbenzene pendant by multi-step organic synthesis to make high molecular weight and narrow polydisperse soluble polypeptides. This ROP process was successfully demonstrated for two b-sheet forming polypeptides such as poly(ʟ-serine), poly(ʟ-cysteine) and poly(ʟ-tyrosine). It was established that a-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 b-sheet secondary structures in poly(ʟ-serine)s. Furthermore, the scope of this approach was expanded for the building inaccessible block copolymer macromolecular architectures. 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.
Anticancer Drug Deliver to Tumors
Macromolecular drug delivery platform came into existence in 1975 wherein the conventional chemotherapeutic drugs are being replaced by their nano-formulations. These formulations were developed to overcome the limitations of the small molecule drugs including reduced bioavailability, systemic toxicity, early clearance, and immunogenicity. Several factors such as imperfect polymer design, instability of prodrug nano-formulations under blood circulation, premature release of cargoes prior reaching the target, fast excretion via renal filtration apparatuses, and most decisively non-biodegradability under physiological conditions are noticed to be bottlenecks limiting the efficacies under in vivo. Self-assembled nanoparticles of polymers (or small molecules) in aqueous medium are resultant of multi-molecular association and exits as aggregated micellar nanoparticles (AMNp). AMNps are susceptible to undergo disassembly below their critical micellar concentration (CMC, or critical association constant). As a consequence, a high concentration of injected drug formulation (above CMC) inevitably experience 100 to 1000 times dilution under blood steam. Hence, designing new branched macromolecular architectures imbibed with single polymer chain or unimolecular micelle nanoparticle (UMNp) self-assembly is an elegant strategy for in vivo administration of drug molecules. The UMNp, in principle, would be devoid of the destabilization against concentration gradient under in vivo. In this case, the increase (or decrease) in the polymer concentration would simply vary the number of nanocarriers in solutions, as in the case of natural proteins in aqueous medium. This fundamental problem at chemistry-biology interface is addressed here by carefully structure-engineering lysosomal-enzyme biodegradable star-AMNp and nano-compartmentalized star-UMNp in a single system (for the first time) and study their drug delivering capability in vitro and in vivo mice xenograft tumor models as shown in Figure 2.
Figure 2. (a) Structural-engineering of unimolecular micelles in star block copolymers by modulating amphiphilicity and self-assembly aspects. (b) Encapsulation capability of unimolecular micelles. (c) In-vitro evaluation of aggregated and unimolecular micelles for biodegradation in living cells. (d) Tumor xenograft models for the live-animal bioimaging and their ability to suppress the solid tumor.
The star polymers were built using two aliphatic polycaprolactone (PCL) segments having slow-biodegradable hydrophobic PCL chains and relatively fast-biodegradable anionic-charged carboxylic PCL chains, as core and shell, respectively. A 6-arm star-architecture was designed with a fixed hydrophobic PCL core and variable chain length anionic-cum-hydrophilic carboxylic PCL shell. With increase in the shell length, the star polymers were found to undergo self-assembly transformation from AMNp to UMNP as shown in Figure 2a. The core-shell star-UMNp of < 25 nm in size exhibited excellent encapsulation capabilities for clinical anticancer drug doxorubicin (DOX), fluorescent dyes such as HPTS and Sulforhodamine-B, deep-tissue penetrable near-infra red fluorescent dye ICG (water soluble) and IR-780 (water insoluble) to study biodistribution in live-animals (see Figure 2b). The star-UMNp exhibited excellent drug action in multiple cancer cell lines at substantially low IC50 values. The efficacy of the UMNp platform is further validated in Mia PaCa 2 tumor-bearing mice model (see Figure 2c). This effort opens new avenue for UMNp as an efficient platform in drug delivery applications and also provides the direct account on its superiority in tumor regression.
Figure 3: Schematic illustrating the application of star unimolecular micellar nano scaffold for the delivery of MLN8237 and targeting of AURKA-RalA crosstalk in an in vivo model. (A) Nanotherapeutic design strategy for the efficient delivery of MLN8237 in Ras-independent and Ras-dependent xenograft tumors in mice. (B) RalA signaling regulated by its phosphorylation at Ser194 residue by AURKA could affect anchorage-independent growth and tumorigeneses. (C) Cellular uptake of UMNp and enzymatic release of MLN8237 (NPMLN) causes specific AURKA inhibition, which disrupts RalA phosphorylation (pSer194) and suppresses anchorage-independent growth and tumorigenesis.
