Exogenous application of nanocarrier-mediated double-stranded RNA manipulates physiological traits and defence response against bacterial diseases.

Stability and delivery are major challenges associated with exogenous double-stranded RNA (dsRNA) application into plants. We report the encapsulation and delivery of dsRNA in cationic poly-aspartic acid-derived polymer (CPP6) into plant cells. CPP6 stabilizes the dsRNAs during long exposure at varied temperatures and pH, and protects against RNase A degradation. CPP6 helps dsRNA uptake through roots or foliar spray and facilitates systemic movement to induce endogenous gene silencing. The fluorescence of Arabidopsis GFP-overexpressing transgenic plants was significantly reduced after infiltration with gfp-dsRNA-CPP6 by silencing of the transgene compared to plants treated only with gfp-dsRNA. The plant endogenous genes flowering locus T (FT) and phytochrome interacting factor 4 (PIF4) were downregulated by a foliar spray of ft-dsRNA-CPP6 and pif4-dsRNA-CPP6 in Arabidopsis, with delayed flowering and enhanced biomass. The rice PDS gene targeted by pds-dsRNA-CPP6 through root uptake was effectively silenced and plants showed a dwarf and albino phenotype. The NaCl-induced OsbZIP23 was targeted through root uptake of bzip23-dsRNA-CPP6 and showed reduced transcripts and seedling growth compared to treatment with naked dsRNA. The negative regulators of plant defence SDIR1 and SWEET14 were targeted through foliar spray to provide durable resistance against bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Overall, the study demonstrates that transient silencing of plant endogenous genes using polymer-encapsulated dsRNA provides prolonged and durable resistance against Xoo, which could be a promising tool for crop protection and for sustaining productivity.

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

A localized hydrogel-mediated chemotherapy causes immunogenic cell death via activation of ceramide-mediated unfolded protein response.

Treatment of triple-negative breast cancer (TNBC) is challenging because of its "COLD" tumor immunosuppressive microenvironment (TIME). Here, we present a hydrogel-mediated localized delivery of a combination of docetaxel (DTX) and carboplatin (CPT) (called DTX-CPT-Gel therapy) that ensured enhanced anticancer effect and tumor regression on multiple murine syngeneic and xenograft tumor models. DTX-CPT-Gel therapy modulated the TIME by an increase of antitumorigenic M1 macrophages, attenuation of myeloid-derived suppressor cells, and increase of granzyme B+CD8+ T cells. DTX-CPT-Gel therapy elevated ceramide levels in tumor tissues that activated the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-mediated unfolded protein response (UPR). This UPR-mediated activation of apoptotic cell death led to release of damage-associated molecular patterns, thereby activating the immunogenic cell death that could even clear the metastatic tumors. This study provides a promising hydrogel-mediated platform for DTX-CPT therapy that induces tumor regression and effective immune modulation and, therefore, can be explored further for treatment of TNBC.

Hydrophobicity of Cholic Acid-Derived Amphiphiles Dictates the Antimicrobial Specificity.

The unique structural components of cell membranes of Gram-positive bacteria, Gram-negative bacteria, and mycobacteria provide an excellent therapeutic target for developing highly specific antimicrobials. Here, we report the synthesis of nine cholic acid (CA)-derived amphiphiles, where three hydroxyl groups of CA were tethered to dimethylamino pyridine and the C24-carboxyl group was conjugated with different alkyl chains. Structure-activity investigations revealed that amphiphile 1 harboring a methyl group has antimicrobial activity against mycobacterial species. On the other hand, amphiphile 7 containing an octyl chain was selective against Gram-positive and Gram-negative bacilli. Biochemical assays confirmed the selective membrane permeabilization abilities of amphiphiles 1 and 7. Importantly, we demonstrate the selective actions of amphiphiles in clearing biofilms, intracellular bacteria, and wound infections. Therefore, for the first time, we show that the unique structural features of CA-derived amphiphiles dictate selective activity against specific bacterial species.

