Acan downregulation in parvalbumin GABAergic cells reduces spontaneous recovery of fear memories.

While persistence of fear memories is essential for survival, a failure to inhibit fear in response to harmless stimuli is a feature of anxiety disorders. Extinction training only temporarily suppresses fear memory recovery in adults, but it is highly effective in juvenile rodents. Maturation of GABAergic circuits, in particular of parvalbumin-positive (PV+) cells, restricts plasticity in the adult brain, thus reducing PV+ cell maturation could promote the suppression of fear memories following extinction training in adults. Epigenetic modifications such as histone acetylation control gene accessibility for transcription and help couple synaptic activity to changes in gene expression. Histone deacetylase 2 (Hdac2), in particular, restrains both structural and functional synaptic plasticity. However, whether and how Hdac2 controls the maturation of postnatal PV+ cells is not well understood. Here, we show that PV+- cell specific Hdac2 deletion limits spontaneous fear memory recovery in adult mice, while enhancing PV+ cell bouton remodeling and reducing perineuronal net aggregation around PV+ cells in prefrontal cortex and basolateral amygdala. Prefrontal cortex PV+ cells lacking Hdac2, show reduced expression of Acan, a critical perineuronal net component, which is rescued by Hdac2 re-expression. Pharmacological inhibition of Hdac2 before extinction training is sufficient to reduce both spontaneous fear memory recovery and Acan expression in wild-type adult mice, while these effects are occluded in PV+-cell specific Hdac2 conditional knockout mice. Finally, a brief knock-down of Acan expression mediated by intravenous siRNA delivery before extinction training but after fear memory acquisition is sufficient to reduce spontaneous fear recovery in wild-type mice. Altogether, these data suggest that controlled manipulation of PV+ cells by targeting Hdac2 activity, or the expression of its downstream effector Acan, promotes the long-term efficacy of extinction training in adults.

Effects of heterocyclic-based head group modifications on the structure-activity relationship of tocopherol-based lipids for non-viral gene delivery.

Gene therapy, a promising strategy for the delivery of therapeutic nucleic acids, is greatly dependent on the development of efficient vectors. In this study, we designed and synthesized several tocopherol-based lipids varying in the head group region. Here, we present the structure-activity relationship of stable aqueous suspensions of lipids that were synthetically prepared and formulated with 1,2-dioleoyl phosphatidyl ethanolamine (DOPE) as the co-lipid. The physicochemical properties such as the hydrodynamic size, zeta potential, stability and morphology of these formulations were investigated. Interaction with plasmid DNA was clearly demonstrated through gel binding and EtBr displacement assays. Further, the transfection potential was examined in mouse neuroblastoma Neuro-2a, hepatocarcinoma HepG2, human embryonic kidney and Chinese hamster ovarian cell lines, all of different origins. Cell-uptake assays with N-methylpiperidinium, N-methylmorpholinium, N-methylimidazolium and N,N-dimethylaminopyridinium head group containing formulations evidently depicted efficient cell uptake as observed by particulate cytoplasmic fluorescence. Trafficking of lipoplexes using an endocytic marker and rhodamine-labeled phospholipid DHPE indicated that the lipoplexes were not sequestered in the lysosomes. Importantly, lipoplexes were non-toxic and mediated good transfection efficiency as analyzed by ß-Gal and GFP reporter gene expression assays which established the superior activity of lipids whose structures correlate strongly with the transfection efficiency.

Systemic delivery of stable siRNA-encapsulating lipid vesicles: optimization, biodistribution, and tumor suppression.