Targeting Aurora Kinase A (AURKA) to modulate RalA activation offers a promising strategy for tumor suppression in Ras-independent and Ras-dependent cancers. However, the clinical use of the AURKA inhibitor MLN8237 (Alisertib) is limited by its hydrophobicity and poor water solubility. To overcome these limitations, here, we developed an enzyme-biodegradable unimolecular micelle (UMM) nanoparticle (NPMLN) to deliver MLN8237 and evaluated its therapeutic efficacy in tumor xenograft models (this work was done in collaboration with Dr. Nagaraj Balasubramanian at IISER Pune). The star-UMM was built having hydrophobic and hydrophilic biodegradable polycaprolactone backbones in the core and shell, respectively; thus, upon degradation, the polymers were completely chopped into small entities, leaving limited to no cytotoxicity to the living system. NIR dye-loaded star UMM probes from our lab established that the UMM was stable under circulation for more than 96 hours, non-toxic, non-hemolytic, and non-immunogenetic. In the present investigation, the unique biodegradable PCL UMM is explored for the delivery of AURKA inhibitor into in vivo tumor xenograft model, for the first time, in the literature. The star UMM core-shell NP design is explored for the loading of a very high content of MLN8237 (DLC = 4.8 %) to produce stable nanoformulation for in vivo administration in a tumor xenograft model. This six-arms-based UMM was designed according to the procedure described in our previous study and it is used to deliver MLN8237 (NPMLN), as shown in (Figure 3A). With improved drug loading, NPMLN allowed efficient uptake and specific targeting of AURKA at lower concentrations than previously reported without affecting AURKB. Enhanced bioavailability and retention of NPMLN improved its therapeutic potential in targeting AURKA and pSer194RalA for regressing tumor growth in Ras-independent (SKOV3) and Ras-dependent (MIA PaCa-2) cancers (Figure 3B and 3C). Therefore, this study offers novel insight into AURKA-RalA crosstalk in the presence and absence of oncogenic Ras, while suggesting a method to increase bioavailability and reduce toxicity and side effects caused by high doses of MLN8237.
Drug Delivery Across Blood Brain Barrier
Drug delivery across the tightly regulated vasculature of the blood-brain barrier (BBB) in treating tumors and neurodegenerative diseases has been a major bottleneck. The BBB maintains the homeostatic balance by regulating the transport of small-molecule nutrients and ions through the vasculature into the brain; thus, restricting the transportation of larger size and extraneous species across this biological barrier. Uncontrollable glomerular renal filtration of the smaller (< 15 nm) nanocarriers, splenic and hepatic filtration of bigger NPs (> 200 nm) in the body limits the drug NP formulation concentration in the intravenous administration, which in turn reduces the bioavailability of NP for BBB crossing. Thus, the next generation BBB crossing nanocarriers are mandatorily designed to be substantially stable to evade disassembly in body fluidics in vivo, tiny-size sub-nanometer objects (< 50 nm) for penetrating the tightly regulated t-junction, high drug loading content, reduced cardiotoxicity, and most importantly biodegradable for safe usage in BBB research. Star-block copolymers provide excellent structural control to build well-defined core-shell nanoparticles. Persistent to their three-dimensional globular core-shell geometry, star polymers often exist as unimolecular micelles which is highly desirable for in vivo drug administration to maintain the drug NP against concentration gradient in the bloodstream. Here, these unique features of the star-polymer unimolecular micelles (star-UMNp) are explored for BBB research based on biodegradable polymer nanovector, for the first time, and the proof-of-concept was successfully demonstrated in vivo for clinically important anticancer drug doxorubicin (DOX) and brain-tissue penetrable near-infrared (NIR) biomarker IR-780. This new strategy is shown in Figure 4.
Figure 4. New Strategy for Blood Brain Barrier Breaching: Design and development of biodegradable star block copolymer unimolecular micelles (UMM) for brain specific delivery.
The in vivo biodistribution data established the supremacy of the star-UMM platform for crossing the BBB with reduced side-effect of cardiotoxicity. Further, microtubule assisted protein 2 (MAP 2), neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) immunostaining were employed to mark the different cell types across the brain tissue to ascertain the neural uptake of star-UMM. The present approach opens up new avenue of research opportunities based on biodegradable star polymer macromolecules as potential futuristic single molecular like star-UMM nanoparticle to breach blood brain barrier and useful for long-term brain-specific drug delivery. The work gave a fundamental insight into the effect of polymer topology on all aspects of self-assembly, in vitro cellular internalization and in vivo biodistribution and pharmacokinetics. These polycaprolactone block copolymers were non-immunogenic with no hemolysis to the RBCs and no toxicity to the tissues. The surface charge variation led to difference in the endocytosis mechanism across different cell types.