Polyaspartate-derived synthetic antimicrobial polymer enhances the activity of rifampicin against multidrug-resistant Pseudomonas aeruginosa infections.

Infections caused by multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa) pose major challenges for treatment due to the acquired, adaptive, and intrinsic resistance developed by the bacteria. Accumulation of mutations, the ability to form biofilms, and the presence of lipopolysaccharides in the outer bacterial membranes are the key mechanisms of drug resistance. Here, we show that a polyaspartate-derived synthetic antimicrobial polymer (SAMP) with a hexyl chain (TAC6) is an effective adjuvant for a hydrophobic antibiotic, rifampicin. Our in vitro studies demonstrated that the combination of TAC6 and rifampicin is effective against clinically isolated multidrug-resistant strains of P. aeruginosa. Membrane permeabilization studies showed that TAC6 allows the permeabilization of bacterial membranes, and the accumulation of rifampicin inside the cells, thereby enhancing its activity. The combination of TAC6 and rifampicin can also degrade the P. aeruginosa biofilms, and therefore can mitigate the adaptive resistance developed by bacteria. We further demonstrated that the combination of TAC6 and rifampicin can clear P. aeruginosa-mediated wound infections effectively. Therefore, our study showed polyaspartate-derived SAMP to be an effective antibiotic adjuvant against P. aeruginosa infections.

A Localized Chimeric Hydrogel Therapy Combats Tumor Progression through Alteration of Sphingolipid Metabolism.

Rapid proliferation of cancer cells assisted by endothelial cell-mediated angiogenesis and acquired inflammation at the tumor microenvironment (TME) lowers the success rate of chemotherapeutic regimens. Therefore, targeting these processes using localized delivery of a minimally toxic drug combination may be a promising strategy. Here, we present engineering of a biocompatible self-assembled lithocholic acid-dipeptide derived hydrogel (TRI-Gel) that can maintain sustained delivery of antiproliferating doxorubicin, antiangiogenic combretastatin-A4 and anti-inflammatory dexamethasone. Application of TRI-Gel therapy to a murine tumor model promotes enhanced apoptosis with a concurrent reduction in angiogenesis and inflammation, leading to effective abrogation of tumor proliferation and increased median survival with reduced drug resistance. In-depth RNA-sequencing analysis showed that TRI-Gel therapy induced transcriptome-wide alternative splicing of many genes responsible for oncogenic transformation including sphingolipid genes. We demonstrate that TRI-Gel therapy targets the reversal of a unique intron retention event in ß-glucocerebrosidase 1 (Gba1), thereby increasing the availability of functional Gba1 protein. An enhanced Gba1 activity elevates ceramide levels responsible for apoptosis and decreases glucosylceramides to overcome drug resistance. Therefore, TRI-Gel therapy provides a unique system that affects the TME via post-transcriptional modulations of sphingolipid metabolic genes, thereby opening a new and rational approach to cancer therapy.

Cholic Acid-Peptide Conjugates as Potent Antimicrobials against Interkingdom Polymicrobial Biofilms.

Interkingdom polymicrobial biofilms formed by Gram-positive Staphylococcus aureus and Candida albicans pose serious threats of chronic systemic infections due to the absence of any common therapeutic target for their elimination. Herein, we present the structure-activity relationship (SAR) of membrane-targeting cholic acid-peptide conjugates (CAPs) against Gram-positive bacterial and fungal strains. Structure-activity investigations validated by mechanistic studies revealed that valine-glycine dipeptide-derived CAP 3 was the most effective broad-spectrum antimicrobial against S. aureus and C. albicans CAP 3 was able to degrade the preformed single-species and polymicrobial biofilms formed by S. aureus and C. albicans, and CAP 3-coated materials prevented the formation of biofilms. Murine wound and catheter infection models further confirmed the equally potent bactericidal and fungicidal effect of CAP 3 against bacterial, fungal, and polymicrobial infections. Taken together, these results demonstrate that CAPs, as potential broad-spectrum antimicrobials, can effectively clear the frequently encountered polymicrobial infections and can be fine-tuned further for future applications.