Lipid-based nanoparticles are considered as promising candidates for delivering siRNA into the cytoplasm of targeted cells. However, in vivo efficiency of these nanoparticles is critically dependent on formulation strategies of lipid-siRNA complexes. Adsorption of serum proteins to lipid-siRNA complexes and its charge determine siRNA degradation and serum half-life, thus significantly altering the bioavailability of siRNA. To address these challenges, we developed a formulation comprising dihydroxy cationic lipid, N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium chloride (DHDEAC), cholesterol, and varying concentrations of 1,2-distearoryl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol-2000)] (DSPE-PEG 2000). Using an ethanol dilution method, addition of these lipids to siRNA solution leads to formation of stable and homogeneous population of siRNA-encapsulated vesicles (SEVs). Biodistribution of these SEVs, containing 5 mol % of DSPE-PEG 2000 in xenograft mice, as monitored by live animal imaging and fluorescence microscopy, revealed selective accumulation in the tumor. Remarkably, four intravenous injections of the modified vesicles with equimolar amounts of siRNA targeting ErbB2 and AURKB genes led to significant gene silencing and concomitant tumor suppression in the SK-OV-3 xenograft mouse model. Safety parameters as evaluated by various markers of hepatocellular injury indicated the nontoxic nature of this formulation. These results highlight improved pharmacokinetics and effective in vivo delivery of siRNA by DHDEAC-based vesicles.

Conformation-dependent binding and tumor-targeted delivery of siRNA by a designed TRBP2: Affibody fusion protein.

Efficiency of systemically delivered siRNA in gene silencing is compromised due to lack of target-specific delivery and rapid clearance of siRNA by in vivo elimination pathways. We designed a fusion protein consisting of a dsRNA binding domain of transactivation response RNA binding protein (TRBP2) fused to ErbB2 binding affibody (AF) for target specific delivery of siRNA. Designated as TRAF, the fusion protein is stable and binds efficiently and specifically to siRNA, forming homogenous non-aggregated and nuclease-resistant particles that efficiently and selectively transport siRNA into HER-2 overexpressing cancer cells and tissues. Administration of siRNA by TRAF into cells resulted in significant silencing of chosen genes involved in cell proliferation viz. AURKB and ErbB2. Noticeably, intravenous administration of TRAF:siRNA against these genes resulted in remarkable tumor suppression in the SK-OV-3 xenograft mouse model. Our results establish the potential of engineered proteins for specific and systemic delivery of siRNA for cancer therapy. FROM THE CLINICAL EDITOR: The use of siRNA in one of many novel treatments in cancer therapy. However, a major challenge for using siRNA is the lack of specificity and rapid RNA clearance. In this article, the authors designed a tumor targeting fusion protein, which can deliver siRNA specifically. In the experimental xenograft model, it was shown that intravenous administration of this resulted in significant tumor suppression. The results seem to hold promise in future clinical studies.

Synthesis and transfection efficiency of cationic oligopeptide lipids: role of linker.

In the design of new cationic lipids for gene transfection, the chemistry of linkers is widely investigated from the viewpoint of biodegradation and less from their contribution to the biophysical properties. We synthesized two dodecyl lipids with glutamide as the backbone and two lysines to provide the cationic headgroup. Lipid 1 differs from Lipid 2 by the presence of an amide linkage instead of an ester linkage that characterizes Lipid 2. The transfection efficiency of lipoplexes with cholesterol as colipid was found to be very high with Lipid 1 on Chinese Hamster Ovary (CHO) and HepG2 cell lines, whereas Lipid 2 has shown partial transfection efficiency on HepG2 cells. Lipid 1 was found to be stable in the presence of serum when tested in HepG2 and CHO cells albeit with lower activity. Fluorescence-based dye-binding and agarose gel-based assays indicated that Lipid 1 binds to DNA more efficiently than Lipid 2 at charge ratios of >1:1. The uptake of oligonucleotides with Lipid 1 was higher than Lipid 2 as revealed by confocal microscopy. Transmission electron microscopy (TEM) images reveal distinct formation of liposomes and lipoplexes with Lipid 1 but fragmented and unordered structures with Lipid 2. Fusion of Lipids 1 and 2 with anionic vesicles, with composition similar to plasma membrane, suggests that fusion of Lipid 2 was very rapid and unlike a fusion event, whereas the fusion kinetics of Lipid 1 vesicles was more defined. Differential scanning calorimetry (DSC) revealed a high T(m) for Lipid 1 (65.4 °C) while Lipid 2 had a T(m) of 23.5 °C. Surface area-pressure isotherms of Lipid 1 was less compressible compared to Lipid 2. However, microviscosity measured using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed identical values for vesicles made with either of the lipids. The presence of amide linker apparently resulted in stable vesicle formation, higher melting temperature, and low compressibility, while retaining the membrane fluid properties suggesting that the intermolecular hydrogen bonds of Lipid 1 yielded stable lipoplexes of high transfection efficiency.

