Santosh Yadav

Multifunctional self-assembled cationic peptide nanostructures efficiently carry plasmid DNA in vitro and exhibit antimicrobial activity with minimal toxicity

In this study, a modified dehydropeptide, Boc-FΔF-εAhx-OH, was conjugated with an aminoglycoside antibiotic, neomycin, to construct a multifunctional conjugate, Pep-Neo. The amphiphilic conjugate (Pep-Neo) was able to self-assemble into cationic nanostructures in an aqueous solution at low concentrations. Nanostructure formation was evidenced by TEM and dynamic light scattering analyses. The average hydrodynamic diameter of the self-assembled Pep-Neo nanostructures was found to be ∼279 nm with a zeta potential of +28 mV. The formation of nanostructures with a hydrophobic core and cationic hydrophilic shell resulted in an increased local concentration of cationic charge (ca. in 50% aqueous methanol, i.e. disassembled structure, zeta potential decreased to +17.6 mV), leading to efficient interactions with negatively charged plasmid DNA (pDNA). The size and zeta potential of the resulting Pep-Neo/pDNA complex were found to be ∼154 nm and +19.4 mV, respectively. Having been characterized by physicochemical techniques, the complex was evaluated for its toxicity and ability to deliver nucleic acid therapeutics. The flow cytometry results on MCF-7 cells revealed that Pep-Neo/pDNA complex transfected ∼27% cells at a w/w ratio of 66.6 while the standard transfection reagent, Lipofectamine, could transfect only ∼15% cells. MTT and hemolysis assays showed the non-toxic nature of the projected conjugate at various concentrations. Further, these nanostructures were shown to encapsulate hydrophobic drugs in the core. Finally, Pep-Neo nanostructures showed efficient antibacterial activity against different strains of Gram-positive and -negative bacteria. Interestingly, unlike neomycin, which is highly effective against Gram-negative bacteria, these nanostructures showed considerably high efficiency against Gram-positive strains, highlighting the promising potential of these nanostructures for various biomedical applications.

Azobenzene-aminoglycoside: Self-assembled smart amphiphilic nanostructures for drug delivery.

Here, we have designed and synthesized a novel cationic amphiphilic stimuli-responsive azobenzene-aminoglycoside (a small molecule) conjugate, Azo-AG 5, and characterized it by UV and FTIR. Light responsive nature of Azo-AG 5 was assessed under UV-vis light. Self- assembly of Azo-AG 5 in aqueous solutions into nanostructures and their ability to act as drug carrier were also investigated. The nanostructures of Azo-AG 5 showed average hydrodynamic diameter of ∼ 255 nm with aminoglycoside moiety (neomycin) and 4-dimethylaminoazobenzene forming hydrophilic shell and hydrophobic core, respectively. In the hydrophobic core, eosin and aspirin were successfully encapsulated. Dynamic light scattering (DLS) measurements demonstrated that the nanoassemblies showed expansion and contraction on successive UV and visible light irradiations exhibiting reversible on-off switch for controlling the drug release behavior. Similar behavior was observed when these nanostructures were subjected to pH-change. In vitro drug release studies showed a difference in UV and visible light-mediated release pattern. It was observed that the release rate under UV irradiation was comparatively higher than that observed under visible light. Further, azoreductase-mediated cleavage of the azo moiety in Azo-AG 5 nanoassemblies resulted in the dismantling of the structures into aggregated microstructures. Azo-AG 5 nanostructures having positive surface charge (+9.74 mV) successfully interacted with pDNA and retarded its mobility on agarose gel. Stimuli responsiveness of nanostructures and their on-off switch like behavior ensure the great potential as controlled drug delivery systems and in other biomedical applications such as colon-specific delivery and gene delivery.

Synthesis and evaluation of N-(2,3-dihydroxypropyl)-PEIs as efficient vectors for nucleic acids

Branched polyethylenimine (bPEI, 25 kDa) has been widely used as an efficient delivery vector for nucleic acids in vitro. However, its charge-associated toxicity has limited its in vivo applications. In an attempt to control its toxicity, it was reacted with varying amounts of glycidol (2,3-epoxy-1-propanol) to obtain a small series of hydrophilic polymers, 2,3-dihydroxypropyl-grafted-polyethylenimines (DHP-g-P). The resulting polymers were characterized by (1)H-NMR and subjected to interaction with negatively charged pDNA, which yielded complexes in the size range of ~171-190 nm with a zeta potential of ∼+33-39 mV. Acid-base titration revealed no effect of substitution on the buffering capacity of the modified polymers. Grafting of 2,3-dihydroxypropyl groups on bPEI significantly improved the cell viability (i.e. almost non-toxic) as well as the DNA release properties of these modified polymers compared to native bPEI. Formation of a relatively loose DHP-g-P25/pDNA complex (the best working system in terms of transfection efficiency) resulted in the efficient nuclear release of pDNA for transcription, a prerequisite for efficient transfection. Subsequently, upon evaluation of their ability to transfer nucleic acids in vitro, the DHP-g-P/pDNA complexes exhibited higher gene transfection efficiency with one of the formulations, DHP-g-P25/DNA complex, displaying ~2.7 folds higher GFP expression than bPEI and ~2.3-3.5 folds higher than the selected commercial transfection reagents used in this study. Further to quantify the extent of GFP positive cells, FACS analysis was performed, which revealed DHP-g-P25/DNA mediated gene expression in ~51% cells outcompeting bPEI, Superfect™, Fugene™ and Lipofectamine™. Sequential delivery of GFP-specific siRNA resulted in ~78% suppression of the target gene compared to ~49% achieved by Fugene™. All these results demonstrate the potential of these polymers for in vivo gene delivery.