Targeted SHP-1 Silencing Modulates the Macrophage Phenotype, Leading to Metabolic Improvement in Dietary Obese Mice.

Chronic over-nutrition promotes adipocyte hypertrophy that creates inflammatory milieu leading to macrophage infiltration and their phenotypic switching during obesity. The SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1) has been identified as an important player in inflammatory diseases involving macrophages. However, the role of SHP-1 in modulating the macrophage phenotype has not been elucidated yet. In the present work, we show that adipose tissue macrophage (ATM)-specific deletion of SHP-1 using glucan particle-loaded siRNA improves the metabolic phenotype in dietary obese insulin-resistant mice. The molecular mechanism involves AT remodeling via reducing crown-like structure formation and balancing the pro-inflammatory (M1) and anti-inflammatory macrophage (M2) population. Therefore, targeting ATM-specific SHP-1 using glucan-particle-loaded SHP-1 antagonists could be of immense therapeutic use for the treatment of obesity-associated insulin resistance.

A nanogel based oral gene delivery system targeting SUMOylation machinery to combat gut inflammation.

Poor success rates and challenges associated with the current therapeutic strategies of inflammatory bowel disease (IBD) have accelerated the emergence of gene therapy as an alternative treatment option with great promise. However, oral delivery of nucleic acids (NAs) to an inflamed colon is challenged by multiple barriers presented by the gastrointestinal, extracellular and intracellular compartments. Therefore, we screened a series of polyaspartic acid-derived amphiphilic cationic polymers with varied hydrophobicity for their ability to deliver NAs into mammalian cells. Using the most effective TAC6 polymer, we then engineered biocompatible and stable nanogels composed of polyplexes (TAC6, NA) and an anionic polymer, sodium polyaspartate, that were able to deliver the NAs across mammalian cells using caveolae-mediated cellular uptake. We then utilized these nanogels for oral delivery of PIAS1 (protein inhibitor of activated STAT1), a SUMO 3 ligase, encoding plasmid DNA since PIAS1 is a key nodal therapeutic target for IBD due to its ability to control NF-κB-mediated inflammatory signaling. We show that plasmid delivery using TAC6-derived nanogels diminished gut inflammation in a murine colitis model. Therefore, our study presents engineering of orally deliverable nanogels that can target SUMOylation machinery to combat gut inflammation with very high efficacy.

Deciphering the Role of Intramolecular Networking in Cholic Acid-Peptide Conjugates on the Lipopolysaccharide Surface in Combating Gram-Negative Bacterial Infections.

The presence of lipopolysaccharide and emergence of drug resistance make the treatment of Gram-negative bacterial infections highly challenging. Herein, we present the synthesis and antibacterial activities of cholic acid-peptide conjugates (CAPs), demonstrating that valine-glycine dipeptide-derived CAP 3 is the most effective antimicrobial. Molecular dynamics simulations and structural analysis revealed that a precise intramolecular network of CAP 3 is maintained in the form of evolving edges, suggesting intramolecular connectivity. Further, we found high conformational rigidity in CAP 3 that confers maximum perturbations in bacterial membranes relative to other small molecules. Interestingly, CAP 3-coated catheters did not allow the formation of biofilms in mice, and treatment of wound infections with CAP 3 was able to clear the bacterial infection. Our results demonstrate that molecular conformation and internal connectivity are critical parameters to describe the antimicrobial nature of compounds, and the analysis presented here may serve as a general principle for the design of future antimicrobials.

Oral Delivery of Cholic Acid-Derived Amphiphile Helps in Combating Salmonella-Mediated Gut Infection and Inflammation.