Structure-activity relationship of serotonin derived tocopherol lipids.

Tocopherol-based lipids are widely used for nucleic acid delivery. Using tocopherol molecules, we designed and synthesized 5-HT functionalized lipids by tethering 5-hydroxytryptamine (5-HT), a small molecule ligand as the head group to a natural amphiphilic molecule namely α-tocopherol (Vitamin E). This is with the aim of delivering nucleic acids specifically into cells expressing the serotonin receptors (5-hydroxytryptamine[5-HT]) which are abundant in the central nervous system. In order to achieve target recognition, we adopted an approach wherein two structurally different lipid molecules having serotonin as the head group was conjugated to tocopherol via different linkers thus generating lipids with either free -NH2 or -OH moiety. The corresponding lipids designated as Lipid A (Tocopheryl carbonate serotonin-NH2) and Lipid B (Tocopheryl 2-hydroxy propyl ammonium serotonin-OH), were formulated with co-lipids 1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-sn-3-phosphatidylcholine (DOPC) and evaluated for their ability to deliver plasmid DNA through reporter gene expression assays in vitro. Furthermore, the physicochemical characteristics and cellular interactions of the formulations were examined using serotonin-receptor enriched cells in order to distinguish the structural and functional attributes of both lipids. Cell-based gene expression studies reveal that in comparison to Lipid A, a formulation of Lipid B prepared with DOPE as the co-lipid, contributes to efficient uptake leading to significant enhancement in transfection. Specific interactions explored by molecular docking studies suggests the role of the hydroxyl moiety and the enantiospecific significance of serotonin- conjugated tocopherol lipids in recognizing these receptors thus signifying a promising lipid-based approach to target the serotonin receptors in the central nervous system.

Delivery of BACE1 siRNA mediated by TARBP-BTP fusion protein reduces ß-amyloid deposits in a transgenic mouse model of Alzheimer's disease.

Systemic delivery of nucleic acids to the central nervous system (CNS) is a major challenge for the development of RNA interference-based therapeutics due to lack of stability, target specificity, non-permeability to the blood-brain barrier (BBB), and lack of suitable carriers. Using a designed bi-functional fusion protein TARBP-BTP in a complex with siRNA, we earlier demonstrated knockdown of target genes in the brain of both AßPP-PS1 (Alzheimer's disease, AD) and wild-type C57BL/6 mice. In this report, we further substantiate the approach through an extended use in AßPP-PS1 mice, which upon treatment with seven doses of ß-secretase AßPP cleaving Enzyme 1 (BACE1) TARBP-BTP:siRNA, led to target-specific effect in the mouse brain. Concomitant gene silencing of BACE1, and consequent reduction in plaque load in the cerebral cortex and hippocampus (greater than 60%) in mice treated with TARBP-BTP:siRNA complex, led to improvement in spatial learning and memory. The study validates the efficiency of TARBP-BTP fusion protein as an efficient mediator of RNAi, giving considerable scope for future intervention in neurodegenerative disorders through the use of short nucleic acids as gene specific inhibitors.

Restoration of p53 Function in Ovarian Cancer Mediated by Gold Nanoparticle-Based EGFR Targeted Gene Delivery System.

