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The work demonstrated a successful synthesis of nitric oxide (NO)-releasing material and its antibacterial effect on Gram-negative Escherichia coli (E. coli), Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA). The polymeric support composed of thermosensitive Pluronic F68 having good biocompatibility and branched polyethylenimine (BPEI) housed N-diazeniumdiolates (NONOates) which could store and release NO under appropriate physiological condition. The developed F68-BPEI-NONOates releases a sufficient amount of NO under physiological condition to elicit effective killing of E. coli, S. aureus and MRSA. The antibacterial ability of the released NO was compared to untreated control or unmodified F68 polymer by using confocal microscopy; F68-BPEI-NONOates demonstrated excellent antibacterial activity with in vitro low cytotoxicity. TEM investigation also revealed the destruction of bacteria membrane caused by NO. The effectiveness of F68-BPEI-NONOates against resistant strains such as MRSA provides a very simple but highly efficient strategy to combat drug-resistant bacterial infections.

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The work employs carbon dot (CD) which has been emerging as a fluorescent nanomaterial with excellent biocompatibility and perceived as a promising alternative to quantum dot (QD), to monitor the association/dissociation of polymeric carrier/plasmid DNA (pDNA) complex during transfection. To shed light on the underlying post-endosomal events and provide the insight to design rational and efficient gene delivery vector, the adopted strategy exploited the quenching of the fluorescence of CD by Au nanoparticles. The surface of CD and Au was modified with highly cationic polymer, polyethylenimine (PEI) and subsequent treatment with non-labeled pDNA gave rise to quenched delivery complex. High salt concentration triggered the dissociation of the complex with accompanied fluorescence recovery arising due to the increase in distance between CD and Au. The studies revealed the potential of the developed CD-PEI/Au-PEI/pDNA ternary nano-assembly as a highly efficient hybrid transfecting agent with high cell viability under the optimum condition. The changes occurred at the intracellular level during transfection especially post-endosomal step were monitored by fluorescence measurement using fluorescence microscope. This nano-assembly system was found to be very effective at monitoring the carrier/pDNA dissociation in a non-labeled manner, thus provides efficient strategy to study the mechanistic aspect of polymer-mediated pDNA delivery.

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A photoresponsive drug releasing system based on mesoporous silica nanoparticles (MSN) was developed. A model drug was decorated on the surfaces via singlet oxygen sensitive linkers and a singlet oxygen-generating photosensitizer was loaded in the mesopores of the MSN. An on-demand drug releasing system was successfully developed by irradiation with long-wavelength light.

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This work demonstrates the judicious application of a prodrug delivery strategy to achieve a highly improved anticancer drug effect of cisplatin using mesoporous silica nanoparticles. Our effort primarily addressed several pressing needs to overcome various impediments such as toxicity concerns, rapid inactivation, and low drug efficiency of cisplatin prodrug. The developed delivery system utilizes fluorescent mesoporous silica nanoparticles as a template to host the cisplatin prodrug through a reducible linkage. The inactive oxidized Pt(iv) complex installed on the surface of the mesoporous silica nanoparticles in the prodrug conferred stability to cisplatin; however, in the reductive environment of cancer cell lines the active cisplatin form was regenerated. Prodrug-conjugated nanoparticles showed 63 times lower IC50 value than that of cisplatin in HeLa cell line. The delivery system not only demonstrates enhanced cellular uptake but also shows a high drug effect which should diminish the associated side effects. Furthermore, a new, easy and inexpensive fluorescent based Pt quantification method has been adopted instead of the commonly used ICP-based quantification method and this strategy of quantification could be elaborated to monitor fluorescent prodrug nanoparticles during real-time diagnosis.