A major impediment to developing effective antimicrobials against Gram-negative bacteria like Salmonella is the ability of the bacteria to develop resistance against existing antibiotics and the inability of the antimicrobials to clear the intracellular bacteria residing in the gastrointestinal tract. As the critical balance of charge and hydrophobicity is required for effective membrane-targeting antimicrobials without causing any toxicity to mammalian cells, herein we report the synthesis and antibacterial properties of cholic acid-derived amphiphiles conjugated with alkyl chains of varied hydrophobicity. Relative to other hydrophobic counterparts, a compound with hexyl chain (6) acted as an effective antimicrobial against different Gram-negative bacteria. Apart from its ability to permeate the outer and inner membranes of bacteria; compound 6 can cross the cellular and lysosomal barriers of epithelial cells and macrophages and kill the facultative intracellular bacteria without disrupting the mammalian cell membranes. Oral delivery of compound 6 was able to clear the Salmonella-mediated gut infection and inflammation, and was able to combat persistent, stationary, and multi-drug-resistant clinical strains. Therefore, our study reveals the ability of cholic acid-derived amphiphiles to clear intracellular bacteria and Salmonella-mediated gut infection and inflammation.

Emerging biomedical applications of polyaspartic acid-derived biodegradable polyelectrolytes and polyelectrolyte complexes.

Polyelectrolytes (PELs) - polymers with charged repeat units - have emerged as a useful class of polymers for biomedical applications due to their high aqueous solubility, low aggregation propensity and the opportunity they afford for polyvalent interactions with surfaces. Biodegradability and biocompatibility of PELs are important prerequisites for their utilization in in vivo applications. PELs that can be chemically functionalized with ease prove advantageous for creating diverse biomaterials. Polyaspartic acid (PASA) is a modular and biocompatible synthetic PEL that has all these features. It also shows many positive biomedical attributes such as bone-tissue targeting, muco-adhesive behavior and extended blood circulation time. Cationic PELs derived from PASA are rapidly internalized by mammalian and bacterial cells, and hence have immense utility in therapeutic delivery applications. Polyelectrolyte complexes (PECs) and multilayers (PEMs) formed from PASA PELs have further expanded their biomedical utility. This mini-review highlights some recent literature examples of unique biomedical applications of PELs, PECs and PEMs prepared through the molecular engineering of PASA. It discusses biomineralization modulators, anti-mycobacterial agents, underwater adhesives, mucoadhesive drug and gene delivery agents, and cell encapsulants fabricated using PASA derived PELs.

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 (TAC1) 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 TAC1 to induce membrane lysis and to bind to the genomic material of mycobacteria, thereby inducing mycobacterial cell death. TAC1 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 TAC1, 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.

Injectable, Self-Healing Chimeric Catechol-Fe(III) Hydrogel for Localized Combination Cancer Therapy.

Conventional intravenous or oral administration of a combination of chemotherapeutics displays poor bioavailability and induces undesirable systemic toxicity. Therefore, localized delivery of such chemotherapeutic combinations using polymeric hydrogels is expected to help in enhancing drug efficacy and reducing systemic toxicity. In this manuscript, we have utilized a chitosan-catechol based hydrogel (CAT-Gel) assembled through catechol-Fe(III) coordinative interactions for localized combination therapy in murine lung and breast cancer models. CAT-Gel offers a unique blend of material properties such as injectability and self-healing along with useful biological attributes like their noncytotoxic and nonhemolytic nature. The amphipathic nature of this hydrogel enabled us to incorporate a recipe of hydrophilic doxorubicin hydrochloride (DOX) and hydrophobic docetaxel (DTX) anticancer drugs. Rheology studies confirmed the self-healing nature of this chimeric hydrogel even after drug loading. CAT-Gel was retained for more than 40 days in mice upon subcutaneous injection. The sequential and sustained release of the entrapped DOX and DTX from the hydrogel resulted in synergistic therapeutic effect with increased median survival against murine lung and breast cancer models. Therefore, CAT-Gel provides a new coordinatively assembled biocompatible scaffold for localized delivery of chemotherapeutic drugs.

