Manohar Mahato | IGIB - Academia.edu (original) (raw)

Papers by Manohar Mahato

Abstract The success of nonviral gene therapy mainly depends on the design and development of saf... more Abstract The success of nonviral gene therapy mainly depends on the design and development of safe and effective gene delivery systems. Nonviral vectors have attracted the attention of researchers, as they offer several advantages over their viral counterparts. Among various nonviral vectors, polyethylenimines have been the most widely used in in vitro and in vivo gene transfer studies. Cationic nanoparticles have been shown to enhance the efficiency of the transfection. These are compact and help in overcoming various biological and cellular barriers. Due to their smaller size, these are less susceptible to reticuloendothelial (RES) clearance and exhibit better uptake and internalization into the cells/tissues. To exploit these properties of the nanoparticles, the present chapter highlights the synthesis of nanoparticles of polyethylenimine (linear and branched) and their ability to effect gene transfer across the biological systems.

Colloids and Surfaces B: Biointerfaces, Sep 1, 2013

Recent advancements in polymeric gene delivery have raised the potential of gene therapy as treat... more Recent advancements in polymeric gene delivery have raised the potential of gene therapy as treatment for various acquired and inherited diseases. Here, we report on the synthesis and characterization of N-ethyl-N'-(3-dimethylaminopropyl)-guanidinyl-polyethylenimine (sGP) polymers and investigation of their capability to carry DNA and siRNA in vitro. Zinc triflate-mediated activation of primary amines of branched polyethylenimine (bPEI) followed by reaction with varying amounts of N-ethyl-N'-(3dimethylaminopropyl) carbodiimide (EDAC) resulted in the generation of a small series of trisubstituted guanidinyl-modified polyethylenimine polymers. Determination of primary amines on modified polymers by TNBS assay revealed 62-84% of the attempted conjugation of EDAC onto bPEI. These modified polymers were shown to condense plasmid DNA and retard its mobility on 0.8% agarose gel. Further, these polymers were evaluated for their capability to carry pDNA into the cells by performing transfection assay on various mammalian cells. All the modified polymer/pDNA complexes exhibited significantly higher levels of gene expression with one of the complexes, sGP3/pDNA complex, displayed ∼1.45 to 3.0 orders of magnitude higher transfection efficiency than that observed in the native bPEI and the commercial transfection reagent, Lipofectamine TM. The efficacy of sGP3 polymer was further assessed by siRNA delivery, which resulted in ∼81% suppression of the target gene. In conclusion, these studies demonstrate the potential of these substituted guanidinyl-modified PEIs as efficient gene delivery vectors.

Journal of Polymer Science Part A, Mar 23, 2012

Highly toxic polyallylamine (PA) was reacted with a varying amount of a novel linker, 6-(N,N,N 0 ... more Highly toxic polyallylamine (PA) was reacted with a varying amount of a novel linker, 6-(N,N,N 0 ,N 0-tetramethylguanidinium chloride) hexanoic acid (Tmg-HA), to prepare a series of tetramethylguanidinium-PA (Tmg-PA) polymers, which were used as vectors for gene transfection. The extent of attachment of the linker, Tmg-HA, to the PA backbone was determined by 2,4,6-trinitrobenzene sulfonic acid assay. The modified polymers (Tmg-PAs), when complexed with pDNA, exhibited good condensation ability. The nanoparticles, so formed, were characterized by their size and zeta potential and were subsequently evaluated for their toxicity and transfection ability on various mammalian cells, viz., HeLa, CHO, and HEK 293 cells. Mobility shift assay revealed that on increasing the percent substitution of Tmg-HA onto PA (from Tmg-PA1 to Tmg-PA6), relatively higher amounts of modified polymers were required to retard the mobility of a fixed amount of DNA. Besides, Tmg-PA polymers provided sufficient protection (ca. 84-88%) to bound DNA against nucleases and one of the formulations, Tmg-PA2 (ca. 15% substitution) displayed the highest transfection efficiency outcompeting the commercial transfection reagent, Lipofectamine TM with minimal cytotoxicity. More impressively, the transfection efficiency increased despite recording a decrease in the buffering capacity of the grafted polymers suggesting that buffering capacity is not the sole parameter in determining the gene delivery efficiency of a vector system. V