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Polymeric gene delivery vectors show great potential for the construction of the ideal gene delivery system. These systems harness their ability to incorporate versatile functional traits to overcome most impediments encountered in gene delivery: from the initial complexation to their target-specific release of the therapeutic nucleic acids at the cytosol. Among the numerous multifunctional polymers that have been designed and evaluated as gene delivery vectors, polymers with redox-sensitive (or bioreducible) functional domains have gained great attention in terms of their structural and functional traits. The redox environment plays a pivotal role in sustaining cellular homeostasis and natural redox potential gradients exist between extra- and intracellular space and between the exterior and interior of subcellular organelles. In some cases, researchers have designed the polymeric delivery vectors to exploit these gradients. For example, researchers have taken advantage of the high redox potential gradient between oxidizing extracellular space and the reducing environment of cytosolic compartments by integrating disulfide bonds into the polymer structure. Such polymers retain their cargo in the extracellular space but selectively release the therapeutic nucleic acids in the reducing space within the cytosol. Furthermore, bioreducible polymers form stable complex with nucleic acids, and researchers can fabricate these structures to impart several important features such as site-, timing-, and duration period-specific gene expression. Additionally, the introduction of disulfide bonds within these polymers promotes their biodegradability and limits their cytotoxicity. Many approaches have demonstrated the versatility of bioreducible gene delivery, but the underlying biological rationale of these systems remains poorly understood. The process of disulfide reduction depends on multiple variables in the cellular redox environment. Therefore, the quest to unravel various issues such as the site and time of disulfide bond reduction during the cellular uptake and trafficking have stimulated a number of interesting studies which have employed disulfide compounds with a variety of reducible linkers. Such studies help researchers understand not only how modifications made to disulfides can alter their thiol-disulfide exchange characteristics but also to decipher the effect of the induced changes on the dynamics of the redox environment. This Account discusses current research trends and recent progress in the disulfide chemistry enabling novel and versatile designs of reducible polymeric gene delivery systems. We present strategies for the introduction of disulfide bonds into polymers. These representative examples and their respective outcomes elaborate the benefit and efficiency of disulfides at the individual stages of gene delivery.

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Graphene oxide (GO) has attracted an increasing amount of interest because of its potential applications in biomedical fields such as biological imaging, molecular imaging, drug/gene delivery, and cancer therapy. Moreover, GO could be fabricated by modifying its functional groups to impart specific functional or structural attributes. This study demonstrated the development of a GO-based efficient gene delivery carrier through installation of polyethylenimine, a cationic polymer, which has been widely used as a nonviral gene delivery vector. It was revealed that a hybrid gene carrier fabricated by conjugation of low-molecular weight branched polyethylenimine (BPEI) to GO increased the effective molecular weight of BPEI and consequently improved DNA binding and condensation and transfection efficiency. Furthermore, this hybrid material facilitated sensing and bioimaging because of its tunable and intrinsic electrical and optical properties. Considering the extremely high transfection efficiency comparable to that of high-molecular weight BPEI, high cell viability, and its application as a bioimaging agent, the BPEI-GO hybrid material could be extended to siRNA delivery and photothermal therapy.

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This review will cover the current strategies that are being adopted to efficiently deliver small interfering RNA using nonviral vectors, including the use of polymers such as polyethylenimine, poly(lactic-co-glycolic acid), polypeptides, chitosan, cyclodextrin, dendrimers, and polymers-containing different nanoparticles. The article will provide a brief and concise account of underlying principle of these polymeric vectors and their structural and functional modifications which were intended to serve different purposes to affect efficient therapeutic outcome of small-interfering RNA delivery. The modifications of these polymeric vectors will be discussed with reference to stimuli-responsiveness, target specific delivery, and incorporation of nanoconstructs such as carbon nanotubes, gold nanoparticles, and silica nanoparticles. The emergence of small-interfering RNA as the potential therapeutic agent and its mode of action will also be mentioned in a nutshell.

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In recent years, numerous research activities have been devoted to the controlled release of nitric oxide (NO) due to its potential as a restenosis inhibitor which inhibits the proliferation of vascular smooth muscle cells, the apoptosis of vascular endothelial cells, and aggregation of platelets. This work has demonstrated the development of a novel NO-conjugated gel system comprising of thermosensitive Pluronic F127, branched polyethylenimine (BPEI), and diazeniumdiolates (NONOates). Synthesis of conjugated Pluronic-BPEI-NONOates involved coupling of activated F127 to BPEI followed by the installation of NONOates at the secondary amine sites of branched PEI backbone under high pressure. NO-conjugated gel system, F127-BPEI-NONOates, reduced the initial burst of NO release and prolonged NO release. Furthermore, F127-BPEI-NONOates polymer coated on cell culture dish displayed much higher increase of endothelial cell proliferation and reduction of smooth muscle cell proliferation than that exhibited by non-NO releasing control. Such an NO-releasing device can operate locally and has a great potential in several biomedical applications due to high biocompatibility imparted by the conjugated F127.

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This work demonstrated the development of multifunctional silica nanotubes (SNT) by functionalization of their inner void and outer surface with magnetic-fluorescence nanocomposites and cationic polymers, respectively. The successful construction of BPEI-SNT was established by electron energy loss spectroscopy in conjugation with standard analytical tools. The mean fluorescence intensity in a FACS assay, a luciferase gene expression assay and a confocal fluorescence study demonstrated the efficacy of BPEI-SNT as a gene delivery vector. Endocytotic uptake was also demonstrated by the colocalization of LysoTracker Red(®) and green fluorescent quantum dots. Moreover, enhanced magnetic resonance imaging revealed the potential of the BPEI-SNT nanocomposite to act as a dual-modality nano-device.