Targeted gene delivery of wild type tumor suppressor gene p53 is a promising approach to inhibit the progression of ovarian cancer. Although several gene delivery vehicles have been reported earlier, there is paucity for targeted delivery of wild type p53 to ovarian cancer using gold nanoparticles. As it is well-known that EGFR (epidermal growth factor receptor) is overexpressed in ovarian cancer, in this study we hypothesized that the FDA approved monoclonal antibody C225 (cetuximab) that targets EGFR could be used for targeted delivery of wild type p53 gene. With this impetus, we devised an approach wherein cationic gold nanoparticles (AuNPs) were employed to generate gold nanoparticle-based drug delivery system (DDS, Au-C225-p53DNA where p53DNA is pCMVp53 plasmid) that was formulated and characterized by biochemical and biophysical methods. The nanoconjugate complexed with DNA (Au-C225-p53DNA) is serum-stable and protects the bound DNA from digestion by DNase-I. Additionally, in vitro reporter gene expression assays demonstrated efficient and specific gene transfection in EGFR overexpressing SK-OV-3 cells. Further, the intraperitoneal administration of Au-C225-p53DNA in SK-OV-3 xenograft mouse model displayed significant tumor targeting and tumor regression. Altogether, these studies indicated a promising nanoparticle-based approach for targeting ovarian cancers caused by mutated p53.

Engineered fusion protein-loaded gold nanocarriers for targeted co-delivery of doxorubicin and erbB2-siRNA in human epidermal growth factor receptor-2+ ovarian cancer.

Designed recombinant proteins comprising functional domains offer selective targeting of cancer cells for the efficient delivery of therapeutic agents. The efficacy of these carriers can be further enhanced by conjugating engineered proteins to nanoparticle surfaces. However, recombinant protein-loaded nanoparticle-based drug delivery systems are not well addressed for ovarian cancer therapy. In the present study, using a combinatorial approach, we designed and fabricated a drug delivery system by combining gold nanoparticles (AuNPs) with an engineered bi-functional recombinant fusion protein TRAF(C) (TR), loaded with an anticancer drug, namely doxorubicin (DX), and erbB2-siRNA (si), to mediate target specific delivery into SK-OV-3, a model human ovarian cancer cell line over expressing HER2 receptors (i.e. human epidermal growth factor receptor-2). The nanoparticle-based targeted drug delivery system, designated as TDDS (Au-TR-DX-si), was found to be stable and homogenous as revealed by physicochemical and biochemical studies in vitro. In addition, TDDS was functional upon evaluation in vivo. Intraperitoneal administration of TDDS at 2.5 mg kg-1 of DX and 0.25 mg kg-1 of erbB2 siRNA into SK-OV-3 xenograft nude mice, revealed target specific uptake and consequent gene silencing resulting in significant tumor suppression. We attribute these results to specific co-delivery of erbB2 siRNA and DX mediated by TDDS into SK-OV-3 cells via HER2 receptors. Additionally, the biodistribution of TDDS, as quantitated by ICP-OES, confirmed tumor-specific accumulation of AuNPs primarily in tumor tissues, which firmly establishes the efficacy of the nanomedicine-based combinatorial approach for the treatment of ovarian cancer in a non-toxic manner. Based on these findings, we strongly believe that the nanomedicine-based combinatorial approach can be developed as a universal strategy for treatment of HER2+ ovarian cancers.

Cell biological and biophysical aspects of lipid-mediated gene delivery.

Cationic lipids are conceptually and methodologically simple tools to deliver nucleic acids into the cells. Strategies based on cationic lipids are viable alternatives to viral vectors and are becoming increasingly popular owing to their minimal toxicity. The first-generation cationic lipids were built around the quaternary nitrogen primarily for binding and condensing DNA. A large number of lipids with variations in the hydrophobic and hydrophilic region were generated with excellent transfection efficiencies in vitro. These cationic lipids had reduced efficiencies when tested for gene delivery in vivo. Efforts in the last decade delineated the cell biological basis of the cationic lipid gene delivery to a significant detail. The application of techniques such as small angle X-ray spectroscopy (SAXS) and fluorescence microscopy, helped in linking the physical properties of lipid:DNA complex (lipoplex) with its intracellular fate. This biological knowledge has been incorporated in the design of the second-generation cationic lipids. Lipid-peptide conjugates (peptoids) are effective strategies to overcome the various cellular barriers along with the lipoplex formulations methodologies. In this context, cationic lipid-mediated gene delivery is considerably benefited by the methodologies of liposome-mediated drug delivery. Lipid mediated gene delivery has an intrinsic advantage of being a biomimetic platform on which considerable variations could be built to develop efficient in vivo gene delivery protocols.