Springer eBooks, 2018

Gene therapy offers a great potential for the treatment of genetic diseases as well as acquired d... more Gene therapy offers a great potential for the treatment of genetic diseases as well as acquired diseases by means of delivering therapeutic nucleic acids inside the cell. To deliver nucleic acids, broadly two strategies have been employed by using viral vectors and non-viral vectors. The viral vectors exhibited high transduction efficacy both in vitro and in vivo. The non-viral vectors composed of mainly cationic polymers and lipids which provide efficient condensing capability against negatively charged nucleic acids and low cytotoxicity. Till date, >2300 clinical trials for gene therapy are going on worldwide, approximately 70% using viral vectors and remaining with non-viral vectors. The immunogenicity, non-targeting abilities are the biggest hurdles in terms of safety and efficiency for successful therapy with these vectors. These two classes of vectors have their own advantages as well as disadvantages which hinder their therapeutic endpoint in clinical trials. Now, researchers have made attempts to form virus encapsulated in chemical vectors which are called as hybrid vectors. These hybrid vectors have immense potential to evade host immune system by masking the immunogenic epitopes present on viral vectors. The molecules or scaffold which is used for encapsulating virus enhance their targeting ability and sustained release to targeted tissue. The hybrid vectors, combination of viral and chemical vectors, form a new class of gene delivery vectors which overcome the limitations of each vector and simultaneously augment desirable features such as targeting ability, low immunogenicity, cytotoxicity, higher payload, and ability to deliver more than one transgene. The hybrid vectors should retain characteristics of the each vector in order to achieve optimal tissue targeting and gene delivery with minimal toxicity. To achieve therapeutic endpoint with the hybrid vectors, development of such hybrid vectors requires extensive understanding of physicochemical properties after coating virus with chemical analogues and their optimal ratio as well. These aspects will be discussed in this chapter.

Nature Communications

Transfusion of healthy red blood cells (RBCs) is a lifesaving process. However, upon storing RBCs... more Transfusion of healthy red blood cells (RBCs) is a lifesaving process. However, upon storing RBCs, a wide range of damage-associate molecular patterns (DAMPs), such as cell-free DNA, nucleosomes, free-hemoglobin, and poly-unsaturated-fatty-acids are generated. DAMPs can further damage RBCs; thus, the quality of stored RBCs declines during the storage and limits their shelf-life. Since these DAMPs consist of either positive or negative charged species, we developed taurine and acridine containing electrospun-nanofibrous-sheets (Tau-AcrNFS), featuring anionic, cationic charges and an DNA intercalating group on their surfaces. We show that Tau-AcrNFS are efficient in scavenging DAMPs from stored human and mice RBCs ex vivo. We find that intermittent scavenging of DAMPs by Tau-AcrNFS during the storage reduces the loss of RBC membrane integrity and reduces discocytes-to-spheroechinocytes transformation in stored-old-RBCs. We perform RBC-transfusion studies in mice to reveal that intermi...

Metal Allergy, 2018

Metals are a group of elements which are ubiquitous in modern life. They are used in the fields o... more Metals are a group of elements which are ubiquitous in modern life. They are used in the fields of cosmetics, water purification, medicine, paint, food products, pesticides, and almost innumerable others. As the use of metals has increased in recent decades, so has human exposure to these elements. Metals such as mercury, lead, arsenic, nickel, and others have been implicated in negatively affecting human homeostasis by causing chronic inflammatory diseases, among other serious conditions. Both acute and chronic metal toxicity in vital organs could arise from local or systemic exposure to numerous metals. Although some metals have health benefits, overaccumulation of metals in body tissues can result in deleterious, toxic effects. Most exposure to metals occurs via cutaneous, inhalation, or oral routes. At the highest risk of negative effects of exposure are pregnant women and children. To ameliorate or prevent the toxic effects of metals, chelating agents and barrier creams are used widely in medical practice today. In this chapter, we will discuss preventing metal toxicity from overexposure via chelation therapy and skin barrier creams.

Gene and Cell Therapy: Biology and Applications, 2018

Bulletin of Materials Science, 2020

Design of amphiphiles to develop robust self-assembled soft nanomaterials, such as micelles and h... more Design of amphiphiles to develop robust self-assembled soft nanomaterials, such as micelles and hydrogels is an interesting subject. A series of cationic amphiphilic compounds were synthesized comprising 1-ethoxy (3-pentadecyl) benzene as the hydrophobic tail. The second carbon of ethoxy was linked to quaternary head groups (trimethyl ammonium bromide (PEA), triethyl ammonium bromide (PETE), pyridinium bromide (PEPy), N-methyl morpholino bromide (PENM), N-methyl piperidine bromide (PENP)). Inclusion of benzene ring leads to a significant decrease in critical micellar concentration (CMC) as compared to other cationic surfactants, such as cetyl trimethyl ammonium bromide (CTAB). Interestingly, at higher concentration, these cationic amphiphiles were forming soft hydrogels with critical gelation concentration (CGC) from 3 to 10% (w/v). The small-angle X-ray scattering (SAXS) analysis of xerogel revealed the formation of self-assembled lamellar patterns of molecules. Further, the morphology of xerogels were also seen under a scanning electron microscope (SEM) which correlates with SAXS data. The SAXS and SEM data confirms the formation of worm-like micellar structures and entangle themselves to form a hydrogel. The cytotoxicity assay was done on HDFA, HeLa and HEK cell lines, haemolysis assay showed better haemocompatibility than CTAB. The synthesized surfactants exhibited up to 3-fold higher solubilization capability against hydrophobic molecules than CTAB.