Synthesis and Evaluation of Tetramethylguanidinium-Polyethylenimine Polymers as Efficient Gene Delivery Vectors

Previously, we demonstrated that 6-(N,N,N′,N′-tetramethylguanidinium chloride)-hexanoyl-polyethylenimine (THP) polymers exhibited significantly enhanced transfection efficiency and cell viability. Here, in the present study, we have synthesized a series of N,N,N′,N′-tetramethylguanidinium-polyethylenimine (TP1-TP5) polymers via a single-step reaction involving peripheral primary amines of bPEI and varying amounts of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). These polymers were found to interact efficiently with negatively charged pDNA and formed stable complexes in the size range of ~240–450 nm. Acid-base titration profiles revealed improved buffering capacity of TP polymers as compared to bPEI. Transfection and cytotoxicity assays performed with TP/pDNA complexes on HEK293, CHO, and HeLa cells showed significantly higher transfection efficiency and cell viability with one of the complexes, TP2/pDNA complex, exhibited the highest transfection efficiency (~1.4–2.3-fold) outcompeting native bPEI and the commercially available transfection reagent, Lipofectamine 2000. Compared to previously reported THP polymers, the transfection efficiency of TP/pDNA complexes was found to be lower, as examined by flow cytometry. These results highlight the importance of the hydrophobic C-6 linker in THP polymers in forming compact nanostructures with pDNA, which might lead to efficient uptake and internalization of the complexes; however, the projected TP polymers offer an advantage of their rapid and economical one-step synthesis.

Amphiphilic azobenzene-neomycin conjugate self-assembles into nanostructures and transports plasmid DNA efficiently into the mammalian cells

The present study demonstrates the use of self-assembled nanostructures of cationic amphiphilic azobenzene-neomycin (a small molecule) conjugate, Azo-Neo, as delivery vector for plasmid DNA. These nanostructures efficiently condensed nucleic acid and formed more compact nanoassemblies. DLS analysis showed size and zeta potential of the resulting Azo-Neo/pDNA nanoassemblies ∼153.7nm and +7.26mV, respectively. The nanoassemblies were characterized by physicochemical techniques and evaluated for its toxicity and ability to deliver nucleic acid therapeutics. The flow cytometry results on MCF-7 and HEK293T cells revealed that Azo-Neo/pDNA nanoassemblies transfected ∼31% and 23% cells, respectively, at a w/w ratio of 250, while the standard transfection reagent, bPEI/pDNA complex, could transfect only ∼21% and 29% cells, respectively, at its best w:w ratio of 2.3. MTT and hemolysis assays showed the non-toxic nature of the projected nanoassemblies and nanostructures, respectively, at various concentrations. Further, Azo-Neo nanostructures showed efficient antibacterial activity against different strains, laboratory strain of Staphylococcus aureus (MTCC 740) as well as MRSA strains (Staphylococcus aureus ATCC 33591, ATCC 43300 and ATCC 700699). These results ensure the great potential of these nanostructures in gene delivery and antimicrobial applications.

Enhanced antibacterial activity of tetramethylguanidinium-conjugated linear polyethylenimine polymers

ABSTRACT In the present study, varying amounts of tetramethylguanidinium moiety have been conjugated to linear polyethylenimine to obtain linear polyethylenimine-tmg (LPTG) polymers. Incorporation of hydrophobic and highly basic moiety in the polymeric backbone resulted in the significant improvement in the antibacterial activity which was confirmed by zone of inhibition and MIC assays. Further, the results of transmission electron microscopy and confocal studies revealed that the projected LPTG polymers possessed higher antibacterial activity than the native polymer. In addition, these modified polyethylenimine (PEI) polymers were capable of reducing auric chloride into stable gold nanoparticles. These polyamine-stabilized gold nanoparticles can be used in various biomedical applications. GRAPHICAL ABSTRACT

Core/shell nanoassembly of amphiphilic naproxen-polyethylene glycol: synthesis, characterisation and evaluation as drug delivery system

Small molecule-based amphiphiles self-assemble into nanostructures (micelles) in aqueous medium which are currently being explored as novel drug delivery systems. Here, naproxen-polyethylene glycol (N-PEG), a small molecule-derived amphiphile, has been synthesised, characterised and evaluated as hydrophobic drug carrier. 1H, 13C Nuclear magnetic resonance (NMR), mass spectrometry (MS) and Fourier-transform infrared spectroscopy (FTIR) confirmed the formation of N-PEG and dynamic light scattering (DLS) revealed the formation of nano-sized structures of ∼228 nm. Transmission electron microscope (TEM) analysis showed aggregation behaviour of the structures with average size of ∼230 nm. Biodegradability aspect of the micellar-structured N-PEG was demonstrated by lipase-mediated degradation studies using DLS and TEM. High encapsulation efficiency followed by release in a sustained manner of a well-known anticancer drug, doxorubicin, demonstrated the feasibility of the new drug delivery system. These results advocate the promising potential of N-PEG micelles as efficient drug delivery system for specific delivery to cancerous cells in vitro and in vivo.