Antimicrobial Polymers and Diagnostics
Treatment of infectious diseases caused by bacteria and virus are emerging as the major challenges in addressing the healthcare issues and society at the large. Continually increasing multi-drug resistance and steadfast mutations of microorganisms severely obstruct the treatment with existing drugs or antibiotics, and this also further limit the existing diagnostic methods in the fast detection of infectious diseases. This emphasis that the next generation candidates are essential to be smart multitasking probes having capability to exhibit both therapeutics and diagnostics together (theranostics) in singe dose of drug administration. It is also important to note that the probe molecules come in direct contact with the healthy tissues (or cells), proteins and red blood cells (RBCs) while testing their activity which further introduce unwanted toxicity issues. Therefore, the incorporation of biodegradation property is essential component in theranostic probe design for safer biomedical applications especially in treating the bacterial infections. To address this important problem, we have developed new biodegradable theranostic antibacterial nanoprobes” in enzymatic-biodegradable cationic polycaprolactone (PCL) platform. The theranostic nanoprobe was successfully employed for direct visualization and qualitative determination of bactericidal activity by the newly developed fluorescent assay methodology which could distinguish live and dead bacteria in real case scenario. Hence, the new approach reports new biodegradable antibacterial polymer (as drug) as well as fluorescent imaging probe to real-time visualization and treatment of infections as shown in Figure 5a.
Figure 5: (a) Structural engineering of new cationic polycaprolactone based biodegradable fluorescent theranostic nano-probes for direct visualization and estimation of antibacterial activity. (b) Prokaryotic and eukaryotic biological species and their diverse membrane architectures. (c) Tweaking polymer topology of linear and star macromolecules having identical chemical compositions, mass, size, surface charge and their effect on biological systems
The cationic polymer fluorescent theranostic nanoprobe was built on aliphatic polyester backbone (as shown in Figure 5a); thus, it is readily susceptible to undergo two types of enzymatic-biodegradation: (a) by bacteria-secreted lipase enzyme bio-degradation of the polymer backbone during their encounter with microorganism for antimicrobial activity, and (b) by lysosomal enzymes at the intracellular compartments of endothelial cells in the event of their unwanted encounter with mammalian cells in healthy tissues. The cationic polymers were found to be non-toxic to mammalian cells and they barely interacted with the RBCs and are largely non-hemolytic in blood. The loading of anionic fluorescent marker HPTS yielded green-fluorescent theranostic polymer probe to differentiate the live or dead bacteria by green or orange-color (co-stained with PI) fluorescence, respectively. Bio-imaging protocols in confocal microscope analysis enabled the quantitative estimation of the degree of the bacterial killing by direct visualization. Selective photo excitation experiments in confocal microscope were coupled with time-dependent incubation assay to establish the proof-of-concept with very good accuracy. This new concept and the custom designed theranostic probe has very good potential for long-term implication in diagnostics and therapeutics in treating the infectious diseases. Cationic anti-microbial polymers direct encounter with prokaryotic and eukaryotic species are important problem to be addressed for safe treatment of microbial infections. Single-celled prokaryotic microbes like E. coli (gram-negative bacteria), and eukaryotic red-blood cells (RBCs) and mammalian cells are present in our biological body. Therefore. it is essential to design next generation macromolecular nanosystems that are less toxic or non-toxic to eukaryotic species while employing them for antimicrobial activity in prokaryotic species (Figure 5b). Prokaryotic cell membranes (for e.g., E. coli) are multi-layered, rigid, and anionic in nature (x = -22 ± 3.0 mV); thus, the selection of cationic polymers having appropriate amphiphilicity become a natural choice to electrostatically adhere and persuade membrane disruption in bacterial cells. The RBC membrane is slightly less negative (x = -15 ± 0.1 mV) compared to microbial membranes whereas the mammalian cell membranes are zwitterionic (x = neutral), and therefore, the cationic polymers need to navigate through these membranes potential difference to avoid unwanted toxicity towards RBCs and healthy cells or tissues. We have reported an elegant strategy to tweak the topology of one dimensional linear di-block and three-dimensional star di-block cationic polymer having identical chemical compositions, molecular weights, size, and charge; however, they differ substantially in their biological action in vitro and in vivo towards prokaryotic and eukaryotic species (Figure 5c).
Development of Intracellular FRET BioProbes
Fluorescent polymer nano-assemblies are emerging as important class of biomaterials for delivering therapeutic agents at the intracellular compartments and simultaneously probing the macromolecular nano-carriers accumulation at tumor micro-environments by bioimaging techniques. The AIE chromophores are excellent system to exhibit strong Fluorescence characteristics in aqueous medium; hence, the development of new the AIE-assisted stimuli-responsive fluorescent polymersomes would open up new opportunities to study the real-time drug delivery process and would also open up new avenue for many unexplored biological applications. Our research group has engineered bio-resourced based polysaccharide polymersomes by conjugating plant-based hydrophobic unit 3-pentadecyl phenol (PDP, from cashew nut shell liquid) as vesicular directing component on the dextran backbone to bring the appropriate geometry for polymersome self-assembly in aqueous medium. The PDP hydrophobic handle is very versatile in producing enzyme, pH and GSH responsive polymersome depending upon the choice of the chemical linkages chosen for the stimuli-responsiveness at the cellular level. These polysaccharide polymersomes were found to be efficient host for delivering water-soluble doxorubicin, water insoluble camptothecin, and also metal based cisplatin anticancer drugs together in single nanoplatform. Fluorescence resonance energy transfer (FRET) is one of the widely explored photophysical process in fluorescent polymers to study the polymer chain interactions, stimuli-responsive drug release, and so on so forth. The FRET concept is also brought in the present strategy to engineer next generation smart enzyme-responsive fluorescent polysaccharide polymersome FRET probes for cancer research as shown in Figure 6.