Nanomedicine, 2019

Lifelong systemic immunosuppression remains the biggest challenge in vascularized composite allot... more Lifelong systemic immunosuppression remains the biggest challenge in vascularized composite allotransplantation (VCA) due to the adverse effects it causes. Since VCA is a life-enhancing procedure as compared with solid organ transplant which is life-saving; one needs to weigh the benefits and risks carefully. Thus, there is a huge unmet clinical need to design biomaterial-based vehicles that can deliver drugs more efficiently, topically and locally to eliminate adverse effects of systemic immune suppression. This review discusses several biomaterial-based systems that have been carefully designed, conceived and attempted to make VCA a more patient compliant approach. Variety of promising preclinical studies has shown the feasibility of the approaches, and clinical trials are required to bridge the gap. Several challenges for the future and new approaches have been discussed.

Colloids and surfaces. B, Biointerfaces, Jan 20, 2017

In the recent studies, it has been demonstrated that incorporation of unnatural amino acid, α,β-d... more In the recent studies, it has been demonstrated that incorporation of unnatural amino acid, α,β-dehydrophenylalanine, in small peptides results in stable self-assembled nanostructures with different sizes and shapes. Here, we have replaced the natural amino acid, phenylalanine, from our earlier reported work on self-assembled peptide, Boc-Pro-Phe-Gly-OMe, with a constrained dehydro amino acid, α,β-dehydrophenylalanine, to study its influence on self-assembled nanostructures. Dehydrotripeptide, Boc-Pro-ΔPhe-Gly-OMe, self-assembled into nanostructures in aqueous solutions and formed hydrophobic matrix with improved encapsulation efficiency of hydrophobic molecules. The hydrodynamic size of peptide nanostructures from DLS study was found to be ∼257nm. The morphology and size of the loaded nanoparticles were also determined by TEM. To improve aqueous dispersibility the projected nanostructures for efficient use in drug delivery, self-assembled dehydropeptide nano carriers were further s...

Advances in Nanomedicine for the Delivery of Therapeutic Nucleic Acids, 2017

Scientific reports, Jan 21, 2016

To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of... more To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Be...

Colloids and Surfaces B: Biointerfaces, 2015

Here, we have designed and synthesized a novel cationic amphiphilic stimuli-responsive azobenzene... more 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 ∼255nm 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 (+9.74mV) 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.

Pharmaceutical Nanotechnology, 2015

Polyamidoamine dendrimers (PAMAM) are being used as efficient vectors for delivery of nucleic aci... more Polyamidoamine dendrimers (PAMAM) are being used as efficient vectors for delivery of nucleic acids to the cells. However, these dendrimers cause a significant amount of cytotoxicity. In order to improve its transfection efficiency and cell viability, surface amine groups of PAMAM were converted into guanidinium (Gn) and tetramethylguanidi-nium (TMG) moieties. These modified PAMAM dendrimers interacted with negatively charged plasmid DNA efficiently and formed stable complexes as revealed by dynamic light scattering analysis. PAMAM/pDNA, PAMAM-TMG/pDNA and PAMAM-Gn/pDNA complexes were found to be in the range of 175-250 nm with zeta potential in the range of +21-37 mV. Further, these modified dendrimers did not display toxicity rather it decreased a bit when tested in HEK293, HeLa and MCF-7 cells. Among these modified dendrimers, PAMAM-Gn/pDNA complex displayed the highest transfection efficiency in both the cell lines HEK293 and MCF-7.

Current Topics in Medicinal Chemistry, 2015

J. Mater. Chem. B, 2014

An amphiphilic peptide–aminoglycoside (Pep–Neo) conjugate has been synthesized, self-assembled in... more An amphiphilic peptide–aminoglycoside (Pep–Neo) conjugate has been synthesized, self-assembled into nanostructures and evaluated for its multifunctional properties.

BioMed Research International, 2014

Previously, we demonstrated that 6-(N,N,N′,N′-tetramethylguanidinium chloride)-hexanoyl-polyethyl... more 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 ef...

Molecular BioSystems, 2012

Molecular BioSystems, 2013