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The work demonstrated the successful delivery of gene to mouse brain overcoming the blood-brain barrier (BBB) through expedient vector construct having RVG peptide as targeting ligand for neuronal cells. The newly developed delivery vector was designed to impart bioreducibility for greater intracellular pDNA release, higher serum stability and efficient complexing ability by incorporating disulfide linkage, PEG and low molecular weight polyethylenimine, respectively. The physiochemical properties of the polyplex, its cytotoxicity and the in vitro transfection efficiency on Neuro2a cell were studied prior to the successful in vivo study. In vivo fluorescence assay substantiated the permeation of the pDNA loaded polymeric vector through the BBB. The RVG-mediated target-specific cellular uptake of polymeric vector was established conclusively by competitive assay.

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The work demonstrated development of multifunctional gene carrier which has incorporated reducible moiety, tumor targeting ligands as well as PEG to achieve efficient release of pDNA, enhanced tumor-specificity and long circulation, respectively. In our successful one-pot synthesis of multifunctional polymer, low molecular weight branched polyethylenimine (BPEI) was thiolated with propylene sulfide, and mixed with alpha-Maleimide-omega-N-hydroxysuccinimide ester polyethylene glycol (MAL-PEG-NHS, MW: 5000), and cyclic NGR peptide. The structural elucidation of the cNGR conjugated reducible BPEI containing disulfide bond (BPEI-SS-PEG-cNGR), was done by NMR and GPC study. Complex formation as well as reducible property of the polymer was confirmed by gel retardation assay. In order to achieve efficient tumor targeting, we have used cNGR peptide which is known to bind to CD13 overexpressed in neovasculature endothelial cells. Tumor target-specificity of polymer was established by carrying out competitive inhibition assay with free cNGR peptide. Cellular uptake of polymers was evaluated by confocal laser scanning microscope (CLSM). Finally, addition of free cNGR and buthionine sulfoximine (BSO) reduced transfection efficiency synergistically, which implied that multifunctional polymer-mediated gene transfection took place tumor-specifically and via GSH-dependent pathway.

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The work demonstrated the development of thermally cross-linked superparamagnetic nanomaterial which possessed polyethylene glycol moiety and covalently linked branched polyethylenimine (BPEI), and exhibited highly efficient magnetofection even under serum conditioned media. The study showed its high anti-biofouling, cell viability and serum stability and thus revealed a potential magnetic nanoparticle-mediated targeted gene delivery system. This superparamagnetic particle mediated rapid and efficient transfection in primary vascular endothelial cells (HUVEC) successfully inhibits expression of PAI-1 which is responsible for various vascular dysfunctions such as vascular inflammation and atherosclerosis and thereby provides a potential strategy to transfect highly sensitive HUVEC. The sequential steps for the enhanced magnetofection had been studied by monitoring cellular uptake with the aid of confocal microscopy.

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The work demonstrates a judicious approach to achieve hepatocyte cell-targeted highly efficient gene delivery by utilizing a supramolecular complex of galactosylated cucurbituril and dextran-spermine conjugates.

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Despite the immense potential of non-viral delivery system in gene therapy its application has been impaired greatly by various impediments having contrasting traits. Therefore it is an absolute necessity to develop some non-viral vectors which are endowed with special characteristics to act differently in intracellular as well as extracellular compartments to surmount these inter-conflicting hurdles. Such smart polymers should serve some specific purposes by adjusting their structural or functional traits under the influence of stimuli such as temperature, light, salt concentration or pH. Among all these stimuli-responsive polymers pH-responsive polymers have attracted major attention and great impetus has been directed towards utilizing the subtle yet significant change in pH value within the cellular compartments. This review is intended to provide a comprehensive account of the development of pH-responsive polymeric vectors based on their structural features and consequent functional attributes to achieve efficient transfection. The underlying modes of actions relating to structure and differential pH environment have also been discussed in this review.