Figure 6. Development of AIE-active polysaccharide polymersomes and their enzyme-responsive FRET fluorescent probes for monitoring intracellular level delivery in live-cells.
Briefly, fluorescent polysaccharide polymersomes were tailor-made by conjugating AIE-active TPE chromophore and renewable PDP acid together on the dextran backbone via aliphatic ester linkage for enzymatic biodegradation. Thus, the fluorescent polymersome in the present investigation is designed with enzyme-trigger to study the real-time delivering capabilities of the fluorescent polymersomes which will be more useful for futuristic applications. Water soluble (Rose Bengal) and water insoluble (Nile red) biocompatible fluorophores were chosen as FRET acceptors and encapsulated successfully in the core and layer of the polymersome, respectively. Steady state and time-resolved photophysical studies were carried out to confirm the occurrence of the FRET process and Forster distance in the polymersome nano-assemblies. The placement of FRET acceptor in the polymersome compartment and energy terms are found to be directly correlated to the efficiency of the FRET probe action. In vitro cellular uptake studies in breast cancer MCF 7 cell line and wild type MEF cell lines established the non-toxicity of the FRET probes and lyso-tracker staining studies further evident for the internalization of the FRET probes in the lysosomal compartments of the cells and their enzyme-trigger stimuli-responsiveness. Live-cell confocal microscope imaging was deployed to directly visualize and quantify the enzyme-trigger function of FRET probes.
ESIPT Probe for Lysosome Biogenesis
Inter-organelle fusion involving endosomes and lysosomes as well as endo-lysosome-mediated lysosome fission are crucial processes that play a key role in regulating cellular homeostasis. Aberrations in either fusion or fission are implicated in many diseases. For instance, during malignant transformation, enhanced lysosome biogenesis increases the number, size, and activity of lysosomes. An increase in lysosomes assists cancer cells by enhancing catabolism and trapping drugs, thus preventing them from reaching intracellular targets and causing multi-drug resistance (MDR). Furthermore, cargo uptake and degradation involving endosomes-lysosomes fusion are integral to the nanoparticle-assisted delivery of therapeutic drugs. While understanding this intricate process can aid in disease prognosis, tracking such events with minimal disturbance remains elusive due to the scarcity of single-component synthetic probes capable of distinctly and simultaneously labelling both endosomes and lysosomes. Past efforts have focused on developing genetic transfection-assisted Fluorescent protein-tag-specific probes. However, these techniques are limited by permeabilization steps, transfection efficiency, cell line dependency, expensive plasmid designs, unstable fluorescence after fixation, and reliance on two or more fluorescent dyes, among other challenges. Employing two dyes necessitates separate excitation sources, which can sometimes lead to spectral or excitation cross-talk, posing challenges when paired with similarly excitable fluorescent drugs or dyes during multiplex imaging.
Figure 7: (a) Probe design and single cell showing both endosomes (red) and lysosomes (green). (b) Selected time snaps of a single cell recorded every 61 seconds showing transient fusion (marked in square ROI- X) and complete fusion (marked in square ROI-Y). (c) Zoomed-in time snapshots of ROI-X showing transient fusion marked by arrow.
Synthetic probes, in contrast, minimally disturb cells and are more versatile. Here, we have reported an amphiphilic donor–π–acceptor-conjugated imine-based single-component micellar probe, Nano-emitter as shown in Figure 7. The favorable orientation of the π-conjugated amphiphile in its micellar self-assembly stabilizes Excited State Intramolecular Proton Transfer (ESIPT) and fluoresces red at endosomal pH. Its hydrolysis to amine, PEG-Naph, at lysosomal pH, illuminates the lysosomes fluorescent green, with both forms excitable using a 405 nm confocal laser. The two-color labelling of endosomes and lysosomes enables the tracking of their fusion and lysosome-biogenesis processes using a single excitation laser. Its single wavelength excitation enabled its coupling with fluorescent anti-cancer drugs in multiplex time-lapse imaging, where we investigated the role of fusion and fission processes in lysosome-mediated doxorubicin sequestration in MCF-7 cells. Our results show that endosomes and endo-lysosomes also sequester doxorubicin, apart from previously recognized lysosomes.