A designed recombinant fusion protein for targeted delivery of siRNA to the mouse brain.

RNA interference represents a novel therapeutic approach to modulate several neurodegenerative disease-related genes. However, exogenous delivery of siRNA restricts their transport into different tissues and specifically into the brain mainly due to its large size and the presence of the blood-brain barrier (BBB). To overcome these challenges, we developed here a strategy wherein a peptide known to target specific gangliosides was fused to a double-stranded RNA binding protein to deliver siRNA to the brain parenchyma. The designed fusion protein designated as TARBP-BTP consists of a double-stranded RNA-binding domain (dsRBD) of human Trans Activation response element (TAR) RNA Binding Protein (TARBP2) fused to a brain targeting peptide that binds to monosialoganglioside GM1. Conformation-specific binding of TARBP2 domain to siRNA led to the formation of homogenous serum-stable complex with targeting potential. Further, uptake of the complex in Neuro-2a, IMR32 and HepG2 cells analyzed by confocal microscopy and fluorescence activated cell sorting, revealed selective requirement of GM1 for entry. Remarkably, systemic delivery of the fluorescently labeled complex (TARBP-BTP:siRNA) in ΑßPP-PS1 mouse model of Alzheimer's disease (AD) led to distinctive localization in the cerebral hemisphere. Further, the delivery of siRNA mediated by TARBP-BTP led to significant knockdown of BACE1 in the brain, in both ΑßPP-PS1 mice and wild type C57BL/6. The study establishes the growing importance of fusion proteins in delivering therapeutic siRNA to brain tissues.

Design of cholesterol arabinogalactan anchored liposomes for asialoglycoprotein receptor mediated targeting to hepatocellular carcinoma: In silico modeling, in vitro and in vivo evaluation.

We have developed active targeting liposomes to deliver anticancer agents to ASGPR which will contribute to effective treatment of hepatocellular carcinoma. Active targeting is achieved through polymeric ligands on the liposome surface. The liposomes were prepared using reverse phase evaporation method and doxorubicin hydrocholoride, a model drug, was loaded using the ammonium sulphate gradient method. Liposomes loaded with DOX were found to have a particle size of 200nm with more than 90% entrapment efficiency. Systems were observed to release the drug in a sustained manner in acidic pH in vitro. Liposomes containing targeting ligands possessed greater and selective toxicity to ASGPR positive HepG2 cell lines due to specific ligand receptor interaction. Bio-distribution studies revealed that liposomes were concentrated in the liver even after 3h of administration, thus providing conclusive evidence of targeting potential for formulated nanosystems. Tumor regression studies indicated greater tumor suppression with targeted liposomes thereby establishing superiority of the liposomal system. In this work, we used a novel methodology to guide the determination of the optimal composition of the targeting liposomes: molecular dynamics (MD) simulation that aided our understanding of the behaviour of the ligand within the bilayer. This can be seen as a demonstration of the utility of this methodology as a rational design tool for active targeting liposome formulation.

Structural changes in DNA mediated by cationic lipids alter in vitro transcriptional activity at low charge ratios.

Lipid/DNA complexes or Lipoplexes have been characterized by various biochemical and biophysical methods to understand the physical basis of transfection. Here we have addressed the effect of cationic liposomes, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), on transcription of DNA templates in vitro. Transcriptional activity of DNA-dependent RNA polymerase at DNA templates complexed with the cationic lipid varied as a function of charge ratio of lipid/DNA. At low charge ratios of 0.3:1 lipid/DNA and up to 1:1, we observed stimulation in transcription, while at higher charge ratios of lipid/DNA 3:1, complete inhibition in the activity occurred. Cetyl tri-methyl ammonium bromide (CTAB), a cationic detergent, and polyethylenimine (PEI), a cationic polymer, also bring about similar changes although to a lesser extent. The stimulation in transcription motivated us to probe into the molecular nature of the lipid/DNA interactions by absorbance spectroscopy and circular dichroism (CD). Upon interaction with lipids, hyperchromicity and susceptibility to micrococcal nuclease has increased, which suggests that the DNA was partially denatured. On complexation with the cationic lipid (DOTAP), the magnitude of the positive band in CD spectra decreased, accompanied with a red shift, as a function of charge ratio. Results from spectroscopic and enzyme assays suggest that at low charge ratios DNA may be partially unwound.