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Novel star-shaped copolymers consisting of multiarm polyethylene glycol and low molecular weight linear polyethylenimines (MAPEG-LPEIs) with a high transfection efficiency and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. The cationic polymers were prepared by conjugating low molecular weight linear PEI (2.5 kDa) to six-arm PEG-NHS (10 kDa) in two different compositions. Two copolymers, MAPEG-LPEI(3) and MAPEG-LPEI(6) with molecular weights of 17.5 kDa and 25 kDa respectively, were synthesized. The MAPEG-LPEI(3)/pDNA and MAPEG-LPEI(6)/pDNA polyplexes are stably dispersed in aqueous media with a narrowly distributed size range of <200 nm as determined by dynamic light scattering. Furthermore, these polyplexes showed different surface charges depending upon the relative proportion of MAPEG and LPEI. Moreover, these polyplexes can protect pDNA from enzymatic degradation in serum containing media up to 24 h. These polyplexes were able to efficiently transfect luciferase-coded reporter gene into HeLa cancer cells and showed considerable gene transfection efficacy even in 50% serum-conditioned media in vitro. MAPEG-LPEI(6) exhibited higher transfection activity than that of MAPEG-LPEI(3) at the same weight ratios. Furthermore, MAPEG-LPEI/pDNA polyplexes were less toxic than LPEI/pDNA complexes as determined by MTT assay. These favorable results could be attributed to the combined effect of low molecular weight LPEI and multiarm PEG. The special structural features of the multiarm star-shaped central PEG core play an important role in achieving higher transfection efficiency as it imparts higher charge density to polyplexes and prevents the unwanted aggregation of the smaller polyplex particles. These two important factors contributed toward enhanced gene transfection. On the other hand, LPEI provides low cytotoxicity and effective complexation with pDNA in the designed architecture. Therefore it is possible to achieve enhanced gene transfection by using these two components, namely, pivotal multiarm PEG core and LPEI, in optimal ratio as observed in the case of MAPEG-LPEI(6).

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The work demonstrates the development of acid-labile temperature-responsive sol-gel reversible polymer for enhanced in vivo myocardium and skeletal muscle gene delivery. In this report, multi-block copolymers (MBCPs) synthesized from pluronic and di-(ethylene glycol) divinyl ether (DEGDVE) were used as a delivery vehicle for controlled and sustained release of plasmid DNA (pDNA) in in vitro as well as in vivo experiments. The non-ionic MBCP/pDNA complex showed remarkable transfection efficiencies against the myocardium cells as well as muscle cells in vivo, which is otherwise very difficult to achieve by using cationic polymers. In in vitro experimental settings, this intelligent stimuli-responsive polymer is shown to improve the transfection efficiency of branched polyethylenimine (BPEI)/pDNA complex when used together. The effect of MBCP on the surface charge and particle size of its various complexes with pDNA and BPEI was also studied. The release profile of pDNA from the MBCP gel was investigated and pH of the degraded polymer was also monitored to ascertain its non-cytotoxicity arising due to the increased acidity as observed with other PLGA-based polymers. The rapid sol-gel transition of MBCP under thermal stimuli with concomitant release of pDNA under acidic stimulation has potential for site specific, efficient and controlled transfection of therapeutic gene. In short, MBCP provides the silver lining in combat against the hurdles encountered in transfection to myocardial or other muscle cells.

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Nanometre-scaled DNA machine based on molecular recognition properties of DNA has now become a powerful tool in nanodevices, miniaturized structure, and nanofabrication. The common principle behind designing a DNA nanomachine is DNA strand exchange or rearrangement, which is solely controlled by the stabilization through associative and dissociative forces arising from base pair interaction of DNA molecules. Thus, highly effective DNA reaction actuator will make DNA nanomachine more flexible, controllable, and powerful device. Here, we report the novel polymer-mediated platform in proton-driven DNA strand rearrangement actuation. This cationic low molecular weight water-soluble chitosan (LMWSC) exhibited pH-dependent complexation with oligodeoxynucleotides (ODN). It formed complex with ODN only at low pH and accelerated the DNA strand exchange (or rearrangement) reaction between dsDNA and its complementary ssDNA at pH 5.0. However, no complexation was observed between LMWSC and ODN at neutral pH. We assume that at physiological pH, LMWSC is not protonated enough for formation of complex with ODN. Therefore, it can not diminish the electrostatic repulsion among the negatively charged DNA strands of the three-stranded intermediate formed during the strand exchange reaction. In contrast, LMWSC becomes positively charged at acidic pH, and it stabilizes the three-stranded intermediate by spreading out the accumulated counter-ions and increasing the entropy of the system. This fascinating observation prompted us to believe that this intelligent proton-driven DNA reaction actuator has a potential for the precise control of DNA nanomachine and would be applied for operating and controlling nanoscaled machine.

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A simple synthesis of chiral spironucleosides and spirobisnucleosides is described. Intramolecular 1,3-dipolar nitrone cycloaddition reaction of d-glucose-derived precursors having olefin at C-3 and nitrone at C-5, C-1, or C-2 (in nor-series) furnished bisisoxazolidinospirocycles 4-7, 11, and 12 in good yields. Reductive ring opening of the isoxazolidine moieties in 4-6 followed by construction of a nucleoside base upon the generated amino groups smoothly yielded spirobisnucleosides 17 and 18 and spironucleosides 20 and 21.

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