Cell Permeating Nano-Complexes of Amphiphilic Polyelectrolytes Enhance Solubility, Stability, and Anti-Cancer Efficacy of Curcumin.

Many hydrophobic drugs encounter severe bioavailability issues owing to their low aqueous solubility and limited cellular uptake. We have designed a series of amphiphilic polyaspartamide polyelectrolytes (PEs) that solubilize such hydrophobic drugs in aqueous medium and enhance their cellular uptake. These PEs were synthesized through controlled (∼20 mol %) derivatization of polysuccinimide (PSI) precursor polymer with hydrophobic amines (of varying alkyl chain lengths, viz. hexyl, octyl, dodecyl, and oleyl), while the remaining succinimide residues of PSI were opened using a protonable and hydrophilic amine, 2-(2-amino-ethyl amino) ethanol (AE). Curcumin (Cur) was employed as a representative hydrophobic drug to explore the drug-delivery potential of the resulting PEs. Unprecedented enhancement in the aqueous solubility of Cur was achieved by employing these PEs through a rather simple protocol. In the case of PEs containing oleyl/dodecyl residues, up to >65000× increment in the solubility of Cur in aqueous medium could be achieved without requiring any organic solvent at all. The resulting suspensions were physically and chemically stable for at least 2 weeks. Stable nanosized polyelectrolyte complexes (PECs) with average hydrodynamic diameters (DH) of 150-170 nm (without Cur) and 220-270 nm (after Cur loading) were obtained by using submolar sodium polyaspartate (SPA) counter polyelectrolyte. The zeta potential of these PECs ranged from +36 to +43 mV. The PEC-formation significantly improved the cytocompatibility of the PEs while affording reconstitutable nanoformulations having up to 40 wt % drug-loading. The Cur-loaded PECs were readily internalized by mammalian cells (HEK-293T, MDA-MB-231, and U2OS), majorly through clathrin-mediated endocytosis (CME). Cellular uptake of Cur was directly correlated with the length of the alkyl chain present in the PECs. Further, the PECs significantly improved nuclear transport of Cur in cancer cells, resulting in their death by apoptosis. Noncancerous cells were completely unaffected under this treatment.

Robust, self-healing hydrogels synthesised from catechol rich polymers.

Coordinative interactions between polymer-bound catechols and metal ions are the basis for numerous bio-inspired soft materials. Here, we demonstrate rapid access to catechol rich polymers through reductive amination (RA) strategy. We employed chitosan to exemplify the utility of this protocol. Controlled grafting of catechol pendants (from as low as 18 mol% to as high as 80 mol%) onto chitosan was readily achieved in aqueous medium under ambient conditions by RA protocol. Because of the high density of catechol units grafted onto chitosan, we could accomplish the gelation of water even in acidic medium in the presence of transition metal ions, or on addition of chemical oxidant such as NaIO4. Increasing the mol% of catechol in polymer decreased the amounts of Fe(iii) or NaIO4 required to yield gels. UV-vis and Raman studies indicated the presence of mono-complex between Fe(iii) and catechol in the gels formed through coordinative crosslinking. Highly ductile hydrogels exhibiting excellent load bearing ability were obtained under these conditions. Electrostatic repulsions between the cationic polymer chains presumably prevented the gel to collapse upon the application of load. These gels were also completely self-healing due to the reversible nature of their coordinative interactions. Gels formed at higher pH are brittle and less resilient compared to those formed at lower pH. Ductility and self-healing ability of coordinative cross-linked gels are superior to those formed by oxidative crosslinking. We conclude that RA strategy offers rapid and easy access to catechol-rich systems, and retention of basic amine functionalities allows the preparation of robust bio inspired soft materials.