Cationic lipids and cationic ligands induce DNA helix denaturation: detection of single stranded regions by KMnO4 probing.

Cationic lipids and cationic polymers are widely used in gene delivery. Using 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as a cationic lipid, we have investigated the stability of the DNA in DOTAP:DNA complexes by probing with potassium permanganate (KMnO4). Interestingly, thymidines followed by a purine showed higher susceptibility to cationic ligand-mediated melting. Similar studies performed with other water-soluble cationic ligands such as polylysine, protamine sulfate and polyethyleneimine also demonstrated melting of the DNA but with variations. Small cations such as spermine and spermidine and a cationic detergent, cetyl trimethylammonium bromide, also rendered the DNA susceptible to modification by KMnO4. The data presented here provide direct proof for melting of DNA upon interaction with cationic lipids. Structural changes subsequent to binding of cationic lipids/ligands to DNA may lead to instability and formation of DNA bubbles in double-stranded DNA.

Chemically tunable cationic polymer-bonded magnetic nanoparticles for gene magnetofection.

This study evaluates the efficiency of novel non-viral vectors consisting of super paramagnetic iron oxide nanoparticles functionalized with the chemically tunable cationic polymer for in vitro gene magnetofection. The cationic polymer, poly(vinyl pyridinium alkyl halide), with a reactive alkoxysilyl group at one terminal of the polymer (VPCmn, m = length of the side chain and n = polymerization degree), was grafted onto the surface of iron oxide nanoparticles through a silane coupling reaction. The VPCmn grafted-magnetic nanoparticles (Mag-VPCmn) were quaternized with various alkyl halides such as methyl iodide (m = 1), ethyl bromide (m = 2), butyl bromide (m = 4), hexyl bromide (m = 6) and octyl bromide (m = 8). Mag-VPCmn quaternized with a shorter alkyl chain (m = 1, 2, 4 and 6) were water dispersible, but that quaternized with a longer alkyl chain (m = 8) was precipitated in water. The surface of water dispersible Mag-VPCmns was positively charged in pH ranging from 2 to 11, and is stable for more than one month in this pH range. The complexes of Mag-VPCmns and nucleoside molecules with various N/P ratios were evaluated using gel electrophoresis, surface charge (ζ-potential) measurement, and particle size measurement. In vitro transfection experiments were assayed in human embryonic kidney 293 cells (HEK293 cells) using pmaxGFP plasmid as a reporter gene. Gene expression was found to be strongly influenced by the length of the side alkyl chains. Higher transfection efficiencies were observed with longer alkyl chains (C6 > C4 > C2 ≥ C1), indicating that hydrophobic side chains were effective in increasing the transfection efficiency.

Liposomes for targeting hepatocellular carcinoma: use of conjugated arabinogalactan as targeting ligand.

Present study investigates the potential of chemically modified (Shah et al., 2013) palmitoylated arabinogalactan (PAG) in guiding liposomal delivery system and targeting asialoglycoprotein receptors (ASGPR) which are expressed in hepatocellular carcinoma (HCC). PAG was incorporated in liposomes during preparation and doxorubicin hydrochloride was actively loaded in preformed liposomes with and without PAG. The liposomal systems with or without PAG were evaluated for in vitro release, in vitro cytotoxicity, in vitro cell uptake on ASGPR(+) cells, in vivo pharmacokinetic study, in vivo biodistribution study, and in vivo efficacy study in immunocompromised mice. The particle size for all the liposomal systems was below 200 nm with a negative zeta potential. Doxorubicin loaded PAG liposomes released significantly higher amount of doxorubicin at pH 5.5 as compared to pH 7.4, providing advantage for targeted tumor therapy. Doxorubicin in PAG liposomes showed superior cytotoxicity on ASGPR(+) HepG2 cells as compared to ASGPR(-), MCF7, A549, and HT29 cells. Superior uptake of doxorubicin loaded PAG liposomes as compared to doxorubicin loaded conventional liposomes was evident in confocal microscopy studies. Higher AUC in pharmacokinetic study and higher deposition in liver was observed for PAG liposomes compared to conventional liposomes. Significantly higher tumor suppression was noted in immunocompromised mice for mice treated with PAG liposomes as compared to the conventional liposomes. Targeting ability and superior activity of PAG liposomes is established pre-clinically suggesting potential of targeted delivery system for improved treatment of HCC.

Bioinspired peptides as versatile nucleic acid delivery platforms.

Non-viral gene therapy approaches have strongly established the utility of peptides as integral constituents of delivery platforms devised for efficient transfer of therapeutic molecules into cells. Among these, cell-penetrating peptides (CPPs), encompassing a family of short peptide sequences and their chimeric derivatives, have gained versatility through the addition of de novo peptide ligands primarily to facilitate cell-specific nucleic acid delivery in vitro and in vivo. The review illustrates the structural requirements of a noteworthy peptide TAT-PTD and other derivatives chiefly to exemplify their implication in gene therapy. An overview of the emerging concept and recent explorations will be presented through unique examples which form a facet in nanotechnology-based cancer therapy. Finally the basis for the utility of CPPs in plants will be discussed in view of its biotechnological potential.

Gene delivery into human cancer cells by cationic lipid-mediated magnetofection.

In this study, a combination of magnetic nanoparticles (MNPs) together with cationic lipid N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium chloride formulated with colipid cholesterol, upon magnetofection, enhanced DNA uptake into human glioblastoma-astrocytoma, epithelial-like cell line U-87 MG, hepatocellular carcinoma Hep G2, cervical cancer HeLa and breast cancer MDA-MB-231 cells. Having confirmed this, we monitored uptake of plasmid DNA mediated by ternary magnetoplexes by fluorescence microscopy, flow cytometry and reporter gene expression assays in the presence and absence of a magnetic field. Our observations clearly indicate enhanced transfection efficiency in vitro, upon magnetofection, in the presence of serum as seen from ß-Gal reporter gene expression. The observed activity in serum suggests the suitability of MNPs for in vivo applications. Further, we measured the transverse relaxation time (T2) and obtained T2-weighted MRI images of treated U-87 MG cells. T2 determined for MNP-VP-Me22 and MNP-VP-Et22 corresponds to 22.6±0.8 ms and 36.0±2.1 ms, respectively, as compared to 47±1.7 ms for control, suggesting their applicability in molecular imaging. Our results collectively highlight the potential of lipid-based approach to augment magnetic-field guided-gene delivery using MNPs and additionally towards developing intracellular molecular probes for magnetic resonance imaging.

Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate.

Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMVß-gal, resulted in enhanced ß-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of ß-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P<0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.

Targeted liposomes to deliver DNA to cells expressing 5-HT receptors.

Cell targeted delivery of drugs, including nucleic acids, is known to enhance the therapeutic potential of free drugs. We used serotonin (5-HT) as the targeting ligand to deliver plasmid DNA to cells specifically expressing 5-HT receptor. Our liposomal formulation includes the 5-HT conjugated targeting lipid, a cationic lipid and cholesterol. DNA-binding studies indicate that the targeting 5-HT-lipid binds DNA efficiently. The formulation was tested and found to efficiently deliver DNA into CHO cells stably expressing the human serotonin(1A) receptor (CHO-5-HT(1A)R) compared to control CHO cells. Liposomes without the 5-HT moiety were less efficient in both cell lines. Similar enhancement in transfection efficiency was also observed in human neuroblastoma IMR32 and hepatocellular carcinoma (HepG2) cells. Cell uptake studies using CHO-5-HT(1A)R cells by flow cytometry and confocal microscopy clearly indicated that the targeting liposomes through 5-HT moiety may have a direct role in increasing the cellular uptake of DNA-lipid complexes. To our knowledge this is the first report that demonstrates receptor-targeted nucleic acid delivery into cells expressing 5-HT receptor.